CN115068477A

CN115068477A - Application of rapamycin derivative in preparing antitumor drugs

Info

Publication number: CN115068477A
Application number: CN202210644188.8A
Authority: CN
Inventors: 陈晓明; 应加银; 黄捷; 陈夏琴; 李夸良; 刘颖
Original assignee: Fujian Institute of Microbiology
Current assignee: Fujian Institute of Microbiology
Priority date: 2022-06-09
Filing date: 2022-06-09
Publication date: 2022-09-20
Anticipated expiration: 2042-06-09
Also published as: CN115068477B

Abstract

The invention relates to the field of antitumor drugs, in particular to application of a rapamycin derivative in preparing an antitumor drug. The rapamycin derivative is 40-O- (3- (4-piperidinecarboxylate) -1H-1,2, 3-triazole-1-yl)) ethyl rapamycin. According to the invention, the rapamycin derivative has strong antitumor activity through in vitro anticancer activity model screening, shows strong inhibition effect on various cancer cell strains, and particularly has significantly improved activity on gastric cancer cell strains compared with a control drug rapamycin. By researching the in vivo and in vitro tumor inhibition activity of the compound, a theoretical basis is provided for continuously and deeply developing more excellent rapamycin mTOR targeted drugs.

Description

Application of rapamycin derivative in preparing antitumor drugs

Technical Field

The invention relates to the field of antitumor drugs, in particular to application of a rapamycin derivative in preparing an antitumor drug.

Background

mTOR is a silk/threonine kinase, which is the center of cell growth, proliferation and metabolism, and it receives information on hormones, growth factors, trophic factors and cellular energy levels to regulate cell growth, proliferation and metabolism. In cells, mTOR C1 consisting of mTOR and associated proteins and the upstream PI3K and AKT form a classical PI3K/AKT/mTOR signaling pathway, participate in biological processes such as gene transcription, protein translation, ribosome synthesis and the like, and play an important role in cell growth, apoptosis, metabolism, autophagy and angiogenesis. In the PI3K/AKT/mTOR signaling pathway, phosphorylation of PIP2 by growth factor-activated PI3K produces PIP3, which in turn phosphorylates AKT by PIP3, activated AKT relieves the TSC-1/TSC-2 from inhibiting mTORC1, allowing mTORC1 to activate and act on downstream substrates. p70S6K1 and 4EBP1 are the most direct and important downstream substrates for mTORC 1. After phosphorylation of mTOR, p70S6K1 phosphorylates S6, drives translation of 5' terminal oligopyrimidine mRNA, and promotes translation elongation and ribosome biosynthesis. 4EBP1 is activated by phosphorylation of mTOR when isolated from eIF4E, facilitating translation of the coding protein which regulates cell proliferation and metabolism. Thus, over-activation of mTOR increases the synthesis of a variety of proteins associated with tumorigenesis and progression, such as cyclin D1, which promotes cell cycle progression, HIF, which drives expression of the pro-angiogenic factor VEGF, etc.

Rapamycin (rapamycin, also called sirolimus) which is the first mTOR inhibitor is a secondary metabolite produced by streptomyces hygroscopicus, and has been used as an immunosuppressant and a stent coating drug for rejection reaction of organ transplantation and treatment of restenosis of coronary artery. In the research on the immunosuppressive mechanism of rapamycin in the early 90 s, scientists found 289kDa of mTOR in mammalian cells, and the multifunctional role of rapamycin in tumor resistance and the like was recognized with the disclosure of the function of mTOR and the signal transduction pathway of mTOR. Rapamycin binds to mTORC1 after forming a complex with FKBP12, and the inhibition of mTOR activity blocks mTOR signaling pathway information transfer, leads apoptosis of tumor cells, and promotes autophagy and cycle arrest. The study of rapamycin for treating cancers such as leukemia, renal cancer, liver cancer, breast cancer, non-small cell lung cancer, bladder cancer, etc. is in clinical trial. The antitumor activity of rapamycin attracts the attention of pharmaceutical workers in various countries, and local modification and alteration are carried out on the rapamycin structure, so that a series of valuable derivatives are obtained. Temsiriolimus (trade name: Torisel) developed by Hui's pharmaceutical (Wyeth) was approved by the FDA for the treatment of advanced kidney cancer, and Everolimus (trade name: Afinitor) developed by Novartis was approved by the FDA for the treatment of advanced kidney and breast cancer, etc. In conclusion, a plurality of anti-cancer drugs have been successfully developed and have achieved satisfactory clinical effects by modifying and modifying the rapamycin structure. Therefore, the development of structural modification of rapamycin to obtain rapamycin mTOR targeted antitumor drugs with stronger antitumor activity is always the direction of efforts of drug researchers.

