CN113350356A - 2,4, 7-trisubstituted pyrimidoindole compound with anti-tumor and drug-resistant effects - Google Patents
2,4, 7-trisubstituted pyrimidoindole compound with anti-tumor and drug-resistant effects Download PDFInfo
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Abstract
The invention relates to a 2,4, 7-trisubstituted pyrimidoindole structure compound, which can inhibit the growth of various drug-resistant tumor cells, and particularly can efficiently inhibit the proliferation of adriamycin-resistant breast cancer, cisplatin-resistant lung cancer and cisplatin-resistant liver cancer cells. In addition, the compound of the invention can inhibit the clone formation of drug-resistant tumor cells at a very low concentration and inhibit the in vitro excretion function of P glycoprotein and ABCG2 transporter. The invention also relates to a preparation method of the compound and application of the compound in tumor treatment or adjuvant therapy.
Description
Technical Field
The invention relates to a 2,4, 7-trisubstituted pyrimidoindole compound, a preparation method thereof and application thereof in tumor drug resistance treatment.
Background
In recent years, tumors have become one of the leading causes of human death. With the continuous discovery of antineoplastic drugs such as cisplatin, adriamycin, paclitaxel, etc., chemotherapy has become one of the main methods for treating malignant tumors. However, after a period of clinical chemotherapy with cisplatin, adriamycin and paclitaxel, serious problems of acquired tumor resistance are usually inevitable, and the tumor resistance becomes a main cause of failure of clinical treatment and poor prognosis.
The compound is a 2,4, 7-trisubstituted pyrimidoindole structure, can inhibit the growth of adriamycin, cis-platinum and taxol-resistant breast cancer cells, lung cancer cells and liver cancer cells, and particularly can efficiently kill the adriamycin-resistant breast cancer cells. In addition, the compound of the invention can inhibit the cloning formation of the adriamycin-resistant breast cancer cells at a very low concentration, which has important clinical significance for inhibiting the growth of cancers. The invention also finds that the compound can inhibit the discharge function of P glycoprotein of tumor cells and a transporter, thereby inhibiting the discharge of the tumor cells to the medicine and improving the intracellular concentration of the chemotherapeutic medicine. Therefore, the compounds have wide application prospect in tumor treatment or adjuvant therapy.
Numerous studies have shown that tumor cells (e.g., breast, pancreatic, prostate) highly express the sigma-2 receptor (more than 10-fold higher than surrounding normal tissues) during the course of canceration, and therefore, compounds with high affinity for the sigma-2 receptor are able to selectively bind to tumor cells that highly express the sigma-2 receptor. Currently, breast cancer diagnosis mainly depends on radiography (such as CT, with radiation side effect), observation, biopsy (requiring invasive sampling), and other means. The main disadvantages of these methods are their high cost, low accuracy (high false positives, high false negatives) and great physical damage.
The invention relates to a series of compounds with fluorescent structures, high affinity activity and selectivity on sigma-2 receptors, which can be effectively and selectively bound to the sigma-2 receptors and can be applied to diagnosis of tumors by utilizing the characteristic of fluorescence emission. The compounds can antagonize the binding of A beta in nerve cells, prevent the signal transduction and cytotoxicity caused by the A beta, and have the functions of diagnosis, treatment or improvement of AD.
Disclosure of Invention
The invention mainly relates to a 2,4, 7-trisubstituted pyrimidoindole compound, a preparation method thereof and application thereof in tumor drug resistance treatment.
The invention also provides processes and intermediates useful for preparing the compounds of the invention.
The invention also provides compositions comprising a pharmaceutically acceptable carrier and at least one of the compounds of the invention or isomers, prodrugs, pharmaceutically acceptable salts thereof.
The compounds of the invention are useful in the treatment of drug resistant tumors.
The compound can be used for preparing a medicinal composition for treating drug-resistant tumors.
The compounds of the invention are useful for killing drug-resistant tumor cells.
The compounds of the invention are useful for inhibiting the clonogenic formation of drug-resistant tumor cells.
