CN112390765A - Multi-target antitumor retinoid derivative and synthesis method and application thereof - Google Patents

Abstract

The invention provides a multi-target antitumor retinoid derivative and a synthesis method and application thereof. Based on the multi-target pharmaceutical theory as guidance, Am580 is used as a mother nucleus and combined with the structural characteristics thereof, a furazan nitrogen (Furoxans) NO donor is selected as an NO source, a series of different donors are obtained by chemically modifying the NO donor, and then a series of novel multi-target retinoic acid derivatives are synthesized by coupling a lipid or amido bond with the mother nucleus. The structural general formula of the multi-target antitumor retinoid derivative provided by the invention is as shown in formula (I):

Description

Multi-target antitumor retinoid derivative and synthesis method and application thereof

Technical Field

The invention belongs to the technical field of drug synthesis, and particularly relates to a multi-target antitumor retinoid derivative, a synthesis method and application thereof, in particular to application of the multi-target antitumor retinoid derivative as an anti-leukemia drug.

Background

Retinoic acid (Retinoic acid) class of compounds, which were originally metabolites of vitamin A found in humans, regulate proliferation, differentiation and apoptosis of many cell types in vivo and in vitro. Retinoids have widely different structures and generally consist of three parts: a polar tail, a hydrophobic head and a conjugated chain connecting the head and the tail. Many lead compounds having biological activity have been found by chemically modifying them, and these compounds are widely used for the treatment of diseases such as skin diseases and cancers.

Am580 was first synthesized by Kagechika et al in 1986, and has a strong selective affinity for retinoic acid alpha receptor (RAR α), thereby inducing the synthesis of IL-4, IL-5 and IL-13, inhibiting the synthesis of IL-12 and IFN γ, and more importantly inducing APL cell differentiation in vitro 7 times as much as natural all-trans retinoic acid (ATRA). Due to its good antitumor activity and lower side effects compared to ATRA, it is of great interest to researchers. Am580 and ATRA have the following structural formulas:

nitric Oxide (NO) was initially recognized as a toxic and harmful gas, and since the discovery of its mechanism of action in the human body, research on this signaling molecule has been expanding into various fields since the last 90 s. NO is a short-lived and lipophilic important messenger and effector molecule that participates in a variety of pathophysiological processes in the human body and plays a vital role in the processes of the circulatory system, nervous system, immune system, and the development and progression of cancer. High concentration of NO has definite cytotoxicity, and can generate active nitrogen series (RNS including NO, peroxynitrite ion, hydroxyl radical and the like) by reacting with superoxide anion, molecular oxygen and the like under physiological conditions, and induce apoptosis or necrosis of tumor cells through multiple sites. NO donors (NO donor) refer to compounds capable of releasing NO in vivo after non-metabolism or metabolism, and the compounds can overcome the defects of difficult carrying, difficult quantification, short half-life period and the like of NO. Through development for more than thirty years, research of scientific researchers greatly enriches the types of NO donor compounds, and the existing donor types can be classified into the following types according to chemical structure classification: nitrates, furazan nitroxides, azodialenium salts, oximes, NO-metal complexes, S-nitrosothiols, and the like.

In recent years, many pharmaceutical chemists connect a compound with definite antitumor activity with an NO donor through a connecting group, combine the structure of a known drug or a known active compound with various NO donors through various connecting groups by utilizing a prodrug principle to prepare a prodrug, achieve the effect of multi-target point synergistic treatment by releasing NO in vivo and the compound with antitumor activity, increase the antitumor activity of the drug, and achieve good effect at present.

New retinoid compounds are continuously developed, and have profound influence on the research on molecular mechanisms of the retinoid compounds for inducing cell differentiation, apoptosis and regulating cell proliferation and the development of new ideas in treating related diseases playing a main role in retinoic acid receptors. How to chemically modify NO donors and explore and develop a new tretinoin derivative with multiple target points so as to obtain a new anti-tumor medicine with higher activity, lower toxic and side effects and better anti-tumor effect, which becomes a technical problem to be solved urgently.

