CN115894490B - Hydroxy evodiamine and erlotinib split drug containing indoloquinone unit, and preparation method and application thereof - Google Patents
Hydroxy evodiamine and erlotinib split drug containing indoloquinone unit, and preparation method and application thereof Download PDFInfo
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- CN115894490B CN115894490B CN202211328516.XA CN202211328516A CN115894490B CN 115894490 B CN115894490 B CN 115894490B CN 202211328516 A CN202211328516 A CN 202211328516A CN 115894490 B CN115894490 B CN 115894490B
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- erlotinib
- evodiamine
- indoloquinone
- hydroxy
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- 238000002360 preparation method Methods 0.000 title claims abstract description 9
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- HMXRXBIGGYUEAX-SFHVURJKSA-N Evodiamine Natural products CN1[C@H]2N(CCc3[nH]c4ccccc4c23)C(=O)c5ccccc15 HMXRXBIGGYUEAX-SFHVURJKSA-N 0.000 claims abstract description 18
- TXDUTHBFYKGSAH-SFHVURJKSA-N Evodiamine Chemical compound C1=CC=C2N(C)[C@@H]3C(NC=4C5=CC=CC=4)=C5CCN3C(=O)C2=C1 TXDUTHBFYKGSAH-SFHVURJKSA-N 0.000 claims abstract description 5
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a hydroxy evodiamine and erlotinib split drug containing an indoloquinone unit, a preparation method and application thereof, wherein the biological activity of the hydroxy evodiamine and erlotinib split drug with the indoloquinone unit is evaluated through in vitro experiments, and the hydroxy evodiamine and erlotinib split drug has strong antiproliferative activity on non-SMALL CELL lun lung cancer (NSCLC) cells and shows time-dose dependence. In addition, the effect of the combined administration of the hydroxyl evodiamine, the erlotinib, the hydroxyl evodiamine and the erlotinib is not the same as that of the drug prepared by combining the hydroxyl evodiamine and the erlotinib and having the indoloquinone unit. Furthermore, the normal LO2 cells are treated by the split drug of the hydroxy evodiamine with the indoloquinone unit and the erlotinib, the toxicity is obviously lower than that of the hydroxy evodiamine and the erlotinib, and the toxicity is far lower than that of the evodiamine prodrug containing the indoloquinone unit disclosed in the prior art.
Description
Technical Field
The invention belongs to the medicine technology, and in particular relates to a preparation method and application of a hydroxy evodiamine and erlotinib split medicine containing an indoloquinone unit.
Background
The pathological mechanism of non-small cell lung cancer has been found to express up to 50% of the Epidermal Growth Factor Receptor (EGFR) in most patients. EGFR tyrosine kinase inhibitor (EGFR-TKI) represented by erlotinib can prevent EGFR phosphorylation, thereby blocking downstream signal transduction, and playing a role of targeting anti-tumor [ Zhang Hui, zhang Shucai. EGFR gene targeting therapy of non-small cell lung cancer, development [ J ]. J. 2017,20 (01) of Chinese lung cancer, 61-65 ]. Clinical studies have shown that erlotinib can extend survival in NSCLC patients, but unfortunately, patients develop drug resistance after short periods of use resulting in disease progression. Many mechanisms are involved in the development of EGFR-TKI resistance, including EGFR secondary mutations and the like [ Chen Rui, zhao Da, wang Lina ]. Non-small cell lung cancer EGFR-TKI resistance mechanism and treatment strategy [ J ]. Tumor prevention and control research [ 2017, 44 (03): 225-230 ]. The combination of traditional Chinese medicine and chemical medicine is researched, but the pharmacokinetic property is complex, and the rationality of medicine compatibility is required to be confirmed by synergistic administration.
Disclosure of Invention
The invention discloses a preparation method and application of a hydroxy evodiamine and erlotinib split drug containing an indoloquinone unit. Chemotherapy is still currently the means of choice for cancer treatment. Although the chemotherapeutic drugs have good treatment effect, the toxic and side effects are large, and drug resistance can be generated. Many natural plant extracts have proven to be effective in the treatment of malignant tumors, whose mechanism of action is different from that of traditional chemotherapeutics, and have gradually attracted attention from the tumor pharmaceutical community. However, both chemotherapeutic agents and natural plant extracts are directed to only a single drug target, and compensatory modulation is likely to occur. The invention reports the design, synthesis and preliminary drug research of the split drug of the hydroxy evodiamine and erlotinib containing the indoloquinone unit. The effect of the synthesized compounds on cell activity and apoptosis of the human NSCLC cell line was studied and the mechanism was further investigated.