Disclosure of Invention

The invention aims to solve the technical problem of providing an application of a rapamycin derivative in preparing an anti-tumor medicament.

The invention is realized in the following way:

the rapamycin derivative is 40-O- (3- (4-piperidine ethyl formate) -1H-1,2, 3-triazole-1-yl)) ethyl oxygen rapamycin, and the structural formula of the rapamycin derivative is as follows:

the invention provides application of the rapamycin derivative in preparing an antitumor drug.

Further, the antitumor drug includes a drug that inhibits tumor growth or inhibits tumor cell proliferation.

Further, the anti-tumor drug includes a drug that promotes or induces apoptosis of tumor cells.

Further, the tumor includes human non-small cell lung cancer, human gastric cancer, human bladder cancer, human melanoma, human pancreatic cancer, human renal cancer, human prostate cancer, human neuroblastoma, human colon cancer, breast cancer, human cervical cancer, and human nasopharyngeal cancer.

Furthermore, the tumor cells comprise human non-small cell lung cancer A549, human gastric cancer AGS, human bladder cancer 5637, human melanoma A375, human pancreatic cancer BXPC-3, human renal cancer cell ACHN, human prostate cancer cell PC-3, human neuroblastoma U251, human colon cancer HCT116, breast cancer cell T47D, cervical cancer CASKI and nasopharyngeal cancer CNE-2.

The invention has the following advantages: according to the invention, the rapamycin derivative has strong antitumor activity through in vitro anticancer activity model screening, shows strong inhibition effect on various cancer cell strains, and is remarkably improved in activity compared with a control drug rapamycin. By researching the in vivo and in vitro tumor inhibition activity of the compound, a theoretical basis is provided for continuously and deeply developing more excellent rapamycin mTOR targeted drugs.

Drawings

The invention will be further described with reference to the following examples with reference to the accompanying drawings.

FIG. 1 shows the change in tumor volume in groups of nude mice.

FIG. 2 shows the expression of CD34 (x 400) in nude mouse transplanted with gastric cancer AGS cell tumor.

FIG. 3 shows the CD34 expression analysis of nude mouse transplanted tumor tissue; group ratio to Control, P < 0.05; group # vs. Rapamycin, P < 0.05.

FIG. 4 shows the expression of CC3 (x 400) in nude mouse transplanted tumor tissue with gastric cancer AGS cells.

FIG. 5 shows the expression analysis of nude mouse transplanted tumor tissue CC 3; p <0.05 for Control group ratio.

Detailed Description

The examples are intended to illustrate, but not to limit, the scope of the invention. The nuclear magnetic resonance hydrogen spectrum of the compound prepared by the invention is measured by Bruker ARX-300, and the mass spectrum is measured by Agilent 1100 LC/MSD; all reagents used were analytically or chemically pure.