Disclosure of Invention

The invention aims to solve the technical problems and provides a multi-target antitumor retinoid derivative, a synthetic method and application thereof. Based on the multi-target pharmaceutical theory as guidance, Am580 is used as a mother nucleus and combined with the structural characteristics thereof, a furazan nitrogen (Furoxans) NO donor is selected as an NO source, a series of different donors are obtained by chemically modifying the NO donor, and then a series of novel multi-target retinoic acid derivatives are synthesized by coupling a lipid or amido bond with the mother nucleus.

One of the objects of the present invention is to provide a multi-target antitumor retinoid derivative having the following general structural formula (i):

wherein R1 is selected from the following groups with the carbon atom number of 2-4: a diol residue, a methylenebenzenediol residue, or an amino acid residue; or, R1 is selected from a linking group having the following structural formula (ii):

wherein R5 is selected from the following groups with the carbon atom number of 2-6: a diol residue, a diamine residue, an ethanolamine residue, or a pentyne residue;

wherein R2 is phenyl or benzenesulfonyl.

The above-mentioned C2-4 diol residue and C2-6 diol residue mean a partial residue of C2-4 diol from which 2 hydroxyl hydrogen atoms have been removed, for example, O (CH)₂)₂O、O(CH₂)₃O、O(CH₂)₄O、O(CH₂)₅O、O(CH₂)₆O。

The diamine residue as described above means a partial group of diamine left by removing one hydrogen atom from each of two amino groups, e.g., O (CH)₂)₂NH。

The ethanolamine residue as mentioned above means a partial group obtained by removing one hydrogen atom from each of the alcoholic hydroxyl group and the amino group, for example, O (CH)₂)₂NH。

The above-mentioned nitrate residue means a group remaining after removal of one hydrogen atom from the hydroxyl group of 3-hydroxy-propylene nitrate, for example, OCH₂CH₂(ONO₂)CH₂ONO₂。

The invention also aims to provide a synthetic method of the multi-target anti-tumor retinoid derivative, which comprises the following steps:

(1) dissolving 3- (chloromethyl) -4-phenyl-1, 2, 5-oxadiazole 2-oxide in an organic solvent, and carrying out catalytic reaction with the raw material to obtain an intermediate product, wherein the raw material comprises any one of benzenediol, dihydric alcohol, alcohol amine, diamine or various amino acids;

(2) dissolving the intermediate product in the step (1) in an organic solvent, and reacting with an agonist Am580 under the action of a catalyst to obtain a target product shown in the structural general formula (I).

Further, the catalyst for catalyzing the reaction in step (1) comprises potassium carbonate or a mixture of cesium carbonate and potassium iodide.

Further, the organic solvent in the step (2) includes tetrahydrofuran or dichloromethane.

Further, the catalyst in step (2) comprises any one of the following mixtures: a mixture of EDC and DMAP, a mixture of EDC and HOBT, and a mixture of DMAP and DCC.

The target product (I) obtained from the raw materials and the synthetic route thereof are shown as follows:

the specific preparation method of the key intermediates required for preparing the target product (I) in the invention is as follows:

(1) taking 3-phenyl-prop-2-ene-1-ol (23) as an initial raw material, catalyzing and converting the initial raw material by sodium nitrite and acetic acid into 3- (hydroxymethyl) -4-phenyl-1, 2, 5-oxadiazole 2-oxide (24), and reacting the product with thionyl chloride to obtain a key intermediate 3- (chloromethyl) -4-phenyl-1, 2, 5-oxadiazole 2-oxide (2);

(2) thiophenol (10) is taken as an initial raw material and reacts with chloroacetic acid to obtain 2- (thiophenyl) acetic acid (11), and the product is oxidized by hydrogen oxide and nitric acid sequentially to obtain an intermediate 2- (benzenesulfonyl) acetic acid (12) and a 3, 4-bis (benzenesulfonyl) -1,2, 5-oxadiazole 2-oxide (13).

