CN113563336B - Evodiamine prodrug containing indoloquinone unit and preparation method and application thereof - Google Patents

Abstract

The invention relates to an evodiamine prodrug containing indoloquinone units, a preparation method and application thereof. The invention synthesizes a series of evodiamine derivatives with strong activity of indoloquinone units, has strong antiproliferative activity on non-small cell lung cancer (NSCLC), and has dose and time dependence. In vitro experiments evaluate the biological activity of the compounds, and the synthesized compounds have stronger inhibitory activity on lung cancer strains. Through molecular docking analysis, the binding affinity of the ligand and the active site of the target protein is predicted, and the interaction capacity with the protein is stronger.

Description

Indoloquinone unit-containing evodiamine prodrug and preparation method and application thereof

Technical Field

The invention belongs to the medicine technology, and particularly relates to an evodiamine prodrug containing indoloquinone units, and a preparation method and application thereof.

Background

Generally, the traditional Chinese medicine has the functions of synergism and attenuation. At present, TCM is an important component of The comprehensive treatment of cancer and plays an indispensable role in The various stages of lung cancer treatment [ Qi, F., L. ZHao, A. Zhou, et al., the additives of The use of traditional Chinese medicine as an additional therapeutic in The lung cancer treatment, 2015.9 (1): p.16-34 of cancer treatment of The dependent tertiary stage of cancer]. Various Chinese Herbal Medicines (CHM), including herbal compounds and extracts thereof, have been shown to play important roles in anticancer therapy through a variety of pathways and targets [ Li, Z.Y., W.C. Huang, R.S. Tu, et al, sophoraflavanone G inclusions in Human Leukemedia Cells and Blocks MAPK activation. Am J Chin Med, 2016.44 (1): p.165-76.Yang, M.Y., C.H. Hung, C.H. Chang, et al,Solanum nigrum Suppress Angiogenesis-Mediated Tumor Growth Through Inhibition of the AKT/mTOR Pathway. Am J Chin Med, 2016. 44(6): p. 1273-1288.Fu, S., Y. Yang, D. Liu, et al., Flavonoids and Tannins from Smilax china L. Rhizome Induce Apoptosis Via Mitochondrial Pathway and MDM2-p53 Signaling in Human Lung Adenocarcinoma Cells. Am J Chin Med, 2017. 45(2): p. 369-384]. Natural active compounds also play an important role in the plasticity of the treatment of non-small cell lung cancer and are increasingly recognized.

Traditional Chinese medicine, including herbal compounds and extracts thereof, have been shown to play a key role in cancer treatment through a variety of pathways and targets. Evodiamine (EVO) is extracted fromEuodiae FructusThe quinolone alkaloid extracted from the Chinese herbal medicines has low toxicity and has long-term proved to have anti-lung cancer activity. However, it lacks specificity and is limited by undesirable toxicity and side effects.

Disclosure of Invention

The invention discloses an evodiamine prodrug containing indoloquinone units, and a preparation method and application thereof. Evodiamine (EVO) is a low-toxicity natural indole alkaloid, and is widely present in traditional Chinese medicine fructus evodiae. Some researches report the antiproliferative, apoptosis-promoting and anti-invasive effects of evodiamine on cancer cells, and the prior art uses the evodiamine as a lead compound, carries out systematic structure optimization and structure-activity relationship research, designs and synthesizes a plurality of compounds, and screens out some high-activity compounds, such as 10-hydroxyl evodiamine, but the performance of the currently disclosed evodiamine prodrug needs to be improved. The invention reports the design, synthesis and preliminary drug research of an evodiamine prodrug containing indoloquinone units. The effect of the synthesized compound on the activity and apoptosis of human NSCLC cell lines is studied, and the mechanism is further studied.

The invention adopts the following technical scheme:

an evodiamine prodrug containing indoloquinone units, which has the following chemical structural formula:

n is 2 to 4.

The invention discloses an indoloquinone carboxylic acid compound, which has the following chemical structural formula:

n is 2 to 4.

The invention discloses a preparation method of the indoloquinone carboxylic acid compound, which is characterized in that hydroxyindoloquinone reacts with an anhydride compound to prepare the indoloquinone carboxylic acid compound.

The invention discloses a preparation method of the evodiamine prodrug containing indoloquinone units, wherein an indoloquinone carboxylic acid compound is prepared by reacting hydroxyindoloquinone with an anhydride compound; and then reacting the indoloquinone carboxylic acid compound with hydroxyl evodiamine to obtain the evodiamine prodrug containing indoloquinone units.

The chemical structural formula of the hydroxyindoloquinone is as follows:

the acid anhydride compound is diacid anhydride or dithio malonic anhydride; preferably, the chemical formula of the dianhydride is as follows:

n' is 0 to 4.

The chemical structural formula of the hydroxyl evodiamine is as follows:

in the invention, hydroxyindoloquinone and an anhydride compound react in a solvent at room temperature in the presence of an organic catalyst to obtain an indoloquinone carboxylic acid compound; preferably, the reaction time is 12 to 24 hours; the organic catalyst is a pyridine compound such as DMAP, 4-dimethylaminopyridine. Preferably, the molar ratio of the hydroxy indoloquinone to the acid anhydride compound to the organic catalyst is 1: 3: 1.

In the invention, an indoloquinone carboxylic acid compound and hydroxyevodiamine react in a solvent at room temperature in the presence of an organic catalyst and an amine ligand to obtain an evodiamine prodrug containing indoloquinone units. Preferably, the reaction time is 24 to 48 hours; the organic catalyst is a pyridine compound such as DMAP, 4-dimethylaminopyridine; the amine ligand is a carbodiimide, such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI). Preferably, the mol ratio of the indoloquinone compound to the hydroxyevodiamine to the organic catalyst to the amine ligand is 1: 5.

