CN103948587A - Dual-channel inhibitor using Raf/MEK/ERK and PI3K/Akt as targets - Google Patents

Abstract

The invention discloses a dual channel inhibitor targeting Raf/MEK/ERK and PI3K/Akt, the chemical name is 1-(2-amino)ethyl-3-(3-(4-methoxy) Phenyl)propenylindol-2-one hydrochloride, a compound of formula (I): , and also discloses the preparation method of the compound and the application in the preparation of antitumor drugs. The dual-channel inhibitor of the present invention has the following advantages: 1) increasing potency and reducing the occurrence probability of drug resistance; 2) reducing toxicity and improving patient compliance. Therefore, the design and development of dual channel inhibitors could provide the cancer research community with a new chemical tool and may be developed as a new anticancer agent.

Description

A dual-channel inhibitor targeting Raf/MEK/ERK and PI3K/Akt

technical field

The present invention relates to a dual-channel inhibitor targeting Raf/MEK/ERK and PI3K/Akt, specifically, the present invention provides 1-(2-amino)ethyl-3-(3-(4-methyl Oxygen)phenyl)propenylindol-2-one hydrochloride and preparation method thereof, can be used as a dual-channel inhibitor with Raf/MEK/ERK and PI3K/Akt as dual targets, can be used for treating cancer, Belongs to the field of medicinal chemistry technology

Background technique

Malignant tumor is a major disease that seriously endangers human health. Infiltration and metastasis are one of its basic characteristics, and they are also the main reason for the death of patients caused by clinical treatment failure. At present, there is a lack of effective anti-tumor metastasis drugs in clinic. However, most of the existing drugs have poor tumor selectivity, low curative effect, and large toxic and side effects. The study of effective anti-cancer and anti-metastasis drugs has become an urgent problem to be solved in tumor chemotherapy. The signal transduction research on tumor metastasis and the design of chemical small molecule compounds based on targets are one of the effective ways to solve these problems.

Specific expression of signaling pathways, such as Raf/MEK/ERK (extracellular signal-regulated kinase) and PI3K (phosphatidylinositol (-3) kinase)/Akt cascade, can promote the survival and growth of cancer cells. The Raf/MEK/ERK pathway is one of the evolutionarily conserved mitogen-activated protein kinase pathways, which play an important role in promoting cell proliferation and inhibiting apoptosis. After activation by growth factors, serum, cytokines, and osmotic stress, ERK can phosphorylate and regulate many substrates, such as cytoskeletal proteins, kinases, and transcription factors. These responses ultimately alter gene expression and cell proliferation, differentiation, and survival processes. Studies by Roberts, P.J. et al. have shown that abnormal activation of this pathway at different levels is closely related to the generation of many human tumors, especially melanoma, breast cancer, ovarian cancer and leukemia (Oncogene 2007, 26, 3291-3310). The PI3K/Akt signaling pathway plays an important role in the development of cancer. Genetic aberrations are more common in the PI3K/Akt pathway than in other pathways during cancer development, signaling through apoptotic precursors and apoptotic factors, mTOR, GSK-3 (Glycogen Synthesis Kinase-3) , p53 and other downstream effectors regulate many basic functions of cells such as cell growth, proliferation, survival, apoptosis, metabolism, etc. The phosphatase and tensin homologous gene (PTEN) deleted on human chromosome 10 is a negative regulator of PI3K/Akt, which plays a role by dephosphorylating PIP3. Paz-Ares, L. and other studies have confirmed that PTEN can lose its activity in human primary or metastatic tumors through genetic or acquired modification (Clin.Transl.Oncol.2009,11,572-579), and it is found in many human cancers Mutations and/or activation of PI3K and Akt can be detected.