The specific synthetic route is as follows:

the invention provides a synthesis method of a multi-target anti-tumor retinoid derivative with another structure, which comprises the following steps:

(1) dissolving thiophenol in ethanol, and obtaining 2- (benzenesulfonyl) acetic acid by base catalysis;

(2) oxidizing and nitrifying the product obtained in the step (1) to obtain 3, 4-bis (benzenesulfonyl) -1,2, 5-oxadiazole 2-oxide, and reacting with dihydric alcohol with the carbon atom number of 2-4 to obtain an oxazole oxide intermediate;

(3) and (3) carrying out base catalysis on the intermediate obtained in the step (2) in a solvent to obtain the compound shown in the general structural formula (I).

Specifically, the base for catalysis in the step (1) and the step (3) is sodium hydroxide.

Specifically, the solvent in the step (3) is dichloromethane.

The synthetic route of the synthetic method is as follows:

the invention also aims to provide application of the multi-target antitumor retinoid derivatives, and particularly application of the multi-target antitumor retinoid derivatives in preparation of medicines for preventing or treating various leukemias.

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

based on the multi-target pharmaceutical theory as guidance, Am580 is used as a mother nucleus and combined with the structural characteristics thereof, a furazan nitrogen (Furoxans) NO donor is selected as an NO source, a series of different donors are obtained by chemically modifying the NO donor, and then a series of novel multi-target retinoic acid derivatives are synthesized by coupling a lipid or amido bond with the mother nucleus.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments, and it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.

Example 1

Synthesis of 3- (hydroxymethyl) -4-phenyl-1, 2, 5-oxadiazole 2-oxide (24)

Cinnamyl alcohol (1.34g, 10mmol) is dissolved in acetic acid (20mL) under the protection of argon, and NaNO is slowly added in portions in an ice-water bath₂(2.07g, 30mmol), after the reaction was warmed to room temperature and reacted for 4h, the reaction was diluted with ice water and extracted with ethyl acetate (3X 30 mL). The combined organic phases were washed with brine and then over anhydrous Na₂SO₄Drying and concentration under reduced pressure gave an orange oily liquid which was then purified by silica gel column chromatography (hexane: ethyl acetate 5:1) to give 24(0.55g, 24.9%) as a yellow solid, Mp 64-66 ℃. 1H NMR (CDCl3,400MHz): delta 7.85-7.82(m,2H),7.61-7.54(m,3H),4.77-4.76(d,2H, J ═ 4.89Hz),2.82(s, 1H).

Example 2

Synthesis of 3- (chloromethyl) -4-phenyl-1, 2, 5-oxadiazole 2-oxide (2)

In a 250mL flask, compound 24(2.00g, 0.01mol) was dissolved in dichloromethane (80mL), anhydrous pyridine (2mL) was added, thionyl chloride (2mL, 0.027mol) was added dropwise under ice bath conditions, and the mixture was stirred at room temperature overnight, followed by washing with ice water (80mL), separation of an organic layer, adjustment of pH to 7 with a saturated sodium bicarbonate solution, washing with saturated brine, drying over anhydrous sodium sulfate, filtration, and concentration to give orange-yellow oil 2(4.20g, 87%).

Example 3

3- ((4-hydroxyphenoxy) methyl) -4-phenyl-1, 2, 5-oxadiazole 2-oxide (4a),

3- ((3-hydroxyphenoxy) methyl) -4-phenyl-1, 2, 5-oxadiazole 2-oxide (4b),

general procedure for the Synthesis of 3- ((2-hydroxyphenoxy) methyl) -4-phenyl-1, 2, 5-oxadiazole 2-oxide (4c)

Compound 2(3.00g,0.014mol) was dissolved in 120mL of acetonitrile, and 3a (2.2g,0.018mol), or 3b-c (0.018mol), anhydrous potassium carbonate 10.90g, potassium iodide 2.00g, and the mixture was added thereto and reacted at room temperature for 4 hours. Filtering, evaporating solvent to obtain reddish brown liquid, extracting with diethyl ether (50 ml. times.3), mixing diethyl ether layers, and concentrating to obtain pale white solids 4a, 4b, 4c (4.22g, yield 98%), Mp 61-63 deg.C.