The invention adopts the following technical scheme:
A medicine containing indoloquinone unit and composed of hydroxyevodiamine and erlotinib has the chemical structural formula as follows:
The invention discloses an indoloquinone intermediate, which has the following chemical structural formula:
The invention prepares the indoloquinone intermediate, and reacts with hydroxy evodiamine and erlotinib to prepare the hydroxy evodiamine and erlotinib split drug containing indoloquinone units.
The invention discloses a preparation method of the indoloquinone intermediate, which is characterized in that an ethoxycarbonylmethyl group is connected to the N-1 position of a small indoloquinone molecule, the position is ensured to be used as an open reaction site, so that the coupling reaction with a long chain group is promoted, a dimethyl diamine is connected to the C-3 position, the influence of steric hindrance is eliminated, and the stability of a target compound is ensured. Specifically, an azidoamine chain compound 11-azido-3, 6, 9-trioxaundecane-1-amine is coupled with a small molecule of indoloquinone to obtain an indoloquinone compound 9; the compound 9 is activated by 4-nitrophenyl chloroformate and then condensed with N, N-dimethyl ethylenediamine to obtain a compound 10; compound 10 was reacted with triphosgene to give the indoloquinone intermediate.
The chemical structural formula of the indoloquinone small molecule is as follows:
The chemical structural formula of compound 9 is as follows:
The chemical formula of compound 10 is as follows:
the chemical structural formula of the hydroxyl evodiamine is as follows:
The chemical structural formula of erlotinib is as follows:
According to the invention, the indoloquinone intermediate is connected with hydroxy evodiamine through esterification and condensation, and is connected with erlotinib through click reaction, so that the hydroxy evodiamine and erlotinib split medicine containing the indoloquinone unit is obtained.
The invention discloses application of the indoloquinone intermediate in preparing the hydroxy evodiamine and erlotinib split drug containing the indoloquinone unit; also discloses the application of the hydroxy evodiamine and erlotinib split drug containing the indoloquinone unit in preparing antitumor drugs. Preferably, the tumor is lung cancer.
The invention successfully designs and synthesizes the split drug of the hydroxyl evodiamine and erlotinib containing the indoloquinone unit, and then evaluates the bioactivity of the split drug in vitro experiments. The synthesized compound has stronger inhibition activity to A549, H460, PC-9 and PC-9/GR. As a specific experiment: MTT results show that the split medicine can remarkably inhibit cell viability and proliferation, and is dose-dependent and time-dependent, so that the compound has a strong antiproliferative effect. Apoptosis is an important target for anti-tumor therapy, and the influence of compounds on tumor cell apoptosis and cycle is further detected by Annexin V/PI staining flow cytometry. A549 cells were treated with different compounds at the same concentration 48 h, the cells were harvested and stained with Annexin V-FITC and Propidium Iodide (PI). In this study, the split drug of hydroxyevodiamine and erlotinib containing indoloquinone units can induce apoptosis of a549 cells by inducing apoptosis to inhibit proliferation of tumors, and arrest cell cycle in the G0/G1 phase.
Drawings
FIG. 1 is a synthetic route to an indoloquinone intermediate;
FIG. 2 is a synthetic route for a split drug of hydroxy evodiamine and erlotinib containing an indoloquinone unit;
FIG. 3 shows the cytotoxicity results of different compounds on A549, H460, PC-9 and PC-9/GR cells using the MTT assay;
FIG. 4 shows apoptosis results of Compound 13; different compounds were used at the same concentration on a549 cells.
Detailed Description
The invention bonds different medicines in a chemical way to obtain a novel compound with reduced toxic and side effects and added or synergistic pharmacological actions, and has obvious effect improvement compared with the combined medicine. The raw materials and reagents involved in the reaction were all analytically pure except for the specific descriptions, and were purchased from SIGMA ALDRICH TRADING co. The final product is purified by conventional methods such as common silica gel column chromatography, rapid silica gel column chromatography, recrystallization and the like. The nuclear magnetic resonance spectrometer is Bruker AVANCE400 and Bruker AVANCE500 of Germany, TMS is used as an internal standard, DMSO-d 6 or CDCl 3 is used as a solvent, and the chemical shift (d) and the coupling constant (J) units are ppm and Hz respectively. ESI mass spectrometry was performed by Aglient 7250& JEOL-JMS-T100LP AccuTOF mass spectrometer, TLC analysis was performed on silica gel GF254 (China Qingdao ocean chemistry), and silica gel column chromatography was performed on 60G silica gel (China Qingdao ocean chemistry). The human NSCLC cell lines H460 and A549 were both purchased from Shanghai ATCC cell bank, PC-9 and its resistant strain PC-9/GR were purchased from Shanghai Gejia Biotechnology Co. The specific experimental operation and test of the invention are conventional technology.