The compounds of the invention are useful for inhibiting the efflux function of P glycoprotein.
The compounds of the invention are useful for inhibiting the in vitro excretion function of the ABCG2 transporter.
The compounds of the present invention may be used alone, in combination with other compounds of the present invention, or in combination with one or more other drugs.
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FIG. 1 Effect of compound XH003 on the clonogenic formation of MCF-7/ADR cells and their parental cells
Detailed Description
A first aspect of the present disclosure relates to the use of compound XH003, or a pharmaceutically acceptable salt, or ester, or prodrug thereof, in the manufacture of a medicament for the treatment of a drug-resistant tumour:
in one embodiment, the drug comprises compound XH003, or an isomer, prodrug, pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable adjuvants or excipients.
In one embodiment, the drug-resistant tumor is selected from one or more of an doxorubicin-resistant breast cancer, a cisplatin-resistant lung cancer, and a cisplatin-resistant liver cancer cell.
In a typical embodiment, the drug-resistant tumor is selected from an doxorubicin-resistant breast cancer.
In one embodiment, the treatment-resistant tumor is formed by inhibiting the cloning of tumor cells.
In one embodiment, the treatment-resistant tumor is by inhibiting P glycoprotein efflux function.
In one embodiment, the treatment-resistant tumor is functional by inhibiting ABCG2 transporter excretion in vitro.
In one embodiment, the therapeutic-resistant tumor is functional by simultaneously inhibiting tumor cell cloning, inhibiting P glycoprotein efflux, and inhibiting ABCG2 transporter efflux in vitro.
Example (b):
example 1: synthesis of 2-benzyl-4- (6, 7-dimethoxy-1, 2, 3, 4-tetrahydroisoquinoline-2-ethylamino) -7- (2-methyltetrazol-5-yl) pyrimido [4, 5] indole (XH003)
1.15 Synthesis of (3-nitrophenyl) tetrazole (B001)
30.0g (202.69mmol) of m-nitrobenzonitrile, 14.5g (222.96mmol) of sodium azide and 45.7g (202.69mol) of zinc bromide are put into a 1000ml flask, 500ml of water is added, the mixture is refluxed and reacted for 24h at 100 ℃, then the temperature is reduced to room temperature, reaction liquid is poured into 500ml of water, 100ml of 3N hydrochloric acid is added, ethyl acetate (3 multiplied by 200ml) is used for extraction, an organic phase is collected, ethyl acetate is dried in a decompression mode to obtain white solid, 800ml of 0.25mol/L NaOH is added, stirring is carried out, white flocculent zinc hydroxide is re-precipitated after the solid is dissolved, suction filtration is carried out, the pH of filtrate is adjusted to 5 by 3.0mol/L of hydrochloric acid, a large amount of white solid is precipitated, 34.8g of dried white product is obtained, and the yield is 90%.
The structure of the product is confirmed:1H NMR(DMSO-d6,ppm)δ8.85-8.86(t,1H),8.48-8.51(dt,1H)，8.43- 8.46(dq,1H),7.91-7.95(t,1H)。
synthesis of 1.22-methyl-5- (3-nitrophenyl) tetrazole (B002)
Dissolving intermediate B00133.0 g (172.64mmol) in 150ml DMF, adding anhydrous potassium carbonate 28.63g (207.17mmol) and iodomethane 29.41g (207, 17mmol), reacting at 90 deg.C for 1h, cooling to room temperature until the reaction liquid turns dark yellow, slowly dropping the reaction liquid into 1L water to precipitate a large amount of white solid, filtering, and drying to obtain white solid. Recrystallization from ethyl acetate and petroleum ether gave 26g of product, 72.2% yield.
