CN106905184B - Nitrogen mustards compound containing benzamide group and its preparation method and application - Google Patents

Abstract

The nitrogen mustard compound containing benzamide group and its preparation method and use relate to the field of medicinal chemistry. The compounds have the ability to inhibit the proliferation of cancer cells and achieve the purpose of treating cancer. In particular, it has excellent activity of inhibiting the proliferation of human skin melanoma A375, human liver cancer cell HepG2, human liver cancer cell SMMC7721, human lung cancer cell A549 and human lung cancer cell H1299. The compound has the structure shown in the following general formula I, wherein Z, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ and X ₈ are as defined in the specification of the present application.

Description

Nitrogen mustard compound containing benzamide group and preparation method and use thereof

technical field

The invention relates to the field of medicine, in particular to a nitrogen mustard compound containing a hydroxamic acid group and a preparation method and application thereof.

Background technique

Chlorambucil drugs, such as chlorambucil, bendamustine hydrochloride, etc., are the first alkylating antitumor drugs that have been used clinically and have achieved outstanding curative effects. These drugs kill cells by directly destroying their DNA. Such drugs are not selective for cancer cells, so they have strong toxic and side effects, which limit their clinical application.

Histone deacetylase (HDAC) and histone acetyltransferase (HAT) are a class of mutually antagonistic proteases widely present in eukaryotic cells. Acetylation to bring it into equilibrium. Acetylation and deacetylation of histones are a way to regulate gene transcription. The degree of acetylation of histones affects gene expression by affecting the structure of chromatin. HDACs are overexpressed in tumor cells, thereby inhibiting the expression of certain tumor suppressor genes. In recent years, HDAC inhibitors have become important targets for anti-tumor effects. Among them, a class of HDAC inhibitors with a benzamide structure has attracted attention due to their good oral bioactivity and anti-tumor activity. Many amide HDAC inhibitors are in the clinical research stage. Among them, Tacedinaline (CI-994) shows a certain inhibitory activity against HDAC and has a broad-spectrum anti-tumor activity, becoming the first amide HDAC inhibitor to enter clinical trials. CI-994 is currently in phase II clinical trials in combination with gemcitabine for the treatment of patients with solid tumors such as non-small cell lung cancer and colon cancer.

The above formula is the design strategy of the compound structure of this patent

At present, targeted therapy has become an important direction of cancer treatment, and the treatment mode of one drug and one target is mostly adopted. However, tumors are different from general diseases. Their growth and survival not only depend on the transduction of a receptor or a signaling pathway, which makes a strategy that simply acts on one target cannot kill tumor cells completely, and is easy to produce drug resistance. Therefore, the combination of multiple drugs has become the main method of clinical treatment of cancer. Although the expected therapeutic effect can be achieved to a certain extent, due to the easy interaction of multiple drugs with each other, such as affecting the absorption and metabolism of drugs, even if There is no interaction, and these drugs usually cannot be used in combination at the doses when they are used alone. For this reason, drug researchers draw on the principle of multi-drug combination and use the method of combining two or more drugs. The pharmacophore is assembled into a molecule, and the molecule itself or its metabolites act on two or more targets, thereby producing a synergistic effect to improve the efficacy.

The inventor of the present patent creatively introduced a benzamide structure with HDAC inhibitory activity into the structure of chlorambucil, and synthesized nitrogen mustard compounds containing benzamide groups, which also have nitrogen mustard groups. The antitumor activity of the group, but also the inhibitory activity of HDAC. The evaluation results of in vitro anti-tumor cell activity, effect on tumor cell monoclonal formation, effect on tumor cell cycle and effect on tumor cell apoptosis show that the compound in this patent has higher anti-tumor activity than the parent drug chlorambucil 4 to 20 times more potent and 9 to 46 times more potent than the parent drug bendamustine hydrochloride.

