CN113149965B - Erlotinib derivative and application thereof in preparation of IDO1 activity inhibition drugs and antitumor drugs - Google Patents

Abstract

The invention provides an erlotinib derivative and application thereof in preparation of IDO1 activity inhibition drugs and antitumor drugs, and belongs to the technical field of drug synthesis. The erlotinib derivative provided by the invention has a good inhibition effect on IDO1, and the 1,2, 3-triazole structure can form a stronger action effect with Fe ions in heme so as to competitively inhibit the activity of IDO1 enzyme; the compound has good inhibition effect on wild lung cancer tumor cells, also has inhibition effect on mutant lung cancer tumor cells, and has remarkable universality of tumor cell inhibition activity compared with erlotinib.

Description

Erlotinib derivative and application thereof in preparation of IDO1 activity inhibition drugs and antitumor drugs

The present invention claims priority from a chinese patent application entitled erlotinib derivatives and methods for their preparation having killing properties against wild-type lung cancer tumor cells filed by the chinese patent office at 21/04 in 2020, application No. 202010315872.2, which is incorporated herein by reference in its entirety.

Technical Field

The invention belongs to the technical field of synthesis of medicaments with antitumor activity, and particularly relates to an erlotinib derivative and application thereof in preparation of medicaments for inhibiting IDO1 activity and antitumor medicaments.

Background

Erlotinib (Erlotinib), also known as Tarceva (Tarceva), was co-developed by three companies, Genetech, OSI, Roche, and was produced by the Roche pharmaceutical factory as a selective inhibitor of the first epidermal growth factor tyrosine kinase. The medicine is approved to be on the market in 4 months in 2006, 11 months in 2004 and 9 months in 2005 respectively in China, America and Europe. Meanwhile, the combination of erlotinib hydrochloride and gemcitabine approved for treating pancreatic cancer in 2005 is the first drug approved for advanced treatment of pancreatic cancer in recent years. At present, the method is mainly suitable for the metastatic non-small cell lung cancer or the local advanced two-way or three-way treatment after two or more chemotherapy schemes fail; erlotinib contains a quinazoline backbone. Quinazoline derivatives exist in natural alkaloids, the quinazoline skeleton is also an important structure in medicinal chemistry, and a plurality of compounds containing the quinazoline structure show good anticancer, antibacterial and anti-inflammatory effects. The 1,2, 3-triazole is an important pesticide and medicine pharmacophore and has good biocompatibility and aromaticity. For years, 1,2, 3-triazole is usually used as an important pharmacophore, pharmacodynamic molecules of different substrates are linked together through click chemical reaction, and a better effect is shown through structural modification. Erlotinib, as an anticancer drug, has already developed drug resistance and adverse reactions.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of erlotinib derivatives in the preparation of drugs and antitumor drugs for inhibiting IDO1 activity.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides an erlotinib derivative, which has a structural formula as follows:

wherein n is 1 or 2, R1 and R2 and R3 and R4 are different substituents.

Preferably, the erlotinib derivative has the structural formula:

the invention provides application of the erlotinib derivative in preparing a medicament for inhibiting IDO1 activity.

The invention provides application of the erlotinib derivative in preparation of antitumor drugs.

The invention provides application of the erlotinib derivative in preparation of a medicine for preventing and/or treating lung cancer.

Preferably, the lung cancer comprises non-small cell lung cancer.

The invention provides application of the erlotinib derivative in preparation of a medicine for inhibiting wild lung cancer tumor cells.

The invention provides application of the erlotinib derivative in preparation of a medicament for inhibiting mutant lung cancer tumor cells.

The invention has the beneficial effects that: the erlotinib derivative is an erlotinib-1, 2, 3-triazole compound; the compound has good inhibition effect on IDO1, and a 1,2, 3-triazole structure can form a stronger action effect with Fe ions in heme so as to competitively inhibit IDO1 enzyme activity; the compound has good inhibition effect on wild lung cancer tumor cells, also has inhibition effect on mutant lung cancer tumor cells, and has remarkable universality of tumor cell inhibition activity compared with erlotinib.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of the compound obtained in example 1;

FIG. 2 shows that the compound obtained in example 1 induces apoptosis; a is PC-9 cells, B is H460 cells, and C is Hoechst stains (Hoechst stains).

FIG. 3 shows that the compound obtained in example 1 induces cell cycle arrest; a is PC-9 cells, B is H460 cells, and C is qPCR results.

FIG. 4 shows the results of the inhibition of PC-9 tumor growth in example 1; a is a tumor image, B is a tumor growth curve, C is tumor weight, and D is the change rate of mouse body weight;

FIG. 5 shows the results of example 1 in inhibiting the growth of 4T1 tumor; a is tumor image, B is tumor growth curve, C is tumor weight, and D is tumor HE staining.

Detailed Description

The invention provides an erlotinib derivative, which has a structural formula as follows:

wherein n is 1 or 2, R1 and R2 and R3 and R4 are different substituents.

