CN110577504B

CN110577504B - Hydrogen peroxide responsive azonium diol salt compound and application thereof

Info

Publication number: CN110577504B
Application number: CN201910875765.2A
Authority: CN
Inventors: 傅俊杰; 尹健; 孟婷婷; 胡静
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-09-17
Filing date: 2019-09-17
Publication date: 2021-05-28
Anticipated expiration: 2039-09-17
Also published as: CN110577504A

Abstract

The invention discloses a hydrogen peroxide-responsive azonium diol salt compound and application thereof, belonging to the field of pharmaceutical chemistry. The compound has high stability, and the alpha-ketoamide structure of the compound can specifically respond to H₂O₂The NO is released, different structures can realize specific inhibition on different types of tumor cells, the toxicity on normal cells is low, and the NO can be used as a novel NO donor type medicament with high tumor cell selectivity and low toxic and side effects, and has good market prospect.

Description

Hydrogen peroxide responsive azonium diol salt compound and application thereof

Technical Field

The invention relates to a hydrogen peroxide-responsive azonium diol salt compound and application thereof, belonging to the field of pharmaceutical chemistry.

Background

Nitric Oxide (NO) has a wide range of physiological activities and also plays an important role in the anti-tumor field. A number of studies have shown that NO has a dual effect on tumor cells: the low-concentration NO can induce the mutation of apoptosis related genes (such as p53), increase the drug resistance of tumor cells, promote the regeneration and metastasis of tumor blood vessels and promote the growth of the tumor cells; the high-concentration NO can up-regulate the expression of a cancer suppressor gene p53, promote the degradation of anti-apoptosis proteasome, increase the permeability of a mitochondrial membrane, release cytochrome C (Cyt-C), induce apoptosis and cell cycle arrest and exert antitumor activity.

The NO donor is a compound capable of releasing NO in vivo through enzymatic or non-enzymatic action, and can be used as a transport form and a storage form of NO in vivo, so that the half-life of NO is prolonged, and the controlled release of NO is realized. As NO donors, nitrates, azoniadiols (NONOates), nitrosothiols, furannitroxides, and the like have been mainly reported. Among them, O of azonium dialcohol salts²The site atom can form a stable prodrug after structural modification, activate and release NO under specific conditions, and realize targeted and controllable release of NO, thereby drawing extensive attention to people, and the specific NO release mechanism is as follows:

currently reported prodrugs of azoniadiolate NO donors: 1) prodrugs activated by various enzymes (e.g., esterases, glycosidases, glutathione transferases, beta-lactamases, P450 enzymes, etc.); 2) light (ultraviolet, near infrared, visible, etc.) activated prodrugs; 3) activated oxygen cluster (hydrogen peroxide, peroxynitrite, etc.) activated prodrugs.

Hydrogen peroxide (H)₂O₂) Is an important active oxygen cluster, and some researches show that H in tumor cells₂O₂The concentration is as high as 5 mu M to 1.0mM and is far higher than that of normal cells (<1 μ M). Thus, H₂O₂Activated azonium dialkoxides enable the selective release of NO in tumor cells. H has been reported so far₂O₂The activated azonium dialkoxides are all based on H₂O₂The response phenylboronic acid ester structure has the advantages of single structure, poor stability and H₂O₂The problem of insufficient specificity, and the lack of more detailed research on the antitumor activity. The invention firstly converts H into H₂O₂The responsive alpha-ketoamide structure is used for modifying azonium dialkoxide prodrug, and a brand new H is developed₂O₂Responsive azonium dialkoxide prodrugs having stability and H compared to reported structures₂O₂The specificity is greatly improved. We also showed the relationship between NO release, antitumor activity, structure activity (different R)₁、R₂The influence of NO release and antitumor activity), antitumor mechanism, etc.

Disclosure of Invention

The invention synthesizes a brand new alpha-keto amide modified azonium diol salt prodrug. In the absence of H₂O₂The stability is high when the catalyst exists; h₂O₂When present, the alpha-ketoamide structure can be via H₂O₂Nucleophilic attack, rearrangement, hydrolysis (as shown in FIG. 1) to produce CO₂P-nitrobenzoic acid and aniline, and the aniline is eliminated by 1, 6-to release NO. Such prodrug pair H₂O₂Has high specificity and almost no response to the active oxygen group peroxynitrite. The invention also relates to the NO release, the anti-tumor activity and the structure-activity relationship (different R) of the prodrug₁、R₂The influence of NO release and antitumor activity), antitumor mechanism, etc.