Notably, these two signaling pathways can cooperate with each other to regulate apoptosis and survival of mutant cells. Both signaling pathways can phosphorylate and regulate many common downstream effectors including factors that regulate cell survival and apoptosis, such as CREB, Bad, Bim, and caspase9. More and more evidence shows that the interaction between these two pathways can interfere with each other and there is a feedback regulation mechanism. Studies by Carracedo, A. et al. have confirmed that in metastatic cancer patients, cancer cell lines and animal models of prostate cancer, mTOR inhibits The application of drugs can activate the Raf/MEK/ERK pathway, which suggests that there is a feedback regulation mechanism between Raf/MEK/ERK and PI3K/Akt (J. Clin. Invest. 2008, 118, 3065-3074). However, the presence of this mechanism may lead to resistance to drugs that target a single pathway. This conclusion was also confirmed by the frequent activation of Raf/MEK/ERK and PI3K/Akt in human advanced prostate cancer patients. More importantly, in vivo and in vitro experiments have shown that the concomitant disruption of these two pathways can synergistically activate the apoptosis of cancer cells, suggesting that people can simultaneously target these two pathways for clinical drug treatment research. However, as far as we know, all current combined target therapies are combined drugs that use inhibitors targeting Raf/MEK/ERK and PI3K/Akt respectively, and combined drugs will increase the dosage of drugs, which has a negative effect on the human body. Increased toxicity, and increased the probability of drug resistance. However, no monomeric small molecule substances that can simultaneously inhibit these two pathways have been found.

Contents of the invention

In view of the deficiencies in the prior art, the object of the present invention is to provide a dual-channel inhibitor targeting Raf/MEK/ERK and PI3K/Akt, the chemical name is 1-(2-amino)ethyl-3 -(3-(4-methoxy)phenyl)propenylindol-2-one hydrochloride, the dual-channel inhibitor improves potency and reduces the probability of drug resistance, reduces toxicity, improves patient compliance.

Another object of the present invention is to provide a preparation method of the dual-channel inhibitor and its application in the preparation of antitumor drugs.

To achieve the above object, the present invention adopts the following technical solutions:

A dual-channel inhibitor targeting Raf/MEK/ERK and PI3K/Akt with the chemical name 1-(2-amino)ethyl-3-(3-(4-methoxy)phenyl)propene Indol-2-one hydrochloride, a compound of formula (I):

The preparation method of the dual-channel inhibitor comprises the following reaction steps:

1) Synthesis of N-phenylethylenediamine (2):

With toluene as solvent, 2-bromoethylamine hydrobromide and aniline (1) were heated to reflux for 14-18h, cooled, extracted, washed and purified to obtain compound (2) N-phenylethylenediamine; 2-bromo The molar ratio of ethylamine hydrobromide and aniline (1) is 1: (1.5～2.5);

2) Synthesis of N-tert-butoxycarbonyl-N'-phenylethylenediamine (3):

Using methanol as a solvent, mix N-phenylethylenediamine (2) and di-tert-butyl carbonate prepared in step 1) and react for 3-5 hours to obtain compound (3) N-tert-butoxycarbonyl-N' -Phenylethylenediamine; The molar ratio of N-phenylethylenediamine (2) and di-tert-butyl carbonate is 1: (1～1.5);

3) Synthesis of N-(2-(N'-chloroacetyl) phenylamino) ethyl tert-butyl carbamate (4):

Dissolve N-tert-butoxycarbonyl-N'-phenylethylenediamine (3) prepared in step 2) in dichloromethane, add triethylamine and cool to 0oC, add chloroacetyl chloride dropwise under stirring, reaction 2 -4h, the reaction solution was poured into ethyl acetate, washed, and dried to obtain compound (4); N-tert-butoxycarbonyl-N'-phenylethylenediamine (3), triethylamine, chloroacetyl chloride The molar ratio is (1~1.5):(1~1.5):1;

4) Synthesis of N-(2-oxoindol-1-yl) ethyl tert-butyl carbamate (5):

Under the protection of nitrogen, add anhydrous triethylamine and anhydrous toluene to palladium acetate, 2-(di-tert-butylphosphino)biphenyl, step 3) to compound (4), and react at 70-90oC 14 -18h, cooled, diluted with ethyl acetate, filtered, concentrated, purified to obtain compound (5) N-(2-oxoindol-1-yl) ethyl carbamate tert-butyl alcohol ester; palladium acetate, 2- The molar ratio of (di-tert-butylphosphino)biphenyl, compound (4), anhydrous triethylamine and anhydrous toluene is 2:1:(1～2):(2～4):(1～3) ;

5) Synthesis of 5-(4-methoxy)phenylmethylene-2,2-dimethyl-1,3-dioxazolidine-4,6-dione (7):

Using ethanol as a solvent, mix p-methoxybenzaldehyde (6), cyclo(sub)isopropyl malonate (Merberine's acid), and piperidine, stir and react overnight at room temperature, filter, and wash to obtain compound (7) ; The mol ratio of p-methoxybenzaldehyde (6) and cyclo(sub)isopropyl malonate (Magnus acid) is (1～1.5): (1～1.5);

