CN1858040B - 5,8-disubstituted quinazoline and its preparing method and use - Google Patents

Abstract

其中R ₁代表芳基胺、烷基胺或烷氧基，R ₂代表硝基或氨基或磺酰氨基或烷氧基，这类具有5，8-二取代-4-羟基喹唑啉结构的新化合物展示非常高的抑制肿瘤细胞生长的活性，尤其对于EGFR或ErbB-2高表达的乳腺癌细胞和卵巢癌细胞的生长具有显著的抑制效果，半抑制浓度IC ₅₀能达到0.01μM。本发明也揭示了这类化合物的制备过程。The invention discloses 5, 8-disubstituted quinazolines, a preparation method and application thereof, and the compounds with general formula 1 are as follows.

Wherein R ₁ represents arylamine, alkylamine or alkoxy, R ₂ represents nitro or amino or sulfonylamino or alkoxy, this type has 5,8-disubstituted-4-hydroxyquinazoline structure The new compound exhibits a very high activity of inhibiting the growth of tumor cells, especially for the growth of breast cancer cells and ovarian cancer cells with high expression of EGFR or ErbB-2, and the half-inhibitory concentration IC ₅₀ can reach 0.01 μM. The invention also discloses the preparation process of such compounds.

Description

5,8-disubstituted quinazolines and their preparation and use

technical field

The present invention relates to multi-substituted quinazoline novel compounds, their synthesis and their anti-tumor cell growth activity, more specifically containing 5,8-disubstituted-4-hydroxyquinazoline compounds as highly effective targeting epidermal growth factor receptors Synthesis and application of antineoplastic drugs for EGFR and HER2. the

Background technique

The Epidermal Growth Factor Receptor (EGFR) family belongs to the type I receptor tyrosine kinase superfamily and is an important regulator of cell growth, differentiation and survival. Its members include: erbB-1 (EGFR, HER1 ), erbB-2 (HER2), erbB-3 (HER3) and erbB-4 (HER4), which are similar in structure, all consist of an extracellular ligand binding region, a single-chain transmembrane region and a highly conserved protein tyrosine kinase district composition. This structure not only has the function of a receptor, but also has the ability to directly convert extracellular signals into intracellular effects, and is a novel transmembrane transmission method. Once specific ligands such as EGF or TGF-α are bound, the receptors can be activated through autophosphorylation of the corresponding tyrosine kinases, thereby initiating a chain reaction of intracellular signaling. These signal transmissions include: the activation of the Ras kinase protein and the mitogenic kinase protein MAPK, which in turn activate a variety of proteins in the nucleus, including the key cycle protein D1 for cell cycle proliferation, resulting in DNA synthesis, cell growth, A series of important cellular events such as differentiation and survival occur (Jorissen, R.N.; Walker, F.W.; Pouliot, N.; Garrett, T.P.J.; Ward, C.W.; Burgess, A.W. Epidermal growth factor receptor: mechanism of activation and signaling. Exp. Cell Res. 2003, 284, 31-53). Overactivation of growth factor receptors leads to uncontrolled proliferation of cells, resulting in various types of hyperproliferative diseases, such as non-small cell lung cancer, leukemia, breast cancer, brain cancer, atherosclerosis and regeneration after angioplasty. narrow. Inhibition of growth factor receptor tyrosine kinases has been shown to regulate uncontrolled cell replication and thus has become a target for novel antineoplastic drug therapy (Ritter, C.A. and Arteaga, C.L. The epidermal growth factor receptor-tyrosine kinase: a promising therapeutic target in solid tumors. Semin. Oncol. 2003, 30, 3-11). the

At present, many synthetic compounds have the activity of inhibiting cell epidermal growth factor receptor tyrosine kinase (EGFR-PTK), especially quinazoline compounds have been studied most deeply, among which ZD1839 was approved by FDA in 2003 for the treatment of non- Small cell lung cancer (Ranson, M. Epideraml growth factor receptor tyrosine kinase inhibitors. British J. Cancer 2004, 90, 2250-2255.). However, the quinazoline compounds reported in the literature as tyrosine kinase inhibitors mainly focus on 4-substituted anilino-6,7-disubstituted quinazoline structural types (Bridges, A.J.Chemical inhibitors of proteinkinases.Chem.Rev.2001 , 101, 2541-2571.), we designed and synthesized a new series of 4-hydroxy-5,8-disubstituted quinazoline compounds through molecular model calculations, and found small anti-tumor activity by optimizing the structure of the substituent molecular drug.

Contents of the invention

The purpose of the present invention is to search for a new class of quinazoline compounds with anti-tumor activity and inhibit cell epidermal growth factor receptor signal transduction. the

Another object of the present invention is to provide a preparation method of a class of 5,8-disubstituted-4-hydroxyquinazoline compounds. the

Another object of the present invention is to provide the medical application of 5,8-disubstituted-4-hydroxyquinazoline compound. the

The present design is based on crystal structures of EGFR tyrosine kinase proteins interacting with small molecule inhibitors (Wood, E.R.; Truesdale, A.T.; McDonald, O.B.; Yuan, D.; Hassell, A.; Dickerson, S.H.; Ellis, B.; Pennisi, C.; Horne, E.; Lackey, K.; Alligood, K.J.; protein conformation, inhibitor off-rate, and receptor activity in tumor cells. Cancer Res. 2004, 64, 6652-6659), through the synthesis method of a focused compound library, that is, with the pharmacophore quinazoline ring as the core structure, through The aryl nucleophilic substitution reaction introduces different substituents at the 5-position of the quinazoline ring, and introduces different 8-position substituents in conjunction with the nitration reaction, reduction reaction and sulfonylation reaction, and synthesizes a series of 5,8-di Substituting 4-hydroxyquinazoline as a new compound, the inhibitory activity of each compound on the growth of tumor cells overexpressed by epidermal growth factor was measured at the cell level. the