SUMMARY OF THE INVENTION

The present invention relates to the design and synthesis of a class of compounds. In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a nitrogen mustard compound containing a hydroxamic acid group with better antitumor activity and a preparation method and application thereof. The reason why the present invention can solve the above-mentioned problems is to realize through the following technical solutions:

In the first aspect of the present invention, there is provided a compound represented by the general formula I or a pharmaceutically acceptable salt or a solvate thereof or a prodrug molecule thereof:

in,

X ₁ , X ₂ , X ₃ , X ₄ are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, amino, hydroxyl, mercapto, carboxyl, Alkoxy, cycloalkoxy, halogenated aryl, alkoxycarbonyl, halogen, cyano, amido, thiocyano, isothiocyano, ureido, sulfo;

Z is selected from alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, the aforementioned alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkyne group is optionally unsubstituted or substituted with one or more substituents each independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, amino , hydroxyl, mercapto, carboxyl, alkoxy, cycloalkoxy, halogen, cyano, nitro, nitroso, sulfo;

X ₅ , X ₆ , X ₇ , X ₈ are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, amino, hydroxyl, mercapto, carboxyl, Alkoxy, cycloalkoxy, haloaryl, alkoxycarbonyl, halogen, cyano, amido, thiocyano, isothiocyano, ureido, sulfo.

In a preferred embodiment of the first aspect of the invention, the compound is preferably selected from the following characteristics:

X ₁ , X ₂ , X ₃ , X ₄ are each independently selected from hydrogen, alkyl, alkoxy, cycloalkoxy, alkoxycarbonyl, halogen, cyano, nitro, nitroso, amido;

Z is selected from alkyl, aryl, alkenyl, and alkyne, and the above-mentioned alkyl, aryl, and alkenyl are optionally unsubstituted or substituted by one or more substituents, each of which is independently selected from alkyl, Cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, amino, hydroxyl, mercapto, carboxyl, alkoxy, cycloalkoxy, halogen, cyano, sulfo;

X ₅ , X ₆ , X ₇ , X ₈ are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, amino, hydroxyl, mercapto, alkoxy, cycloalkoxy ,halogen.

In the most preferred embodiment of the first aspect of the present invention, the following specific compounds are provided:

Table 1 The present invention synthesizes a part of the compounds

In the second aspect of the present invention, there is provided a pharmaceutical composition, the pharmaceutical composition comprising: the compound of the first aspect of the present invention or a pharmaceutically acceptable salt or a solvate or a prodrug molecule thereof.

In the third aspect of the present invention, there is provided the use of the above-mentioned compound of general formula I, a pharmaceutically acceptable salt thereof, a solvate or a prodrug molecule thereof in the preparation of a drug for the treatment or prevention of anti-tumor and anti-cancer .

In a preferred embodiment of the third aspect of the present invention, the tumor or cancer is selected from the group consisting of melanoma, gastric cancer, cervical cancer, ovarian cancer, liver cancer, lung cancer, nasopharyngeal cancer, colon cancer, rectal cancer, lymphoma, blood cancer , bone marrow cancer, brain cancer, skin cancer, bone cancer, nasopharyngeal cancer, pancreatic cancer, kidney cancer, thyroid cancer, prostate cancer, bladder cancer, esophageal cancer, breast cancer.

In the fourth aspect of the present invention, there is provided a method for preparing the compound of general formula I, the method comprising the steps of:

(a) changing the carboxyl group of compound 1 into an acid chloride to obtain compound 2;

(b) Compound 2 is reacted with compound 3 to obtain compound 4

(c) Reduction of the nitro group of compound 4 to amino group to obtain compound 5

wherein Z, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ and X ₈ are as defined in general formula I of compounds provided by the first aspect of the present invention.

In a preferred embodiment of the fourth aspect of the present invention, the reagent used to change the carboxyl group of compound 1 into an acid chloride in the reaction step (a) is selected from a combination of a chlorinating reagent and N,N-dimethylformamide (DMF) , wherein the chlorination reagent is selected from: thionyl chloride, oxalyl chloride, phosphorus oxychloride, phosphorus trichloride, phosphorus pentachloride, sulfonyl chloride, phosphorus pentachloride; Reaction step (c) used nitro reducing reagent is selected from The combination of self-active metal and acid, wherein the active metal is selected from: zinc, iron and tin, and the acid is selected from: hydrochloric acid, sulfuric acid, propionic acid, acetic acid, formic acid, phosphoric acid;

In the fifth aspect of the present invention, there is provided another method for preparing the compound of general formula I, the method comprising the steps of:

Compound P is reacted with compound Q under the action of peptide condensing agent to obtain compound M

wherein Z, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ and X ₈ are as defined in general formula I of compounds provided by the first aspect of the present invention.

In a preferred embodiment of the fifth aspect of the present invention, the peptide condensing agent used in the reaction is selected from:

Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate,

Benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate,

N,N'-dicyclohexylcarbodiimide,

O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid.