6) Synthesis of 1-(2-amino)ethyl-3-(3-(4-methoxy)phenyl)propenylindole-2-one hydrochloride:

With dichloromethane as solvent, add compound (7), in the presence of acid, drop sodium borohydride at 0oC, react to obtain compound (8); the mol ratio of compound (7) and sodium borohydride is 1:(3 ~4);

Using tetrahydrofuran as a solvent, add compound (8), phenylsilane and triethylamine, react for 15-30min, wash and purify to obtain compound (9); the molar ratio of compound (8), phenylsilane and triethylamine is 1: (2~4): (1.5~3);

Add compound (9) and piperidine to the ethanol suspension of compound (5), heat to 70-90oC for 1-2h, cool the reaction solution to room temperature, and filter to obtain compound (10); compound (9), piperidine The molar ratio of pyridine to compound (5) is (1～2):(0.2～0.4):1;

Dissolve the Boc-protected compound (10) in ethyl acetate, then add dioxane-HCl, react for 1-3h, a suspension appears, filter, wash, and obtain; compound (10) and dioxane-HCl The molar ratio is (1～2):(1～2).

In the step (1), the operations of extraction, washing, and purification are specifically: adding water and 50% KOH aqueous solution, standing for stratification; washing the aqueous phase with saturated NaCl and extracting it several times with dichloromethane; combining the organic phases After that, it was washed with saturated NaCl, dried over anhydrous Na ₂ SO ₄ , filtered, evaporated to dryness, and purified by column chromatography.

In the step (3), the addition method of chloroacetyl chloride is: first dropwise add 80% of the added amount of chloroacetyl chloride, after reacting for 1.5-3 hours, then add dropwise the remaining 20% of chloroacetyl chloride, and react for 30-60 minute.

In the step (4), the specific operations of filtering, concentrating and purifying are as follows: filter with diatomaceous earth, and spin the filtrate to dry and concentrate to obtain a crude product, which is purified by silica gel chromatography.

In the step (6), the acid used in the preparation of the compound (8) is glacial acetic acid.

The synthetic route is as follows:

The application of the dual-channel inhibitor of the present invention in the preparation of antitumor drugs.

Compared with single-channel inhibitors, the dual-channel inhibitors of the present invention have the following advantages: 1) increase the potency and reduce the occurrence probability of drug resistance; 2) reduce the toxicity of antitumor drugs and improve patient compliance. Therefore, the design and development of dual channel inhibitors could provide the cancer research community with a new chemical tool and may be developed as a new anticancer agent.

Detailed ways

The present invention will be further described in conjunction with the examples. It should be noted that the following descriptions are only for explaining the present invention and not limiting its content.

Example 1

1). The synthesis of N-phenylethylenediamine (2):

2-Bromoethylamine hydrobromide (0.1 mmol) and aniline (1) (0.2 mmol) were added to 40 ml of toluene, heated to reflux for 16 hours, and cooled. Add 60ml of water and 20ml of 50% KOH aqueous solution, and let stand to separate layers. The aqueous phase was washed with saturated NaCl and extracted three times with dichloromethane. The combined organic phases were washed with saturated NaCl, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to dryness. The pure compound (2) was obtained by column chromatography with a yield of 60%. ¹ H (400MHz, MeOH): δ7.10-7.08 (t, J = 3.4Hz, 2H), 6.67-6.60 (m, 3H), 3.23-3.20 (t, J = 6.08Hz, 2H), 2.90-2.87 (t, J=6.24Hz, 2H).

2). Synthesis of N-tert-butoxycarbonyl-N'-phenylethylenediamine (3):

N-phenylethylenediamine (2) (1.0 mmol) and di-tert-butyl carbonate (1.2 mmol) were dissolved in 15 ml of methanol, stirred for 4 hours, and concentrated under reduced pressure to obtain a brown oil. The pure white solid compound (3) was obtained by column chromatography with a yield of 92%. ¹ H NMR (400MHz, CDCl ₃ ): δ7.25-7.18(m, 2H), 6.81-6.6(m, 2H), 3.38-3.36(d, J=7.6Hz, 2H), 3.27-3.23(t, J=8.0Hz, 2H), 1.48(s, 9H).