Preferably, the erlotinib derivative has the structural formula:

in the invention, the preparation method of the erlotinib derivative comprises the following steps:

(1): dissolving a certain amount of 3, 4-bis (2-methoxyethyl) ethyl benzoate in dichloroethane, uniformly stirring at room temperature, adding N-iodosuccinimide, reacting at room temperature until the raw materials completely react, filtering the reaction solution, pouring the filtrate into ice water, adding saturated sodium hydroxide solution, separating an organic phase, adjusting the pH of the organic phase to be neutral by using dilute hydrochloric acid solution, separating the organic phase again, drying by using anhydrous magnesium sulfate, and concentrating to obtain 6-iodine-3, 4-bis (2-methoxyethyl) ethyl benzoate; the feeding amount molar ratio of the ethyl 3, 4-bis (2-methoxyethyl) benzoate to the N-iodosuccinimide is 1: 1.1-2.

(2): adding a certain amount of 6-iodine-3, 4-bis (2-methoxyethyl) ethyl benzoate, tris (triphenylphosphine) cobalt chloride, 1, 8-diazabicycloundece-7-ene and cesium carbonate into anhydrous toluene in a closed reaction bottle with a water separator, stirring uniformly, displacing gas for three times in the reaction bottle by nitrogen, then slowly heating to reflux, cooling to room temperature after reflux stirring reaction for a period of time, slowly dropwise adding toluene solution in which azidotrimethylsilane and potassium iodide are dissolved, heating to 70 ℃ after dropwise adding, keeping the temperature for reaction for a period of time to obviously observe the color deepening of the reaction liquid, thereby judging that iodine on a benzene ring is displaced, heating to reflux after observing the color fixation of the reaction liquid, then adding aqueous solution in which ammonium sulfamate is dissolved into the water separator, the method comprises the following steps of (1) refluxing toluene, enabling the toluene to enter a water separator to be mixed with water, slowly dropwise adding a mixed solution into a reaction liquid through the water separator, continuously circulating through a reflux system, continuously reacting for a period of time, cooling to room temperature, dropwise adding dilute hydrochloric acid into a filtrate to enable the pH value of the filtrate to reach 1-2, slowly stirring at 10 ℃, gradually separating out solids, filtering, recrystallizing in acetone to further remove the color introduced by iodine, filtering again, collecting a filter cake, and drying to obtain ethyl 4, 5-bis (2-methoxyethyl) -2 aminobenzoate hydrochloride; the molar ratio of the charging amount of the 6-iodine-3, 4-bis (2-methoxyethyl) ethyl benzoate to the charging amount of azidotrimethylsilane to the charging amount of potassium iodide to the charging amount of ammonium sulfamate is 1:1.2:0.1: 1.5.

(3): adding a certain amount of 4, 5-bis (2-methoxyethyl) -2-aminobenzoic acid ethyl ester hydrochloride into formamide, stirring uniformly at room temperature, then adding formamide solution dissolved with ammonium formate, slowly heating to a certain temperature under the nitrogen atmosphere, reacting until the raw materials completely react, cooling to room temperature, adding ethyl acetate into the reaction solution, stirring for a period of time, then adding water, separating out an organic phase, and concentrating to obtain 6, 7-bis (2-methoxyethoxy) quinazolin-4-one; the feeding amount molar ratio of the 4, 5-di (2-methoxyethyl) -2-aminobenzoic acid ethyl ester hydrochloride to the ammonium salt is 1: 3; the reaction temperature is 130-160 ℃.

(4): adding a certain amount of 6, 7-bis (2-methoxyethoxy) quinazoline-4-ketone and N, N-dimethylformamide into thionyl chloride, slowly heating to reflux, after the reaction is finished, evaporating unreacted thionyl chloride in vacuum, then adding a saturated sodium bicarbonate solution at 0-10 ℃, extracting for multiple times by using dichloromethane after stirring, combining organic phases, washing once by using saturated saline solution, washing for multiple times by using water, drying by using anhydrous sodium sulfate, and concentrating to obtain 6, 7-bis (2-methoxyethoxy) quinazoline-4-chlorine.

(5): adding a certain amount of 6, 7-di (2-methoxyethoxy) quinazoline-4-chlorine into isopropanol, stirring uniformly at room temperature, then adding m-aminophenylacetylene, stirring uniformly again, heating for refluxing, reacting for a period of time, cooling to room temperature, generating a large amount of solid, filtering, and drying to obtain erlotinib.

(6): adding a certain amount of erlotinib and an azide into a solution of tert-butyl alcohol, water and tetrahydrofuran, adding a certain amount of copper sulfate pentahydrate and sodium ascorbate, heating to 70 ℃ for reaction until the erlotinib is monitored to be completely reacted, adding dichloromethane into a reaction solution, filtering the reaction solution, separating an organic phase, and concentrating to obtain a product; the feeding amount mass ratio of the erlotinib to the azide to the copper sulfate pentahydrate to the sodium ascorbate is 2:2:1: 2.