Example 4

Synthesis of 4-phenyl-3- ((4- ((4- (5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalene-2-carboxamido) benzoyl) oxy) phenoxy) methyl) -1,2, 5-oxadiazole 2-oxide (1a)

Compound Am580(1.05g, 3mmol) was dissolved in THF (30ml) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (0.96g, 5mmol) and 4-Dimethylaminopyridine (DMAP) (0.12g,1mmol) were added and the reaction stirred at room temperature for 2h to give active intermediate solution A and compound 4a dissolved in THF (30ml) to give solution B. Slowly dripping the solution A into the solution B at room temperature, keeping the same temperature for reacting for 6h after dripping is finished, then concentrating under reduced pressure to about 25mL volume, adding distilled water (200mL) under stirring, standing for 5h after stirring for 0.5h to precipitate a solid, performing suction filtration to obtain a crude product, performing column chromatography (petroleum ether: ethyl acetate: 3:1) separation on the crude product, and concentrating the eluent under reduced pressure to obtain a white solid 1a (1.36g, 73.5%) and Mp 140-. 1HNMR (300MHz, DMSO-d6) δ:1.24(s,6H),1.26(s,6H),1.65(s,4H),5.26(s,2H),7.13(m,2H),7.29(m,3H),7.63(m,4H),7.69(d, J ═ 2.4Hz,1H),7.84(m.2h),8.15(d, J ═ 8.4Hz,2H),8.24(d, J ═ 8.4Hz,2H),10.34(s, 1H).

Example 5

Synthesis of Compound 6a

Respectively dissolving a compound 2(2.10g and 10mmol) and a compound 5a (10mmol) in 60mL of anhydrous DMF, adding cesium carbonate (3.30g and 10mmol) and potassium iodide (1.7g and 10mmol), stirring at room temperature for reaction for 3h, pouring the reaction liquid into 150mL of water under stirring, adding ether for extraction for 3 times (100mL multiplied by 3), combining organic layers, washing with a saturated sodium bicarbonate solution and a saturated sodium chloride solution in sequence, drying with anhydrous sodium sulfate, carrying out suction filtration, concentrating the filtrate to remove the solvent, and obtaining an oily intermediate product 6 a.

Example 6

Synthesis of Compound 7a

Compound 6a (1.0g, 2.5mmol) was added to a solution of trifluoroacetic acid and dichloromethane (15mL, 5.0mol/L), respectively, stirred at room temperature for 6h, then concentrated under reduced pressure to remove the solvent to give a crude product, which was subjected to column chromatography (ethyl acetate: methanol ═ 5:1), and concentrated under reduced pressure to give 3- ((glycogenoxy) methyl) -4-phenyl-1, 2, 5-oxadiazole 2-oxide (7a) (0.48g, 78.3%) as a yellow oil.