3). Synthesis of tert-butyl alcohol N-(2-(N'-chloroacetyl)anilino)ethylcarbamate (4):

Compound (3) (1.2mmol) was dissolved in 40ml of dichloromethane, triethylamine (1.2mmol) was added and cooled to 0oC, and chloroacetyl chloride (0.8mmol) was added dropwise with stirring. After reacting for 3 hours, continue to add chloroacetyl chloride (0.2 mmol) dropwise. After reacting for 30 minutes, pour the reaction solution into 20 ml of ethyl acetate, and wash with water, saturated ammonium chloride solution, saturated sodium bicarbonate solution, and brine successively. Dry over anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain brown oily compound (4), yield 48%. ¹ H NMR (400MHz, CDCl ₃ ): δ7.48-7.41(m, 3H), 7.28-7.27(d, J=6.6Hz, 2H), 3.87-3.83(m, 4H), 3.35-3.34(d, J=5.44Hz, 2H), 1.4165(s, 9H).

4). Synthesis of N-(2-oxoindol-1-yl) ethyl tert-butyl carbamate (5):

The oven-dried two-necked flask was cooled to room temperature, equipped with electromagnetic stirring, and palladium acetate (1.0 mmol), 2-(di-tert-butylphosphino)biphenyl (0.5 mmol) and compound (4) (1.0 mmol) were added. Continue to pass nitrogen gas to evacuate the gas in the device, and add anhydrous triethylamine (1.5 mmol) and anhydrous toluene (1.0 ml) respectively under nitrogen protection. Place the two-necked flask in an oil bath preheated to 80oC. After 16 hours of reaction, the heating was stopped, cooled to room temperature, and diluted with ethyl acetate (10 ml). The mixture was filtered through celite, and the filtrate was spin-dried and concentrated to obtain a crude product. The crude product was purified by silica gel chromatography to obtain compound (5) with a yield of 65%. ¹ H NMR (400MHz, CDCl ₃ ): δ7.28-7.21(m, 2H), 7.04-7.00(t, J=7.5Hz, 1H), 6.96-6.93(d, 7.8Hz, 1H), 4.14-4.07 (d, J=7.2Hz, 2H), 3.86-3.82 (t, J=8.0Hz, 2H), 3.39-3.35 (q, J=5.7Hz, 2H), 1.40 (s, 9H).

5). Synthesis of 5-(4-methoxy)phenylmethylene-2,2-dimethyl-1,3-dioxazolidine-4,6-dione (7):

Dissolve p-methoxybenzaldehyde (6) (1.1 mmol) in 20 ml of ethanol, and add cyclo(ylidene)isopropyl malonate (Merz's acid) (1.2 mmol) and 5 drops of piperidine. The reaction was stirred overnight at room temperature. The reaction mixture was filtered under reduced pressure and washed with ethanol and methanol to obtain pure product (7). Yield 51%. ¹ H NMR (300MHz, CDCl3): 8.38(s, 1H), 8.24-8.21(d, J=9.3Hz, 2H), 6.99-6.97(d, J=9.3Hz, 2H), 3.91(s, 3H) ,1.79(s,6H).

6). Synthesis of 1-(2-amino)ethyl-3-(3-(4-methoxy)phenyl)propenylindole-2-one hydrochloride:

Compound (7) (1mmol) was dissolved in 30ml of dichloromethane, 4ml of glacial acetic acid was added, and sodium borohydride (3.2mmol) was added at 0oC. After reacting for 1 hour, the reaction solution was added to 20 ml of dichloromethane, and then washed with brine. The organic phase was dried with anhydrous Na ₂ SO ₄ , and the solvent was evaporated to obtain compound (8);

Compound (8) (1 mmol) was dissolved in tetrahydrofuran, and phenylsilane (3 mmol) and triethylamine (2 mmol) were added respectively. The reaction solution was stirred at room temperature for 15 minutes, then 10 ml of water was added to the reaction solution, and stirred for 15 min; the reaction solution was added ether (50 ml), washed with water (2×50 ml), brine (50 ml), separated the organic phase, and used Washed with brine, and then purified by flash column chromatography (n-hexane/ethyl acetate=10/1) to obtain a colorless oil (9);

Compound (9) (1.5 mmol) and piperidine (0.3 mmol) were added to a suspension of compound (5) (1 mmol) in ethanol (15 ml). Heat to 80oC to react for 1.5 hours, cool the reaction solution to room temperature, and filter to obtain a precipitate, which is compound (10);

The Boc-protected compound (10) (1 mmol) was dissolved in ethyl acetate (3 mL), then dioxane-HCl (1 mmol) was added, and the mixture was stirred for 2 hours. A suspension appeared, filtered and washed with ethyl acetate and ether to give 1-(2-amino)ethyl-3-(3-(4-methoxy)phenyl)propenylindole-2-one hydrochloride , the code name is LD12, and the yield is 38%.