Example 1

Preparation of mono, rapamycin derivatives

Step A: preparation of C-40- (2-bromoethyl) oxy rapamycin

Bromoethanol 12.50g (100mmol) was added to a 100mL dichloromethane solution, after the addition was complete, cooled to-30 deg.C, 2, 6-lutidine 16.10g (150mmol) was added, followed by dropwise addition of trifluoromethanesulfonic anhydride 33.86g (120mmol), and the reaction was stirred magnetically for 2 hours. The reaction was followed by TLC, 1mL of water was added and stirring was continued for 10 minutes, the reaction solution was poured into 80mL of water, extracted with dichloromethane, the combined extracts were washed with water and dried over anhydrous sodium sulfate. Evaporating to dryness to obtain oily substance, and separating by column chromatography to obtain 17.3g (67.58mmol) of sulfonate side chain product (compound 2) with yield of 67.5%.

Rapamycin (6 g, 6.60mmol) was added to 60mL of toluene solution, 5.1g (20mmol) of the sulfonic acid ester side chain (Compound 2) and 8mL of Diisopropylethylamine (DIPEA) were added, the reaction was heated to 60 ℃ and reacted for 3 hours, after completion of TLC tracing, the reaction mixture was poured into 100mL of water, washed with water, extracted with dilute hydrochloric acid, saturated sodium bicarbonate and saturated brine, and the organic layer was dried over anhydrous sodium sulfate. Evaporation to dryness gave 4.66g (4.6mmol) of oil, i.e. C-40- (2-bromoethyl) rapamycin (compound 3), 69.3% yield, MS: 1044.2(M + Na).

And B: preparation of C-40- (2-azidoethyl) oxy rapamycin

2.04g (2mmol) of C-40- (2-bromoethyl) rapamycin (Compound 3) was gradually added to 30mL of a solution of N, N-dimethylformamide, and an aqueous solution of sodium azide (34.4mmol,2.24g,5mL of H) was added thereto at room temperature ₂ O), after the addition was complete, the reaction was carried out at 60 ℃ for 1 hour. After the reaction is finished, the reaction solution is poured into a large amount of water, ethyl acetate is used for extraction, an extracted organic phase is dried and concentrated under reduced pressure, and column chromatography (PE/Ac: 3:1) is carried out to obtain 1.2g (1.2mmol) of light yellow solid, namely C-40- (2-azidoethyl) oxy rapamycin (compound 4), and the yield is as follows: 61.2%, MS:1006.2(M + Na).

And C: preparation of 40-O- (3- (4-piperidinecarboxylate) -1H-1,2, 3-triazol-1-yl)) ethyl oxygen rapamycin

In a 100mL single-necked flask were added ethyl 4-piperidinecarboxylate (1.56g,10mmol), bromopropyne (1.78g,15mmol), sodium carbonate (2.76g,20mmol), and 100mL of DMF, respectively. Heating and refluxing the reaction liquid for 8 hours, cooling to room temperature, performing TLC tracking detection to detect that the reaction is finished, evaporating the solvent under reduced pressure, extracting with 200mL ethyl acetate, washing the organic phase with 30mL water for 3 times, drying the organic phase with anhydrous sodium sulfate, performing suction filtration, evaporating the solvent under reduced pressure, and performing column chromatography to obtain 1.5g (7.7mmol) of light yellow liquid, namely ethyl 1-propynyl-4-piperidinecarboxylate (compound I), wherein the yield is 76.92 percent, and the MS is 218.2(M + Na).

In a 50mL dry round bottom flask, 0.98g (1mmo1) of C-40- (2-azidoethyl) oxy rapamycin (Compound 4), 2mL DMA, 2mLH were added ₂ 0, and then 0.30mg (1.5mmol) of ethyl 1-propynyl-4-piperidinecarboxylate (compound I) and CuSO4 & 5H were added in that order ₂ O0.8mg (0.032mmol) and sodium ascorbate 22mg (0.11mmol) were stirred at room temperature for 1 hour. TLC (thin layer chromatography) tracking detection reaction is finished, 100mL water is added, 200mL ethyl acetate is used for extraction, 100mL organic phase is washed for 3 times by water, after being dried by anhydrous sodium sulfate, suction filtration and reduced pressure evaporation are carried out to remove the solvent, and then column chromatography separation is carried out to obtain 0.85g (0.73mmol) of white powder, namely 40-O- (3- (4-piperidinecarboxylate) -1H-1,2, 3-triazole-1-yl)) ethyl oxygen rapamycin (compound I), the yield is 73.4%, and MS is 1187.5(M + Na).