Description of drawings

Figure 1: Inhibitory activity of compounds against HeLa nuclear HDAC enzymes

Figure 2: Effects of compounds on the ability of tumor cells to form monoclonal

Detailed ways

The term "alkyl" as used in the present invention refers to a group consisting only of carbon atoms and hydrogen atoms without unsaturation (eg, double bonds, triple bonds or rings), which covers various possible geometrical differences structural and stereoisomeric groups. This group is linked to the rest of the molecule through a single link. As non-limiting examples of alkyl groups, one may cite the following straight-chain or branched groups: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl , n-pentyl and its other seven isomers, n-hexyl and its other sixteen isomers, n-heptyl and its respective isomers, n-octyl and its various isomers, n-nonyl and its various isomers.

The term "cycloalkyl" used in the present invention refers to a saturated non-aryl ring system consisting of at least 3 carbon atoms, which can be monocyclic, bicyclic, polycyclic, or fused, bridged, spiro ring. As non-limiting examples of cycloalkyl groups, the following may be cited: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl; and groups consisting of two or more The above-mentioned monocyclic ring is a fused ring, bridged ring, or spiro ring group formed by a common side and a common carbon atom.

The term "alkenyl" as used in the present invention refers to a group formed in the presence of one or more double bonds in the above-mentioned alkyl groups (other than methyl).

The term "alkynyl" as used in the present invention refers to a group formed in the presence of one or more triple bonds in the above-mentioned alkyl groups (other than methyl).

The term "alkoxy" as used in the present invention refers to a group in which an oxygen atom is attached to the above-mentioned alkyl group and is attached to the rest of the molecule by a single bond through the oxygen atom, which covers all possible geometric isomeric groups with stereoisomeric groups. As non-limiting examples of alkoxy, one may cite the following straight or branched groups: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy and seven other isomers, n-hexyloxy and sixteen other isomers, n-heptoxy and various isomers, n-octyloxy and Various isomers, n-nonyloxy and various isomers.

The term "aryl" as used in the present invention refers to an aromatic ring system consisting of at least 6 carbon atoms, and the ring system may be monocyclic, bicyclic, or polycyclic, wherein the bicyclic and multicyclic rings may be connected by a single ring through a single bond form or fused form. As non-limiting examples of aryl groups, one may cite the following groups: phenyl, naphthyl, anthracenyl, phenanthrenyl, indenyl, pyrenyl, perylene, pentvalenyl, hepvalenyl, triphenylene, Tetraphenyl, penfenyl, pentacyl, tetra-o-phenylene, hexenyl, hexaphenyl, coronyl, trinaphthylene, hepfenyl, heptaphenyl, egg phenyl, biphenyl base, binaphthyl.

The term "heteroaryl" as used in the present invention refers to a 5-14 membered aromatic heterocyclic ring system having one or more heteroatoms independently selected from N, O or S, which may be monocyclic, Bicyclic, polycyclic, wherein bicyclic and multicyclic can be formed by single ring connected by single bond or fused. As non-limiting examples of heteroaryl groups, the following groups may be cited: oxazolyl, isoxazolyl, imidazolyl, furyl, indolyl, isoindolyl, pyrrolyl, triazolyl, triazinyl , tetrazolyl, thienyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzofuranyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzothienyl, benzofuranyl, carbazolyl, isoquinolinyl, quinazolinyl, naphthyridinyl, purinyl, thiadiazolyl, indolazinyl, phenazinyl, coumarinyl, pyridopyridyl, pyridopyridazinyl, imidazopyridyl, imidazopyridazinyl; and groups formed from the above-mentioned heteroaryl groups by single bond connection or fusion.

The term "heterocyclyl" as used in the present invention refers to a non-aromatic 3-18 membered ring system consisting of carbon atoms and heteroatoms independently selected from N, O or S, which may be monocyclic, bicyclic, or polycyclic, may also be fused, bridged, spiro, and may optionally contain one or more double bonds. As non-limiting examples of heterocyclyl groups, the following may be cited: acridinyl, benzodioxanyl, benzopyranyl, chromanyl, dioxolanyl, decahydro Isoquinolinyl, indanyl, indolinyl, isoindolinyl, isochromanyl, morpholinyl, piperazinyl, 2-oxopiperazinyl, octahydroindolyl , octahydroisoindolyl, 4-piperidinone, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, 1-(diphenylmethyl) Piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrrolyl, piperidinyl.

The term "halogen" or "halo" as used herein refers to fluorine, chlorine, bromine or iodine.