The first object of the invention is to provide an azonium diol salt compound containing alpha-ketoamide, which comprises a compound shown in the formula I, wherein the compound is H₂O₂Responsive azonium glycol salt prodrugs:

x is hydrogen, alkyl, halogen, nitro, cyano, trifluoroalkyl, amino, carboxyl, alkoxy, acylamino R_a-CONH-, alkoxyacyl R_aOCO-, wherein R_aSelected from alkyl, haloalkyl, cycloalkyl, halocycloalkyl, alkenyl, haloalkenyl, aryl;

R₁and R₂Is substituted or unsubstituted C_1-12Linear alkyl or alkenyl, substituted or unsubstituted C_3-12Branched alkyl or alkenyl, substituted or unsubstituted C_1-4Arylalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted heteroaryl; or NR₁R₂Is substituted or unsubstituted pyrrolidinyl, piperazinyl, morpholinyl, piperidinyl, prolinol group.

In one embodiment of the invention, said substituted C_1-12Linear alkyl or alkenyl of, C_3-12Branched alkyl or alkenyl of, C_1-4The substituent of arylalkyl, benzyl, heteroaryl, pyrrolidinyl, piperazinyl, morpholinyl, piperidinyl, prolinamido includes halogen, nitro, cyano, trifluoroalkyl, amino, carboxyl, alkoxy.

In one embodiment of the invention, the heteroaryl is an aryl group containing 1 to 3 heteroatoms, including S, N, O.

In one embodiment of the invention, the amino group is-NR_aR_bWherein R is_a、R_bIndependently selected from hydrogen, substituted or unsubstituted C_1-12Alkyl, or NR_aR_bIs substituted or unsubstituted pyrrolidinyl, piperazinyl, morpholinyl, piperidinyl, prolinol group.

In one embodiment of the invention, X is preferably hydrogen, R₁And R₂Is substituted or unsubstituted C_1-12Linear alkyl of (3), substituted or unsubstituted C_3-12A branched alkyl group of (a); or NR₁R₂Is substituted or unsubstituted pyrrolidinyl, piperazinyl, morpholinyl, piperidinyl, prolinol group; represented by the following general formula (II):

in one embodiment of the invention, R₁、R₂Further preferred is substituted or unsubstituted C_1-3Linear alkyl of (2), or NR₁R₂Is substituted or unsubstituted pyrrolidinyl, piperazinyl, morpholinyl, piperidinyl.

The structure of the further preferable scheme of the invention is as follows:

in one embodiment of the invention, the compound of formula (I) is obtained by reacting intermediate 1 with 4-aminobenzyl alcohol to obtain intermediate 2, and then reacting the alcoholic hydroxyl group of intermediate 2 with a differently substituted azonium dialkoxide after bromination;

in one embodiment of the present invention, the method for preparing the compound comprises:

(1) dissolving p-nitroacetophenone in pyridine, and adding selenium dioxide (SeO)₂) Stirring under reflux and nitrogen protection to obtain intermediate 1, wherein, the p-nitroacetophenone and SeO₂The molar ratio is 1 (1-2), wherein 1:1.5 is preferred.

(2) Taking N, N-Dimethylformamide (DMF) as a reaction solvent, adding the intermediate 1, p-aminobenzyl alcohol, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) and N, N-Diisopropylethylamine (DIPEA), and stirring at room temperature to obtain an intermediate 2, wherein the molar ratio of the intermediate 2 is 1 (0.8-1.5) to (1-2), and the preferred ratio is 1:1:1.5: 1.5.

(3) Dichloromethane (DCM) is used as a reaction solvent, and the intermediate 2 and carbon tetrabromide (CBr) are added₄) And triphenylphosphine (PPh)₃) Stirring the mixture at room temperature under the protection of nitrogen to obtain an intermediate 3, wherein the molar ratio of the intermediate 3 to the intermediate is 1 (1-2) to (1-2), and the intermediate is preferably 1:1.5: 1.5.