¹ H NMR (400MHz, DMSO): δ8.14(s, 1H), 7.65-7.64(d, J=7.48, 1H), 7.35-7.31(t, J=7.72, 1H), 7.24-7.20(t, J=8.48,3H),7.10-7.06(t,J=7.60,1H),6.89-6.85(m,3H),4.00-3.96(t,J=6.52,2H),3.01-2.95(m,4H) ,2.88-2.85(t,J=6.92,2H),2.51-2.49(m,3H);

¹³ C NMR (100MHz, DMSO): 167.0, 157.7, 142.1, 141.9, 140.9, 140.6, 132.6, 129.3, 129.2, 129.1, 128.7, 127.2, 123.5, 122.2, 121.8, 121.6, 119.4, 110.8, 108 , 37.0, 36.8, 36.8, 36.6, 33.4, 32.8, 30.4, 30.4, 29.3.

Example 2

1). The synthesis of N-phenylethylenediamine (2):

2-Bromoethylamine hydrobromide (0.1 mmol) and aniline (1) (0.25 mmol) were added to 40 ml of toluene, heated to reflux for 18 hours, and cooled. Add 60ml of water and 20ml of 50% KOH aqueous solution, and let stand to separate layers. The aqueous phase was washed with saturated NaCl and extracted three times with dichloromethane. The combined organic phases were washed with saturated NaCl, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to dryness. The pure compound (2) was obtained by column chromatography with a yield of 62%.

2). Synthesis of N-tert-butoxycarbonyl-N'-phenylethylenediamine (3):

N-phenylethylenediamine (2) (1.0 mmol) and di-tert-butyl carbonate (1.5 mmol) were dissolved in 15 ml of methanol, stirred for 5 hours, and concentrated under reduced pressure to obtain a brown oil. The pure white solid compound (3) was obtained by column chromatography with a yield of 90%.

3). Synthesis of tert-butyl alcohol N-(2-(N'-chloroacetyl)anilino)ethylcarbamate (4):

Compound (3) (1.5mmol) was dissolved in 40ml of dichloromethane, triethylamine (1.0mmol) was added and cooled to 0oC, and chloroacetyl chloride (0.8mmol) was added dropwise with stirring. After reacting for 1.5 hours, continue to add chloroacetyl chloride (0.2 mmol) dropwise. After reacting for 60 minutes, pour the reaction solution into 20 ml of ethyl acetate, and wash with water, saturated ammonium chloride solution, saturated sodium bicarbonate solution, and brine successively. It was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain compound (4) as a brown oil with a yield of 46%.

4). Synthesis of N-(2-oxoindol-1-yl) ethyl tert-butyl carbamate (5):

The oven-dried two-necked flask was cooled to room temperature, equipped with electromagnetic stirring, and palladium acetate (1.0 mmol), 2-(di-tert-butylphosphino)biphenyl (0.5 mmol) and compound (4) (1.0 mmol) were added. Continue to pass nitrogen gas to evacuate the gas in the device, and add anhydrous triethylamine (1.5 mmol) and anhydrous toluene (1.0 ml) respectively under nitrogen protection. Place the two-necked flask in an oil bath preheated to 90oC. After 14 hours of reaction, the heating was stopped, cooled to room temperature, and diluted with ethyl acetate (10 ml). The mixture was filtered through celite, and the filtrate was spin-dried and concentrated to obtain a crude product. The crude product was purified by silica gel chromatography to obtain compound (5) with a yield of 62%.

5). Synthesis of 5-(4-methoxy)phenylmethylene-2,2-dimethyl-1,3-dioxazolidine-4,6-dione (7):

Dissolve p-methoxybenzaldehyde (6) (1.5 mmol) in 20 ml of ethanol, and add cyclo(ylidene)isopropyl malonate (Merberine's acid) (1.0 mmol) and 5 drops of piperidine. The reaction was stirred overnight at room temperature. The reaction mixture was filtered under reduced pressure and washed with ethanol and methanol to obtain pure product (7). Yield 53%.