The preparation method of the erlotinib derivative is characterized in that the reaction equation in the preparation process is as follows:

the invention provides application of the erlotinib derivative in preparing a medicament for inhibiting IDO1 activity.

The invention provides application of the erlotinib derivative in preparation of antitumor drugs.

The invention provides application of the erlotinib derivative in preparation of a medicine for preventing and/or treating lung cancer.

Preferably, the lung cancer comprises non-small cell lung cancer.

The invention provides application of the erlotinib derivative in preparation of a medicine for inhibiting wild lung cancer tumor cells.

The invention provides application of the erlotinib derivative in preparation of a medicament for inhibiting mutant lung cancer tumor cells.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Adding erlotinib 1g and 3, 5-dibromo benzyl azide 1g into solution of tert-butyl alcohol 10mL, water 10mL and tetrahydrofuran 10mL, and adding copper sulfate pentahydrate 0.5g and sodium ascorbate 10mL, heating to 70 ℃ for reaction until TLC monitors complete reaction of erlotinib, adding dichloromethane into the reaction solution, filtering the reaction solution, separating out an organic phase, and concentrating to obtain 1.21g of a product; ¹ H NMR(400Hz,DMSO-d ₆ ):9.56(s,1H),8.71(s,1H),8.48(s,1H),8.27(s,1H),7.91(d,J＝8.0Hz,2H),7.86(d,J＝4.0Hz,1H),7.63(s,2H),7.56(d,J＝8.0Hz,1H),7.46(t,J ₁ ＝8.0Hz,J ₂ ＝8.0Hz,1H),7.23(s,1H),5.69(s,2H),4.33-4.28(m,4H),3.81-3.74(m,4H),3.38(s,3H),3.36(s,3H)； ¹³ C NMR(101Hz,DMSO-d ₆ ):156.83,154.07,153.40,148.56,147.44,147.21,140.82,140.54,133.73,131.21,130.67,129.53,123.23,122.41,120.84,119.29,109.43,108.67,103.68,70.60,70.53,68.82,68.51,58.87,58.82,52.01。

example 2

Detection of IDO1 inhibitory Activity

From CO ₂ Taking out the human cervical carcinoma Hela cell culture dish with viability from the incubator, and respectively carrying out the following operations: the method comprises the steps of performing aseptic operation beside an alcohol lamp, opening a dish cover, sucking out culture solution into a waste liquid jar, washing the culture solution in a culture bottle twice by using 2mL PBS, digesting by using 0.25% trypsin, stopping digestion when cell gaps are increased and cells are changed into small circle shapes after observation, blowing the bottom of the culture bottle by using a pipette to enable the cells to fall off, transferring the obtained cell suspension into an aseptic centrifuge tube, setting a centrifuge to be 1000r/min, centrifuging for 3min, slowly pouring supernatant in the centrifuge tube, adding 2-5 mL culture solution, and counting the cells under an inverted microscope. According to the counting result, viable human cervical carcinoma Hela cells growing in logarithmic phase are paved in 96-well cell culture plates according to the number of 50000 cells per well, are cultured for 5-6h by RPMI1640 containing 10% fetal bovine serum, and 100 mu L of a test compound (the concentration is 0.2 mu mol/L,0.4 mu mol/L,0.8 mu mol/L,1.6 mu mol/L,3.2 mu mol/L,6.4 mu mol/L, 12.8 mu mol/L and 25.6 mu mol/L respectively) diluted by a culture medium and a recombinant human-derived interferon gamma (the final concentration is 100ng/mg) are added to activate the expression of IDO1 in the Hela cells. After the operation is finished, the 96-hole cell culture plate is put into a cell culture box with the temperature of 37 ℃ and rich in 5 percent carbon dioxide for culturing for 18 hours, and then certain amount of the cell culture plate is usedThe reaction was stopped with trichloroacetic acid in an amount of 3.05N and then incubated at 50 ℃ for 30 minutes. After the cell culture solution is precipitated, the supernatant is taken and is colored by p- (N, N-dimethyl) benzaldehyde, and then the absorbance at 480nm is detected by a multifunctional microplate reader. The group treated with IFN γ -only medium without drug was taken as 100% (At), and the group treated with DMSO-only medium 0.1% was taken as a blank 0% (Ab); the absorbance at different conditions was calculated according to the following formula: absorbance% (a-Ab)/(At-Ab), a: drug treatment +100ng/mL IFN γ, Ab: blank control, At: no drug contained 100ng/mL IFN γ alone; generation of the IC Using GraphPad Prism 8.0 software ₅₀ Inhibition curve of values. The objective compound obtained in example 1 was 0.587. mu. mol/L.