(4) Taking DMF as a reaction solvent, adding an intermediate 3, azonium dialkoxides with different structures and sodium bicarbonate (NaHCO)₃) A, B, C, D is obtained by stirring at 0 ℃ under the protection of nitrogen, and the molar ratio is 1 (1-2) to (1-2), preferably 1:1.5:1.

A second object of the invention is the use of the compounds of formula (I) as antitumor agents.

The third purpose of the invention is to provide an anti-tumor preparation, which contains the compound with the structure shown in the formula I.

In one embodiment of the invention, the formulation further comprises a pharmaceutical carrier and/or a pharmaceutical excipient.

In one embodiment of the invention, the dosage form of the preparation comprises injection, freeze-dried powder injection for injection, controlled release injection, liposome injection, suspension, implant, suppository, capsule, tablet, pill and oral liquid.

In one embodiment of the invention, the drug carrier includes microcapsules, microspheres, nanoparticles, and liposomes.

In one embodiment of the invention, the pharmaceutical excipients comprise solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retardants.

In one embodiment of the invention, the pharmaceutical excipients comprise microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.

The invention has the beneficial effects that:

compared with reported phenylboronate azoniadialkoxide, the compound of the invention has high stability and H resistance₂O₂The specific response effect is good, different structures can realize selective inhibition on different tumor cells, a safe, effective and strong-universality novel anti-tumor drug is provided, and the variety and potential biological value of azonium diol NO donors are enriched.

In the absence of H₂O₂Under the condition, the prodrug shows excellent stability in PBS solution, GSH solution and bovine plasma liquid with different pH values, and is obviously superior to corresponding phenylboronate azonium dialkoxide. At H₂O₂Under the action of (3), the prodrug is rapidly degraded to release NO, and the NO release amount is related to the structure of the azonium dialkoxide part. Importantly, the prodrugs are specificSexual response H₂O₂To another active oxygen group peroxynitrite (ONOO)^-) There is little response. In contrast, phenylboronate azoniadialkoxides respond in addition to H₂O₂Also responds significantly to ONOO^-The specificity is insufficient.

Cytotoxic tests show that the synthesized partial azoniadiol salt prodrug has specific inhibition effect on different tumor cells. For example, compound D has a particularly pronounced inhibitory effect on lung cancer cell A549 (IC)₅₀0.88 μ M), compound B had a strong inhibitory activity against melanoma cell B16 (4.27 μ M). Importantly, the compounds have low toxicity to normal colon epithelial cells CCD 841CON, and have prominent tumor cell selectivity, which indicates that the compounds have good safety.

The compound D is taken as an example to carry out anti-tumor mechanism research, and the result shows that the compound D can induce G2/M phase retardation of tumor cells, induce apoptosis of the tumor cells and reduce mitochondrial membrane potential of the tumor cells.

The results show that the azonium diol antitumor prodrug synthesized by the invention has potential clinical value.

Drawings

FIG. 1 shows an azonium dialkoxide prodrug of α -ketoamide structure in H₂O₂A mechanism diagram of NO release by degradation under the action of the catalyst;

FIG. 2 is a drawing of Compound A¹H NMR and¹³C NMR(CDCl₃)；

FIG. 3 is a drawing of Compound B¹H NMR and¹³C NMR(CDCl₃)；

FIG. 4 is a drawing of Compound C¹H NMR and¹³C NMR(CDCl₃)；

FIG. 5 is a drawing of Compound D¹H NMR and¹³C NMR(DMSO)；

FIG. 6 shows the stability of Compounds A and 4 under different conditions;

FIG. 7 shows HPLC monitoring of test compounds (100 μ M) versus H at room temperature₂O₂And ONOO^-(ii) a response of (d); wherein, A) compound D or 5 and H₂O₂(10eq.) incubation in PBS 7.4/DMSO (v, 1:1) mixed solution; B) compound D and varying concentrations of H₂O₂Incubating in PBS 7.4/DMSO (v: v, 1:1) mixed solution for 1 h; (C) compounds D or 5 with H₂O₂Or ONOO^-(10eq.) incubation in PBS 7.4/DMSO (v: v, 1:1) mix for 1 h;