The invention discloses application of the indoloquinone carboxylic acid compound in preparing the evodiamine prodrug containing indoloquinone units; also discloses the application of the evodiamine prodrug containing the indoloquinone unit in preparing antitumor drugs. Preferably, the tumor is lung cancer.

The invention successfully designs and synthesizes a series of evodiamine prodrugs containing indoloquinone units, and then evaluates the biological activity of the prodrugs in vitro experiments. The synthesized compound has stronger inhibition activity on A549 and H358 strains. As a specific experiment: MTT results show that the prodrug of the invention can obviously inhibit cell viability and proliferation, and is dose-dependent and time-dependent, which indicates that the compound has stronger anti-proliferation effect. Apoptosis is an important target for anti-tumor therapy, and the influence of compounds on tumor cell apoptosis is further detected by Annexin V/PI staining flow cytometry. A549 cells were treated with different concentrations of compound for 24 h, harvested and stained with Annexin V-FITC and Propidium Iodide (PI). Annexin V is a phospholipid-binding protein with strong affinity for phosphatidylserine, which appears on the cell surface as a general indicator of apoptosis. In this study, evodiamine prodrugs containing indoloquinone units induced apoptosis in a549 NSCLC cells. In terms of interaction energy, the results showed that compounds 9a, 9b, 9c and 9d had a large interaction with proteins. In general, the active residues of NQO1, including PHE 17, ASN 18, LEU 103, PHE 106, GLY 149, THR 147, GLY 150, TYR 155 and ARG 200, can interact with compounds 9a, 9b, 9c and 9d to varying degrees, and molecular docking assays predict the binding affinity of a ligand at the active site of a target protein.

Drawings

FIG. 1 shows the results of MTT assay for cytotoxicity of various compounds on A549 cells and H358 cells;

FIG. 2 shows the cytotoxicity results of Compound 9b using the MTT method; 9b (0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M and 1. Mu.M) was used at different concentrations on H2122, H358, H23 and A549 cells;

FIG. 3 shows the apoptosis results after treatment of H358 cells with DMSO or different concentrations of compound 9b (0.375, 0.75, 1.5 and 3. Mu.M) (A) stained with annexinv-FITC/PI and analyzed by flow cytometry; (B) detecting apoptosis-related proteins by using Western-blot;

FIG. 4 is a molecular docking simulation assay, the results of three-dimensional docking of compounds 9a (A), 9B (B), 9C (C) and 9D (D) with receptor-ligand interactions of hydrophobic effector regions in the active binding pocket.

Detailed Description

The raw materials and reagents involved in the reaction process of the present invention, except for the specific instructions, were all analytical grade and purchased from Sigma Aldrich tracing co. The final product is purified by common silica gel column chromatography, rapid silica gel column chromatography, recrystallization and other conventional methods. The nuclear magnetic resonance spectrometer is of Bruker AVANCE400 and Bruker AVANCE500 types of Germany company, TMS is taken as an internal standard substance, and DMSO-d ₆ Or CDCl ₃ Chemical shift (d) and coupling constant: (d)J) The units are ppm and Hz, respectively. ESI Mass Spectroscopy by Aglient 7250&The JEOL-JMS-T100LP AccuTOF mass spectrometer adopts silica gel GF254 (Qingdao ocean chemistry, china) for TLC analysis, and adopts 60G silica gel (Qingdao ocean chemistry, china) for silica gel column chromatography.

Human non-small cell lung cancer cell lines H2122, H358, H23 and a549 were purchased from american model culture collection banks (ATCC, manassas, virginia, USA). RPMI 1640 medium, fetal Bovine Serum (FBS) and penicillin-streptomycin (P/S) were purchased from Gibco (Invitrogen Corporation, carlsbad, calif., USA). 3- (4, 5-Dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) was purchased from Thermo Fisher Scientific, beijing Saimer Feishell Scientific, inc. Primary antibodies against GAPDH (# 5174), PARP (# 13684), p-AKT (# 4046), p-ERK (# 4376), t-AKT (# 9272), t-ERK (# 9102), p-mTOR (# 2974), and total mTOR (# 2983) were provided by Cell Signaling Technology (Danvers, MA, USA). Fluorescein secondary antibodies were supplied by LI-COR biosciences (Lincoln, NE, USA). The Annexin V-FITC/PI dead cell apoptosis kit is supplied by BD Biosciences (San Jose, calif., USA).

Synthesis example

5-methoxy-2-methyl-1H-indole-3-aminoaldehyde (2). Under stirring, POCl is added ₃ (0.85mL, 9.28mmol) was added to DMF (N, N-dimethylformamide, 3.0mL,38.9 mmol) cooled to 0 ℃. After addition, vilsmeier reagent was prepared by stirring for 15 minutes at 0 ℃. 5-methoxy-2-methylindole (1.04g, 6.45mmol) is dissolved in 3mL anhydrous DMF and cooled to 0 ℃ and Vilsmeier reagent previously prepared is added. After the addition was complete, the reaction was stirred at 0 ℃ for an additional 30 minutes, checked by TLC spot plates, and after the reaction was complete, the reaction was added to 0 ℃ aqueous 2M NaOH (50 mL), DCM (dichloromethane, 100 mL) was added and isolated by extraction. The aqueous layer was extracted once more with DCM (50 mL). The organic layers were combined, washed with brine and Na ₂ SO ₄ And (5) drying. The solvent was removed and the residue was washed with cooled EtOAc to give 1.05g (86%) of compound 2 as a light brown solid. ¹ H 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); ¹³ C 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, 36.61 ppm。