The synthetic route of the indoloquinone intermediate is shown in figure 1. 5-methoxy-2-methylindole (1) is used as a starting material, a 3-formyl compound 2 is obtained through Vilsmeier reaction treatment, N-1 position is alkylated (3) by utilizing sodium hydride and ethyl bromoacetate, intermediate 3 is subjected to selective nitration, sn/HCl reduction, amine intermediate 5 is generated, fremy's (potassium nitrosopersulfate) is adopted to oxidize the aniline intermediate 5 into quinone (6), 5 equivalents of NaBH 4 is selected to reduce 3-formaldehyde into hydroxyl (7) at 0 ℃, and 1 equivalent of LiOH hydrate is hydrolyzed into ester bonds to obtain a carboxylic acid compound 8. The azidoamine chain compound 11-azido-3, 6, 9-trioxaundecane-1-amine and the carboxylic acid compound 8 can be coupled by using HATU as a catalyst to obtain an indoloquinone compound 9, and the indoloquinone compound can be connected with an alkyne compound through click reaction. In order to improve the stability of the new synthetic compounds and reduce the effect of steric hindrance, a diamine spacer is introduced between the indoloquinone structure and the drug molecule to form a more stable carbamate bond. The compound 9 is activated by 4-nitrophenyl chloroformate and then condensed with N, N-dimethyl ethylenediamine to obtain a compound 10, and secondary amine of the obtained intermediate is converted into chloroformyl (11) by three light gases to improve the reactivity.
Synthesis example
5-Methoxy-2-methyl-1H-indol-3-aminoaldehyde (2).
POCl 3 (0.85 mL,9.28 mmol) was added dropwise to anhydrous DMF (3 mL) at 0deg.C, and the reaction was stirred for 10-15 min to prepare Vilsmeier reagent. 5-methoxy-2-methylindole (1.04 g,6.45 mmol) was dissolved in DMF (3 mL), and the Vilsmeier reagent prepared previously was added dropwise at 0deg.C and the reaction was continued with stirring for 30min. After the reaction was completed, the reaction was slowly added to an ice-cooled 2M NaOH solution, CH 2Cl2 was extracted 3 times, the organic phases were combined, washed with saturated NaCl solution, dried over anhydrous Na 2SO4, concentrated under reduced pressure to remove the solvent and the crystals were washed with ethyl glacial acetate to give light brown solid 2 (1.05 g, 86%).
1H NMR (400 MHz, Chloroform-d) δ 10.04 (s, 1H), 7.74 (d, J = 2.2 Hz, 1H), 7.23 (d, J = 8.8 Hz, 1H), 6.88 (dd, J = 8.8, 2.5 Hz, 1H), 3.88 (s, 3H), 2.72 (s, 3H);13C NMR (101 MHz, Chloroform-d) δ 184.47, 162.71, 156.40, 147.94, 130.22, 126.91, 113.07, 111.82, 102.88, 55.87, 12.11 ppm.
2- (3-Formyl-5-methoxy-2-methyl-1H-indol-1-yl) acetic acid ethyl ester (3)
A NaH suspension (0.564 g in 60% mineral oil, 14.11 mmol) was added to a solution of compound 2 (1.78 g,9.40 mmol) in DMF (3 mL) under nitrogen and stirred at room temperature for 2h. Ethyl bromoacetate (1.25 ml,11.28 mmol) was then added dropwise while maintaining the temperature at 0 ℃, the reaction was allowed to warm to room temperature after the addition was completed, stirring was continued for 2 hours, and TLC monitored for completion of the reaction; then extracted with water, CH 2Cl2, washed with saturated NaCl solution, dried over anhydrous Na 2SO4, concentrated under reduced pressure and purified by column chromatography on silica gel eluting with EtOAc/petroleum ether (1:2, rf=0.25) to give compound 3 as an off-white solid (1.97 g, 97.0%).
1H NMR (400 MHz, Chloroform-d) δ 10.12 (s, 1H), 7.79 (d, J = 2.4 Hz, 1H), 7.08 (d, J = 8.9 Hz, 1H), 6.88 (dd, J = 8.9, 2.5 Hz, 1H), 4.75 (s, 2H), 4.21 (q, J = 7.1 Hz, 2H), 3.87 (s, 3H), 2.60 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 184.40, 167.29, 156.89, 147.80, 131.57, 126.52, 115.06, 113.39, 109.65, 103.35, 62.29, 55.96, 44.98, 14.20, 10.43 ppm.