The column chromatography uses petroleum ether and acetone as eluent.

The conformational characteristics of compound I are as follows:

¹ H NMR(500MHz,CDCl ₃ )δ7.90(s,1H),6.39(dd,J＝14.8,10.8Hz,1H),6.35–6.26(m,1H),6.14(dd,J＝15.1,10.2Hz,1H),5.97(d,J＝10.6Hz,1H),5.54(dd,J＝15.0,8.7Hz,1H),5.41(d,J＝9.9Hz,1H),5.28(d,J＝5.1Hz,1H),5.16(d,J＝4.3Hz,1H),4.52(t,J＝4.9Hz,2H),4.18(d,J＝5.4Hz,1H),4.13(dt,J＝7.0,4.8Hz,3H),3.99–3.92(m,2H),3.87(s,1H),3.84(s,2H),3.76(d,J＝5.6Hz,1H),3.66(t,J＝7.6Hz,1H),3.59–3.54(m,1H),3.43(d,J＝10.7Hz,1H),3.38(s,1H),3.36(s,2H),3.33(s,3H),3.13(d,J＝3.0Hz,3H),3.02(dd,J＝7.6,3.1Hz,4H),2.75–2.66(m,2H),2.57(dd,J＝16.8,6.5Hz,1H),2.34(d,J＝12.5Hz,4H),2.05(s,2H),2.02–1.96(m,3H),1.94–1.83(m,5H),1.76(s,4H),1.74(s,1H),1.65(s,4H),1.61(d,J＝5.3Hz,3H),1.47(s,5H),1.32(d,J＝4.6Hz,2H),1.28(s,1H),1.26(s,3H),1.25(s,2H),1.23(s,1H),1.21(s,1H),1.18(s,1H),1.15(s,2H),1.09(d,J＝6.7Hz,3H),1.05(d,J＝6.4Hz,3H),0.99(d,J＝6.4Hz,3H),0.95(d,J＝6.5Hz,3H),0.90(d,J＝6.7Hz,3H)。

¹³ C NMR(125MHz,CDCl ₃ )δ215.20,208.21,192.75,174.41,169.26,166.73,140.60,140.00,136.04,135.73,133.53,130.20,130.00,129.43,126.56,126.47,98.47,84.73,84.28,83.13,82.73,77.27,77.16,75.52,68.24,67.17,60.55,60.41,59.28,57.27,55.89,51.25,50.80,46.57,44.21,41.50,40.85,40.53,40.20,38.94,38.27,35.83,35.08,33.79,33.11,32.79,31.49,31.19,29.89,29.69,27.21,27.05,25.27,21.49,21.07,20.66,16.24,15.97,15.85,14.20,13.64,13.25,10.18。

synthetic route to Compounds I

II, in-vitro experiment:

1. test cell

Human non-small cell lung cancer A549, human gastric cancer AGS, human bladder cancer 5637, human melanoma A375, human pancreatic cancer BXPC-3, human renal cancer cell ACHN, human prostate cancer cell PC-3, human neuroblastoma U251, human colon cancer HCT116, human breast cancer cell T47D, human cervical cancer CASKI, and human nasopharyngeal cancer CNE-2 were purchased from the Shanghai cell bank of the Chinese academy of sciences.

2. Experimental procedure

2.1 sample preparation

The samples were dissolved in DMSO respectively to a solubility of 10mol/L and then diluted respectively to final concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 10 and 20u mol/L.