5-methoxy-1, 2-dimethyl-1H-Indole-3-carbaldehyde (3). Compound 2 (1.78g, 9.40mmol) was added to a suspension of NaH (0.564g in 60% mineral oil, 14.11 mmol) under nitrogen. After stirring the mixture at room temperature for 2 hours, it was cooled to 0 ℃ with an ice water bath. Ethyl bromoacetate (1.25mL, 11.28mmol) was added thereto, and the reaction was warmed to room temperature and stirred for 2 hours. Water (50 mL) was added to the mixture and extracted with DCM (40mL X3). The organic layers were combined, washed with water (50 mL) and saturated brine (50 mL), then Na ₂ SO ₄ And (5) drying. After removal of the solvent, the residue was purified on column chromatography eluting with EtOAc petroleum ether (1: 2, rf = 0.25) to give 1.97g (97.0%) of compound 3 as an off-white solid. ¹ H NMR (400 MHz, Chloroform-d) δ 10.09 (s, 1H), 7.79 (d, J = 2.4 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 6.91 (dd, J = 8.8, 2.5 Hz, 1H), 3.89 (s, 3H), 3.68 (s, 3H), 2.66 (s, 3H); ¹³ C NMR (101 MHz, Chloroform-d) δ 183.86, 162.56, 156.60, 147.92, 131.88, 126.31, 112.84, 109.97, 102.97, 55.88, 29.73, 10.42 ppm。

4-amino-5-methoxy-1, 2-dimethyl-1H-indole-3-carbaldehyde (5). Compound 3 (1.09g, 5.38mmol) was dissolved in acetic acid (88.7 mL), a nitric acid solution (3.2 mL nitric acid in 18.8mL acetic acid) was added at 15 deg.C, and after the addition was complete, the mixture was allowed to warm to room temperature and stirring continued for an additional 2 hours. The reaction mixture was then poured into ice/water mixture (200 ml), extracted with DCM (150mL × 3), washed with water (200 ml) and brine (200 ml) and washed with Na ₂ SO ₄ And (5) drying. The solvent was removed to give a mixture of 4-and 6-nitro groups, which was used directly in the next step without further purification. The mixture (1.25 g) was dissolved in 80mL of ethanol, tin pellets (1.67g, 14.1 mmol) were added, followed by HCl (3.0M, 16mL). Heated to reflux for 1 hour. Water (20 mL) was added and the reaction solution was quenched with NaHCO ₃ (aq) the solution was neutralized and extracted with DCM (200 mL. Times.3). The combined organic layers were washed with water (100 mL) and brine (50 mL), and Na ₂ SO ₄ And (5) drying. After removal of the solvent, column chromatography purification was performed, eluting with EtOAc/petroleum ether (1, rf = 0.17)0.33g (two steps, 47% yield) of 5 is obtained as a yellow solid. ¹ H NMR (500 MHz, Chloroform-d) δ9.76 (s, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.45 (d, J = 8.5 Hz, 1H), 5.91 (s, 2H), 3.86 (s, 3H), 3.56 (s, 3H), 2.57 (s, 3H); ¹³ C NMR (101 MHz, Chloroform-d) δ 183.08, 149.82, 141.76, 134.63, 132.34, 115.58, 113.24, 110.13, 96.17, 57.30, 29.91, 10.66 ppm。

5-methoxy-1, 2-dimethyl-4, 7-dioxy-4, 7-dihydro-1H-indole-3-aminoaldehyde (6). Compound 5 (98mg, 0.45mmol) was dissolved in 10mL of acetone, a solution of Freund's salt (0.24g, 0.9mmol) dissolved in sodium dihydrogenphosphate buffer (10mL, 0.3M, pH 6.0) was added, and the reaction was stirred at room temperature for 3 hours. Excess acetone was removed using a rotary evaporator. The resulting residue was extracted with DCM (20 mL. Times.2). The combined organic layers were washed with water (20mL. Times.2) and saturated brine (10 mL), and then with Na ₂ SO ₄ Drying afforded 6 (86 mg) (82% yield) as a red solid without further purification. ¹ H NMR (400 MHz, Chloroform-d) δ 10.55 (s, 1H), 5.71 (s, 1H), 3.94 (s, 3H), 3.85 (s, 3H), 2.62 (s, 3H); ¹³ C NMR (101 MHz, Chloroform-d) δ 188.34, 179.06, 177.87, 159.84, 142.71, 124.96,122.80,120.00, 106.75, 56.77, 32.26, 11.33 ppm。

3- (hydroxymethyl) -5-methoxy-1, 2-dimethyl-1H-indole-4, 7-dione (7). Compound 6 (100mg, 0.43mmol) was dissolved in a mixture of anhydrous methanol (3 mL) and anhydrous tetrahydrofuran (3 mL). And cooled to 0 ℃ with an ice-water bath. To which NaBH was added uniformly in three portions ₄ (0.812g, 2.15mmol), and the reaction was stirred for 8 minutes while maintaining the temperature. After the reaction is finished, 10% NH is used ₄ The aqueous Cl solution quenched the reaction until no more bubbles were formed. The solvent was removed, dissolved and extracted with dichloromethane (20 mL), the organic layers were combined, washed with saturated saline solution, dried over anhydrous sodium sulfate, the solvent was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography petroleum ether/ethyl acetate (1, rf = 0.3) to give the product 57mg of compound 7 (56% yield) as a bright red solid, which was used for the following reaction. ¹ H NMR (400 MHz, Chloroform-d) δ 5.64 (s, 1H), 4.62 (d, J = 7.1 Hz, 2H), 3.89 (s, 3H), 3.83 (s, 3H), 2.23 (s, 3H); ¹³ C NMR (101 MHz, Chloroform-d) δ 179.69, 179.05, 160.11, 135.05, 129.87, 123.20, 122.49, 107.53, 57.00, 56.34, 32.82, 9.97 ppm。