2- (4-Amino-3-formyl-5-methoxy-2-methyl-1H-indol-1-yl) acetic acid ethyl ester (5)
Compound 3 (1.09 g,5.38 mmol) was dissolved in acetic acid (88.7 mL), the temperature was controlled at 10-15deg.C, and nitric acid solution (3.2 mL nitric acid in 18.8mL acetic acid) was added dropwise over 45min, then warmed to room temperature, and the reaction was continued with stirring for 2 hours. The ice/water mixture is used as a water phase to prevent the excessive temperature of the reaction solution when being added, CH 2Cl2 is used for extraction, and then water and saturated NaCl are used for washing in sequence, and anhydrous Na 2SO4 is dried. Concentrating under reduced pressure to obtain mixture of 4-and 6-nitro; the nitro mixture obtained in the previous step (1.25 g) was dissolved in ethanol, tin particles (1.67 g,14.1 mmol), HCl (3.0M, 16 mL) were added sequentially and heated to reflux for 1 hour, and TLC monitored the progress of the reaction. Water, naHCO 3 (aq) was neutralized, CH 2Cl2 extracted, washed with saturated NaCl solution and dried over anhydrous Na 2SO4. After concentration under reduced pressure, the mixture was separated and purified by column chromatography eluting with EtOAc/petroleum ether (1:1, rf=0.17) to give compound 5 as a yellow solid (two steps, 0.33g, 47%).
1H NMR (400 MHz, Chloroform-d) δ 9.80 (s, 1H), 6.82 (d, J = 8.6 Hz, 1H), 6.36 (d, J = 8.6 Hz, 1H), 5.90 (s, 2H), 4.69 (s, 2H), 4.21 (q, J = 7.1 Hz, 2H), 3.85 (s, 3H), 2.55 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 183.62, 167.36, 149.86, 141.87, 134.11, 132.39, 116.25, 113.08, 110.17, 95.66, 62.15, 57.15, 44.90, 14.18, 10.41 ppm.
2- (3-Formyl-5-methoxy-2-methyl-4, 7-dioxo-4, 7-dihydro-1H-indol-1-yl) acetic acid ethyl ester (6)
Compound 5 (98 mg,0.45 mmol) was dissolved in acetone (10 mL), and phosphate buffer (10 mL,0.3M, pH 6.0) in which Fremy's (0.24 g,0.9 mmol) was dissolved was added, and the reaction was stirred at room temperature for 3 hours. Then extracted with water, CH 2Cl2, washed with saturated NaCl solution, dried over anhydrous Na 2SO4, and concentrated under reduced pressure to remove the solvent to give 6 (86 mg, 82%) as a red solid.
1H NMR (500 MHz, Chloroform-d) δ 10.56 (s, 1H), 5.70 (s, 1H), 5.16 (s, 2H), 4.26 (d, J = 7.0 Hz, 2H), 3.84 (s, 3H), 2.56 (s, 3H), 1.31 (t, J = 7.0 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 188.21, 179.00, 177.77, 166.86, 160.10, 142.70, 129.33, 122.93, 120.10, 106.53, 62.49, 56.84, 46.25, 14.25, 11.05 ppm.
Ethyl acetate-2- (3- (hydroxymethyl) -5-methoxy-2-methyl-4, 7-dioxo-4, 7-dihydro-1H-indol-1-yl) (7)
Compound 6 (100 mg, 0.43 mmol) was dissolved in a mixture of anhydrous methanol (6 mL) and tetrahydrofuran (6 mL), naBH 4 (0.812 g,2.15 mmol) was added at 0deg.C, the reaction stirred for 8 min, and a 10% solution of NH 4 Cl was quenched. The solvent was removed, water, extracted with CH 2Cl2, washed with saturated NaCl solution, dried over anhydrous Na 2SO4, concentrated under reduced pressure, and purified by column chromatography on silica gel eluting with petroleum ether/ethyl acetate (1:1, rf=0.3) to give 57mg (56%) of the product as bright red intermediate 7.
1H NMR (500 MHz, Chloroform-d) δ 5.66 (s, 1H), 5.13 (s, 2H), 4.67 (s, 2H), 4.27 (q, J = 7.2 Hz, 2H), 3.85 (s, 3H), 2.21 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 179.38, 178.66, 167.42, 159.99, 134.61, 129.52, 122.87, 122.55, 106.97, 62.21, 56.74, 56.00, 46.62, 14.27, 9.41 ppm.