FIG. 8 shows Compound D in vitro channels H₂O₂Reacting to generate mass spectrograms of the p-nitrobenzoic acid and the p-aminobenzyl alcohol;

fig. 9 is a graphical representation of the results of NO release of compound D in a549 cells;

FIG. 10 is a graph showing the results of Compound D inducing A549 cell cycle arrest;

FIG. 11 is a graph showing the results of Compound D inducing apoptosis of A549 cells;

fig. 12 is a graph showing the results of compound D inducing alterations in mitochondrial membrane potential in a549 cells.

Detailed Description

The invention is described in further detail in the following examples, which are intended to be illustrative only, and the physical data for the exemplified compounds are consistent with the structures specified for those compounds. The examples given do not limit the scope of the invention.

Example 1: synthesis of Compound A

The first step is as follows: synthesis of intermediate 1

The method comprises the following specific operations: p-nitroacetophenone (1g, 6.06mmol) was dissolved in pyridine (10mL), and SeO was added₂(1g, 9.08mmol) of p-nitroacetophenone and SeO₂The molar ratio was 1: 1.5. Introducing nitrogen into the mixture for protection, stirring for 4h at 90 ℃, cooling and filtering after the reaction is finished, washing a filter cake for 3 times by using Ethyl Acetate (EA), collecting an organic layer, washing by using 2M HCl, finally combining the organic layers, adding anhydrous sodium sulfate for drying, distilling under reduced pressure to remove a solvent, and eluting by using column chromatography (PE/EA v/v, 7:3) to obtain a yellow intermediate 1.

The second step is that: synthesis of intermediate 2

The method comprises the following specific operations: intermediate 1(1g, 5.13mmol) was dissolved in tetrahydrofuran THF (10mL), and p-aminobenzyl alcohol (0.64g, 5.13mmol), EDCI (1.475g, 7.7mmol) and DIPEA (1.28mL, 7.7mmol) were added in that order and stirred at room temperature for 1-2 h. After the reaction is finished, washing with water and saturated salt water in sequence, combining organic layers, adding anhydrous sodium sulfate for drying, and removing the solvent by reduced pressure distillation to obtain a dark brown intermediate 2 which is directly subjected to the next reaction.

ESI-MS:299.3[M–H]^-.

The third step: synthesis of intermediate 3

The method comprises the following specific operations: intermediate 2(0.3g, 1mmol) was dissolved in anhydrous DCM, stirred at 0 deg.C under nitrogen protection, and successively added PPh₃(0.395g, 1.5mmol) and CBr₄(0.5g, 1.5mmol), brought to room temperature and stirred for 1 h. After the reaction is finished, the solvent is removed by reduced pressure distillation, and the yellow intermediate 3 (PE/EAv/v, 9:1) is obtained by column chromatography elution.

m.p.133–135℃.

¹H NMR(400MHz,Chloroform-d)δ8.98(s,1H,NH),8.66–8.54(m,2H,ArH),8.41–8.29(m,2H,ArH),7.74–7.63(m,2H,ArH),7.50–7.39(m,2H,ArH),4.51(s,2H,ArCH₂).

¹³CNMR(100MHz,Chloroform-d)δ185.94,157.80,151.10,137.64,136.30,135.35,132.76,130.29,123.71,120.33,32.98.

ESI-MS:361.1[M–H]^-.

The fourth step: synthesis of Compound A

Azo onium diolsSalt (64mg, 0.42mmol) and NaHCO₃(23mg, 0.28mmol) was dissolved in anhydrous DMF (2mL), stirred at 0 ℃ under nitrogen protection, then intermediate 3(100mg, 0.28mmol) was added, and stirring was continued for 1-2 h after returning to room temperature. After the reaction, the reaction product is washed by water and saturated salt water in turn, organic layers are combined, anhydrous sodium sulfate is added for drying, the solvent is removed by reduced pressure distillation, and the target compound A (38%) is obtained by column chromatography elution (PE/EA v/v, 7: 3).

m.p.117–118℃.