The synthetic route of the indoloquinone skeleton compound is shown as follows:

starting from 5-methoxy-2-methylindole (1), intermediate 2 is first treated with Vilsmeier reagent to give 3-formyl intermediate 2, and intermediate 2 is methylated with iodomethane in the presence of sodium hydride to give intermediate 3. The nitration reaction at the desired position 4 is carried out in the presence of a relatively low reaction concentration and a large excess of nitric acid, and the intermediate 4 is used in the next step without purification due to its low solubility in organic solvents. Reduction of the nitro group with Sn/HCl produced the amine intermediate 5, which was then purified using a chromatographic column.

The Friedel-crafts salt is used to oxidize the amine intermediate 5 having an aromatic ring to a quinone structure, intermediate 6 in NaBH ₄ Is reduced into a hydroxyl intermediate 7 in the presence of the acid, and then the hydroxyl of the intermediate 7 is converted into fatty carboxylic acid with different chain lengths by condensation reaction by utilizing anhydride of dicarboxylic acid, such as succinic anhydride, glutaric anhydride, adipic anhydride and dithiomalonic anhydride, so as to obtain target intermediates 8a-d.

Taking the intermediates 8a-8d as raw materials, stirring the intermediates, EDCI and DMAP in dichloromethane, and finally carrying out condensation reaction on the synthesized fatty carboxylic acids with different chain lengths and high-activity evodiamine derivatives to obtain target products 9a-9d:

example one

Synthesis of compound 8 a. Succinic anhydride (51mg, 0.51mmol) was dissolved in DCM (2 mL), adding DMAP (41.5mg, 0.34mmol) was used as a catalyst, and after stirring for 30 minutes in the conventional manner, compound 7 (40mg, 0.17mmol) was added, and stirring was continued at room temperature for 12 hours. Then washed with 10wt% hydrochloric acid (20 mL) and extracted with DCM (20 mL. Times.3). The combined organic layers were washed with saturated brine (20 mL) and Na ₂ SO ₄ And (5) drying. After removal of the solvent, the residue was purified by column chromatography eluting with EtOAc/petroleum ether (1, rf = 0.21) to give compound 8a as a red solid (73% yield).

Synthesis of Compound 8 b. Glutaric anhydride (58.2 mg, 0.51mmol) was dissolved in DCM (2 mL), DMAP (41.5mg, 0.34mmol) was added as a catalyst, and after stirring for 30 minutes conventionally, compound 7 (40mg, 0.17mmol) was added, and stirring was continued at room temperature for 12 hours. Then washed with 10wt% hydrochloric acid (20 mL) and extracted with DCM (20 mL. Times.3). The combined organic layers were washed with brine (20 mL) and Na ₂ SO ₄ And (5) drying. After removal of the solvent, the residue was purified by column chromatography eluting with EtOAc/petroleum ether (1, rf = 0.21) to give compound 8b as a red solid (yield 70%).

Synthesis of compound 8 c. Adipic acid (2.0 g) was dissolved in 6mL of acetyl chloride and refluxed for 1.5 hours. After removal of the solvent, the residue was precipitated in cold ether (20 mL) for 4 h. Adipic anhydride was obtained after the mixture was filtered to remove ether and dried under vacuum for 24 hours. Adipic anhydride (65mg, 0.51mmol) was dissolved in DCM (2 mL), DMAP (41.5mg, 0.34mmol) was added as a catalyst, and after stirring conventionally for 30 minutes, compound 7 (40mg, 0.17mmol) was added and stirring continued at room temperature for 18 hours. Then washed with 10wt% hydrochloric acid (20 mL) and extracted with DCM (20 mL. Times.3). The combined organic layers were washed with saturated brine (20 mL) and Na ₂ SO ₄ And (5) drying. After removal of the solvent, the residue was purified by column chromatography eluting with EtOAc/petroleum ether (1, rf = 0.21) to give compound 8c as a red solid (yield 68%).

Synthesis of Compound 8 d. 3,3' -Dithiodipropionic acid (2.0 g) was dissolved in 6mL of acetyl chloride and refluxed for 1.5 hours. After removal of the solvent, the residue was precipitated in cold diethyl ether (20 mL)And 4 h. The mixture was filtered to remove ether and dried under vacuum for 24 hours to obtain 3,3' -dithiodipropionic anhydride. 3,3' -Dithiomalonic anhydride (98mg, 0.51mmol) was dissolved in DCM (2 mL), DMAP (41.5mg, 0.34mmol) was added as a catalyst, and after stirring for 30 minutes conventionally, compound 7 (40mg, 0.17mmol) was added and stirring was continued at room temperature for 24 hours. Then washed with 10wt% hydrochloric acid (20 ml) and extracted with DCM (20 ml. Times.3). The combined organic layers were washed with saturated brine (20 mL) and Na ₂ SO ₄ And (5) drying. After removal of the solvent, the residue was purified by column chromatography eluting with EtOAc/petroleum ether (1, rf = 0.21) to give compound 8d as a red solid (yield 65%).