Through the following specific embodiments, the above-mentioned content of the present invention will be further explained and described in detail, so that those skilled in the art can more easily understand the present invention, but should not be construed as the subject matter of the present invention. The scope is limited to the following examples and limits any or all rights of the present invention, and should not depart from the spirit of the present invention.

Example 1: Synthesis of some compounds of the present invention

Preparation of N-(2-aminophenyl)-4-{4-[bis(2-chloroethyl)amino]phenyl}butanamide (H104).

Step (1): Weigh chlorambucil (0.30g, 1mmol) and N,N-dimethylformamide (0.1ml) in a 50ml single-necked round-bottomed flask and stir under ice bath, drop liquid with constant pressure Add 2ml of oxalyl chloride dropwise to the funnel, remove the ice and stir at room temperature for 1 hour, spin the reaction system to obtain an oily substance; take another 100ml three-necked round-bottomed flask, add o-nitroaniline (0.14g, 1mmol), tetrahydrofuran (10ml) to it ), triethylamine (0.15g, 1.5mmol), the reaction system was placed in an ice bath and stirred, and the oil obtained by spin-drying was dissolved in 10ml of tetrahydrofuran and added dropwise to the reaction system using a constant pressure dropping funnel. After adding complete, remove the ice, continue stirring at room temperature, after TLC spot plate confirms the completion of the reaction, drop the reaction dropwise into the stirring ice water to precipitate a large amount of solid, and filter to obtain 0.39g of yellow solid (yield 93%)

Step (2): Weigh the yellow solid (0.3g, 0.7mmol), zinc powder (0.09g, 1.4mmol), methanol (10ml) and water (2ml) obtained in step (1) into a 50ml three-necked round bottom flask At room temperature, add dilute hydrochloric acid (3.5ml, 1mol/L) dropwise with a constant pressure dropping funnel, continue stirring at room temperature after the dropwise addition, after the TLC spot plate confirms that the reaction is completed, filter the reaction solution, and the filtrate is spun. After drying, it was extracted with ethyl acetate, washed with ammonia water three times, dried with anhydrous potassium carbonate, filtered, and spin-dried to obtain an oily substance, which was purified by silica gel column chromatography to obtain 0.13 g of a solid (yield 46%); ¹ H NMR ( 400MHz, DMSO-d ₆ ): δ=9.12(s, 1H), 7.17(d, J=7.8Hz, 1H), 7.07(d, J=8.2Hz, 2H), 6.91(t, J=7.5Hz, 1H), 6.74(d, J＝8.0Hz, 1H), 6.69(d, J＝8.2Hz, 2H), 6.56(t, J＝7.5Hz, 1H), 5.04(s, 2H), 3.71(s, 8H), 2.53 (t, J=7.9 Hz, 2H), 2.33 (t, J=7.2 Hz, 2H), 1.85 (m, 2H); ¹³ C NMR (400 MHz, DMSO-d ₆ ): δ=27.3, 33.6, 35.2, 25.6, 52.2, 111.9, 115.9, 116.2, 123.6, 125.3, 125.6, 129.3, 129.9, 141.7, 144.4, 170.9 ppm; C ₂₀ H ₂₅ Cl ₂ N ₃ O, MS(ES+) m/z: 394.15 (M+H) ⁺ .

Another method for the preparation of N-(2-aminophenyl)-4-{4-[bis(2-chloroethyl)amino]phenyl}butanamide (H104).

Take a 100ml single-neck round-bottomed flask, add chlorambucil (0.50g, 1.64mmol), N,N-dimethylformamide (10ml), add BOP (benzotriazole-1-yloxy tri( Dimethylamino) phosphonium hexafluorophosphate) 0.87g (1.97mmol), triethylamine 0.67g (6.6mmol), add o-phenylenediamine 0.213g (1.97mmol) after stirring at room temperature for 10min, continue magnetic stirring at room temperature, The progress of the reaction was followed by a thin layer chromatography plate, and the final reaction time was 2h. After the reaction was completed, 350 ml of ethyl acetate was added to the reaction solution for extraction, poured into a separatory funnel, washed three times with saturated aqueous sodium carbonate solution, and washed twice with saturated sodium chloride. The organic phase was dried with anhydrous magnesium sulfate and filtered. The solid was obtained by rotary evaporation, which was recrystallized from methanol to obtain 0.53 g of an off-white solid (yield 82%).