¹H NMR(400MHz,Chloroform-d)δ8.987(s,1H,NH),8.63–8.59(m,2H,ArH),8.38–8.33(m,2H,ArH),7.73–7.68(m,2H,ArH),7.50–7.41(m,2H,ArH),5.28(s,2H,ArCH₂),3.61–3.44(m,4H,NCH₂×2),1.98–1.90(m,4H,CH₂×2).

¹³C NMR(100MHz,Chloroform-d)δ186.07,157.94,151.12,137.74,136.42,133.65,132.73,129.92,123.69,120.11,74.66,51.06,22.95.

ESI-MS:428.30[M–H]^-.

Example 2: synthesis of Compound B

Azolonium dialkoxide (71mg, 0.42mmol) and NaHCO₃(23mg, 0.28mmol) was dissolved in anhydrous DMF (2mL), stirred at 0 ℃ under nitrogen protection, then intermediate 3(100mg, 0.28mmol) was added, and stirring was continued for 1-2 h after returning to room temperature. After the reaction is finished, water and saturated salt water are sequentially used for washing, organic layers are combined, anhydrous sodium sulfate is added for drying, the solvent is removed through reduced pressure distillation, column chromatography elution (PE/EA v/v, 6:4) is carried out to obtain a crude product, and the crude product is further recrystallized through DCM/EtOH to obtain a target compound B (27%).

m.p.149–150℃.

¹H NMR(400MHz,Chloroform-d)δ9.01(s,1H,NH),8.63–8.57(m,2H,ArH),8.38–8.33(m,2H,ArH),7.74–7.68(m,2H,ArH),7.48–7.42(m,2H,ArH),5.22(s,2H,ArCH₂),3.85–3.80(m,4H,OCH₂×2),3.43–3.37(m,4H,NCH₂×2).

¹³C NMR(100MHz,Chloroform-d)δ186.00,157.91,151.11,137.68,136.62,133.08,132.74,130.00 123.70,120.16,75.15,65.80,51.73.

ESI-MS:428.30[M–H]^-.

Example 3: synthesis of Compound C

Azolonium dialkoxide (100mg, 0.42mmol) and NaHCO₃(23mg, 0.28mmol) was dissolved in anhydrous DMF (2mL), stirred at 0 ℃ under nitrogen protection, then intermediate 3(100mg, 0.28mmol) was added, and stirring was continued for 1-2 h after returning to room temperature. After the reaction, the reaction product is washed by water and saturated salt water in turn, organic layers are combined, dried by adding anhydrous sodium sulfate, decompressed and distilled to remove the solvent, and eluted by column chromatography (PE/EA v/v, 7:3) to obtain a target compound C (47%).

m.p.145–146℃.

¹H NMR(400MHz,Chloroform-d)δ9.01(s,1H,NH),8.60–8.55(m,2H,ArH),8.36–8.30C(m,2H,ArH),7.72–7.67(m,2H,ArH),7.44–7.38(m,2H,ArH),5.20(s,2H,ArCH₂),4.13(q,J＝7.1Hz,2H,OCH₂),3.62(dd,J＝6.0,4.4Hz,4H,CONCH₂×2),3.41–3.28(m,4H,NCH₂×2),1.25(t,J＝7.1Hz,3H,CH₃).

¹³C NMR(100MHz,Chloroform-d)δ185.95,157.90,155.18,151.05,137.63,136.64,132.90,132.68,129.95,123.65,120.14,75.19,61.94,51.16,42.38,14.70.

ESI-MS:499.38[M–H]^-.

Example 4: synthesis of Compound D

Azolonium dialkoxide (76mg, 0.42mmol) and NaHCO₃(23mg, 0.28mmol) was dissolved in anhydrous DMF (2mL), stirred at 0 deg.C under nitrogen, and addedAnd recovering the intermediate 3(100mg, 0.28mmol) to room temperature, and continuing stirring for 1-2 h. After the reaction, the reaction mixture was washed with water and saturated brine, the organic layers were combined, dried over anhydrous sodium sulfate, and then subjected to distillation under reduced pressure to remove the solvent and column chromatography (DCM/MeOH v/v, 95:5) to give the desired compound D (16%).

m.p.142–143℃

¹H NMR(400MHz,d⁶-DMSO)δ11.02(s,1H,NH),8.39(d,J＝8.8Hz,2H,ArH),8.29(d,J＝8.8Hz,2H,ArH),7.71(d,J＝8.3Hz,2H,ArH),7.30(d,J＝8.2Hz,2H,ArH),4.58(s,1H,OH),3.45(s,3H,ArCH₂+HOCH),2.67(d,J＝10.5Hz,2H,NCH₂),2.06(s,2H,NCH₂),1.75–1.62(m,2H,CHCH ₂),1.45–1.33(m,2H,CHCH ₂).