The structure of the product is confirmed:1H NMR(DMSO-d6,ppm)δ8.66-8.67(t,1H),8.40-8.42(dt,1H),8.33-8.35 (dt,1H),7.81-7.85(t,1H),4.46(s,3H)。
1.3 Synthesis of N-hydroxy-N-acetyl-4- (2-methyltetrazol-5-yl) aniline (B003)
Dissolving intermediate B00230 g (146.22mmol) in 350ml tetrahydrofuran at room temperature, adding NI powder 10.0g, cooling to 0 ℃ in an ice bath, dropwise adding hydrazine hydrate 7.3g (143, 49mmol) in batches, stirring for 30min at 0 ℃, slowly heating to room temperature to continue reacting for 24h, wherein the solution becomes light yellow, cooling to 0 ℃ after TLC monitors that raw materials disappear, adding sodium bicarbonate 54g with 4 equivalents, generating gas, stirring for 30mim, dropwise adding acetyl chloride 13.77g (175.46mmol) at 0 ℃, slowly heating to room temperature to react for 2h, performing suction filtration, washing filter cakes with tetrahydrofuran (3 × 50ml), collecting filtrate, and performing reduced pressure spin-drying on the tetrahydrofuran to obtain yellow solid. Recrystallization of the solid from ethyl acetate gave 28g of product in 84% yield.
The structure of the product is confirmed:1H NMR(DMSO-d6,ppm)δ10.83(s,1H),8.43(s,1H),7.82-7.84(d,2H), 7.54-7.58(t,1H),4.45(s,3H),2.28(s,3H)。
synthesis of 1.42-amino-6- (2-methyltetrazole-5-) indole-3-carboxamide (B004)
Dissolving intermediate B00326.0g (111.48mmol) in 500ml of chloroform, dissolving part of solid, adding malononitrile 7.4g (111.48mmol), cooling to 0 ℃, stirring for 30 mm, slowly dropping triethylamine 11.28g (111.48mmol), stirring for 30min after dropping, slowly heating to room temperature, reacting for 1h, precipitating a large amount of solid, performing suction filtration, washing a filter cake with (3 x 50ml) of chloroform, placing the filter cake in a 500ml flask, adding triethylamine 11.28g (111.48mmol), methanol 200ml, performing reflux reaction for 10h, clarifying a reaction liquid, precipitating a large amount of off-white solid, concentrating the filtrate under reduced pressure, and performing suction filtration to obtain 16g of off-white solid, wherein the yield is 58%.
The structure of the product is confirmed:1H NMR(DMSO-d6,ppm)δ10.80(s,1H),7.82(s,1H),7.64-7.68(q,2H), 7.03(s,2H),6.60(s,2H),4.39(s,3H)。
synthesis of 1.52-phenylacetylamino-6- (2-methyltetrazole-5-) indole-3-carboxamide (B005)
B00414.6g (56.34mmol) of B00414 is dissolved in 200ml of DMF, 17.1g (169.03mmol) of triethylamine and 17.42g (112.69mmol) of phenylacetyl chloride are added to react at 37 ℃ for 24h, the reaction solution is slowly dropped into 500ml of water, a large amount of yellow solid is precipitated, and the yellow solid is filtered by suction and dried. The solid was placed in a 500ml flask, 200ml of ethyl acetate was added, refluxed for 6h, and filtered with suction to give 15.0g of an off-white product with a yield of 70.9%.
The structure of the product is confirmed:1H NMR(DMSO-d6,ppm)δ12.23(s,1H),11.76(s,1H),8.29(s,1H),7.96- 8.00(m,1H),7.79-7.81(m,1H),7.26-7.40(m,7H),4.41(s,3H),3.91(s,2H)。
1.62-benzyl-7- (2-methyltetrazol-5-yl) -3H-4-oxo-pyrimidine [4, 5] indole (B006)
Dissolving intermediate B00516.3 g (43.4mmol) in 500ml isopropanol, adding potassium tert-butoxide 29.2g (260.4mmol), refluxing at 90 deg.C for 12h, concentrating the reaction solution under reduced pressure after the starting material point disappears, adding 300ml water, adjusting pH to 6 with 6mol/L hydrochloric acid solution, and filtering to obtain off-white solid. The solid was placed in a 1L flask, 500ml methanol was added, refluxing was carried out for 8h, suction filtration was carried out repeatedly for 3 times to obtain 11.6g of a white product with a yield of 74.8%.