The above product was characterized as follows:

4- ((5-methoxy-1, 2-dimethyl-4, 7-dioxy-4, 7-dihydro-1H-indol-3-yl) methoxy) -4-oxobutanoic acid (8 a): ¹ H NMR (400 MHz, Chloroform-d) δ5.62 (s, 1H), 5.28 (s, 2H), 3.90 (s, 3H), 3.80 (s, 3H), 2.69~2.65 (m, 2H), 2.63~2.59 (m, 2H), 2.27 (s, 3H). ¹³ C NMR (101 MHz, Chloroform-d) δ 179.03, 177.74,177.36, 172.20, 159.83, 138.11, 129.25, 124.96, 115.75,106.79, 57.06, 56.59, 32.55, 29.01, 28.80, 9.65 ppm.

5- ((5-methoxy-1, 2-dimethyl-4, 7-dioxy-4, 7-dihydro-1H-indol-3-yl) methoxy) -5-oxopentanoic acid (8 b): ¹ H NMR (400 MHz, Chloroform-d) δ 5.62 (s, 1H), 5.25 (s, 2H), 3.90 (s, 3H), 3.80 (s, 3H), 2.42~2.37 (m, 4H), 2.28 (s, 3H), 1.97~1.92 (m, 2H). ¹³ C NMR (101 MHz, Chloroform-d) δ 179.04, 178.31, 177.74, 172.98, 159.85, 137.99, 129.32, 121.89, 115.88, 106.79, 56.82, 56.59, 33.24, 33.04, 32.56, 20.02, 9.68 ppm.

6- ((5-methoxy-1, 2-dimethyl-4, 7-dioxo-4, 7-dihydro-1H-indol-3-yl) methoxy) -6-oxohexanoic acid (8 c): ¹ H NMR (400 MHz, DMSO-d ₆ )δ 5.73 (s, 1H), 4.57 (s, 2H), 3.83 (s, 3H), 3.75 (s, 3H), 2.74~2.66 (m, 2H), 2.24 (s, 3H), 2.04~1.93 (m, 1H), 1.23 (s, 3H), 1.10 (t, J = 7.5 Hz, 2H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 177.99, 177.37, 159.19, 137.08, 127.52, 121.67, 120.42, 106.56, 56.37, 53.20, 37.96, 31.87, 30.89, 16.33, 13.61, 9.15 ppm.

3- ((3- ((5-methoxy-1, 2-dimethyl-4, 7-dioxo-4, 7-dihydro-1H-indol-3-yl) methoxy) -3-oxopropyl) disulfanyl) propionic acid (8 d): ¹ H NMR (400 MHz, DMSO-d ₆ )δ5.73 (s, 1H), 4.56 (s, 2H), 3.83 (s, 3H), 3.75 (s, 3H), 2.75~2.65 (m, 2H), 2.24 (s, 3H), 1.23 (s, 2H), 1.10 (t, J = 7.5 Hz, 2H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ177.98, 177.38, 159.20, 142.28, 137.09, 127.53, 121.66, 120.42, 106.55, 56.37, 53.21, 31.88, 16.34, 13.61, 9.15 ppm。

example two

The chemical structural formula of 10-OH-EVO is as follows:

synthesis of Compound 9 a. 10-OH-EVO (15.95mg, 0.05mmol) was dissolved in DCM (2 mL) and DMAP (6.1mg, 0.05mmol) was added to the mixture as a catalyst. After the addition was completed, the mixture was stirred at 0 ℃ and then added with compound 8a (12mg, 0.05mmol) and EDCI (0.25 mmol), followed by stirring at room temperature for 24 hours. The reaction mixture was then washed with 10% hydrochloric acid (20 mL) and extracted with DCM (20 mL. Times.3). The combined organic layers were washed with saturated sodium bicarbonate (20 mL) and saturated brine (20 mL) and Na ₂ SO ₄ And (5) drying. After removal of the solvent, the residue was purified on silica gel with DCM/CH ₃ OH (4%, rf = 0.32) elution provided compound 9a (58% yield) as a light red-brown solid.

Synthesis of Compound 9 b. 10-OH-EVO (15.95mg, 0.05mmol) was dissolved in DCM (2 mL) and DMAP (6.1mg, 0.05mmol) was added to the mixture as a catalyst. After the addition was completed, stirring was performed at 0 ℃ and Compound 8b (17.47mg, 0.05mmol) and EDCI (0.25 mmol) were added and further stirring was performed at room temperature for 24 hours. Then washed with 10% hydrochloric acid (20 ml)The reaction mixture was washed and extracted with DCM (20 mL. Times.3). The combined organic layers were washed with saturated sodium bicarbonate (20 mL) and saturated brine (20 mL) and Na ₂ SO ₄ And (5) drying. After removal of the solvent, the residue was purified on silica gel with DCM/CH ₃ OH (4%, rf = 0.32) elution provided compound 9b (51% yield) as a light red-brown solid.

Synthesis of Compound 9 c. 10-OH-EVO (15.95mg, 0.05mmol) was dissolved in DCM (2 mL) and DMAP (6.1mg, 0.05mmol) was added to the mixture as a catalyst. After the addition was completed, stirring was performed at 0 ℃ and Compound 8c (18.17mg, 0.05mmol) and EDCI (0.25 mmol) were added, followed by stirring at room temperature for 24 hours. The reaction mixture was then washed with 10% hydrochloric acid (20 mL) and extracted with DCM (20 mL × 3). The combined organic layers were washed with saturated sodium bicarbonate (20 mL) and saturated brine (20 mL) and Na ₂ SO ₄ And (5) drying. After removal of the solvent, the residue was purified on silica gel with DCM/CH ₃ OH (4%, rf = 0.32) gave compound 9c (49% yield) as a light red-brown solid.