¹³CNMR(101MHz,d⁶-DMSO)δ187.71,161.60,150.52,137.59,136.25,131.55,129.36,123.94,120.10,61.54,50.72,34.34,30.33.

By using¹H NMR，¹³C NMR and ESI-MS characterize the molecular structure of the compound and the product obtained is stored at 4 ℃.

¹H NMR，¹³The C NMR results are shown in FIGS. 2-5, respectively, and indicate that compounds A, B, C and D were successfully synthesized.

Example 5: prodrug stability study

To achieve selective release of NO, the prodrug is in the absence of H₂O₂Should be sufficiently stable in the presence of the acid. We examined the stability of the prodrug by HPLC in PBS solutions of different pH, in the presence of Glutathione (GSH) and in bovine plasma and compared it with the reported boronate azonium dialkoxides. Taking compound a as an example, the test shows that it has better stability in PBS (pH 5.0,7.4,8.0), GSH and bovine plasma after 48h incubation, and the degradation rate is less than 10%. The phenylboronate compound 4 corresponding to a had poor stability and significant degradation (fig. 6). This experiment demonstrates that the synthesized prodrug of the present invention has high stability, which is significantly superior to compounds that have been reported to be also hydrogen peroxide response mechanisms.

Example 6: h of prodrugs₂O₂Study of response Properties

We performed HPLC and LCMS on the prodrug at H₂O₂The activation behaviour in the presence was examined. Taking compound D as an example, H is added₂O₂The prodrug was almost completely degraded within 1.5h, comparable to the degradation rate of the corresponding phenylboronate compound 5 (fig. 7A). Meanwhile, the formation of p-nitrobenzoic acid and p-aminobenzyl alcohol, degradation by-products, was clearly detected by both HPLC and LC-MS (FIG. 8). In addition, D is for H₂O₂The response of (a) showed concentration dependence (fig. 7B). Importantly, D is on the reactive oxygen species peroxynitrite (ONOO)^-) Hardly responds, and exhibits excellent H₂O₂Specificity, whereas phenylboronic acid ester compound 5 is on ONOO^-There was significant degradation in the presence and insufficient specificity (FIG. 7C).

Example 7: detection of NO release in tumor cells

The log growth phase of human lung adenocarcinoma cells A549 cells was 1X 10⁶cells/well were seeded in 6-well plates at 37 ℃ with 5% CO₂After culturing under the condition until the cells are 50% fused, the cells are synchronized by incubating for 2h with serum-free RPMI1640 medium. Subsequently, the supernatant was discarded and the compound was allowed to act for 48h at final concentrations of 0, 3, 6. mu.M. After the administration of the incubated cells, they were digested with trypsin without EDTA, and centrifuged at 1500rpm at 4 ℃ for 5min to collect the cells. 1.5mL of 5. mu.M DAF-FMDA working solution was added to each sample and incubated at 37 ℃ for 20min in the absence of light. After incubation, centrifugation is carried out at 1500rpm for 10min, the staining reaction solution is carefully discarded, 200. mu.L PBS is added to each well for washing for 2 times, each centrifugation is carried out for 5min, and then 200. mu.L PBS is used for resuspending the cells. The fluorescence signal values of the cells in 10000 cells are detected by a BD small flow cytometer FL1 channel within half an hour to obtain a peak shift curve (when the number of NO in the cells is large, the fluorescence signal is strong,the peak shifts right) and the intracellular NO level is determined.