Example 2: Compound anti-tumor cell proliferation experiment

(1) Experimental cell lines, five cancer cells were selected: human skin melanoma A375, human liver cancer cell HepG2, human liver cancer cell SMMC7721, human lung cancer cell A549 and human lung cancer cell H1299. Before the experiment, five cancer cells were stored in liquid nitrogen. First, the cancer cells were removed, and they were rapidly heated to 37 degrees Celsius in a 37 degrees Celsius water bath. The cell fluid was centrifuged to remove the supernatant, and the cells were resuspended with culture medium. After transfer to 24mL cell culture flasks, each flask was added 6ml of culture based on 37 degrees Celsius, 5% _CO2 incubator culture, (in which human skin melanoma A375, liver cancer cells HepG2 and human liver cancer cells SMMC7721, in 10% In DMEM medium containing fetal bovine serum, subculture with 0.25% trypsin routinely. Human lung cancer cell A549 and human lung cancer cell H1299 are in RPMI-1640 medium containing 10% fetal bovine serum, routinely treated with 0.25% trypsin Subculture after digestion.) When the cell growth reaches about 70%-80% of the culture flask, first wash off the medium with PBS, digest with 0.25% trypsin to remove the cells from the culture flask, add fresh medium and centrifuge, The supernatant medium was removed, and fresh medium was added to resuspend after 1:2 to 3 passages.

(2) When the cell growth is stable, the cancer cells in log phase are digested into single cells with trypsin, and then diluted with fresh medium to a cell density of (3-4)×10 ⁴ cells/ml, in 96 wells In the plate, add 90 μL of cell fluid to each well, incubate for 12 h at 37 degrees Celsius in an incubator containing 5% CO ₂ , and add drugs (pre-diluting the synthesized compounds to a series of different concentrations, and adding different concentrations of compound solutions to the plate). To 96-well plate, each concentration was repeated 3 times), after 72 hours, 10 μL of CCK-8 solution was added to each well of the 96-well plate, and after culturing for 1 hour, the BIO-RAD microplate reader was used to measure the wavelength of 450nm. The absorbance value of , was fitted by Graphpad Prism 5 software to obtain the inhibition curve, and finally the IC ₅₀ value was obtained.

Table 2: Inhibitory effect of the compounds synthesized in the present invention on five different cancer cells

The results in Table 2 show that the chlorambucil compounds containing benzamide groups synthesized by the present invention have strong inhibitory activity against five cancer cells, and the compounds synthesized by the present invention are significantly more effective than the parent drugs chlorambucil and Stronger antitumor effect of bendamustine hydrochloride and CI994. The chlorambucil compound containing hydroxamic acid group synthesized in this patent is 4 to 40 times stronger than the parent drug chlorambucil, and 9 to 64 times stronger than the parent drug bendamustine hydrochloride.

Example 3: Inhibition experiments of compounds on HDAC enzymes

use swiss The HDAC green fluorescence test kit (product number: BML-AK530) produced by Life Sciences Company was used to conduct the HDAC enzyme activity inhibition experiment of HeLa cell nuclear extract, and the kit with product number BML-AK511 was used to conduct the HDAC1 enzyme activity inhibition experiment. The kit with the serial number of BML-AK512 was used for the HDAC2 enzyme activity inhibition test, and the kit with the product serial number of BML-AK516 was used for the HDAC6 enzyme activity inhibition test. The operation was carried out in strict accordance with the kit instructions, 15 μL of HDAC enzyme was thoroughly mixed with 10 μL of the test compound, the substrate and the above mixture were placed at 37 degrees Celsius for 5 minutes to obtain the same starting temperature for the substrate and the enzyme, and then each well was quickly Add 25 μL of substrate, incubate the entire 96-well plate at 37°C for 30-60 minutes, then add 50 μL of stop solution to each well. After that, the 96-well plate was placed at room temperature for 10 minutes, and the fluorescence intensity was measured with a microplate reader. After obtaining the inhibition rate of different concentrations of compounds on HDAC enzyme, the inhibition curve was obtained by fitting the Graphpad Prism 5 software, and the IC ₅₀ value was finally obtained.

The results shown in Figure 1 show that the chlorambucil compound containing hydroxamic acid group synthesized by the present invention has inhibitory activity on the HDAC enzyme of HeLa cell nuclear extract, while the parent compound chlorambucil has no HDAC enzyme activity in HeLa cell nuclear extract. inhibitory activity.