The structure of the product is confirmed:1H NMR(DMSO-d6,ppm)δ12.48(b,1H),12.40(b,1H),8.07-8.20(m, 3H),7.91-7.97(m,1H),7.26-7.41(m,4H),4.43(s,3H),4.04(s,2H)。
synthesis of 72-benzyl-4-chloro-7- (2-methyltetrazol-5-yl) pyrimido [4, 5] indoline (B007)
Taking 6.3g (16.76mmol) of intermediate B0066 to a 250ml flask, adding 100ml of phosphorus oxychloride, refluxing for 12h at 110 ℃, concentrating the reaction solution under reduced pressure to about 20ml, slowly dropping into 500ml of ice water, stirring to separate out yellow solid, adjusting the pH to about 8 with 10% NaOH solution, filtering, drying to obtain 6.1g of yellow product, wherein the yield is 92%.
The structure of the product is confirmed:1H NMR(DMSO-d6,ppm)δ12.90(s,1H),8.32-8.35(d,1H),8.19(s,1H), 8.04-8.07(dd,1H),7.22-7.38(m,5H),4.46(s,3H),4.30(s,2H)。
1.82 Synthesis of- (6, 7-methoxy-3, 4-dihydroisoquinoline) -ethylenediamine
Dissolving bromoethylamine hydrobromide 10.0g (48.81mmol) and triethylamine 7.42g (73.22mmol) in 50ml of ethanol, carrying out ice bath to 0 ℃, dropwise adding Boc-anhydride 12.85g (58.57mmol), slowly heating to room temperature after dropwise adding to react for 15h, after the reaction is finished, carrying out reduced pressure spin-drying on the ethanol, adding 50ml of water, extracting with ethyl acetate (3 × 100ml), collecting an organic phase, drying with anhydrous sodium sulfate, and carrying out reduced pressure spin-drying on the ethyl acetate to obtain a light yellow oily product Boc-bromoethylamine 10.0g, wherein the yield is 91.4%.
Dissolving 2.0g (8.8mmol) of 6, 7-dimethoxy-1, 2, 3, 4-tetrahydroisoquinoline hydrochloride in 25ml dichloromethane, adding 2.64g (26.12mmol) of triethylamine and 2.34g (10.45mmol) of Boc-bromoethylamine, stirring at room temperature overnight, pouring 50ml water into the reaction solution after the reaction, extracting the water layer with 3 × 50ml dichloromethane, collecting the organic phase, drying with anhydrous sodium sulfate, adding 4.0g silica gel powder, purifying the product by silica gel column chromatography (eluent: ethyl acetate: petroleum ether ═ 1:1) to obtain 2.4g of light yellow oily product, adding 20ml dichloromethane, dropping trifluoroacetic acid in 1ml, stirring at room temperature for 6h, spinning dichloromethane under reduced pressure, adding 20ml water, adjusting pH to 10 with 10% sodium hydroxide solution, extracting with dichloromethane (3 × 50ml), collecting the organic phase, drying with anhydrous sodium sulfate, spinning dichloromethane under reduced pressure to obtain 1.6g of light yellow oily product, the yield was 95%. Directly used for the next reaction.
1.9 Synthesis of the target product XH003
Taking 0.4g (1.07mmol) of intermediate B007in 10ml DMSO, adding 0.3g (2.14mmol) of anhydrous potassium carbonate and 0.5g (2.14mmol) of 2- (6, 7-methoxy-3, 4-dihydroisoquinoline) -ethylenediamine, reacting at 90 ℃ for 8h, dropping the reaction liquid into ice water after the reaction is finished, precipitating a light yellow solid, carrying out suction filtration, drying, and recrystallizing the light yellow solid ethyl acetate to obtain XH0030.18g with the yield of 29%.