Example 3

Inhibition of cell proliferation Activity assay

From CO ₂ The lung cancer cells H460, H1975, H1299, A549 and PC-9 culture dishes are taken out from the incubator and respectively carried out as follows: aseptic operation is carried out beside an alcohol lamp, a dish cover is opened, culture solution is sucked out of a waste liquid cylinder, the culture solution in a culture bottle is washed twice by 2mL PBS, 0.25% trypsin is used for digestion, digestion is stopped when observation shows that cell gaps are increased and cells are changed into small circle shapes, a liquid transfer gun is used for blowing the bottom of the culture bottle to enable the cells to fall off, the obtained cell suspension is transferred into an aseptic centrifuge tube, a centrifuge is set to be 800r/min and 3min for centrifugation, then supernatant in the centrifuge tube is poured slowly, 2-5 mL culture solution is added, and cell counting is carried out under an inverted microscope. According to the counting result, the obtained product is prepared into 1 × 10 culture solution ⁵ cells/mL of single cell suspension, then seeded in 6-well plates, and 2mL per well. Placing 6-well plate at 37 deg.C and 5% CO ₂ Culturing in an incubator for 24 h.

Inoculating 100 mu L of tumor cell suspension into 96 holes at 1500-2000 cells/hole 24h in advance; removing the original culture medium, adding 100 μ L of culture medium containing different test drugs (final concentration of 1 μ M, 2 μ M, 4 μ M, 8 μ M or 16 μ M), culturing for 48 hr, repeating each group for 3 times, and using 0.1% DMSO as control; removing the drug-containing medium, adding 100 μ L of the diluted complete medium1X Cell Counting Kit-8(CCK-8) reagent, and placing a 96-well plate in an incubator for incubation for 1-4 h; detecting the absorbance at 450nm by using a SynergyHTX multifunctional microplate reader; and (3) calculating the inhibition rate by using the absorbance, wherein the calculation formula is as follows: inhibition rate ═ [ (Ac-As)/(Ac-Ab)]X 100%; as, experimental wells (drug treatment); ac, control wells (0.1% DSMO treatment); ab, blank (no cells). Half Inhibitory Concentration (IC) of drug on cell growth was determined using Graph Pad Prism 8.0 software ₅₀ )。

The half inhibitory concentrations of the target compound obtained in example 1 were 5.18. mu. mol/L, 3.31. mu. mol/L, 7.06. mu. mol/L, 5.80. mu. mol/L, and 4.38. mu. mol/L, respectively; erlotinib was 6.04. mu. mol/L, 12.67. mu. mol/L, 22.31. mu. mol/L, 10.20. mu. mol/L, 0.21. mu. mol/L, respectively.

Example 4

Apoptosis detection

Apoptosis analysis was performed by flow cytometry using the AnnexinV/PI apoptosis kit according to the manufacturer's instructions. 1mL of PC-9 or H460 cell suspension (4X 10) 24H in advance ⁴ ～6×10 ⁴ Per well) in 12-well plates; the original medium was removed and treated with 1mL of complete medium containing different concentrations of the objective compound obtained in example 1 (final concentration of 4. mu.M, 8. mu.M or 12. mu.M) for 48h, respectively, using 0.1% DMSO (complete medium dilution) as a control; cells were collected and washed with PBS; resuspend cells with 100 μ L of 1XAnnexinV binding buffer containing 1 μ LPI and 1 μ LFITCAnnexinV, incubate for 15min at room temperature in the dark; add 200 μ L of 1xannexin v bininglbuffer to each tube and perform flow cytometry analysis; the percentage of apoptotic cells was analyzed using FlowJo V10 software.

The apoptosis rates of PC-9 cells treated with the objective compound obtained in example 1 were 10.24% (4. mu.M), 14.45% (8. mu.M) and 35.74% (12. mu.M), respectively; the apoptosis rates of H460 cells were 9.78% (4. mu.M), 47.97% (8. mu.M) and 72.37% (12. mu.M), respectively.

Example 5

Herster staining (Hoechst statins) to detect apoptosis

100 μ L of PC-9 cell suspension (3000/well) was plated in 96-well plates and grown for 24 h; the samples were treated with complete medium containing the target compound obtained in example 1 at various concentrations (final concentration of 4. mu.M, 8. mu.M or 12. mu.M) for 24 hours, respectively, and 0.1% DMSO (complete medium dilution) was used as a control; removing the cell culture medium, adding 100 mu L of culture medium containing 1X Hoechst 33342 dye for replacement, and incubating the cells in the incubator for 5-15 min; washed twice with PBS; observed and imaged under a NikonTi-U microscope.

The PC-9 cells treated with the compound obtained in example 1 exhibited typical apoptotic characteristics such as nuclear condensation, chromatin fragmentation and cell shrinkage, as compared to the cells treated with 0.1% DMSO, indicating that the compound obtained in example 1 did induce apoptosis of PC-9 cells.