2- (3- (Hydroxymethyl) -5-methoxy-2-methyl-4, 7-dioxo-4, 7-dihydro-1H-indol-1-yl) acetic acid (8)
Compound 7 (100 mg,0.325 mmol) was dissolved in a mixture of methanol (6 mL) and tetrahydrofuran (6 mL), cooled to 0deg.C in an ice-water bath, and an aqueous LiOH solution (14.3 mg, 0.3411 mmol) was added dropwise with stirring, warmed to room temperature, and the reaction was continued with stirring for 1 hour. After the reaction was completed, 1M HCl was adjusted to weak acidity (ph=6.0), and after the solvent was removed by concentration under reduced pressure, the mixture was separated and purified on a silica gel column, and EtOAc/CH 3 OH (1:1, rf=0.33) was eluted to obtain 0.89mg (60%) of compound 8 as a bright red solid.
1H NMR (400 MHz, DMSO-d6) δ 5.66 (s, 1H), 4.68 (s, 2H), 4.49 (s, 2H), 3.73 (s, 3H), 2.09 (s, 3H); 13C NMR (101 MHz, Deuterium Oxide) δ 179.67, 179.19, 174.49, 159.68, 138.52, 128.75, 121.05, 120.11, 106.37, 56.77, 53.62, 48.93, 8.33 ppm.
N- (2- (2- (2- (2-azidoethoxy) ethoxy) ethyl) -2- (3- (hydroxymethyl) -5-methoxy-2-methyl-4, 7-dioxo-4, 7-dihydro-1H-indol-1-yl) acetamide (9)
To anhydrous DMF (3 mL) was added compound 8 (84 mg,0.30 mmol), TEA (50 mg,0.493 mmol) and 11-azido-3, 6, 9-trioxaundecan-1-amine (98.2 mg,0.45 mmol) under nitrogen. HATU (136.8 mg,0.36 mmol) was added at 0 ℃ and the reaction stirred at room temperature for 18 hours. CH 2Cl2 extraction, washing with water, 2% dilute hydrochloric acid, saturated sodium carbonate, naCl solution in this order, drying over anhydrous Na 2SO4, concentrating under reduced pressure and separating and purifying CH 2Cl2/ CH3 OH (2:1, rf=0.23) on a silica gel column to give 40.6mg (32%) of red solid 9.
1H NMR (400 MHz, Chloroform-d) δ 6.70 (s, 1H), 5.60 (s, 1H), 4.96 (s, 2H), 4.59 (s, 2H), 3.79 (s, 3H), 3.62 – 3.40 (m, 18H), 2.96 (s, 2H), 2.24 (s, 3H), 1.95 (s, 2H); 13C NMR (101 MHz, Chloroform-d) δ 178.89, 178.32, 165.98, 159.52, 135.47, 128.69, 122.69, 122.23, 106.66, 70.34, 70.26, 70.15, 70.05, 69.96, 69.54, 56.38, 55.53, 50.42, 48.05, 39.24, 9.20ppm. MS (ESI, positive) found(M+Na) 502.19, calc (C21H29N5O8 m/z)479.20.
(1- (14-Azido-2-oxo-6, 9, 12-trioxo-3-azatetradecyl) -5-methoxy-2-methyl-4, 7-dioxo-4, 7-dihydro-1H-indol-3-yl) methyl-12- (methylamino) ethyl) carbamate (10)
Compound 9 (45 mg,0.094 mmol) and pyridine (76. Mu.L) were dissolved in anhydrous CH 2Cl2 (1.0 mL), the solution was cooled to 0deg.C in an ice-water bath, a solution of CH 2Cl2 (0.5 mL) in which 4-nitrophenyl chloroformate (189 mg,0.94 mmol) was slowly added while stirring, warmed to room temperature, and the reaction was continued for 1 hour. CH 2Cl2 extraction, washing with water and saturated NaCl solution, drying anhydrous Na 2SO4, concentrating under reduced pressure, purifying and separating on silica gel column, eluting with CH 2Cl2/ CH3 OH (100:3, rf=0.64), to obtain red yellow solid compound 9a（45.2 mg,76%）.1H NMR (400 MHz, Chloroform-d) δ 8.27 (d, J = 9.2 Hz, 2H), 7.39 (d, J = 9.1 Hz, 2H), 5.65 (s, 1H), 5.47 (s, 2H), 5.02 (s, 2H), 3.83 (s, 3H), 3.61 – 3.35 (m, 18H), 2.39 (s, 3H).