As shown in FIG. 9, the same concentration of compound A, B, C, D showed a difference in NO release when applied to cells, with D having the highest NO release and A having the lowest NO release (FIG. 9A). This result indicates a structural difference (R) in the azonium dialkoxide moiety of the prodrug₁And R₂) Will affect the compound H₂O₂Final NO release after activation. Prodrug D containing 4-hydroxypiperidine structure releases NO optimally in A549 cells. With 20mM NAC (H)₂O₂Scavenger) or 50 μ M C-PTIO (NO scavenger) pre-treated cells for 1h, both significantly inhibited compound D-induced intracellular NO release (fig. 9B).

Example 8: in vitro anti-tumor Activity assay

Screening cell strains: acute myelocytic leukemia cell HL-60, human lung adenocarcinoma cell A549, human colon cancer cell HCT-116, melanin and human normal colon epithelial cell CCD 841 CON.

The test method comprises the following steps:

(1) digesting and counting cells, preparing cell suspension, and adding 100 mu L of cell suspension into each hole of a 96-hole cell culture plate; the 96-well cell culture plate is placed at 37 ℃ and 5% CO₂Culturing for 24h in an incubator; diluting the drug with culture medium to required working solution concentration, adding 100 μ L corresponding drug-containing culture medium into each well, and setting up negative control group; the 96-well cell culture plate is placed at 37 ℃ and 5% CO₂Culturing for 72 hours in an incubator; carrying out MTT (methyl thiazolyl tetrazolium) staining on a 96-well plate, wherein lambda is 490nm, and determining an OD (optical density) value; 1) adding 20 mu L of MTT (5mg/mL) into each well, and continuously culturing for 4h in the incubator; 2) discarding the supernatant, adding 150 μ L DMSO into each well, and mixing gently by shaking for 10 min; 3) lambda is 490nm, the OD value of each well is read by a microplate reader, and the inhibition rate and the half-inhibition concentration IC are calculated₅₀。

As shown in Table 1, the azonium diol salt compounds A-D showed varying degrees of inhibitory effects on HCT-116, A549, HL-60 and B16, and exhibited certain cell line specificity. For example, compound D is particularly prominent for a549 cell Inhibition (IC)₅₀0.88 μ M), compound B had less inhibitory activity than D on a549 cells, but inhibited B16 cellsHas strong effect (IC)₅₀4.27 μ M). The results show that the alpha-ketoamide structure and the azonium dialkoxide structure jointly determine the cytotoxicity and the cell specificity of the prodrug, and also suggest that the high-activity anti-tumor prodrug aiming at different tumor cells is expected to be obtained through different structural changes, and the high-activity anti-tumor prodrug has stronger universality.

TABLE 1 MTT test results

Example 9: cell cycle arrest assay

Will be 1 × 10⁶cells/well A549 cells were seeded in 6 well plates at 1.5 mL/well and cultured for 24h to effect compound D or 6. mu. MJS-K at final concentrations of 0, 3, 6. mu.M for 48h, or cells were pretreated with 20mM NAC or 50. mu. M C-PTIO for 1h followed by the addition of 6. mu.M compound D for 48 h. After the drug treatment, cells are digested by trypsin, centrifuged at 4 ℃ and 1000rpm for 5min, added with 1ml LPBS to clean the cells, centrifuged and the supernatant is discarded, the steps are repeated, 400 mul of PI (propidium iodide) is added to stain uniformly, the solution is protected from light at 4 ℃ for 30min, the solution is detected on a computer, and red fluorescence at the excitation wavelength of 488nm is recorded.

As shown in FIG. 10, the concentration of compound D increased, the cells in the G2/M phase gradually increased (30-50%), while the cells in the G0/G1 and S phases decreased significantly, and the blocking effect of the G2/M phase was slightly higher than that of the positive control JS-K. NAC or C-PTIO pretreatment significantly attenuated the cell G2/M phase block induced by Compound D. Thus, compound D is seen to pass through H₂O₂And NO-dependent means induce G2/M phase arrest in tumor cells.