Example 6

Cell cycle arrest assay

Cell cycle analysis was performed by flow cytometry using a cell-cycle staining kit (cell-cycle staining kit) according to the manufacturer's instructions. 2mL of cell suspension of PC-9 or H460 (2X 10) was added 24H in advance ⁵ ～3×10 ⁵ Per well) in 6-well plates; the original medium was removed and treated with 0.1% DMSO (complete medium dilution) as a control for 24H (PC-9) or 12H (H460) with 2mL of complete medium containing the compound obtained in example 1 (final concentration 4. mu.M, 8. mu.M or 12. mu.M), respectively; cells were collected and washed with PBS; premixing the DNA stabilizing solution and the Permeabilization solution according to a ratio of 100:1, adding 300 mu L of mixed solution into each sample, carrying out vortex oscillation for 5-10 s, mixing uniformly, and incubating for 30min at room temperature in a dark place; selecting the lowest sample loading speed to carry out detection on a flow cytometer; the distribution of cellular DNA content and its periodic phases was analyzed using ModFit LT software.

The target compound obtained in example 1 blocked PC-9 and H460 cells at G0/G1.

Example 7

qPCR detection of cell cycle regulation related gene

1mL of A549 cell suspension (5X 10) ⁴ ～10×10 ⁴ Per well) is inoculated on a 12-well plate to grow for 24 hours; the cells were treated with complete medium containing the objective compound obtained in example 1 at various concentrations (final concentration of 4. mu.M, 8. mu.M or 12. mu.M), respectively, and 0.1% DMSO (complete medium dilution) was used as a controlIrradiating; collecting cells after 16h, washing the cells with PBS, and discarding supernatant; the cell pellet was frozen in a-80 ℃ ultra-low temperature freezer until RNA extraction. Taking out the cell precipitate from a-80 ℃ ultra-low temperature refrigerator; adding 300 mu L of lysine Buffer into each cell sample, forcibly blowing and beating for 10 times by using a pipette, and carrying out vortex oscillation for more than 10s to fully crack the cells; adding 300 μ L of anhydrous ethanol into the lysed cells, inverting or vigorously pipetting for 10 times, and adding the liquid into a centrifugal column; centrifuging at 4000Xg for 1min, and discarding the waste liquid in the collecting pipe; adding 500 mu L of Wash Buffer into the RNA column, centrifuging for 1min at 12000Xg, and discarding waste liquid in a collecting pipe; centrifuging once in a 12000Xg empty tube; putting the RNA column into a clean 1.5mL centrifuge tube without RNase, opening the cover and airing for 2 min; adding 20-50 mu L ddH to the central part of the membrane of the RNA column ₂ O, standing for 2min at room temperature; centrifuging at 12000Xg for 1min, and measuring the concentration of RNA; RNA was frozen in an ultra low temperature freezer at-80 ℃ until the next experiment. The reaction solution was prepared as in table 1: placing the prepared reaction solution in a PCR instrument for reaction (42 ℃ for 2 min); preparing a reaction solution according to the table 2, and adding 10 mu L of the prepared reaction solution into the reaction solution in the previous step; the well-prepared reaction solution was placed in a PCR instrument for reaction (37 ℃, 15min, 85 ℃, 15 s). Amplifying in a fluorescent quantitative PCR instrument according to the following conditions: holding Stage (95 ℃, 30s), Cycling Stage (95 ℃,5 s; 60 ℃, 34s), 40 cycles; melt Curve Stage (95 ℃, 15 s; 60 ℃,1 min). The primer sequences are shown in Table 3. The average relative expression amount of mRNA was calculated by the calculation method of 2-. DELTA.Ct.

TABLE 1 reverse transcription System

TABLE 2RT-qPCR amplification System

TABLE 3 primer sequences

Example 8

PC-9 nodulation experiment

Taking the lung cancer tumor cell PC-9 growing in the logarithmic phase, digesting with 0.25% trypsin, collecting the cell, resuspending the cell with PBS, counting, and counting at 3 × 10 ⁶ The density of individual cells/mouse was inoculated subcutaneously in NSG mice; when the subcutaneous tumor volume grew to a measurable amount, it was randomly divided into 3 groups: a control group, the target compound group obtained in example 1, and an erlotinib group; with ddH containing 5% DMSO and 30% PEG300 ₂ O dissolving the erlotinib and the target compound group to a final concentration of 2.5 mg/ml; the control group was administered with a solvent (ddH containing 5% DMSO and 30% PEG 300) in accordance with the weight of mice at a dose (25mg/kg) corresponding to the intraperitoneal injection of different drugs given at a frequency of 1 time per day ₂ O), the target compound group and the erlotinib group are administered with the compound target compound group or erlotinib, respectively, at a final concentration of 25 mg/kg; tumor volume and mouse body weight were measured: from the administration on day 1, the length and width of the tumor were measured 1 time every 2 days with a vernier caliper, and the body weight of the mouse was measured, and the tumor volume was calculated by the formula: tumor volume (mm) ³ ) V is length × width/2; dosing was stopped when the mouse graft had grown to a diameter of no more than 15mm, NSG mice were sacrificed by cervical dislocation, and the graft was dissected and weighed.