2.2 cell culture

Respectively seeding tumor cells in exponential growth period in 96-well plate (cell concentration is 10) ⁵ 100 ul/ml), culturing for 24hr, adding 100 ul/well fresh culture medium with drug, setting 3 multiple wells for each concentration, setting blank control well (adding culture medium only) as negative control, and setting 3 multiple wells. The culture was continued for 72hr, and the culture was terminated.

2.3SRB detection ¹

Adding 10% of the cultured cells into each wellTCA 50ul, fixed at 4 ℃ for 1 hr. Washing with distilled water for 5 times, naturally air drying, adding 4mg/ml SRB solution 50ul per well, dyeing at room temperature for 30min, discarding supernatant, and washing with 1% acetic acid for 5 times to remove non-specifically bound dye. 150ul of 10m mol/L Tris solution is added into each hole, the mixture is shaken for 5 minutes, an OD value is measured by a microplate reader at the wavelength of 540, and the inhibition rate is calculated. Calculating IC by conversion of inhibition ratio using SPSS software ₅₀ The value is obtained.

3. Results and analysis

Thirdly, in vivo experiment: experiment for inhibiting human gastric cancer AGS (angiomatosis) transplanted tumor in nude mice

In early experiments, the compound I is found to be capable of effectively inhibiting the proliferation of gastric cancer cells AGS, SGC7901 and MGC80-3 in vitro, inducing apoptosis and blocking cell cycle in G ₀ G ₁ Meanwhile, the compound has obvious inhibition effect on the phosphorylation levels of P-mTOR, P-P70S6K1 and P-4EBP in an mTOR signaling pathway. Next we investigated whether compound I has activity in treating gastric cancer in vivo.

1 materials of the experiment

1.1 Experimental animals

Nude mice, male and female, 3-4 weeks old, and 15-20g in body weight, were provided by Shanghai Spiker laboratory animals Co. Animals were kept at constant temperature (25-27 deg.C), constant humidity (45% -50%), and all nude mice were kept in SPF environment. The cage, padding and feed are all subjected to autoclaving treatment. The drinking water, feed and padding after the high-pressure steam sterilization are replaced every 3 days.

1.2 gastric cancer cells

Human gastric cancer cell AGS, purchased from shanghai cell bank of chinese academy of sciences.

2 method

2.1 cell culture: in vitro assay

2.2 preparation of nude mouse tumor-bearing model:

establishing human gastric cancer AGS cell nude mouse transplantation tumor model by adopting subcutaneous injection tumor formation method, taking 0.2ml AGS cell suspension (about containing 3 multiplied by 10) ⁶ Individual gastric cancer cells) were injected subcutaneously into the left flank of the nude mouse. After inoculation, the feed is normally fed and watered every day, and the feed can move freely.

2.3 grouping and drug treatment method:

when the tumor diameter was as large as about 10mm, 7 male nude mice bearing tumor were divided into 3 groups randomly for 3 females. Group a blank control group (vehicle) (n ═ 1+ 1); group B Rapamycin experimental group (n ═ 3+ 1); group C FIM X-145 experimental group (n ═ 3+ 1). The administration is carried out once a day for 20 times. After the experiment was completed, the animals were sacrificed and the transplanted tumors were completely isolated.

The following treatments were performed:

group A: blank control group (propylene glycol: tween 20: ethanol ═ 18:1:1)

Group B: 10mg/kg Rapamycin (propylene glycol: Tween 20: EtOH 18:1:1)

Group C: 10mg/kg of Compound I (propylene glycol: Tween 20: EtOH 18:1:1)

2.4 general observations:

observing the general condition of the nude mice, measuring the volume of the nude mice transplanted tumor one day before injecting the drug, then measuring the volume of the tumor every other day in the whole experiment process, measuring the diameter of the tumor by using a vernier caliper, and respectively measuring in two mutually perpendicular directions, wherein the calculation formula of the tumor volume is as follows: v ═ 0.5ab ² A represents the major diameter of the tumor, and b represents the major diameter of the tumor. The tumor growth curves of the groups were plotted according to the change in the measured tumor volume, and the growth inhibition rate of the tumor was calculated. Tumor growth inhibition rate ═ 100% (1-change in tumor volume in treatment group/change in tumor volume in control group).