A class of 5,8-disubstituted quinazoline compounds of the present invention has the following structure 1

RO-，RNH-，RR’N- Among them, R ₁ :

RO-, RNH-, RR'N-

R ₂ : -NO ₂ , -NHSO ₂ R, -NRSO ₂ R, -NHR, -OR

R ₃ , R ₄ : F, Cl, Br, I and other halogens, or CF ₃ , RO-, RNH-, R ₂ N-

R and R' are independently C ₁ -C ₁₂ linear or branched or cyclic alkyl containing saturated and unsaturated hydrocarbon chains, C ₆ -C ₁₂ aryl or heterocyclic aryl, carboxyl or hydroxyl or aromatic Alkyl group substituted by a functional group such as a group or a halogen, an aryl group substituted by a functional group such as a carboxyl group or a hydroxyl group or an aryl group or a halogen

The synthesis of this type of compound is shown in the following reaction formula:

Specifically, 5,8-disubstituted-4-hydroxyquinazoline starts from 3-chloro-2-methylaniline, through amino protection, oxidation, N-protection group removal, cyclization into quinazoline ring, nitration It is synthesized by 6-step reaction with nucleophilic substitution reaction of aromatic ring. First, the commercially available raw material 3-chloro-2-methylaniline is dissolved in an inert solvent such as halogenated alkanes, toluene, benzene, tetrahydrofuran, dioxane or ether, acetic acid or N,N-dimethylformamide and other solvents N-protecting group, the protecting group is formyl, acetyl or substituted acetyl, CBZ, BOC, benzyl or substituted benzyl, FMOC, which are commonly used to protect amino groups, and the temperature is 0°C to room temperature, or heated to solvent reflux; Then benzyl is oxidized into benzoic acid by heating and refluxing in potassium permanganate aqueous solution (oxidizing reagent can also be potassium dichromate, chromium trioxide, sodium periodate, IBX, Dess-Martin reagent, fresh manganese dioxide, Hydrogen peroxide, the reaction temperature is controlled at 50°C to 120°C, and the solvents are halogenated hydrocarbons, toluene, acetonitrile, acetic acid and water). Remove the N-protecting group under certain conditions (for N-protected formyl, acetyl or substituted acetyl, CBZ, BOC remove the protecting group under acidic conditions, for N-protected formyl, benzyl or substituted The benzyl group is deprotected by catalytic hydrogenation, and the N-protected FMOC is deprotected under alkaline conditions. The solvent is halogenated hydrocarbon, tetrahydrofuran, 1,4-dioxane, methanol, ethanol, and the temperature is room temperature. to solvent reflux temperature), the obtained 2-amino-6-chlorobenzoic acid and formamide are cyclized to quinazoline at room temperature-175°C, and then further nitrated (nitrating reagent can be fuming nitric acid and concentrated sulfuric acid at 0°C ~ room temperature, or bismuth subnitrate and thionyl chloride, or potassium nitrate, lanthanum nitrate and concentrated nitric acid, or sodium nitrate, lanthanum nitrate and concentrated hydrochloric acid, solvent water, halogenated hydrocarbon or tetrahydrofuran, the temperature is 0 ℃ ~ room temperature) to obtain 8-nitro-5-chloroquinazoline, amine or alcohol carry out aryl nucleophilic substitution reaction to the 5-chlorine of quinazoline as nucleophile and obtain different 5-substituting group-8-nitro-quinazoline compounds ( The reaction solvent is an inert solvent such as tetrahydrofuran, diethyl ether, toluene, acetonitrile, 1,4-dioxane or N,N-dimethylformamide at 40°C to 100°C for nucleophilic substitution reaction), and the 8-position nitrate The group can be further reduced to amino (reducing agent is Pd/C hydrogenation, iron powder and acetic acid, iron powder and ammonium chloride, zinc powder and ammonium chloride, hydrazine hydrate, ammonium formate, solvent water, ethanol, methanol or acetonitrile, temperature At 40°C~100°C) and further converted into sulfonamide. the

Epidermal Growth Factor Receptor (EGFR and HER-2) Tyrosine Kinase Inhibitory Activity Test

Extraction of EGFR and HER-2 enzymes: Bab~to-Bac insect baculovirus expression system was used to express active tyrosine kinase protein, and the protein was affinity purified with Ni-NTA column. Aliquot and store at -70°C. Dilute the enzyme reaction substrate Poly(Glu, Tyr) _4:1 with potassium-free PBS to the optimal concentration, coat the microtiter plate, and react at 37°C for 12-16 hours. Discard the liquid in the well. Wash the plate three times, and dry the plate in a 37°C oven for 1-2 hours. Add 10 μL of a certain concentration of protein tyrosine kinase to each well, set up a corresponding no-enzyme control, and then add 80 μL of a certain concentration of ATP solution diluted with reaction buffer to start the reaction. At the same time, 10 μL of the compound to be tested was added at different concentrations, and 10 μL of a certain concentration of DMSO solution was used as the corresponding negative control, and the mixture was reacted on a shaker at 37° C. for 1 hour. Wash the plate three times with T-PBS, 15 minutes each time. Add 100 μl/well of antibody PY99 (1:1000) and react on a shaker at 37°C for 0.5 hours. Wash the plate three times with T-PBS, add horseradish peroxidase-labeled goat anti-mouse IgG (1:2000) 100 μl/well, and react on a shaker at 37°C for 0.5 hours. Wash the plate three times with T-PBS, add 2mg/ml OPD chromogenic solution 100μl/well, and react in the dark at 25°C for 1-10 minutes. Add 50 μl/well of 2M H ₂ SO ₄ to stop the reaction, and read with VERSAmax, a wavelength-adjustable microplate microplate reader, with a wavelength of 490 nm.