The target compound and erlotinib showed good tumor growth inhibition compared to the control group to which the solvent was administered. Furthermore, statistical analysis showed no significant difference in tumor growth rate with target compound or erlotinib treatment. Tumors were dissected 19 days after inoculation and weighed. After administration of the target compound or erlotinib, tumor weight was significantly reduced and no significant difference was detected between the two groups. In addition, no significant difference was found in body weight change between the three groups after the administration.

Example 9

Mouse breast cancer cell 4T1 tumor formation experiment

Tumor cells grown in log phase 4T1 were harvested by 0.25% trypsin digestion, harvested and resuspended in PBS, counted at 0.75X 10 ⁶ The density of individual cells/mouse was inoculated subcutaneously in BALB/c mice; when the subcutaneous tumor volume grew measurably, it was randomly divided into 4 groups: a control group, the target compound group obtained in example 1, an erlotinib group, and an Epacadostat group; with ddH containing 5% DMSO and 30% PEG300 ₂ Dissolving erlotinib, the target compound group or Epacadostat to a final concentration of 2.5 mg/ml; the drugs were administered to the mice at the corresponding doses (25mg/kg) by intraperitoneal injection, and the control group was administered with a solvent (ddH containing 5% DMSO and 30% PEG 300) ₂ O), target compound group, erlotinib group and Epacadostat group are administered with target compound group (qd), erlotinib (qd) or Epacadostat (bid) respectively at a final concentration of 25 mg/kg; the length and width of the tumor were measured 2 times a week starting from the day 1 dosing using a vernier caliper, and the tumor volume was calculated as: tumor volume (mm) ³ ) V ═ length × width/2; stopping administration when the transplanted tumor of the nude mouse grows to the diameter of no more than 15mm, carrying out cervical dislocation to kill BALB/c mice, dissecting the transplanted tumor and weighing, and reserving part of tumor tissues for HE staining.

The target compound and Epacadostat significantly inhibited the growth of the 4T1 tumor and also reduced the weight of the final tumor. Although statistical analysis showed that erlotinib inhibited the growth of 4T1 tumor at day 14 post-inoculation, the tumor volume did not change significantly compared to the control group given solvent. In addition, it was found using HE staining that tissue necrosis phenomena such as nuclear lysis and nuclear fragmentation are more easily observed in tumor tissues exposed to the target compound.

Example 10

Dissolving 3g of ethyl 3, 4-bis (2-methoxyethyl) benzoate in 30mL of dichloroethane, stirring at room temperature for 30min, then adding 2.5g of N-iodosuccinimide in portions,5g of each batch is separated by 10min, the last batch of N-iodosuccinimide is added and then reacts for 2h at room temperature, TLC monitors that the raw materials completely react, the reaction liquid is filtered, the filtrate is poured into 30mL of ice water, 8mL of saturated sodium hydroxide solution is added, an organic phase is separated, the pH of the organic phase is adjusted to be neutral by dilute hydrochloric acid solution, the organic phase is separated again, the mixture is dried by anhydrous magnesium sulfate and then concentrated to obtain 2.9g of 6-iodine-3, 4-bis (2-methoxy ethyl) ethyl benzoate; ¹ H NMR(400Hz,DMSO-d6):7.26(s,1H),7.11(s,1H),4.15(t,J1＝4.0Hz,J2＝4.0Hz,2H),4.11(t,J1＝4.0Hz,J2＝4.0Hz,2H),3.82-3.77(m,2H),3.74(t,J1＝4.0Hz,J2＝4.0Hz,2H),3.59-3.55(m,2H),3.39(s,3H),3.37(s,3H),1.28(t,J1＝4.0Hz,J 2＝4.0Hz,3H)；LC-MS(ESI)：425[M+H] ⁺

example 11

Dissolving 3g of ethyl 3, 4-bis (2-methoxyethyl) benzoate in 30mL of dichloroethane, stirring for 30min at room temperature, adding 4.6g of N-iodosuccinimide in batches with a time interval of 1.15g for each batch for 10min, reacting for 1h at room temperature after the last batch of N-iodosuccinimide is added, monitoring the complete reaction of raw materials by TLC, filtering the reaction solution, pouring the filtrate into 50mL of ice water, adding 10mL of saturated sodium hydroxide solution, separating out an organic phase, adjusting the pH of the organic phase to be neutral by using dilute hydrochloric acid solution, separating out the organic phase again, drying by using anhydrous magnesium sulfate, and concentrating to obtain 3.7g of ethyl 6-iodo-3, 4-bis (2-methoxyethyl) benzoate; ¹ HNMR(400Hz,DMSO-d6):7.26(s,1H),7.11(s,1H),4.15(t,J1＝4.0Hz,J2＝4.0Hz,2H),4.11(t,J1＝4.0Hz,J2＝4.0Hz,2H),3.82-3.77(m,2H),3.74(t,J1＝4.0Hz,J2＝4.0Hz,2H),3.59-3.55(m,2H),3.39(s,3H),3.37(s,3H),1.28(t,J1＝4.0Hz,J2＝4.0Hz,3H)；LC-MS(ESI)：425[M+H]+。