Example 7

Synthesis of Compound 1g

Compound Am580(1.0g, 2.8mmol) was dissolved in THF (30m L), EDC (0.96g, 5mmol) and 1-Hydroxybenzotriazole (HOBT) (0.68g, 5mmol) were added and the reaction was stirred at room temperature for 2h to give a first solution of the active intermediate, and intermediate 7a (2.8mmol) was dissolved in THF (60mL) respectively to give a second solution. Dropwise adding the active intermediate solution I into the solution II under stirring at room temperature, after dropwise addition within 1h, continuing to stir at room temperature for reaction for 6h, then concentrating under reduced pressure, evaporating to remove the solvent, adding diethyl ether (200m L) into the residue to dissolve, and sequentially dissolving with HCl solution (50ml, 0.1mol/L), saturated NaCl solution, saturated NaHCO solution₃Washing the solution, drying over anhydrous sodium sulfate, concentrating under reduced pressure to dryness to obtain a crude product, separating and purifying by column chromatography (petroleum ether: ethyl acetate: 2:1), and concentrating under reduced pressure to obtain white solid powder 4-phenyl-3- ((((((((4- (5,5,8, 8-tetramethyl-5, 6,7, 8-tetrahydronaphthalene-2-carboxamido) benzoyl) glycyl) oxy) methyl) -1,2, 5-oxadiazole 2-oxide (1g) (1.36g, 83.5%), Mp178-181 ℃. 1HNMR (300MHz, DMSO-d6) δ:1.24(s,6H),1.25(s,6H),1.65(s,4H),4.08(d, J ═ 5.7Hz,2H),5.22(s,2H),7.18(d, J ═ 8.7Hz,1H),7.59(m,4H) 7.74(m,1H),7.96(m,2H),7.98(q, J ═ 8.4Hz,4H),9.17(t, J ═ 5.7Hz,1H),10.210(s, 1H).

Example 8

Synthesis of 3- ((4- (methoxycarbonyl) phenoxy) methyl) -4-phenyl-1, 2, 5-oxadiazole 2-oxide (8)

Dissolving compound 2(5.00g, 23.7mmol) and methyl p-hydroxybenzoate (3.65g, 24.0 mmol 1) in DMF (120mL), adding anhydrous potassium carbonate (13.30g, 96 mmol 1) and potassium iodide (3.98g, 24 mmol 1), stirring at room temperature for 10h, pouring the reaction solution into 500mL water, stirring for 30min, precipitating a solid, filtering, dissolving the filter cake in 200mL diethyl ether, washing with saturated sodium bicarbonate solution and saturated sodium chloride solution in turn, drying with anhydrous sodium sulfate, filtering, collecting the filtrate, concentrating the filtrate, removing the solvent to obtain a crude product, separating with silica gel column chromatography (petroleum ether: ethyl acetate ═ 5:1), and distilling under reduced pressure to remove the solvent to obtain a white crystalline solid 8(6.6g, 85.5%), Mp 97-98 ℃. 1HNMR (300MHz, DMSO-d6) delta: 3.82(s,3H),5.31(s,2H),7.13(m,2H),7.56(m,3H),7.81(m,2H),7.92(m, 2H).

Example 9

Synthesis of Compound 9a

Dissolving a compound 8(10.0g, 30.6mmo1) in DMF (200mL) and water (200mL), adding lithium hydroxide monohydrate (12.8g, 306mmo1), stirring at room temperature for reaction for 10 hours, then dropwise adding acetic acid under vigorous stirring to adjust the pH value to be about 3.5, standing at room temperature for 6 hours, carrying out suction filtration, washing a filter cake to be neutral in pH, carrying out suction drying to obtain a crude product, and recrystallizing the crude product with methanol to obtain a white intermediate solid; then, dissolving the intermediate (1.0g, 3.2mmo1) in anhydrous THF (60mL), adding CDI (1.6g, 10mmol), and reacting at 50 ℃ for 3h to obtain an active intermediate solution I; the diol residue, diamine residue, ethanolamine residue, pentyne residue, etc. (20mmol) were dissolved in THF (50mL) to obtain a second solution. And slowly dropwise adding the active intermediate solution I into the solution II under stirring at room temperature, reacting at the same temperature for 10 hours, stopping the reaction, concentrating under reduced pressure to remove the solvent, adding 150mL of water under stirring, standing at room temperature for 5 hours, performing suction filtration to obtain a crude product, performing column chromatography (petroleum ether: ethyl acetate: 2:1) separation on the crude product, and concentrating the eluent under reduced pressure to dryness to obtain a pure product.