N, N' -methyl ethylenediamine (82 mg,0.93 mmol) was dissolved in anhydrous tetrahydrofuran (8 mL), and compound 9a (200mg,0.31mmol,2mL THF) was slowly added dropwise at 0℃and after 30 minutes, was allowed to warm to room temperature overnight. Saturated NaHCO 3、 CH2Cl2 extraction, brine wash, dry Na 2SO4. Purification by column chromatography on silica eluting with CH 2Cl2 / CH3 OH (2:1, rf=0.18) afforded compound 10 as a reddish brown oil (98 mg, 56%).
1H NMR (400 MHz, Chloroform-d) δ 6.84 (s, 1H), 5.59 (s, 1H), 5.21 (s, 2H), 4.99 (s, 2H), 3.78 (s, 3H), 3.67 (s, 6H), 3.63 – 3.37 (m, 18H), 2.86 (s, 2H), 2.60 (s, 2H), 2.32 (s, 3H); 13C NMR (101 MHz, Chloroform-d) δ187.70, 178.95, 177.76, 166.45, 159.90, 128.95, 106.69, 70.81, 70.76, 70.68, 70.45, 70.15, 69.63, 56.66, 50.82, 48.47, 47.33, 39.65, 32.03, 29.81, 9.71 ppm. MS (ESI, positive) found(M+H) 594.28, calc (C26H39N7O9, m/z) 593.64.
EXAMPLE-preparation of Indoloquinone intermediate
Compound 10 (57 mg,0.096 mmol) was dissolved in anhydrous CH 2Cl2 (3.0 mL) and cooled in an ice-water bath. TEA (142. Mu.L) and triphosgene (28.5 mg,0.096 mmol) were added under stirring, the reaction was continued with stirring at 0℃for 30min, and after completion of the reaction, the solvent was removed by concentration under reduced pressure and dried in vacuo for 1 hour to give Compound 11, which was used in example two without purification.
EXAMPLE two preparation of hydroxy evodiamine and erlotinib Split drug containing Indoloquinone Unit
The chemical structural formula of the hydroxyl evodiamine is as follows:
The chemical structural formula of erlotinib is as follows:
The hydroxyl in the hydroxyl evodiamine is taken as a strong electron withdrawing group to carry out esterification reaction with chloroformyl to obtain the indole quinone compound (12) connected with the evodiamine. In the presence of copper sulfate pentahydrate and sodium ascorbate, connecting an azide group in a compound 12 with an alkynyl part in erlotinib through click reaction to obtain a target compound 13; the reaction process is shown in FIG. 2.
( 14-Methyl-5-oxo-5, 7,8,13 b, 14-hexahydroindole [2',3':3,4] pyridin [2,1-b ] quinazolin-10-yl) ethane-1, 2-diacyldi bis (methyl carbamate) (12 )
Compound 11 was dissolved in anhydrous CH 2Cl2 (2 mL) and cooled with an ice-water bath; the hydroxyl evodiamine is dissolved in anhydrous CH 2Cl2 (5.0 mL), TEA (65 mu L) is added, a CH 2Cl2 solution of the compound 11 is dropwise added into the hydroxyl evodiamine under the protection of nitrogen, and the reaction is stirred at room temperature for 24 hours, and TLC monitoring is carried out. CH 2Cl2 extraction, saturated NH 4 C1, water, 5% NaHCO 3, water and saturated NaCl solution are sequentially washed, and anhydrous Na 2SO4 is dried. Concentrating under reduced pressure, separating and purifying by a silica gel column, eluting with CH 2Cl2 / CH3 OH (2% -4%, rf=0.32), and obtaining the compound 12 as pale red brown solid (40.1 mg, 67%).
1H NMR (400 MHz, Chloroform-d) δ 7.56 – 7.32 (m, 2H), 7.24 – 6.59 (m, 3H), 5.62 (s, 1H), 5.23 (s, 1H), 5.00 (s, 2H), 3.81 (s, 3H), 3.68 (s, 6H), 3.66 – 3.38 (m, 18H), 3.15 – 2.86 (m, 6H), 2.35 (s, 3H), 1.41 (d, J = 4.0 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 188.88, 178.02, 175.35, 173.17, 162.74, 160.12, 156.45, 150.53, 142.85, 138.01, 136.44, 133.06, 128.24, 124.82, 122.15, 114.60, 107.91, 106.47, 96.12, 77.33, 77.22, 77.01, 76.70, 70.64, 70.57, 70.36, 70.04, 56.57, 50.70, 39.51, 29.71 ppm. MS (ESI, positive) found(M+H) 939.97, calc (C46H54N10O12, m/z) 939.40.