example 12

Adding 4.5g of 6-iodine-3, 4-bis (2-methoxyethyl) ethyl benzoate, 0.35g of tris (triphenylphosphine) cobalt chloride, 2g of 1, 8-diazabicycloundecen-7-ene and 3.2g of cesium carbonate into 50mL of anhydrous toluene in a closed reaction bottle with a water separator, stirring uniformly, displacing gas with nitrogen for three times in the reaction bottle, slowly heating to reflux, stirring under reflux for 2h, cooling to room temperature, slowly adding 10mL of toluene solution in which 1.4g of azidotrimethylsilane and 0.17g of potassium iodide are dissolved, heating to 70 ℃ after the addition is completed, keeping the temperature for reaction for 30min, obviously observing the color deepening of the reaction liquid in the reaction process, judging that iodine on a benzene ring is displaced, heating to reflux after observing the color fixation of the reaction liquid, adding 10mL of aqueous solution in which 1.8g of ammonium sulfamate into the water separator, and (2) refluxing toluene, mixing the toluene with water in a water separator, slowly dropwise adding the mixed solution into the reaction solution through the water separator, continuously circulating through a reflux system, continuously reacting for 1h, cooling to room temperature, dropwise adding dilute hydrochloric acid into the filtrate to enable the pH value of the filtrate to reach 1-2, slowly stirring at 10 ℃, gradually separating out solids, filtering, recrystallizing in the mixed solution of acetone and petroleum ether to further remove the color introduced by iodine, filtering again, collecting a filter cake, and drying to obtain 3.0g of 4, 5-bis (2-methoxy ethyl) -2-ethyl aminobenzoate hydrochloride.

Example 13

In a reaction bottle, 3.5g of ethyl 4, 5-bis (2-methoxyethyl) -2-aminobenzoate hydrochloride is added into 20mL of formamide, the mixture is stirred for 10min at room temperature, then 10mL of formamide solution dissolved with 2g of ammonium formate is added, the mixture is slowly heated to 130 ℃ under nitrogen atmosphere, the reaction is carried out for 6h, TLC monitors that the raw materials are completely reacted, the mixture is cooled to room temperature, 35mL of ethyl acetate is added into the reaction solution, the mixture is stirred for 30min, then 15mL of water is added, the stirring is continued for 10min, an organic phase is separated, and 2.7g of 6, 7-bis (2-methoxyethoxy) quinazolin-4-one is obtained after concentration.

Example 14

Adding 3g of 6, 7-bis (2-methoxyethoxy) quinazoline-4-ketone and 1mL of N, N-dimethylformamide into 30mL of thionyl chloride in a reaction bottle, slowly heating to reflux, after the reaction is finished, evaporating unreacted thionyl chloride in vacuum, adding 20mL of saturated sodium bicarbonate solution at the temperature of 0-10 ℃, stirring for 20min, extracting with 10mL of dichloromethane for multiple times, combining organic phases, washing with saturated saline once, washing with water for multiple times, drying with anhydrous sodium sulfate, and concentrating to obtain 2.4g of 6, 7-bis (2-methoxyethoxy) quinazoline-4-chlorine.

Example 15

Adding 3g of 6, 7-bis (2-methoxyethoxy) quinazoline-4-chlorine into 50mL of isopropanol in a reaction bottle, uniformly stirring at room temperature, then adding 1.3g of m-aminophenylacetylene, uniformly stirring again, heating and refluxing, reacting for 1.2h, slowly cooling to room temperature to generate a large amount of solid, continuously cooling to 0 ℃, stirring for 30min, performing suction filtration, and drying to obtain 3.1g of erlotinib.

Example 16

Adding 1g of erlotinib and 1g of 2-fluorobenzyl azide into a solution of 10mL of tert-butyl alcohol, 10mL of water and 10mL of tetrahydrofuran, adding 0.5g of copper sulfate pentahydrate and 1g of sodium ascorbate, heating to 70 ℃, reacting until TLC monitors the complete reaction of erlotinib, adding dichloromethane into the reaction solution, filtering the reaction solution, separating an organic phase, and concentrating to obtain 1.17g of a product; ¹ HNMR(400Hz,DMSO-d6):9.62(s,1H),9.11(s,1H),8.50(s,1H),8.38(s,1H),7.95(dd,J1＝4.0Hz,J2＝8.0Hz,3H),7.69-7.59(m,3H),7.54-7.47(m,2H),7.24(s,1H),4.34-4.29(m,4H),3.81-3.75(m,4H),3.39(s,3H),3.36(s,3H)； ¹³ CNMR(101Hz,DMSO-d6):156.85,154.07,153.40,153.12,148.54,147.47,147.40,140.64,131.92,130.76,129.60,126.53,126.10,123.37,122.73,121.07,119.47,117.77,117.57,109.45,108.67,103.69,70.61,70.54,68.84,68.50,58.87,58.81。