Synthesis of Compound 1m

Compound Am580(1.00g, 2.8mmol) and compound 9a (3mmol) are dissolved in CH₂Cl₂(30ml), DCC (0.62g,3mmol) and DMAP (0.12g,1mmol) are added, the mixture is stirred and reacted for 3h at room temperature, a large amount of white floccule is generated, then the reaction solution is filtered, the filtrate is decompressed and concentrated to be dry to obtain a crude product, the crude product is separated by column chromatography (petroleum ether: ethyl acetate ═ 2:1), and the eluent is decompressed and concentrated to obtain a pure product 1 m.

Example 10

Synthesis of Compound 14a

Compound 13a (13.6mmol) and ethanol (1.00g, 2.7mmol) were dissolved in tetrahydrofuran (20ml), and a 25% aqueous solution of sodium hydroxide (0.5ml) was added dropwise, followed by reaction at room temperature for 1 hour and filtration of the solid. The filtrate was concentrated under reduced pressure, water (50ml) was added to the residue, extraction was performed with ethyl acetate (20 ml. times.3), and the organic phases were combined, washed successively with saturated brine, and dried over anhydrous magnesium sulfate. Then, the reaction mixture was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (ethyl acetate: petroleum ether: 1: 4) to obtain a white powdery solid 14 a.

Example 11

Synthesis of Compound 1d

After a compound Am580(1.00g, 2.8mmol) and a compound 14a (3.0mmol) were dissolved in 30mL of dichloromethane, Dicyclohexylcarbodiimide (DCC) (0.62g,3.0mmol) and DMAP (0.12g,1.0mmol) were added, the mixture was stirred at room temperature for reaction for 3 hours, a large amount of white floc was generated, the reaction solution was filtered under suction, the filtrate was concentrated under reduced pressure to dryness to obtain a crude product, the crude product was subjected to column chromatography (petroleum ether: ethyl acetate: 3:1) to separate the crude product, and the eluent was concentrated under reduced pressure to obtain a pure product 1 d.

Experimental example 1

1NO Release test

Nitrite (NO)₂-) and Nitrate (NO)₃-) is a stable product of NO final metabolism, using the determination of NO₂-and NO₃The amount of-can indirectly reflect the amount of NO produced. NO is very easily oxidized in vivo or in aqueous solution to generate nitrite ions (NO)₂-) capable of diazotizing and coupling with Griess reagent to give a rose-red compound, the concentration of diazo compound formed being determined by the reaction with NO₂The concentration has a linear relationship, and the absorbance value can be measured at a certain wavelength, so that the nitric oxide release amount can be indirectly measured.

1.1 instruments and reagents

The instrument comprises the following steps: ultraviolet visible spectrophotometer (Shimadzu UV-2550 type, Shimadzu Corp.)

Preparing a Griess reagent: sulfanilamide 4g and naphthylethylenediamine hydrochloride 0.2g are weighed and placed in a 100mL brown volumetric flask, and 10mL of 85% phosphoric acid is added and diluted to 100mL with distilled water. And placing in the dark for later use.

Preparation of a phosphate buffer solution (pH 7.4) containing excess L-cysteine: 6.8g of potassium dioxyphosphate is weighed, 395mL of 0.1mol/L sodium oxysulfate solution and 0.6g of L-cysteine are added, and the mixture is dissolved and diluted to 1000mL by water to obtain a phosphate buffer solution with pH 7.4 and containing excess L-cysteine (5 mmol).

Preparing sodium nitrite standard solution: accurately weighing 0.15g of dried sodium hyaluronate (dried at 110 ℃ for 1h), placing in a 100mL volumetric flask, dissolving with appropriate amount of distilled water, diluting to scale, and shaking up. Accurately weighing 0.1mL to 100mL volumetric flasks from the middle, diluting the flasks to the scale with water, and shaking up to obtain 1.5mg/L sodium nitrite standard solution.