(1- (14- (3- ((6, 7-Bis (2-methoxyethoxy) quinazolin-4-yl) amino) phenyl) -1H-1,2, 3-triazol-1-yl) -2-oxo-6, 9, 12-trioxo-3-azatetradecyl) -5-methoxy-2-methyl-4, 7-dioxo-4, 7-dihydro-1H-indol-3-yl) methyl (13)
Compound 12 (33 mg,0.035 mmol) was dissolved in t-butanol (0.3 mL) and water (0.6 mL), erlotinib (17 mg, 0.0070 mmol) was added, after stirring to dissolve, cuSO 4 (1.75 mg,0.028 mL) and sodium ascorbate (2.7 mg,0.014 mmol) were added and the reaction was continued for 1 hour with stirring, TLC monitored the progress of the reaction, silica gel column separation and purification, CH 2Cl2/CH3 OH (2% -3%, rf=0.42) was eluted, affording the objective compound 13 (yield 24%) as a pale-red brown solid.
1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 9.48 (s, 1H), 8.66 (s, 1H), 7.99 (s, 3H), 7.89 (s, 4H), 7.79 (s, 1H), 7.40 (s, 2H), 7.15 (s, 2H), 7.03 (s, 1H), 6.94 (s, 1H), 6.75 (s, 1H), 6.63 (s, 1H), 6.07 (s, 1H), 4.30 (s, 6H), 4.19 (s, 2H), 3.79 (s, 6H), 3.37 (s, 15H), 3.22 (s, 3H), 2.89 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 183.27, 177.44, 176.62, 171.30, 169.45, 166.23, 164.40, 154.37, 148.12, 147.43, 143.98, 142.65, 139.82, 137.30, 133.32, 131.00, 128.82, 126.33, 124.70, 122.50, 119.63, 111.93, 110.54, 103.33, 100.30, 92.66, 83.43, 80.41, 77.41, 70.08, 68.40, 58.27, 52.27, 47.83, 37.93, 36.30, 23.44, 15.86, 8.79 ppm; MS (ESI, positive) found(M+H) 1332.40, calc (C68H77N13O16, m/z) 1331.56; Anal.calcd for C68H77N13O16: C 61.30, H 5.83, N 13.97, O 19.21; found C 60.59, H 6.37, N 13.54, O 19.61.
Example three cytotoxicity experiments
Human NSCLC cell lines A549, H460 and PC-9 cells were incubated in RPMI-1640 medium containing 10% (v/v) fetal calf serum and 1% penicillin/streptomycin (P/S), and PC-9/GR was incubated in DMEM medium containing 10% (v/v) fetal calf serum and 1% penicillin/streptomycin (P/S) at 37℃in A5% CO 2 incubator. When the cell density reaches 70-80%, the subculture is completed. The medium was changed every 3 days.
The in vitro antiproliferative inhibition effect of the target compound 13, the hydroxyl evodiamine, the erlotinib and the hydroxyl evodiamine and the erlotinib on the human NSCLC cell lines A549, H460, PC-9 and the drug-resistant strain PC-9/GR is measured by adopting an MTT method. Cells in the logarithmic growth phase were taken and treated on 96-well cell plates for 24, 48 and 72 h different concentration groups of 1.25, 2.5, 5.0, 10.0 and 20.0. Mu.M were established in each drug group, respectively, wherein 1/2 volumes of hydroxyevodiamine (1.25, 2.5, 5.0, 10.0 and 20.0. Mu.M) were combined with 1/2 volumes of erlotinib (1.25, 2.5, 5.0, 10.0 and 20.0. Mu.M) in the combination administration group. The experimental results are shown in fig. 3, and for the four detected human NSCLC cell lines, the administration of the target compound 13, the hydroxyevodiamine and the erlotinib has obvious inhibition effect, and the cell survival is inhibited in a dose-dependent manner; the in vitro anti-tumor effect of the target compound 13 is stronger than that of the single or combined administration group of the hydroxyevodiamine and erlotinib. In particular compound 13 treatment of a549 for 72 hours with IC 50 of 3.60±0.52 μm; compound 13 treatment of PC-9/GR for 72 hours with IC 50 at 1.32.+ -. 0.01. Mu.M. In addition, the effect of the combined administration experiment of the compound 12 and erlotinib is almost the same as that of the combined administration of the hydroxyevodiamine and erlotinib, and the effect is not as good as that of the compound 13. Further, compound 13 treated normal LO2 cells with an IC 50 of 16.60±0.32 μm for 48 hours, significantly lower toxicity, and much less than the evodiamine prodrugs 9a, 9b, 9c and 9d containing indoloquinone units previously disclosed by the applicant (IC 50 < 9 μm, table 5).