The relative inhibition rate of the drug on the tyrosine kinase protein was determined. the

According to the inhibition rate of each concentration, the half inhibitory concentration IC ₅₀ was calculated by Logit method. Each of the above experiments was repeated twice, and the average IC ₅₀ value of the two experiments was obtained as the final index of inhibitory ability.

Growth inhibitory activity of tumor cells overexpressed by epidermal growth factor receptor (EGFR and HER-2) tyrosine kinases:

According to the cell growth rate, tumor cells in the logarithmic growth phase were seeded in 96-well culture plates at 90 μl/well, adhered to the wall for 24 hours, and then 10 μl/well was administered. Three replicate wells were set up for each concentration. And set the corresponding concentration of normal saline vehicle control and no cell apoptosis well. Tumor cells were cultured at 37°C and 5% CO ₂ for 72 hours, then the culture medium was discarded, the cells were fixed with 10% cold TCA, placed at 4°C for 1 hour, washed 5 times with distilled water, and dried naturally in the air. Then add 100 μl/well of SRB (Sigma) 4 mg/ml solution prepared by 1% glacial acetic acid, stain at room temperature for 15 minutes, remove the supernatant, wash 5 times with 1% acetic acid, and air dry. Finally, 150 μl/well of Tris solution was added, and the OD value was measured with a microplate reader (VERSAmax) at a wavelength of 515 nm. Calculate the inhibitory rate of the test substance on the growth of cancer cells according to the following formula, and calculate the IC ₅₀ value of the half inhibitory dose by the Logit method. Each of the above experiments was repeated twice, and the average IC ₅₀ value of the two experiments was obtained as the final index of inhibitory ability.

Inhibition rate = (OD value of control group - OD value of drug administration group) / OD value of control group × 100%

Two human breast cancer cell lines overexpressing epidermal growth factor receptor tyrosine kinase were used to test the inhibitory effect of compounds on tumor cell growth. The breast cancer cell line SK-BR-3 overexpresses ErbB2 (HER2), and the breast cancer cell line MDA-MB-468 overexpresses EGFR. the

Table 1. Inhibitory effect of compounds 1a-n on tumor cell growth

^aCell line SK-BR-3 (breast cancer) overexpresses erbB2; cell line MDA-MB-468 (breast cancer) overexpresses EGFR.

^b Dose-dependent curves were measured at 5 concentration values; the drug concentration value _IC50 (μM) at which cell growth was inhibited by 50% was obtained from these curves.

^c B17 was used as a positive control (Wang, S.; Yang, D.; Enyedy, I. US Patent US2004023957-A1, 2004.).

Table 2. Inhibitory effects of compounds 1a-n on EGFR and ErbB-2 tyrosine kinase proteins

The pharmacological results at the cellular level show that this type of 5,8-disubstituted 4-hydroxyquinazoline compound is a class of highly active tumor growth inhibitors, and it is effective against tumor cells overexpressing epidermal growth factor receptors EGFR and ErbB-2. The system shows very high inhibitory activity, especially the derivatives substituted by aniline at the 5-position have the best activity, and the 3-position and 4-position substituents of 5-aniline are halogen and electron-donating groups can greatly promote the activity of such compounds. Tumor growth inhibitory activity of 5,8-disubstituted 4-hydroxyquinazoline compounds. The half-inhibitory concentration IC50 of the best compound on the growth of the tumor cell line MDA-MB-468 with high expression of EGFR is less than 0.01 μM, and its inhibitory activity is higher than that of the positive control compound B-17; for the tumor cell line SK-BR with high expression of ErbB-2 The half-inhibitory concentration IC ₅₀ of the growth of -3 was 7.0 μM.

Pharmacological results show that this type of 5,8-disubstituted 4-hydroxyquinazoline compound is a class of highly active tumor growth inhibitors, and has a strong activity of inhibiting the growth of human breast cancer cells and ovarian cancer cells, especially for cell Tumor cell lines overexpressing epidermal growth factor receptor EGFR and ErbB-2 showed very high selectivity, and the half-inhibitory concentration _IC50 of the best compound against the growth of tumor cell line MDA-MB-468 with high expression of EGFR was less than 0.01 μM , the inhibitory activity was higher than that of the positive control compound B-17; the half-inhibitory concentration IC ₅₀ for the growth of tumor cell SK-BR-3 with high ErbB-2 expression was 7.0 μM. The enzyme activity test at the molecular level shows that our compound has a certain inhibitory effect on EGFR tyrosine kinase and ErbB-2 tyrosine kinase.

Enzyme activity tests at the molecular level have shown that these compounds have certain inhibitory effects on EGFR and ErbB-2 tyrosine kinases, and these 5,8-disubstituted 4-hydroxyquinazoline compounds have inhibitory effects on human breast cancer cells and ovarian cancer cells It has a very significant inhibitory effect on the growth of the tumor, and is a class of highly active tumor growth inhibitors with a new structure. the

Detailed ways

The present invention will be further described below in conjunction with embodiment, but does not limit the present invention. the

Example 15-aniline-8-nitro-4 hydroxyquinazoline 1a

Step 1: 3-Chloro-2-methyl-N-acetanilide (compound 2)

3-Chloro-2-methylaniline (2.34 g, 16.50 mmol) was dissolved in 6 ml of dichloromethane, and acetic anhydride (2.53 g, 24.75 mmol) was slowly added dropwise under vigorous stirring. The resulting large amount of gray precipitated acetylated product was filtered through a Buchner funnel and washed with water. The crude product was recrystallized from ethyl acetate:methanol=10:1 to obtain a white crystalline product (2.73 g, 90.2%). ¹ HNMR (DMOS-d ₆ , 400MHz) 9.70(s, 1H), 7.40(d, J=8.40, 1H), 7.37(t, J=8.01, 1H), 1.99(s, 3H); MS(EI) m/z 183 (M ⁺ ); Anal. (C ₉ H ₁₀ ClNO) C, H, N: calcd: 58.86, 5.49, 7.63.