1.2 preparation of the Standard Curve

Precisely sucking 1.0mL of sodium nitrite standard solution, placing the sodium nitrite standard solution in a 10mL volumetric flask, diluting the sodium nitrite standard solution to a scale with distilled water, and shaking up to obtain the sodium nitrite standard working solution with the concentration of 0.15 mg/L. Precisely absorbing 8.0mL of the working solution, placing the working solution in a 20mL test tube with a plug, precisely adding 2.0mL of Grignard reagent, fully mixing the solution, placing the solution at room temperature for 10min, scanning the solution within the range of 500-700nm, and determining an ultraviolet visible scanning spectrum with the maximum absorption wavelength of about 540 nm.

Precisely measuring 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0mL of sodium nitrite standard solution, respectively placing the standard solution in 6 10mL volumetric flasks, diluting the standard solution to a scale with distilled water, shaking up, and preparing sodium nitrite series standard working solution with the concentration of 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5mg/L respectively. Precisely measuring 8.0mL of each of the sodium nitrite series standard working solutions, placing the sodium nitrite series standard working solutions into 6 20mL test tubes with plugs, precisely adding 2.0mL of Grignard reagent, fully mixing, placing at room temperature for 10min, and measuring the absorbance at the maximum absorption wavelength measured before. Drawing a standard curve according to the obtained data, and performing regression calculation on the concentration of the sodium nitrite standard working solution by using the absorbance value A to obtain a regression equation A which is 0.0257C +0.2524 (r)²0.998), the linear relationship is good.

1.3NO Release test

Precisely measuring 1.0mL of the synthesized test sample, placing the test sample in a 100mL volumetric flask, diluting the test sample to a scale with a phosphate buffer solution containing excessive L-cysteine (5mmol/L), and shaking up to make the final concentration of each test sample solution be 10-4 mol/L. Incubating the test solutions at 37 ℃ for 3h, precisely sucking 8.0mL of reaction solution, respectively placing in 20mL test tubes with plugs, precisely adding 2.0mL Grice reagent, mixing well, standing at room temperature for 10min, and measuring absorbance at the maximum absorption wavelength. Calculating the NO of each sample solution according to the obtained standard curve₂-and thereby indirectly reflects the NO release of the respective test sample.

TABLE 1NO Release ratio

2MTT method for measuring activity of anti-tumor cells

2.1 culture of cell lines

Human leukemia HL-60, NB4 and K562 cell lines were transferred to a cell culture flask, and cultured in a medium (RPMI-1640 complete medium containing 10% fetal bovine serum) at 37 ℃ under 5% saturated humidity.

Taking 1 bottle of certain cells in logarithmic growth phase, blowing and beating uniformly, taking cell suspension to prepare a blood cell counting plate smear, counting the number of the cells under an inverted microscope, adding a culture medium to adjust the number of the cells to 10⁵/mL。

2.2 design and handling of the experiments

Taking a 96-well cell culture plate for cell inoculation and drug experiments, and setting a blank control group, a negative control group, a positive control group and a drug experiment group, wherein the blank control group is only added with 150 mu L/hole of cell culture solution, the negative control group is seeded with 100 mu L/hole of cell suspension and is added with 50 mu L/hole of cell culture solution, the positive control group is seeded with 100 mu L/hole of cell suspension and is added with 50 mu L/hole of positive control drug solution, the drug experiment group is seeded with 100 mu L/hole of cell suspension and is added with 50 mu L/hole of compound solution to be detected, and the positive control group and the drug experiment group are respectively set with 5 different final drug concentrations of 0.01, 0.1, 1, 10 and 100 mu moL-1 and are respectively set with 3 parallel compound holes. After the drug addition was complete, the 96-well cell culture plate was placed in CO₂5% CO at 37 ℃ in an incubator₂And culturing for 48h under saturated humidity condition.