Synthesis of Compound 9 d. 10-OH-EVO (15.95mg, 0.05mmol) was dissolved in DCM (2 mL) and DMAP (6.1mg, 0.05mmol) was added to the mixture as a catalyst. After the addition was completed, stirring was performed at 0 ℃ and Compound 8d (21.37mg, 0.05mmol) and EDCI (0.25 mmol) were added, followed by stirring at room temperature for 24 hours. The reaction mixture was then washed with 10% hydrochloric acid (20 mL) and extracted with DCM (20 mL. Times.3). The combined organic layers were washed with saturated sodium bicarbonate (20 mL) and saturated brine (20 mL) and Na ₂ SO ₄ And (5) drying. After removal of the solvent, the residue was purified on silica gel with DCM/CH ₃ OH (4%, rf = 0.32) gave compound 9d (59% yield) as a light red-brown solid.

The above product is characterized as follows:

(5-methoxy-1, 2-dimethyl-4, 7-dioxy-4, 7-dihydro-1H-indol-3-yl) methyl (14-methyl-5-oxy-5, 7,8,13,13b, 14-hexahydroindole [2',3':3,4]Pyridine [2,1-b ]]Quinazolin-10-yl) succinic acid (9 a): ¹ H NMR (400 MHz, Chloroform-d) δ8.26 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.49 (t, J = 7.4 Hz, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H), 6.90 (d, J = 8.6 Hz, 1H), 5.90 (s, 1H), 5.61 (s, 1H), 5.32 (s, 2H), 4.85 (d, J = 12.8 Hz, 1H), 4.12 (q, J = 7.1 Hz, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 2.91 (t, J = 6.5 Hz, 4H), 2.76 (t, J = 6.8 Hz, 2H), 2.49 (s, 3H), 2.27 (s, 3H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ178.17, 177.08, 171.73, 171.36, 164.15, 159.22, 148.68, 146.62,143.58, 138.62, 134.15, 133.44, 132.17, 128.17, 127.96, 125.94, 120.82, 120.29, 119.19, 117.45, 116.00, 111.91, 111.68, 110.27, 106.67, 69.68, 56.41, 36.51, 32.09, 28.91, 28.72, 19.37, 9.09 ppm.MS (ESI, positive) found (M+Na) 659.21, calc (C ₃₅ H ₃₂ N ₄ O ₈ m/z) 636.66.

(5-methoxy-1, 2-dimethyl-4, 7-dioxy-4, 7-dihydro-1H-indol-3-yl) methyl (14-methyl-5-oxy-5, 7,8,13,13b, 14-hexahydroindole [2',3':3,4]Pyridine [2,1-b ]]Quinazolin-10-yl) glutaric acid (9 b): ¹ H NMR (400 MHz, Chloroform-d)δ 8.24 (s, 1H), 8.11 (d, J = 9.1 Hz, 1H), 7.52 – 7.46 (m, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.21 (d, J = 7.9 Hz, 1H), 7.14 (d, J= 8.1 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H), 5.90 (s, 1H), 5.61 (s, 1H), 5.29 (s, 2H), 4.85 (d, J = 12.7 Hz, 1H), 4.12 (q, J = 7.2 Hz, 1H), 3.88 (s, 3H), 3.79 (s, 3H), 2.91 (d, J = 5.4 Hz, 2H), 2.65 (t, J = 7.4 Hz, 2H), 2.49 (s, 3H), 2.47 (d, J = 7.3 Hz, 2H), 2.29 (s, 3H), 2.12 – 2.06 (m, 2H). ¹³ C NMR (101MHz, DMSO-d6) 177.06, 174.65, 172.33, 171.93, 159.64, 159.23, 153.76, 148.70, 143.56, 138.55, 134.15,133.42,131.18,128.24,127.96,125.97,122.87,120.31,119.22,117.49,116.16,114.75,111.90,110.43, 106.68, 99.44,69.66, 56.49, 56.21, 40.80,36.50, 32.44, 32.11, 19.94,19.39, 9.07.MS (ESI, positive) found (M+Na) 673.26, calc (C36H34N4O8 m/z) 650.69.

(5-methoxy-1, 2-dimethyl-4, 7-dioxy-4, 7-dihydro-1H-indol-3-yl) methyl: (14-methyl-5-oxo-5, 7,8,13,13b, 14-hexahydroindole [2',3':3,4]Pyridine [2,1-b ]]Quinazolin-10-yl) adipic acid (9 c): ¹ H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 7.95 (s, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 6.94 (t, J = 7.5 Hz, 1H), 6.61 (dd, J = 8.7, 2.3 Hz, 1H), 6.08 (s, 1H), 5.73 (s, 1H), 4.57 (s, 2H), 3.84 (s, 5H), 3.75 (s, 5H), 2.73 (s, 3H), 2.69 – 2.63 (m, 2H), 2.03 – 1.97 (m, 2H), 0.85 (t, J = 6.6 Hz, 2H). ¹³ C NMR (101 MHz, DMSO-d6) δ 178.03, 177.42, 164.22, 162.28, 159.22, 150.68, 148.62, 137.15, 133.40, 131.10, 130.90, 127.94, 127.54, 126.68, 121.69, 120.43, 119.95, 118.95, 117.01, 110.59, 106.59, 102.10, 69.95, 56.40, 53.21, 41.00, 36.36, 35.76, 31.91, 19.51, 9.20.MS (ESI, positive) found (M+) 664.24, calc (C36H34N4O8 m/z) 664.72.