Step 2: 2-(Acetamide)-6-chlorobenzoic acid (compound 3)

Compound 2 (3.3 g, 18.0 mmol), magnesium sulfate hexahydrate (4.3 g, 38.8 mmol) and water were mixed and heated to 135° C. to reflux. An aqueous solution (100 mL) of potassium permanganate (11.4 g, 72.2 mmol) was slowly added dropwise to the above solution (about 3 hours). After the feeding is completed, heat up to 160°C and reflux for 2 hours, stop heating, and filter with a Buchner funnel after cooling. The filtrate is adjusted to PH=1.0 with 6M aqueous hydrochloric acid solution, and a large amount of white solid product is precipitated. Methanol recrystallization gave white crystalline pure product (2.5 g, 65.7%). ¹ H NMR (DMSO-d ₆ , 400MHz), δ9.70(s, 1H), 7.40(d, J=8.04, 1H), 7.37(t, J=8.01, 1H), 7.31(d, J=8.06 , 1H), 1.99 (s, 3H); MS (EI) m/z 213 (M ⁺ ); Anal. (C ₉ H ₈ ClNO ₃ ) C, H, N, calcd: 50.60, 3.77, 6.56, found: 50.42 , 3.54, 6.45.

Step 3: 5-chloro-4-hydroxyquinazoline (compound 5)

Compound 3 (250 mg, 1.17 mmol) was added to 4 ml of concentrated hydrochloric acid, heated to 80-90°C and stirred for 1.5 hours, the temperature should not be higher than 90°C. Heating was stopped, and white crystals precipitated after cooling. The solid product was collected by filtration and recrystallized from methanol to obtain 2-amino-6-chlorobenzoic acid (4), which was directly used in the next reaction. Add 1.5 ml of formamide to compound 4 (1.2 g, 5.8 mmol), heat, stir at 130°C for 45 minutes, and stir at 175°C for 75 minutes. Heating was stopped, and the translucent solid obtained after cooling was dissolved and stirred with 1.5 milliliters of methylcellulose, and the obtained slurry was poured into 5 milliliters of ice water, and the solid crude product was collected by filtration, and the product was separated and purified by column chromatography to obtain 0.75 grams of White solid product 5, ethyl acetate:petroleum ether=1:1 as developer, yield 67.6%. ¹ H NMR (DMSO-d ₆ , 400MHz), δ12.09(br, 1H), 8.07(s, 1H), 7.71(t, J=8.25, 7.7, 1H), 7.59(d, J=8.0, 1H ), 7.51 (d, J=7.43, 1H); MS (EI) m/z 180 (M ⁺ ); Anal. (C ₈ H ₅ ClN ₂ O) C, H, N, calcd: 53.21, 2.79, 15.51, Found: 53.14, 2.72, 15, 36.

Step 4: 5-chloro-8-nitro-4-hydroxyquinazoline (compound 6)

To compound 5 (155 mg, 0.86 mmol) was added 3 mL of concentrated sulfuric acid, cooled in an ice bath, and fuming nitric acid (3 mL) was slowly added dropwise for about 10 minutes. After the dropwise addition, keep stirring at 0°C for 10 minutes to stop the reaction, pour the reaction solution into crushed ice, neutralize it with 20% aqueous sodium hydroxide solution to adjust the pH to 7, collect the solid product by filtration, and wash twice with a small amount of water. Recrystallization from ethanol-water system gave the product as a pale yellow solid (193.5 mg). ¹ H NMR (DMSO-d ₆ , 400MHz) δ12.68 (br, 1H), 8.27 (d, J=8.86, 1H), 8.22 (s, 1H), 7.75 (d, J=8.85, 1H); MS ( EI) m/z 225 (M ⁺ ); Anal (C ₈ H ₄ ClN ₃ O ₃ ) C, H, N, calcd: 42.59, 1.79, 18.63, found: 42.47, 1.68, 18.60.

Step 5: 5-aniline-8-nitro-4-hydroxyquinazoline (compound 1a)

Compound 6 (100 mg, 0.44 mmol) was dissolved in 18 mL of THF, and 82 mg of aniline (0.88 mmol) was added. Heat the mixed system to reflux for 24 hours (80-90°C), stop the reaction, distill off most of THF under reduced pressure, add water to dilute (15 ml), dissolve with 20% hydrochloric acid to adjust pH=7, and extract twice with ethyl acetate (2 × 20 milliliters), combine the organic phases, dry with anhydrous sodium sulfate, filter, evaporate the solvent under reduced pressure, separate the brown solid product (90 mg, 72.5%) with silica gel column chromatography, and the developer system is acetic acid Ethyl ester: petroleum ether = 1:1. Mp: 233-235°C; ¹ H NMR (DMSO-d ₆ , 300MHz) δ9.88(br, 1H), 8.81(s, 1H), 8.80(d, J=9.08, 1H), 7.34(d, J =8.9, 1H), 7.0-7.2 (m, 5H); MS (EI) m/z 282 (M ⁺ ); Anal. (C ₁₄ H ₁₀ N ₄ O ₃ ) C, H, N, calcd: 59.57, 3.57 , 19.85, found: 59.45, 3.44, 19.98.

The target compounds 1b-1t were prepared in a similar manner. the

Embodiment 2: 5-benzylamine-8-nitro-4 hydroxyquinazoline 1b

Starting material benzylamine (94 mg, 0.88 mmol) gave the product as an orange solid (100 mg, 76.9%). Mp: 182-184°C; ¹ H NMR (DMSO-d ₆ , 300MHz).10.00(br, 1H), 8.68(d, J=9.1, 1H), 7.2-7.3(m, 5H), 6.77(d, J=8.99, 1H), 4.25 (s, 2H); MS (EI) m/z 296 (M ⁺ ); Anal. (C ₁₅ H ₁₂ N ₄ O ₃ ), C, H, N, calcd: 60.81, 4.08 , 18.91, found: 60.72, 4.11, 18.87.