3 results

3.1 Effect of each treatment group on the volume of transplanted tumor of human gastric cancer AGS cell tumor-bearing nude mouse

After the human gastric cancer AGS cell tumor-bearing nude mice are treated by a control group, a Rapamycicn group and a compound I group, the volume of transplanted tumors of the three groups of nude mice is obviously different by visual observation, the long diameter and the wide diameter of the transplanted tumors are measured and recorded once every other day, and the measured result is substituted into a transplanted tumor volume calculation formula. The formula for calculating the tumor volume is as follows: v ═ 0.5ab ² And a represents the long diameter of the tumor, and b represents the wide diameter of the tumor. As shown in FIG. 1, the control transplanted tumor became larger, while the transplanted tumors of Compound I and Rapamycin groups were significantly smaller compared to the blank control (p)<0.05) differences were significant, with compound I group being less than Rapamycin group (p)<0.01) the difference was significant. The compound I group and the Rapamycin group can obviously inhibit the growth of AGS transplanted tumors, the tumor volume is reduced, and the compound I has stronger tumor growth inhibition effect than the Rapamycin.

3.2 changes in the microvascular density (CD34) of human gastric carcinoma AGS cell-bearing tumor-transplanted nude mice

Microvessel density (MVD) was measured as CD34 positivity (fig. 2), results were analyzed by image pro plus, data were compared using one-way analysis of variance and LSD-t test. The results show that compared with the Rapamycin group and the compound I group, the MVD value of the control group is the largest, the MVD value of the nude mouse transplanted tumor of each drug group is obviously reduced, the difference has statistical significance (P <0.05), and the compound I group is lower than that of the Rapamycin treatment group, the difference has significant significance (P <0.05), as shown in figure 3. It is speculated that compound I, Rapamycin may inhibit the growth of transplantable tumors by inhibiting angiogenesis.

3.3 expression of human gastric cancer AGS cell tumor-bearing nude mouse transplanted tumor tissue cell (CC3)

Apoptosis of transplanted tumor cells of human gastric cancer AGS cell tumor-bearing nude mice is measured by CC3 (cleaned Caspase-3) positivity (figure 4), results are analyzed by image pro plus, percentage of CC3 positive cells in total cells is calculated, and data are compared by adopting one-factor analysis of variance and LSD-t test. As shown in the results of FIG. 5, the apoptosis of transplanted tumor cells in the compound I group and the Rapamycin group was significantly different (p <0.05) compared with the control group, and the apoptosis ratio in the compound I group was higher than that in the Rapamycin group (p <0.05), which was statistically significant.

Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.

Claims

1. The application of the rapamycin derivative in preparing the antitumor drug is characterized in that: the rapamycin derivative is 40-O- (3- (4-piperidinecarboxylate) -1H-1,2, 3-triazole-1-yl)) ethyl rapamycin, and the structural formula of the rapamycin derivative is as follows:

2. use according to claim 1, characterized in that: the anti-tumor medicament comprises a medicament for inhibiting tumor growth or tumor cell proliferation.

3. Use according to claim 1, characterized in that: the anti-tumor drug comprises a drug for promoting or inducing apoptosis of tumor cells.

4. Use according to claim 1, characterized in that: the anti-tumor drug includes drugs that inhibit angiogenesis.

5. Use according to claim 1, characterized in that: the anti-tumor drug comprises a drug for inhibiting the growth of a transplanted tumor.

6. Use according to claim 1, characterized in that: the tumor comprises human non-small cell lung cancer, human gastric cancer, human bladder cancer, human melanoma, human pancreatic cancer, human renal cancer, human prostate cancer, human neuroblastoma, human colon cancer, human breast cancer, human cervical cancer and human nasopharyngeal carcinoma.