(5-methoxy-1, 2-dimethyl-4, 7-dioxy-4, 7-dihydro-1H-indol-3-yl) methyl 3- ((3- ((14-methyl-5-oxo-5, 7,8,13,13b, 14-hexahydroindole [2',3':3, 4)]Pyridine [2,1-b ]]Quinazolin-10-yl) oxy) -3-oxopropyl) disulfonyl) propionic acid (9 d): ¹ H NMR (400 MHz, DMSO-d6) δ10.70 (s, 1H), 7.95 (s, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H), 6.94 (t, J = 7.5 Hz, 1H), 6.62 (d, J = 10.9 Hz, 1H), 6.08 (s, 1H), 5.74 (s, 1H), 4.58 (s, 2H), 3.84 (s, 3H), 3.75 (s, 3H), 3.18 (dd, J = 11.9, 8.0 Hz, 2H), 2.89 (d, J = 1.2 Hz, 4H), 2.87 – 2.79 (m, 2H), 2.73 (s, 3H), 2.66 (dd, J = 15.2, 4.5 Hz, 2H), 2.24 (s, 3H), 1.24 (s, 2H). ¹³ C NMR (101 MHz, DMSO-d6) δ 178.03, 177.42, 164.21, 162.28, 159.22, 150.67, 148.62, 137.15, 133.39, 131.10, 130.89, 127.94, 127.54, 126.67, 121.69, 120.43, 119.96, 118.96, 117.02, 112.07, 112.00, 110.59, 106.59, 102.09, 69.94, 64.90, 56.41, 53.20, 40.99, 36.35, 35.75, 31.91, 30.75, 19.50, 9.20。

EXAMPLE three cytotoxicity assays

Human NSCLC cell lines H2122, H358, H23 and A549 were incubated in RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin (P/S) at 37 ℃ and 5% CO ₂ . When the cell density reaches 70-80%, the subculture is completed. The medium was changed every 3 days.

The in vitro anti-tumor cell proliferation activity of the target compound is measured by adopting a conventional MTT method.

Drug concentrations of 20 μ M (compounds dissolved in DMSO) were selected to treat a549 and H358 cells, respectively, for 72H: cells in the logarithmic growth phase were collected, and the cell suspension concentration was adjusted to 3X 10 cells per well using a culture medium ³ Individual cell densities were seeded in 96-well plates in CO ₂ Culturing in an incubator for 12h until a stable monolayer of cells is fully paved on the bottom of the wells, adding 200 μ L of compound with concentration gradient, and arranging three multiple wells in each group. CO 2 ₂ After incubation in an incubator for 72h, 20 μ L of MTT solution was added to each well, incubation was continued for 4h, the culture medium in the wells was discarded (suspension cells were centrifuged at 2000 rrrpm for 12 min and the culture medium in the wells was discarded), 100 μ L of LDMSO was added to each well, and the mixture was shaken slowly on a shaker at 37 ℃ to dissolve formazan crystals sufficiently. The absorbance of the solution at 490nm was measured using a microplate reader. The results are shown in FIG. 1. As can be seen from the figure, compounds 9a, 9b exhibited better cytotoxicity and significantly inhibited the proliferation of NSCLC cells.

Adding the compound 9b into DMSO for dissolving, preparing compound solutions with different concentration gradients, and selecting 4 tumor strains to study the in-vitro anti-tumor activity of the compound 9 b: h2122, H358, H23 and a549. Cells in the logarithmic growth phase were collected, and the cell suspension concentration was adjusted using a culture medium at 5X 10 per well ³ Individual cell densities were seeded in 96-well plates in CO ₂ Incubate for 12h until a stable monolayer of cells is plated on the bottom of the wells, add 200 μ L of compounds with different concentration gradients (no compound in the control group, equal amount of DMSO), and set up three duplicate wells per group. CO 2 ₂ After incubation in an incubator, 20 mu L of MTT solution is added into each hole, the culture is continued for 4h, and the culture solution in the hole is discarded (suspension cells are centrifuged by a 2000Rrpm flat plate for 12 min and then the hole is discardedCulture solution), 100 μ l of LDMSO was added to each well, and slowly shaken on a shaker at 37 ℃ to sufficiently dissolve formazan crystals. The absorbance of the solution at 490nm was measured using a microplate reader and the cell viability was calculated routinely:

survival rate (%) = (OD) _{Experimental group} - OD _{Blank control} )/ (OD _{Control group} - OD _{Blank control} ) ×100%

The IC of the compound was then calculated using Grahpad Prism8 software ₅₀ The value is obtained. Each experiment was performed in triplicate, IC ₅₀ Values were taken as their mean ± Standard Deviation (SD).

The proliferation inhibitory effect of compound 9b on H2122, H358, H23 and a549 cells was observed, fig. 2 shows that compound 9b significantly inhibited proliferation of 4 NSCLC cells by treating cells 24, 48 and 72H with different concentrations of compound 9b, respectively, and the half Inhibitory Concentration (IC) thereof is shown in the figure (IC is shown) ₅₀ ) And (4) data.