Embodiment 3: 5-(3-chloroaniline)-8-nitro-4 hydroxyquinazoline 1c

Starting material 3-chloroaniline (211 mg, 0.88 mmol) gave a yellow solid (110 mg, 79.5%). Mp: 235-237°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ12.90(br, 1H), 11.45(s, 1H), 8.27(s, 1H), 8.23(d, J=9.07, 1H), 7.32(t, J=7.93, 1H), 7.16(d, J=9.07, 1H), 7.16-7.07(m, 2H), 6.95(d, J=7.96, 1H); MS(EI)m /e316 (M ⁺ ); Anal. (C ₁₄ H ₉ ClN ₄ O ₃ ) C, H, N, Calcd: 53.09, 2.86, 17.69. Found: 53.24, 2.97, 17.57.

Embodiment 3: 5-(4-chloroaniline)-8-nitro-4 hydroxyquinazoline 1d

Starting material 4-chloroaniline (112 mg, 0.88 mmol) gave a yellow solid (106 mg, 76.8%). Mp: 241-243°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ11.48 (br, 1H), 8.28 (s, 1H), 8.20 (d, J=9.08, 1H), 7.32 (d, J=8.51, 2H), 7.10 (d, J=9.06, 1H), 6.99 (d, J=8.52, 2H); MS (EI): 317 (M ⁺ +1); Anal. (C ₁₄ H ₉ ClN ₄ O ₃ ), C, H, N, Calcd: 53.09, 2.86, 17.69, Found: 52.86, 3.21, 17.35.

Embodiment 5: 5-(3-methylaniline)-8-nitro-4-hydroxyquinazoline 1e

Starting material 4-methylaniline (95 mg, 0.88 mmol) gave a brownish black solid (185 mg, 71.2%). Mp: 215-217°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ12.85(br, 1H), 11.42(s, 1H), 8.26(s, 1H), 8.18(d, J=9.89, 1H), 7.15(t, J=7.69, 1H), 7.08(d, J=8.79, 1H), 6.93(d, J=9.89, 1H), 6.78(s, 1H), 6.76(d, J=8.79 , 1H), 2.23 (s, 3H); MS (EI) m/e=296 (M ⁺ ); Anal. (C ₁₅ H ₁₂ N ₄ O ₃ ) C, H, NCalcd: 60.81, 4.08, 18.91; Found : 60.92, 4.11, 18.76.

Embodiment 6: 5-methoxy-8-nitro-4-hydroxyquinazoline 1f

To compound 6 (100 mg, 0.44 mmol) was added 10 mL of methanol and 100 mg of sodium methoxide (1.85 mmol) as a solid. The mixed system was heated to reflux at 70°C for 24 hours. Stop the reaction, evaporate most of the methanol solvent, add water to dilute (15 ml), adjust pH=7 with 20% hydrochloric acid solution, extract twice with ethyl acetate (2*20 ml), combine the organic phases, and wash with anhydrous Dry over sodium sulfate, distill off the solvent, and separate by silica gel column chromatography to obtain a white solid product (65 mg, 66.8%). The developing solvent is ethyl acetate:petroleum ether=1:1. MP: 209-210°C, ¹ H MNR (DMSO-d ₆ , 300 MHz) δ 11.28 (br, 1H), 9.13 (s, 1H), 8.67 (d, J=8.79, 1H), 6.83 (d, J =8.66, 1H), 3.90 (s, 3H); MS (EI) m/z 221 (M ⁺ ); Anal. (C ₉ H ₇ N ₃ O ₄ ) C, H, N, calcd: 48.87, 3.19, 19.00 , found: 48.77, 3.23, 18.89.

Embodiment 7: 5-ethoxy-8-nitro-4 hydroxyquinazoline 1g

The preparation method of compound 1g is the same as that of compound 1f. As the starting material, sodium ethoxide (125 mg, 1.85 mmol) and 10 ml of ethanol were used as solvents to obtain 80 mg of white solid with a yield of 77.6%. Mp: 195-198°C; ¹ H NMR (DMSO-d ₆ , 300 MHz) δ11.28 (br, 1H), 9.14 (s, 1H), 8.63 (d, J=8.20, 1H), 6.81 (d, J =8.40, 1H), 4.12 (m, J=6.9, 2H), 1.48 (t, J=6.9, 3H); MS (EI) m/z 235 (M ⁺ ); Anal. (C ₁₀ H ₉ N ₃ O ₄ ) C, H, N, calcd: 51.07, 3.86, 17.87, found: 51.13, 3.68, 17.74.

Embodiment 8: 5-(4-methoxyaniline)-8-nitro-4-hydroxyquinazoline 1h:

The preparation method of compound 1h is the same as that of compound 1a. Starting material 4-methoxyaniline (108 mg, 0.88 mmol) gave a dark green solid (81 mg, 58.9%). Mp: 200-203°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ12.65(br, 1H), δ11.40(s, 1H), 8.24(s, 1H), 8.14(d, J= MS(EI)m /e312 (M ⁺ ); Anal. (C ₁₅ H ₁₂ N ₄ O ₄ ) C, H, N, Calcd: 57.69, 3.87, 17.94; Found: 57.79, 3.98, 17.67.

Embodiment 9: 5-(4-aminoaniline)-8-nitro-4-hydroxyquinazoline 1i

The preparation method of compound 1i is the same as that of compound 1a. Starting material 4-aminoaniline (48 mg, 0.44 mmol) gave a brownish black solid (68 mg, 51.4%). Mp: 223-226°C; ¹ HNMR (DMSO-d ₆ , 300MHz): δ11.28 (br, 1H), 8.21 (s, 1H), 8.08 (d, J=9.06, 1H), 6.90 (d, J = 9.07, 1H), 6.68 (d, J = 8.52, 2H), 6.47 (d, J = 8.79, 2H); MS (EI) m/z M ⁺ = 297; Anal. (C ₁₄ H ₁₁ N ₅ O ₃ ) C, H, N, Calcd: 56.56, 3.73, 23.56, Found: 56.70, 3.46, 23.44.