Example 10: apoptosis detection

Will be 1 × 10⁶cells/well A549 cells were inoculated into 6-well plates, 1.5mL per well, cultured for 24h, and treated with compound D or 6. mu. MJS-K at final concentration of 0, 3, 6. mu.M for 48h, or cells were first administeredAfter 1h pretreatment with 20mM NAC or 50. mu. M C-PTIO, an additional 6. mu.M of Compound D was added for 48 h. After the drug treatment, cells are digested by trypsin, centrifuged at 4 ℃ and 1000rpm for 5min, the supernatant is discarded, 1mLPBS is added to clean the cells, the centrifugation and the discarding of the supernatant are repeated, then 5 mu LannexinV-FITC is added to be mixed evenly and reacted for 15min in a dark place, and the detection is carried out by adopting a flow cytometer.

The test result is shown in FIG. 11, the compound D induces apoptosis of A549 cells in a concentration-dependent manner, and the activity is slightly higher than that of positive control JS-K. NAC or C-PTIO pretreatment significantly impaired the ability of D to induce apoptosis. Thus, compound D is seen to pass through H₂O₂And NO-dependent means to induce apoptosis in tumor cells.

Example 11: mitochondrial membrane potential detection

Cells in logarithmic growth phase at 1X 10⁶cells/well were seeded in 6-well plates at 37 ℃ with 5% CO₂After culturing under the condition until the cells are 50% fused, the cells are synchronized by incubating for 2h with serum-free RPMI1640 medium. Subsequently, the supernatant was discarded and the cells were either treated with compound D or 6. mu. MJS-K at final concentrations of 0, 3, 6. mu.M for 48h, or the cells were pretreated with 20mM NAC or 50. mu. M C-PTIO for 1h followed by 6. mu.M compound D for 48 h. After incubation is finished, the cells are collected and placed in flow tubes respectively, 0.5mL of cell culture solution is added into each tube, 0.5mL of JC-1 staining working solution is added after heavy suspension, the mixture is uniformly mixed and placed in an incubator at 37 ℃ for incubation for 30min, during the incubation period, a proper amount of JC-1 staining buffer solution is prepared according to the proportion that 4mL of distilled water is added into each 1mL of JC-1 staining buffer solution, the mixture is placed in an ice bath, after the incubation is finished, the mixture is centrifuged for 4min at 1000rmp and 4 ℃, supernatant is discarded, 1mL of JC-1 staining buffer solution is added into each tube to wash the cells twice, finally, the JC-1 staining buffer solution is used for heavy suspension of the cells, and the on-machine.

The test result is shown in figure 12, the compound D induces the mitochondrial membrane potential of A549 cells to be reduced in a concentration-dependent manner, and the activity is higher than that of the positive control JS-K. NAC or C-PTIO pretreatment significantly attenuated the ability of D to induce a decrease in mitochondrial membrane potential in cells. Thus, compound D is seen to pass through H₂O₂And NO-dependent manner reduces the mitochondrial membrane potential of tumor cells.

Claims

1. An azonium diol salt compound is a compound shown as a general formula (I):

x is hydrogen; NR (nitrogen to noise ratio)₁R₂Is unsubstituted pyrrolyl, piperazinyl, morpholinyl, piperidinyl.

2. An azonium diol salt compound, wherein the compound is selected from azonium diol salt derivatives having the following structures:

3. a process for the preparation of a compound according to claim 1 or 2, which comprises reacting intermediate 1 with 4-aminobenzyl alcohol to give intermediate 2, brominating the alcoholic hydroxyl group in intermediate 2, and reacting with a different substituted azonium dialkoxide;

4. use of a compound according to claim 1 or 2 for the preparation of an antitumor medicament.

5. An anti-tumor agent comprising the compound of claim 1 or 2.

6. The formulation of claim 5, further comprising a pharmaceutical carrier.

7. The formulation of claim 5, further comprising a pharmaceutical excipient.

8. The preparation of claim 5, wherein the preparation is in a dosage form selected from the group consisting of injection solution, lyophilized powder for injection, controlled release injection, liposome injection, suspension, implant, suppository, capsule, tablet, pill and oral liquid.

9. The formulation of claim 6, wherein the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

10. The formulation of claim 7, wherein the pharmaceutical excipients comprise solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, anti-adhesives, permeation enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, anti-foaming agents, thickening agents, encapsulation agents, humectants, absorbents, flocculating and deflocculating agents, filter aids, and release retardants.

11. The formulation of claim 10, wherein the pharmaceutical excipients comprise microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.