And (3) taking the 96-well cell culture plate, adding 20 mu L of 5mg/mL MTT solution into each well, continuously culturing for 4h, taking out the culture plate, centrifuging for 30min at 2000rpm, removing culture solution in each well, adding 100 mu L of DMSO into each well, oscillating for 15min on a plate oscillator to completely dissolve the formazan crystals, measuring the OD value of each well at the wavelength of 570nm by using a microplate reader, calculating the cell proliferation inhibition rate of each drug at different concentrations, and calculating the half inhibition concentration by using the sps software (IC 50). Wherein the cell proliferation inhibition rate is calculated according to the following formula:

cell proliferation inhibition (%) was ═ (% of negative control wells mean OD value-drug wells mean OD value ÷ (negative control wells mean OD value-blank wells mean OD value) × 100%

The median Inhibitory Concentration (IC) was calculated in this way₅₀) The value is obtained.

TABLE 2 IC of three cell lines₅₀Value of

The results in table 2 show that the compounds obtained by the present invention have better biological activity for both NO-releasing ability and proliferation-inhibiting activity for leukemia HL-60, NB4 and K562 cell lines compared with the positive control. The design idea of the multi-target synergistic medicine has considerable feasibility and practicability, and the design idea can simultaneously act on a plurality of pathological links of the same disease, and the advantage that a plurality of medicine targets play a synergistic treatment role has great significance for the design and development of the anti-tumor medicine.

Claims (9)

1. A multi-target anti-tumor retinoid derivative, which has a general structural formula shown in formula (I):

wherein R is₁Selected from the following groups with 2-4 carbon atoms: a diol residue, a methylenebenzenediol residue, or an amino acid residue; or, R₁A linking group selected from those having the following structural formula (II):

wherein R is₅Selected from the following groups with 2-6 carbon atoms: a diol residue, a diamine residue, an ethanolamine residue, or a pentyne residue;

wherein R is₂Is phenyl or phenylsulfonyl.

2. A method for synthesizing the multi-target anti-tumor retinoid derivative according to claim 1, comprising the steps of:

(1) dissolving 3- (chloromethyl) -4-phenyl-1, 2, 5-oxadiazole 2-oxide in an organic solvent, and carrying out catalytic reaction with corresponding raw materials to obtain an intermediate product, wherein the corresponding raw materials comprise any one of benzenediol, dihydric alcohol, alcohol amine, diamine or various amino acids;

(2) dissolving the intermediate product obtained in the step (1) in an organic solvent, and reacting with an agonist Am580 under the action of a catalyst to obtain a target product shown in a structural general formula (I).

3. The synthesis method according to claim 2, wherein the catalyst for catalyzing the reaction in step (1) comprises potassium carbonate or a mixture of cesium carbonate and potassium iodide.

4. The method of claim 2, wherein the organic solvent in step (2) comprises tetrahydrofuran or dichloromethane.

5. The synthesis method according to claim 2, wherein the catalyst in step (2) comprises any one of the following mixtures: a mixture of EDC and DMAP, a mixture of EDC and HOBT, and a mixture of DMAP and DCC.

6. A method for synthesizing the multi-target anti-tumor retinoid derivative according to claim 1, comprising the steps of:

(1) dissolving thiophenol in ethanol, and obtaining 2- (benzenesulfonyl) acetic acid by base catalysis;

(2) oxidizing and nitrifying the product obtained in the step (1) to obtain 3, 4-bis (benzenesulfonyl) -1,2, 5-oxadiazole 2-oxide, and then reacting with dihydric alcohol with the carbon atom number of 2-4 to obtain an oxazole oxide intermediate;

(3) and (3) carrying out base catalysis on the intermediate obtained in the step (2) in a solvent to obtain the compound shown in the general structural formula (I).

7. The synthesis process according to claim 6, wherein the base for catalysis in step (1) and step (3) is sodium hydroxide.

8. The method of claim 6, wherein the solvent in step (3) is dichloromethane.

9. The use of the multi-target anti-tumor retinoid derivative according to claim 1 or the multi-target anti-tumor retinoid derivative obtained according to the synthesis process of any one of claims 2 to 8, for preparing a medicament for preventing or treating various leukemias.