Table 3: Inhibitory effect of compounds synthesized in the present invention on HDAC enzymes

The results in Table 4 show that the nitrogen mustard compounds containing hydroxamic acid groups synthesized by the present invention have selective inhibitory activity on HDAC2, and the selectivity coefficients of HDAC1/HDAC2 and HDAC6/HDAC2 are all greater than 4.9

Example 4: Compound anti-tumor cell monoclonal formation assay

The human skin melanoma cells A375 in the logarithmic growth phase were plated in 6-well plates, and 3500 cells were plated in each well, and the cells were immediately treated with chlorambucil, CI994 and H104 at 2 μmol/L, 8 μmol/L and 16 μmol/L. , taking the DMSO treatment group as the blank control, after 7 days, remove the medium from each well, add PBS (phosphate buffered saline) to gently wash twice, add 3.7% formaldehyde solution to fix for 20min, and then gently wash twice with PBS , stained with 0.1% crystal violet aqueous solution for 30min, washed 3 times with PBS, observed the experimental results and photographed.

It can be seen from Figure 2 that the compound H104 synthesized by the present invention has stronger anti-tumor cell monoclonal formation ability than the parent drugs chlorambucil and CI994.

Example 5: Compound-induced tumor cell apoptosis assay

The human skin melanoma cells A375 in the logarithmic growth phase were plated in 6-well plates, and 160,000 cells were plated in each well. ) treated cells, treated with DMSO as the control group, collected the medium in each well after 72 h, digested with trypsin to collect the cells in each well, centrifuged at 1000 rpm for 5 min, removed the supernatant after centrifugation, washed twice with PBS, and then added 100 μL of ×binding buffer, 5 μL of Alexa Fluor488annexin and 1 μL of propidium iodide (100 μg/ml), incubate for 15 min at room temperature in the dark, the group stained with Alexa Fluor488annexin and propidium iodide was used as the control group, and the samples were stained with MoFlo XDP Flow cytometry detection.

The results of inducing tumor cell apoptosis show that the compound H104 synthesized in the present invention can induce apoptosis in 20.2%, 62.0% and 64.8% of human skin melanoma cells A375 at concentrations of 2 μmol/L, 8 μmol/L and 16 μmol/L, respectively. , while the parent drug chlorambucil was only able to induce apoptosis in 9.9%, 12.9% and 28.9% of human skin melanoma cells A375 at concentrations of 2 μmol/L, 8 μmol/L and 16 μmol/L. Only 21.6%, 29.6% and 64.0% of human skin melanoma cells could be induced to apoptosis at the concentrations of 8 μmol/L and 16 μmol/L. It can be seen that the compounds synthesized in the present invention have significantly stronger ability to induce tumor cell apoptosis than the parent drugs chlorambucil and CI994.

Example 6: Compounds block tumor cell cycle experiments

A375 human skin melanoma cells in the logarithmic growth phase were plated in 6-well plates, and 160,000 cells were plated in each well. After 72 hours, the culture medium in each well was collected, and the cells in each well were collected by trypsinization, centrifuged at 1000 rpm for 5 min, the supernatant after centrifugation was removed, and washed twice with PBS. The cells were fixed with 70% ethanol and washed with PBS. Twice, RNase was added and incubated at 37°C for 30 min in the dark, and the samples were detected by MoFloXDP flow cytometer.

The results show that the compound H104 synthesized by the present invention can significantly inhibit the human skin melanoma cell A375 in the G2/M phase at a concentration of 8 μmol/L, (the proportion of cancer cells in the G2/M phase in the control group treated with DMSO is 12.6%, After treatment with 8 μmol/L of compound H104, the proportion of cancer cells in G2/M phase increased to 79.5%, while after treatment with chlorambucil, the proportion of human skin melanoma cells A375 in G2/M phase only increased to 47.7% %, after treatment with CI994, the proportion of human skin melanoma cells A375 in the G2/M phase was 10.8%, and there was no significant change).

Claims (4)

1. A compound of formula I: 2. Use of the compound of claim 1 in the preparation of anti-tumor drugs. 3. Use of the compound of claim 1 in the preparation of an anticancer drug. 4. The use according to claim 2 or 3, wherein the tumor is melanoma, and the cancer is selected from gastric cancer, cervical cancer, ovarian cancer, liver cancer, lung cancer, colon cancer, rectal cancer, lymphoma, blood cancer, bone marrow cancer, Brain cancer, skin cancer, bone cancer, nasopharyngeal cancer, pancreatic cancer, kidney cancer, thyroid cancer, prostate cancer, bladder cancer, esophagus cancer, breast cancer.