Example four apoptosis assay

A549 cells were treated for 48h with various concentrations of 9b (0.375, 0.75, 1.5 and 3 μ M) DMSO and treated for apoptosis analysis and western blot analysis using 3% DMSO in drug-free RPMI 1640 medium as a control. The cells were treated according to the instructions of the apoptosis detection kit and stained with annexin V-FITC and PI stain. annexinV with a green fluorescent probe FITC can be specifically bound to Phosphatidylserine (PS) on the cell surface, and PI can stain necrotic cells or cells with loss of cell membrane integrity at the late apoptosis stage. The apoptosis degree of the treated sample can be quantitatively detected by a flow cytometer. And quantitatively analyzing the apoptosis of the NSCLC cells by adopting an Annexin V-FITC and PI double staining kit. Percentage of specific cell populations at different apoptosis stages as shown in figure 3A, compound 9b induced apoptosis of H358 cells in a concentration-dependent manner. As shown in fig. 3A, the apoptosis rates were 12.43%, 16.83%, 21.1% and 48% after exposure to different concentrations (0.375, 0.75, 1.5 and 3 μ M) of compound 9b for 48 h. The percentage of apoptotic cells in the control group was 4.28%.

Example five Western blot analysis

In order to determine the protein levels in the cells, western-blotting was performed. Cell membranes were cultured overnight at 4 ℃ with dilutions of various primary antibodies (including GAPDH, PARP, p-AKT, p-ERK, t-AKT, t-ERK, p-mTOR, and total mTOR antibodies) at 1. Then, the cells were incubated with the secondary antibody at 37 ℃ for 2 hours. The enhanced chemiluminescence reaction mixture was dropped onto the membrane and allowed to react in a dark room for 2 minutes. Protein expression was measured using a LI-COR Odyssey scanner (Belfast, ME, USA) and Western blot for protein expression levels after 48h of treatment with different concentrations of compound 9B, as shown in FIG. 3B.

Example six-molecule simulation

NAD (P) H: the 3 DX-ray crystal structure of quinone oxidoreductase-1 (NQO 1) was combined with coumarin-based inhibitor AS1 (PDB code: 3 JSX) from Protein Databases (PDB). Docking experiments were performed on active compounds 9a, 9b, 9c and 9 d. Receptor-ligand interactions produced by Autodock vina. For docking experiments, the original ligand active site residues were identified and recorded from the PDB coordinates. The binding results were visualized as 3D graphs using Discovery Studio Visualization 4.5 (Accelrys, inc., san Diego, CA 92121, CA, USA). The DOCKER interaction energy (Kcal/mol) values were further evaluated to assess the degree of energy matching. Potential binding patterns and interactions between active compounds 9a, 9b, 9c and 9d were investigated by molecular construction and docking analysis. The calculation of the predicted lowest energy compound is stable intermolecular hydrogen bonding and stacking interactions. The binding values of the compounds were-6.91, -6.52, -7.23 and-6.98 kcal/mol, respectively. As can be seen (fig. 4), the docking results indicate that the compounds can interact with the selected targets to varying degrees.

All the above results are expressed as the mean Standard Deviation (SD) of three independent experiments. The Student t-test was used to analyze the statistically significant difference between drug-exposed cells and control cells, where statistical significance was defined as a p-value of less than 0.05 (p < 0.05).

Conclusion

The research of the invention shows that the evodiamine prodrugs with the indoloquinone unit represent a potential cytotoxic drug with potential new therapeutic value. Therefore, the invention reports a synthetic method of an evodiamine prodrug containing indoloquinone unit for the first time, and the effects of inhibiting the proliferation and inducing apoptosis of human NSCLC cells, and finds that the compound can change the expression of apoptosis-related protein and inhibit the activity of AKT-MTOR and (MAPK)/ER pathways. Overall, this study may provide a strategy for developing new anti-cancer drugs.

Claims (6)

1. An evodiamine prodrug containing indoloquinone units, which has the following chemical structural formula:

n is 2 to 4.

2. The method for preparing the evodiamine prodrug containing the indoloquinone unit as claimed in claim 1, wherein hydroxyindoloquinone reacts with the acid anhydride compound in the presence of an organic catalyst in a solvent at room temperature to obtain an indoloquinone carboxylic acid compound; reacting an indoloquinone carboxylic acid compound with hydroxyevodiamine in a solvent at room temperature in the presence of an organic catalyst and an amine ligand to obtain an evodiamine prodrug containing indoloquinone units; the chemical structural formula of the hydroxyindoloquinone is as follows:

the chemical structural formula of the hydroxyl evodiamine is as follows:

the chemical structural formula of the indoloquinone carboxylic acid compound is as follows:

n is 2 to 4;

the anhydride compound is succinic anhydride, glutaric anhydride, adipic anhydride and dithio-malonic anhydride;

the organic catalyst is pyridine compound; the amine ligand is a carbodiimide.

3. An indoloquinone carboxylic acid compound, which has the following chemical structural formula:

n is 2 to 4.

4. The method for producing an indoloquinone carboxylic acid compound according to claim 3, wherein hydroxyindoloquinone reacts with an acid anhydride compound in a solvent at room temperature in the presence of an organic catalyst to produce an indoloquinone carboxylic acid compound;

the chemical structural formula of the hydroxyindoloquinone is as follows:

the anhydride compounds are succinic anhydride, glutaric anhydride, adipic anhydride and dithio malonic anhydride;

the organic catalyst is a pyridine compound; the amine ligand is a carbodiimide.

5. The use of the indoloquinone carboxylic acid compound of claim 3 in the preparation of a prodrug of evodiamine.

6. The use of the evodiamine prodrug containing an indoloquinone unit of claim 1 in the preparation of an anti-lung cancer medicament.

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