Embodiment 10: 5-(4-benzyloxyaniline)-8-nitro-4-hydroxyquinazoline 1j

The preparation method of compound 1j is the same as that of compound 1a. Starting material 4-benzyloxyaniline (175 mg, 0.88 mmol) gave a tan solid (110 mg, 64.7%). Mp: 220-222°C; ¹ H NMR (DMSO-d ₆ , 300 MHz): δ11.40 (br, 1H), 8.25 (s, 1H), 8.15 (d, J=9.07, 1H), 7.46-7.35 ( m, 5H), 7.02 (d, J=9.07, 1H), 6.94 (s, 4H), 5.05 (s, 1H); MS (EI) m/e388 (M ⁺ ). Anal. (C ₂₁ H ₁₆ N ₄ O ₄ ) C, H, N, Calcd.: 64.94, 4.15, 14.43, Found: 64.82, 4.45, 14.56.

Example 11: 5-pyrroline-8-nitro-4-hydroxyquinazoline 1k

The preparation method of compound 1k is the same as that of compound 1a. Starting material pyrroline (63 mg, 0.88 mmol) afforded a brown solid (90 mg, 78.5%). Mp: 208-210°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ12.30(br, 1H), 8.12(s, 1H), 8.09(d, J=9.07, 1H), 6.72(d, J=9.07, 1H), 3.29 (dt, 4H), 1.89 (dt, 4H); MS (EI) m/e 260 (M ⁺ ); Anal. (C ₁₂ H ₁₂ N ₄ O ₃ ) C, H, N , Calcd: 55.38, 4.65, 21.53, Found: 55.41, 4.35, 21.78.

Example 12: 5-(2H-pyrrole)-8-nitro-4-hydroxyquinazoline 11

The preparation method of compound 11 is the same as that of compound 1a. Starting material 2H-pyrrole (61 mg, 0.88 mmol) gave a brownish black solid (79 mg, 69.8%). Mp: 154-160°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ12.41(br, 1H), 8.11(s, 1H), 7.99(d, J=8.99, 1H), 6.79(d, J=8.99, 1H), 6.79 (d, J=9.06, 1H), 5.91 (s, 2H), 3.98 (s, 4H); MS (EI) m/e257 (M ⁺ -1); Anal. (C _12H10N4O3 )C, H, N, Calcd: 55.81, 3.90, _21.70 , Found: 55.79 _, 3.86, _21.92 .

Embodiment 13: 5-N-(methionine)-8-nitro-4-hydroxyquinazoline 1m

The preparation method of compound 1m is the same as that of compound 1a. The starting material was methionine (131 mg, 0.88 mmol) to obtain a brown solid (76 mg, 51.2%), and the developer system was chloroform:methanol=2:1. Mp: 214-217°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ12.78(br, 1H), 10.34(br, 1H), 8.23(s, 1H), 8.11(d, 1H, J= 9.07), 6.91 (d, 1H, J=9.06), 4.01 (s, 1H), 2.37 (s, 2H), 2.2-1.8 (m, 5H); MS (EI) m/e338 (M ⁺ ); Anal .(C ₁₃ H ₁₄ N ₄ O ₅ S) C, H, N, Calcd: 46.15, 4.17, 16.56. Found: 46.23, 4.25, 16.43.

Embodiment 14: 5-N-(phenylalanine)-8-nitro-4-hydroxyquinazoline 1n

The preparation method of compound 1n is the same as that of compound 1a. Phenylalanine (133 mg, 0.88 mmol) was used as the starting material to obtain a brown solid (74 mg, 49.7%), and the developer system was chloroform:methanol=2:1. Mp: >250°C; ¹ HNMR (DMSO-d ₆ , 300MHz): δ12.80(br, 1H), 11.25(br, 1H), 8.22(s, 1H), 7.91(d, J=9.34, 1H) , 7.25-7.12 (d, 5H), 5.12 (s, 1H); MS (EI) m/e 340 (M ⁺ ); Anal. (C ₁₆ H ₁₂ N ₄ O ₅ ) C, H, N Calcd: 56.47, 3.55, 16.46, Found: 56.22, 3.31, 16.28.

Embodiment 15: 5-(4-(4-hydroxyl-8-nitroquinazoline-5-amino)aniline)-8-nitro-4-hydroxyquinazoline 1o

The preparation method of compound 1o is the same as that of compound 1a. Starting material 1,4-diaminoaniline (24 mg, 0.22 mmol) gave a tan solid (67 mg 62.6%). Mp: >250°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ14.06 (br, 2H), 8.17 (s, 2H), 7.94 (d, J=9.3, 2H), 6.74 (d, J =9.3, 2H), 6.71 (s, 4H); MS (EI) m/e 486 (M ⁺ ); Anal. (C ₂₂ H ₁₄ N ₈ O ₆ ) C, H, N Calcd: 54.33, 2.90, 23.04.

Example 16: 5-(3-Chloro-4-methoxyaniline)-8-nitro-4-hydroxyquinazoline 1p

Same as the preparation method of compound 1a. Starting material 3-chloro4-methoxyaniline (100 mg, 0.68 mmol) gave an orange solid (62 mg, 81.0%). Mp: =216°C; ¹ H NMR (CD ₃ OD, 300MHz): δ8.14(s, 1H), 7.03(d, J=5.4, 2H), 7.01(s, 1H), 6.98(d, J= 9.1, 2H), 6.91 (d, J=9.1, 2H), 3.84 (s, 3H); MS (EI) m/e 346 (M ⁺ ); Anal. (C ₁₅ H ₁₁ ClN ₄ O ₄ ) C, H , N Calcd: 51.96, 3.20, 16.16.