example 17

Adding 1g of erlotinib and 1g of 4-methylphenyl azide into a solution of 10mL of tert-butyl alcohol, 10mL of water and 10mL of tetrahydrofuran, adding 0.5g of copper sulfate pentahydrate and 1g of sodium ascorbate, heating to 70 ℃, reacting until TLC monitors that the erlotinib is completely reacted, adding dichloromethane into a reaction solution, filtering the reaction solution, separating an organic phase, and concentrating to obtain 0.66g of a product; ¹ HNMR(400Hz,DMSO-d6):9.63(s,1H),9.28(s,1H),8.51(s,1H),8.37(s,1H),7.95(d,J＝4.0Hz,2H),7.87(d,J＝8.0Hz,2H),7.67(d,J＝4.0Hz,1H),7.52(t,J1＝8.0Hz,J2＝4.0Hz,1H),7.45(d,J＝4.0Hz,2H),7.25(s,1H),4.34-4.30(m,4H),3.82-3.76(m,4H),3.39(s,3H),3.37(s,3H),2.51(s,3H)； ¹³ CNMR(101Hz,DMSO-d6):156.88,154.09,153.40,148.58,147.70,147.40,140.59,138.83,134.89,131.06,130.75,129.57,122.69,121.02,120.36,120.02,119.46,109.43,108.64,103.69,70.60,70.54,68.84,68.52,58.88,58.82,21.07。

example 18

Adding 0.5g of erlotinib and 0.5g of benzyl azide into a solution of 5mL of tert-butyl alcohol, 5mL of water and 5mL of tetrahydrofuran, adding 0.25g of copper sulfate pentahydrate and 0.5g of sodium ascorbate, heating to 70 ℃, reacting until TLC monitors the complete reaction of erlotinib, adding dichloromethane into a reaction solution, filtering the reaction solution, separating an organic phase, and concentrating to obtain 0.41g of a product; ¹ HNMR(400Hz,DMSO-d6):9.55(s,1H),8.66(s,1H),8.48(s,1H),8.26(s,1H),7.90(d,J＝12.0Hz,2H),7.56(d,J＝4.0Hz,1H),7.45(t,J1＝8.0Hz,J2＝8.0Hz,1H),7.41-7.33(m,5H),7.23(s,1H),5.66(s,2H),4.32-4.28(m,4H),3.80-3.74(m,4H),3.38(s,3H),3.36(s,3H)； ¹³ CNMR(101Hz,DMSO-d6):156.83,154.06,153.39,148.55,147.44,147.11,140.52,136.50,131.39,129.48,129.29,128.66,128.42,122.27,122.12,120.78,119.23,109.43,108.67,103.68,70.60,70.53,68.82,68.50,58.87,58.82,53.52。

example 19

Hela cells were plated in 96-well cell culture plates at 40000 cells per well, cultured for 5-6h in RPMI1640 containing 10% fetal bovine serum, and diluted test compounds (0.1. mu. mol/L, 0.2. mu. mol/L, 0.4. mu. mol/L, 0.8. mu. mol/L, 1.6. mu. mol/L, 3.2. mu. mol/L, 6.4. mu. mol/L) and recombinant human interferon-gamma at a final concentration of 100ng/mg activated IDO1 expression were added. After culturing the cells in a cell culture chamber at 37 ℃ containing 5% carbon dioxide for 20 hours, the reaction was terminated with 5% trichloroacetic acid and incubated at 50 ℃ for 30 minutes. After the cell culture solution is precipitated, taking the supernatant and sending the supernatant to LC/MS for detecting the content of kynurenine in the cell culture solution. Data analysis according to XLfit5 software detected target compound IC 50; the target compound obtained in example 18 was 0.678. mu. mol/L; the target compound obtained in example 17 was 0.342. mu. mol/L.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> southern medical university; guangdong Shengsai Biotech Co., Ltd

<120> erlotinib derivative and application thereof in preparation of IDO1 activity inhibition drugs and antitumor drugs

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 1

agctgtgcat ctacaccgac 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 2

gaaatcgtgc ggggtcattg 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 3

gtgaccagca gcggacaaat 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 4

tggactggag caagacttcg 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 5

gcttttggag tccctgttcg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 6

gcgagtcacc atctcagcaa 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 7

cggtactgaa gtccgggaac 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

catgaatggt gaacgcaggc 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 9

gcagcaggag ctttttgctt 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 10

ccaggtgctg cataactgga 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 11

caatgtggcc gaggactttg 20

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 12

cattctcctt agagagaagt gg 22

Claims (3)

1. An erlotinib derivative having the structural formula:

。

2. use of the erlotinib derivative according to claim 1 for the preparation of a medicament for the prevention and/or treatment of lung cancer.

3. The use of claim 2, wherein the lung cancer is non-small cell lung cancer.

Images