The structure of the product is confirmed:1H NMR(DMSO-d6,ppm)δ12.03(s,1H),8.35-8.37(d,J＝8.2,1H),8.08 (s,1H),7.88-7.90(d,J＝8.2,1H),7.39-7.41(d,J＝7.2,2H),7.27-7.31(d,J＝7.4,3H),7.18-7.22(t, J＝7.3,1H),6.63-6.66(m,2H),4.43(s,3H),4.08(s,2H),3.80-3.85(m,2H),3.69-3.70(6H,2OMe), 3.58(s,2H),2.71-2.76(m,6H)；13C NMR(DMSO-d6,ppm)δ166.61,165.33,157.48,157.20, 147.60,147.34,139.86,136.83,129.62(2C),128.57(2C),127.11,126.47,126.39,122.82,121.84, 121.75,118.51,112.26,110.41,109.14,93.97,57.27,55.95,55.91,55.72,51.12,46.29,38.33, 28.70；LC/MS-m/z:576.4[M+1]+(exact mass:575.3)。
example 2: determination of toxic effect and drug-resistant fold of compound XH003 on MCF-7/ADR, A549/DDP, HepG2/DDP and parent cells thereof
When the cells in good growth state grow to 80%, digesting with pancreatin for 2min, terminating digestion with a culture medium containing 10% FBS to prepare a uniform single cell suspension, inoculating to a 96-well plate at a cell density of 5000 cells/well, and culturing in a cell incubator; 24h after inoculation, cells were grouped as follows: DMEM complete medium treated group (placebo), different XH003 drug concentration groups (1.25, 2.5, 5, 10, 20, 40) and doxorubicin, cisplatin and paclitaxel positive drugs; after the administration treatment is carried out for 24h, 48h and 72h respectively, the cell proliferation condition is detected by using a CCK-8 method, the old culture medium is discarded, 100 mu l of CCK-8 working solution is added, the culture is continued for 1.5h in an incubator, and the absorbance of each hole is measured at the wavelength of 450nm by using an enzyme-labeling instrument.
Survival rate ═ 100% (experimental absorbance-blank well absorbance)/(control absorbance-blank well absorbance) ], drug resistance fold ═ drug resistant cell IC 50/parent cell IC50
The CCK-8 method detects the toxicity of XH003 to the cell proliferation of three drug-resistant cells MCF-7/ADR, A549/DDP, HepG2/DDP and parent cells thereof for 24h, 48h and 72h, and uses Adriamycin (ADR), cisplatin (DDP) and paclitaxel as positive control groups. Results show that compared with positive drugs of adriamycin and cisplatin, XH003 has obvious inhibition effect on the proliferation of three multidrug resistant cells MCF-7/ADR, A549/DDP and HepG2/DDP, and the effect is equivalent to that of parent cells thereof and is concentration dependent; the positive medicines of adriamycin and cisplatin obviously weaken the proliferation inhibition effect on drug-resistant cells MCF-7/ADR, A549/DDP and HepG2/DDP (see tables 1-6).
And calculating the IC50 of 48h between XH003 and positive drug effect resistant cells and parent cells, and comparing the drug resistance times between the XH003 and the positive drug effect resistant cells and the parent cells. The results showed that XH003 exhibited good antitumor multi-drug resistance activity against doxorubicin (fold-resistance of 44.57, 0.95, 371.61 μ M), cisplatin (fold-resistance of 5.55, 6.29, 4.54 μ M), and paclitaxel (fold-resistance of 25.13, 54.86, 37.92 μ M, respectively) (table 7).