TABLE 1 IC 50, μM for different drug pairs A549
TABLE 2 IC 50, μM for different drug pairs H460
TABLE 3 IC 50, μM of different drug pairs PC-9
TABLE 4 IC 50, μM of different drug pairs PC-9/GR
Example four apoptosis assay
To better investigate whether the inhibition of antiproliferative activity of the different compounds observed in the MTT experiments was related to the ability to induce apoptosis, target compound 13 was used at the same dosing concentration (10 μm) with erlotinib alone on a549 cells for 48h and the percentage of apoptotic cells was quantified using annexin v/PI staining. As shown in fig. 4, the percentage of apoptotic cells induced after treatment 48 administration in a549 cells increased: the control group was 2.89%, the erlotinib experimental group was 6.03%, and the compound 13 experimental group was 21.97%. The overall apoptosis rate of the administered group was varied somewhat as compared to the control group, indicating that the target compound 13 could inhibit tumor proliferation by inducing apoptosis.
Conclusion(s)
The drug split is a rapid and effective new drug research and development means, combines the pharmacophores of two drugs into a single molecule through chemical bonding, and is hopeful to obtain new compounds with reduced toxic and side effects and added or synergetic pharmacological effects. According to the invention, indoloquinone small molecules are used as intermediate connectors, hydroxyl evodiamine and erlotinib are spliced based on a splicing design principle to obtain a target compound, and the in-vitro inhibition effect of the target compound on non-small cell lung cancer cell strains is primarily measured.
According to the invention, the inhibition effect of the target compound 13, the hydroxyevodiamine and the erlotinib on the human NSCLC cell lines A549, H460, PC-9 and drug-resistant strains PC-9/GR is researched, and experimental results show that the target compound 13 has good antiproliferative inhibition effect, shows better in-vitro antitumor activity than that of single-drug hydroxyevodiamine and erlotinib, even the combination of the hydroxyevodiamine and the erlotinib, and has good synergistic effect through conventional calculation; the flow experiment result shows that the compound 13 can induce the apoptosis of A549 cells.
Claims (5)
1. A medicine containing indoloquinone unit and composed of hydroxyevodiamine and erlotinib has the chemical structural formula as follows:
。
2. The method for preparing the split drug of the hydroxy evodiamine and erlotinib containing the indoloquinone unit, which is characterized in that the indoloquinone intermediate and the hydroxy evodiamine are subjected to esterification condensation reaction to obtain the evodiamine containing the indoloquinone unit, and then the evodiamine and erlotinib are subjected to click reaction to obtain the split drug of the hydroxy evodiamine and erlotinib containing the indoloquinone unit; the chemical structural formula of the indoloquinone intermediate is as follows:
；
the chemical structural formula of the hydroxyl evodiamine is as follows:
；
The chemical structural formula of erlotinib is as follows:
。
3. the method for preparing the split drug of the hydroxy evodiamine and erlotinib containing the indoloquinone units according to claim 2, wherein the esterification condensation reaction is performed under the catalysis of organic base; the click reaction is carried out in the presence of a copper salt and an organic reducing agent.
4. The method for preparing the split drug of the hydroxy evodiamine and erlotinib containing the indoloquinone unit according to claim 2, wherein the esterification and condensation reaction is carried out for 20-30 hours at room temperature; the click reaction time is 0.5 to 1.5 hours.
5. The use of the split drug of hydroxyevodiamine and erlotinib containing an indoloquinone unit according to claim 1 in the preparation of an antitumor drug.
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| WO2017152756A1 (en) * | 2016-03-11 | 2017-09-14 | 徐州瑞康生物科技有限公司 | Crgd-erlotinib conjugate and preparation method thereof |
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| WO2011002852A2 (en) * | 2009-06-30 | 2011-01-06 | The Regents Of The University Of Michigan | Pro-drug complexes and related methods of use |
| WO2017152756A1 (en) * | 2016-03-11 | 2017-09-14 | 徐州瑞康生物科技有限公司 | Crgd-erlotinib conjugate and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| Design, synthesis and bioactivity evaluation of the combination of evodiamine and erlotinib linked by indolequinone;Binbin Wei等;《Bioorganic & Medicinal Chemistry Letters》;20240118;第99卷;1-6 * |
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