Embodiment 17: 5-(4-carboxyaniline)-8-nitro-4-hydroxyquinazoline 1q

Same as the preparation method of compound 1a. Starting material 4-carboxyaniline (301 mg, 2.2 mmol) gave a bright yellow solid (63 mg, 44.0%). Mp: >250°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ13.8 (br, 1H), 8.22 (s, 1H), 8.03 (d, J=9.3, 2H), 7.80 (d, J = 8.2, 2H), 6.90 (d, J = 9.1, 2H), 6.84 (d, J = 8.2, 2H); Anal. (C ₁₅ H ₁₀ N ₄ O ₅ ) C, H, N Calcd: 55.22, 3.09 , 17.17.

Example 18: 5-pyrrole-8-nitro-4-hydroxyquinazoline 1r

Same as the preparation method of compound 1a. Starting material pyrrole (147 mg, 2.2 mmol) afforded a brownish black solid (75 mg, 66.9%). Mp: >250°C; ¹ H NMR (DMSO-d ₆ , 400MHz): δ10.8(br, 1H), 8.00(s, 1H), 7.88(d, J=9.6, 2H), 6.68(s, 2H ), 6.08 (d, J=9.6, 2H), 5.99 (s, 2H); Anal. (C ₁₂ H ₈ N ₄ O ₃ ) C, H, NCalcd: 56.25, 3.15, 21.87.

Example 19: 5-(3-chloro-4-cyclohexyloxyaniline)-8-nitro-4-hydroxyquinazoline 1s

Same as the preparation method of compound 1a. Starting material 3-chloro-4-cyclohexyloxyaniline (100 mg, 0.45 mmol) gave an ocher solid (72 mg, 79.1%). Mp: =190°C; ¹ H NMR (CD ₃ OD, 300MHz): δ8.17(s, 1H), 8.13(d, J=9.0, 1H), 7.42(d, J=2.7, 1H), 7.27( dd, J = 2.7, 9.0, 1H), 7.22 (d, J = 8.9, 1H), 6.98 (d, J = 9.0, 1H), 4.47 (m, 1H), 1.93-1.23 (m, 10H); MS (EI) m/e 414 (M ⁺ ); Anal. (C ₂₀ H ₁₉ ClN ₄ O ₄ )C, H, N Calcd: 57.90, 4.62, 13.51.

Embodiment 20: 5-(3-chloro-4-hydroxyaniline)-8-nitro-4-hydroxyquinazoline 1t

Same as the preparation method of compound 1a. Starting material 3-chloro-4-hydroxyaniline (100 mg, 0.53 mmol) gave an ocher solid (185 mg, 99.4%). Mp: >250°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ11.34(br, 1H), 10.13(br, 1H), 8.27(s, 1H), 8.13(d, J=8.9, 1H ), 7.03 (m, 2H), 6.85 (m, 2H); MS (EI) m/e332 (M ⁺ ); Anal. (C ₁₄ H ₉ ClN ₄ O ₄ ) C, H, N Calcd: 50.54, 2.73 , 16.84.

Example 21: 5-(4-methoxyaniline)-8-amino-4-hydroxyquinazoline 1u

5-(4-Methoxyaniline)-8-nitro-4-hydroxyquinazoline (10 mg, 0.032 mmol) was dissolved in 5 ml of methanol, 1 mg of 5% palladium-carbon catalyst was added, hydrogenated at normal pressure and temperature After 12 hours, the reaction was stopped, filtered, and the filtrate was sucked dry to obtain a solid crude product, which was recrystallized from methanol to obtain 8 mg of a dark green solid product (92% yield). Mp: >250°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ7.76(s, 1H), 7.18(s, 2H), 6.76(d, J=9.0, 2H), 6.59(d, J =9.0, 2H), 3.65 (s, 3H); MS (EI) m/e282 (M ⁺ ); Anal. (C ₁₅ H ₁₄ N ₄ O ₂ ) C, H, N Calcd: 63.82, 5.00, 19.85.

Example 22: 5-(3-chloroaniline)-8-amino-4-hydroxyquinazoline 1v

5-(3-Chloroaniline)-8-nitro-4-hydroxyquinazoline (10 mg, 0.032 mmol) was dissolved in 5 ml of methanol, 1 mg of 5% palladium-carbon catalyst was added, hydrogenated at normal pressure and temperature, 12 The reaction was stopped after 1 hour, filtered, and the filtrate was sucked dry to obtain a solid crude product, which was recrystallized from methanol to obtain 9 mg of a dark green solid product (98% yield). Mp: >250°C; ¹ H NMR (CD ₃ OD, 300MHz): δ8.01(s, 1H), 7.34(d, J=9.6, 2H), 7.16(d, J=9.0, 1H), 6.86( m, 1H), 6.74 (d, J=9.0, 1H), 6.62 (s, 1H); MS (EI) m/e 286 (M ⁺ ); Anal. (C ₁₄ H ₁₁ ClN ₄ O)C, H, N Calcd: 58.65, 3.87, 19.54.

Example 23: 5-(3-chloro-4-benzyloxyaniline)-8-amino-4-hydroxyquinazoline 1w

Same as the preparation method of compound 1a. Starting material 3-chloro-4-benzyloxyaniline (40 mg, 0.207 mmol) gave an orange solid (47 mg, 60.0%). Mp: =247°C; ¹ H NMR (DMSO-d ₆ , 300MHz): δ8.24 (s, 1H), 8.17 (d, J = 9.0, 1H), 7.68 (d, J = 9.0, 1H), 7.45 (m, 5H); 7.16(s, 1H), 7.05(d, J=9.1, 1H), 6.95(d, J=9.1, 1H), 5.16(s, 2H); Anal.(C ₂₁ H ₁₆ N ₄ O ₄ ) C, H, N Calcd: 64.94, 4.15, 14.43.