TABLE 1 Effect of XH003 on survival of MCF-7/ADR cells
P <0.05, P <0.01 compared to blank group
TABLE 2 Effect of XH003 on survival of MCF-7 cells
P <0.01 in comparison to blank group
TABLE 3 Effect of XH003 on survival of A549/DDP cells
P <0.05, P <0.01 compared to blank group
TABLE 4 Effect of XH003 on A549 cell survival
P <0.01 in comparison to blank group
TABLE 5 Effect of XH003 on HepG2/DDP cell survival
P <0.01 in comparison to blank group
TABLE 6 Effect of XH003 on HepG2 cell survival
P <0.01 in comparison to blank group
TABLE 7 toxic Effect of XH003 on various resistant cells MCF-7/ADR, A549/DDP, HepG2/DDP and their parent cells (IC50) and fold resistance
| XH003 | Adriamycin | Cis-platinum | Paclitaxel | |
| MCF-7/ADR | 5.35±0.42 | 27.19±0.28 | 45.83±1.58 | 18.85±0.45 |
| MCF-7 | 3.79±0.13 | 0.61±0.019 | 8.26±0.40 | 0.75±0.026 |
| Multiple of drug Resistance (RF) | 1.41 | 44.57 | 5.55 | 25.13 |
| A549/DDP | 7.74±0.32 | 0.20±0.06 | 72.67±2.93 | 23.04±0.92 |
| A549 | 4.59±0.35 | 0.21±0.01 | 11.55±0.36 | 0.42±0.05 |
| Multiple of drug Resistance (RF) | 1.69 | 0.95 | 6.29 | 54.86 |
| HepG2/DDP | 8.29±0.04 | 48.31±3.10 | 52.90±0.63 | 20.86±0.43 |
| HepG2 | 4.07±0.25 | 0.13±0.12 | 11.65±0.60 | 0.55±0.09 |
| Multiple of drug Resistance (RF) | 2.04 | 371.61 | 4.54 | 37.92 |
Example 3: the effect of the compound XH003 on the cloning formation of MCF-7/ADR cells and parent cells thereof is to take MCF-7 and MCF-7/ADR cells with good growth, seed 5000 cells/well in a six-well plate after digestion and counting for 24h, and after the cells are attached, divide the MCF-7 and MCF-7/ADR cells into the following groups: complete medium group (control group), XH003 (0.3125, 0.625, 1.25, 2.5, 5. mu.M), drug effect 24h later, replaced complete medium culture for 10 days, every other day dosing. After culturing for 10 days, terminating the culture, removing the culture solution, washing the cells for 2 times by PBS (phosphate buffer solution), fixing the cells by 4% paraformaldehyde for 30min, adding 1% crystal violet for dyeing for 15min, slowly washing the dyeing solution by distilled water, drying, and photographing under an inverted microscope to observe and count the cloned colony cells with more than 50 cells. Clone formation rate (number of clones/number of seeded cells) 100.
The results showed that XH003(1.25, 2.5, 5. mu.M) significantly inhibited the clonogenic activity of MCF-7/ADR and MCF-7 cells (P < 0.05) compared to the blank control, and that at the same concentration, XH003 inhibited the clonogenic activity of MCF-7/ADR as compared to MCF-7 (see FIG. 1).
Example 4: effect of compound XH003 on accumulation of rhodamine 123(Rh123) in MCF-7/ADR cells and their parent cells
To investigate the mechanism of XH003 antitumor resistance, the effect of XH003 on the transport function of P glycoprotein was explored by an experiment in which Rh123 was accumulated in cells. Taking well-grown cells, digesting, centrifuging, resuspending to prepare a cell suspension, inoculating the cell suspension on a 6-well plate at the cell density of 2X 105, and culturing for 24 hours in a cell incubator at 37 ℃; after the cells adhere to the wall, the cells are grouped as follows: (1) a drug-free placebo group; (2) different concentrations of XH003(1.25, 2.5, 5, 10. mu.M); (3) the verapamil group, the positive drug, was 10 μ M. Firstly, discarding an old culture medium, respectively adding different medicines, and continuously culturing for 2 hours in a cell incubator; then, the old culture medium is discarded, 1mL of PBS is added to wash the cells for 2 times, 5 mu g/mL of Rh123 is added to each group of cells, and the cells are incubated for 30 min; after incubation, adding 1ml of PBS to clean cells for 2 times, adding pancreatin to digest and centrifuge, discarding old culture medium, adding 2ml of PBS to fully resuspend cells, rotating at 2000rpm, and centrifuging for 10 min; the supernatant was then discarded, 500. mu.l of PBS was added to resuspend the cells and transferred to a flow tube, and the fluorescence intensity was measured by flow cytometry.
The results show that the concentration of Rh123 in MCF-7/ADR cells and MCF-7 of parent cells thereof is obviously improved by XH003 at concentrations of 2.5, 5 and 10 mu M (P < 0.05) in a concentration-dependent manner compared with a blank control group, and the intracellular accumulation effect of 2.5 mu M XH003 in MCF-7/ADR cells is equivalent to that of a P-gp transporter inhibitor verapamil 10 mu M, which indicates that XH003 can inhibit the transport function of P glycoprotein, thereby increasing the intracellular concentration of chemotherapeutic drugs (see Table 8).
TABLE 8 Effect of XH003 on Rh123 accumulation in MCF-7/ADR cells and their parental cells
P <0.05, P <0.01 compared to blank group
Example 5: effect of Compound XH003 on Rh123 efflux from MCF-7/ADR cells
To further confirm that Rh123 intracellular accumulation was related to XH003 inhibition of P glycoprotein-mediated drug efflux, we performed Rh123 efflux experiments to monitor changes in intracellular concentrations of Rh123 at MCF-7/ADR over 30, 60, 90, 120min for XH 003. After cell digestion and centrifugation, the suspension was resuspended in a cell suspension, seeded at a cell density of 2X 105 onto a 6-well plate, cultured in a cell incubator at 37 ℃ for 24 hours, and the cells were grouped into (1) a drug-free blank control group, (2) XH003(1.25, 2.5. mu.M) at different concentrations, and (3) verapamil group, a positive drug, at 10. mu.M, respectively.
After the cells adhered to the wall, 5. mu.g/mL Rh123 was added and the culture was continued for 2h in the incubator. Then, the cells were washed 2 times with 1ml PBS, and further incubated for 0, 30, 60, 90, and 120min by adding different concentrations of drugs. After the incubation is finished, adding 1ml of PBS to clean the cells for 2 times, adding pancreatin to digest and centrifuge, adding 2ml of PBS to resuspend the cells, rotating at 2000rpm, and centrifuging for 10 min; the supernatant was discarded, 500. mu.l of PBS was added to resuspend the cells and transferred to a flow tube, and the fluorescence intensity was measured by flow cytometry.
The results show that after removal of Rh123, the cell concentrations of Rh123 at 30min were about 76%, 92%, 75% for XH003(1.25, 2.5 μ M) and verapamil (10 μ M), respectively, while the blank was 45%; at 60min, the cell concentrations of XH003(1.25, 2.5. mu.M), verapamil (10. mu.M) and Rh123 in the blank were about 74%, 92%, 34%, 22%, respectively; at 90min, the cell concentrations of XH003(1.25, 2.5. mu.M), verapamil (10. mu.M) and Rh123 of the blank were about 69%, 96%, 35%, 20%, respectively; at 120min, the cell concentrations of XH003(1.25, 2.5. mu.M), verapamil (10. mu.M) and Rh123 in the blank control were about 69%, 90%, 31% and 20%, respectively, and the inhibitory effect of XH003(1.25, 2.5. mu.M) was significantly better than that of verapamil (10. mu.M). The results show that XH003 can inhibit the efflux function of P glycoprotein transporter, reduce the efflux of Rh123 in MCF-7/ADR cells and is concentration-dependent (see Table 9).
TABLE 9 Effect of XH003 on Rh123 efflux from MCF-7/ADR cells
P <0.01 in comparison to blank group
Example 6: effect of Compound XH003 on mitoxantrone accumulation in MCF-7/ADR cells and their parental cells
To explore the effect of XH003 on the transport function of the ABCG2 transporter, MCF-7/ADR and its parental cell, MCF-7, were used to observe the effect of XH003 on mitoxantrone accumulation in cells. Taking well-grown cells, digesting, centrifuging, resuspending to prepare a cell suspension, inoculating the cell suspension on a 6-well plate at the cell density of 2X 105, and culturing for 24 hours in a cell incubator at 37 ℃; after the cells adhere to the wall, the cells are grouped as follows: (1) a drug-free placebo group; (2) different concentrations of XH003(1.25, 2.5, 5, 10. mu.M); (3) positive drug KO 14310. mu.M. Firstly, discarding an old culture medium, respectively adding different medicines, and continuously culturing for 2 hours in a cell incubator; then, discarding the old culture medium, adding 1ml PBS to clean the cells for 2 times, adding 5 mu mol/L mitoxantrone into each group of cells, and incubating for 30 min; after the incubation is finished, adding 1ml of PBS to clean the cells for 2 times, digesting and centrifuging the cells by pancreatin, then adding 2ml of PBS to fully resuspend the cells, and centrifuging at 2000rpm for 10 min; the supernatant was then discarded, 500. mu.l of PBS was added to resuspend the cells and transferred to a flow tube, and the fluorescence intensity was measured by flow cytometry.