Claims (11)

1. a class has 5 of following general formula, 8-disubstituted quinazoline compound,

Wherein, R ₁:

RO-, RNH;

R ₂：-NO ₂；

R ₃, R ₄: be halogen independently, or RO-;

R is independently selected from C ₁-C ₁₂Straight or branched or cyclic alkyl, C ₆-C ₁₂Aryl, wherein cyclic alkyl does not comprise C ₁-C ₂Cyclic alkyl.

2. according to claim 15,8-disubstituted quinazoline compound is characterized in that:

Work as R ₁During for RO-,

R ₂For-NO ₂,

R is selected from methyl, ethyl.

3. a class 5,8-disubstituted quinazoline compound is characterized in that:

Described compound is selected from 5-aniline-8-nitro-4-hydroxyl quinazoline, 5-benzylamine-8-nitro-4-hydroxyl quinazoline, 5-(3-chloroaniline)-8-nitro-4-hydroxyl quinazoline, 5-(4-chloroaniline)-8-nitro-4-hydroxyl quinazoline, 5-(3-monomethylaniline)-8-nitro-4-hydroxyl quinazoline, 5-methoxyl group-8-nitro-4-hydroxyl quinazoline, 5-oxyethyl group-8-nitro-4-hydroxyl quinazoline, 5-(4-anisidine)-8-nitro-4-hydroxyl quinazoline, 5-(4-amido aniline)-8-nitro-4-hydroxyl quinazoline, 5-(4-benzyloxy-aniline)-8-nitro-4-hydroxyl quinazoline, 5-pyrroline-8-nitro-4-hydroxyl quinazoline, 5-(2H-pyrroles)-8-nitro-4-hydroxyl quinazoline, 5-N-(methionine(Met))-8-nitro-4-hydroxyl quinazoline, 5-N-(phenylalanine)-8-nitro-4-hydroxyl quinazoline, 5-(4-(4-hydroxyl-8-nitro-quinazoline-5-amino) aniline)-8-nitro-4-hydroxyl quinazoline, 5-(3-chloro-4-anisidine)-8-nitro-4-hydroxyl quinazoline, 5-(4-carboxyl aniline)-8-nitro-4-hydroxyl quinazoline, 5-pyrroles-8-nitro-4-hydroxyl quinazoline, 5-(3-chloro-4-cyclohexyloxy aniline)-8-nitro-4-hydroxyl quinazoline, 5-(3-chloro-4-hydroxyanilines)-8-nitro-4-hydroxyl quinazoline, 5-(4-anisidine)-8-amino-4-hydroxy quinazoline, 5-(3-chloroaniline)-8-amino-4-hydroxy quinazoline, or 5-(3-chloro-4-benzyloxy-aniline)-8-amino-4-hydroxy quinazoline.

4. as claimed in claim 15, the preparation method of 8-disubstituted quinazoline compound comprises the steps:

3-chloro-2-aminotoluene sets out, through amido protecting, oxidation, take off the N-protected base, become the quinazoline ring, nitrated and aromatic ring nucleophilic substitution reaction gets target compound, reaction formula is as follows:

5. according to claim 45, the preparation method of 8-disubstituted quinazoline compound is characterized in that the N-protected base is formyl radical, ethanoyl, CBZ, BOC, benzyl, FMOC; The inert solvent is halogenated alkane, toluene, benzene, tetrahydrofuran (THF), dioxane or ether, acetic acid or N, dinethylformamide, and temperature of reaction is 0 a ℃～room temperature, or is heated to solvent refluxing.

6. according to claim 45, the preparation method of 8-disubstituted quinazoline compound is characterized in that:

Oxygenant is dichromic acid acid potassium, chromium trioxide, sodium periodate, potassium permanganate solution, IBX, Dess-Martin reagent, and new preparing manganese dioxide, hydrogen peroxide, temperature of reaction is controlled at 50 ℃～120 ℃, and solvent is halohydrocarbon, toluene, acetonitrile, acetate and water.

7. according to claim 45, the preparation method of 8-disubstituted quinazoline compound is characterized in that the N-protected base is that FMOC sloughs protecting group under alkaline condition; solvent halohydrocarbon, tetrahydrofuran (THF), 1; the 4-dioxane, methyl alcohol, ethanol, temperature is that room temperature arrives the solvent refluxing temperature.

8. according to claim 45, the preparation method of 8-disubstituted quinazoline compound is characterized in that:

Cyclization is a quinazoline at 2-amino-6-chloro-benzoic acid and methane amide cyclisation, and the cyclisation temperature is a room temperature to 175 ℃, gradient-heated.

9. according to claim 45, the preparation method of 8-disubstituted quinazoline compound is characterized in that:

Nitrating agent be nitrosonitric acid and the vitriol oil 0 ℃～room temperature, or bismuth subnitrate and thionyl chloride, or saltpetre, lanthanum nitrate and concentrated nitric acid, or SODIUMNITRATE, lanthanum nitrate and concentrated hydrochloric acid, water used in solvent, halohydrocarbon or tetrahydrofuran (THF), temperature is 0 ℃～room temperature.

10. according to claim 45, the preparation method of 8-disubstituted quinazoline compound is characterized in that:

Amine or alcohol carry out the aryl nucleophilic substitution reaction as nucleophilic reagent to the 5-chlorine of quinazoline and obtain different 5-substituting groups-8-position nitro-quinazoline compound, reaction solvent is inert solvent tetrahydrofuran (THF), ether, toluene, acetonitrile, 1,4-dioxane or N, nucleophilic substitution reaction takes place 40 ℃～100 ℃ heating in dinethylformamide.

11. claim 1 or 3 described 5, the application of 8-disubstituted quinazoline compound in preparation treatment human breast cancer and oophoroma tumor medicine.