The results showed that XH003 at concentrations of 2.5, 5 and 10. mu.M and the ABCG2 transporter inhibitor KO143 (10. mu.M) significantly increased the accumulation concentration of mitoxantrone in MCF-7/ADR and MCF-7 cells, which are parental cells thereof (P < 0.05), and that XH003 had an effect on mitoxantrone accumulation in MCF-7 cells equivalent to KO143, indicating that XH003 had a certain inhibitory effect on the transport function of ABCG2 transporter (see Table 10)
TABLE 10 Effect of XH003 on mitoxantrone accumulation in MCF-7/ADR cells
P <0.05, P <0.01 compared to blank group
Example 7: effect of Compound XH003 on mitoxantrone efflux in MCF-7/ADR cells
To further determine that mitoxantrone intracellular accumulation is associated with XH003 inhibition of ABCG2 mediated drug efflux, cell digestion was centrifuged, resuspended to a cell suspension, seeded at a cell density of 2 × 105 in 6-well plates, incubated at 37 ℃ for 24h in a cell incubator, and the cells were grouped into (1) drug-free controls, (2) different concentrations of a011(1.25, 2.5, 5, 10 μ M), (3) positive drug KO143 group 10 μ M, respectively. After the cells are attached to the wall, 5 mu mol/L mitoxantrone is added and the culture is continued for 2h in the incubator. Then, the cells were washed 2 times with 1ml PBS, and further incubated for 0, 30, 60, 90, and 120min by adding different concentrations of drugs. After the incubation is finished, adding 1ml of PBS to clean the cells for 2 times, adding pancreatin to digest and centrifuge, adding 2ml of PBS to resuspend the cells, rotating at 2000rpm, and centrifuging for 10 min; the supernatant was discarded, 500. mu.l of PBS was added to resuspend the cells and transferred to a flow tube, and the fluorescence intensity was measured by flow cytometry.
As a result, as shown in Table 11 below, XH003(1.25, 2.5, 5, 10. mu.M) was found to significantly increase the intracellular mitoxantrone content after removing mitoxantrone, and to have an effect of suppressing efflux superior to that of KO143 (10. mu.M), compared to the control group, and the results showed that XH003 could increase the intracellular mitoxantrone content by suppressing the efflux function of ABCG2 (see Table 11).
TABLE 11 Effect of XH003 on mitoxantrone efflux in MCF-7/ADR cells
P <0.05, P <0.01 compared to blank group.
Claims (6)
1. Use of compound XH003, or a pharmaceutically acceptable salt, or ester, or prodrug thereof, in the manufacture of a medicament for the treatment of a drug resistant tumour:
2. use according to claim 1, wherein the medicament comprises compound XH003, or an isomer, prodrug, pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable adjuvants or excipients.
3. The use according to claim 1, wherein the drug-resistant tumor is selected from one or more of an adriamycin-resistant breast cancer, a cisplatin-resistant lung cancer and a cisplatin-resistant liver cancer cell; preferably, the drug resistant tumor is selected from the group consisting of doxorubicin-resistant breast cancers.
4. The use of any preceding claim, wherein the treatment-resistant tumor is formed by inhibiting the clonal formation of tumor cells.
5. The use of any preceding claim, wherein the treatment-resistant tumor is by inhibiting P glycoprotein efflux function.
6. The use of any preceding claim, wherein the treatment-resistant tumor is dysfunctional in vitro by inhibiting the ABCG2 transporter.
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