6). Synthesis of 1-(2-amino)ethyl-3-(3-(4-methoxy)phenyl)propenylindole-2-one hydrochloride:

Compound (7) (1mmol) was dissolved in 30ml of dichloromethane, 4ml of glacial acetic acid was added, and sodium borohydride (4.0mmol) was added at 0oC. After reacting for 1 hour, the reaction solution was added to 20 ml of dichloromethane, and then washed with brine. The organic phase was dried with anhydrous Na ₂ SO ₄ , and the solvent was evaporated to obtain compound (8);

Compound (8) (1 mmol) was dissolved in tetrahydrofuran, and phenylsilane (4 mmol) and triethylamine (3 mmol) were added respectively. The reaction solution was stirred at room temperature for 15 minutes, then 10 ml of water was added to the reaction solution, and stirred for 15 min; the reaction solution was added ether (50 ml), washed with water (2×50 ml), brine (50 ml), separated the organic phase, and used Washed with brine, and then purified by flash column chromatography (n-hexane/ethyl acetate=10/1) to obtain a colorless oil (9);

Compound (9) (1.0 mmol) and piperidine (0.2 mmol) were added to a suspension of compound (5) (1 mmol) in ethanol (15 ml). Heat to 70oC to react for 2.0 hours, cool the reaction solution to room temperature, and filter to obtain a precipitate, which is compound (10);

The Boc-protected compound (10) (1 mmol) was dissolved in ethyl acetate (3 mL), then dioxane-HCl (1 mmol) was added, and the mixture was stirred for 2 hours. A suspension appeared, filtered and washed with ethyl acetate and ether to give 1-(2-amino)ethyl-3-(3-(4-methoxy)phenyl)propenylindole-2-one hydrochloride , Yield 35%.

Example 3

1). The synthesis of N-phenylethylenediamine (2):

Add 2-bromoethylamine hydrobromide (0.1 mmol) and aniline (1) (0.15 mmol) into 40 ml of toluene, heat to reflux for 14 hours, and cool. Add 60ml of water and 20ml of 50% KOH aqueous solution, and let stand to separate layers. The aqueous phase was washed with saturated NaCl and extracted three times with dichloromethane. The combined organic phases were washed with saturated NaCl, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to dryness. The pure compound (2) was obtained by column chromatography with a yield of 58%.

2). Synthesis of N-tert-butoxycarbonyl-N'-phenylethylenediamine (3):

N-phenylethylenediamine (2) (1.0 mmol) and di-tert-butyl carbonate (1.0 mmol) were dissolved in 15 ml of methanol, stirred for 3 hours, and concentrated under reduced pressure to obtain a brown oil. The pure white solid compound (3) was obtained by column chromatography with a yield of 86%.

3). Synthesis of tert-butyl alcohol N-(2-(N'-chloroacetyl)anilino)ethylcarbamate (4):

Compound (3) (1.0mmol) was dissolved in 40ml of dichloromethane, triethylamine (1.5mmol) was added and cooled to 0oC, and chloroacetyl chloride (0.8mmol) was added dropwise with stirring. After reacting for 2 hours, continue to add chloroacetyl chloride (0.2 mmol) dropwise. After reacting for 45 minutes, pour the reaction solution into 20 ml of ethyl acetate, and wash with water, saturated ammonium chloride solution, saturated sodium bicarbonate solution, and brine successively. Dry over anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain brown oily compound (4), yield 44%.

4). Synthesis of N-(2-oxoindol-1-yl) ethyl tert-butyl carbamate (5):

The oven-dried two-necked flask was cooled to room temperature, equipped with electromagnetic stirring, and palladium acetate (1.0 mmol), 2-(di-tert-butylphosphino)biphenyl (0.5 mmol) and compound (4) (1.0 mmol) were added. Continue to pass nitrogen gas to evacuate the gas in the device, and add anhydrous triethylamine (1.5 mmol) and anhydrous toluene (1.0 ml) respectively under nitrogen protection. Place the two-necked flask in an oil bath preheated to 70oC. After reacting for 18 hours, stop heating, cool to room temperature, add ethyl acetate (10ml) to dilute. The mixture was filtered through celite, and the filtrate was spin-dried and concentrated to obtain a crude product. The crude product was purified by silica gel chromatography to obtain compound (5) with a yield of 59%.

5). Synthesis of 5-(4-methoxy)phenylmethylene-2,2-dimethyl-1,3-dioxazolidine-4,6-dione (7):

Dissolve p-methoxybenzaldehyde (6) (1.0 mmol) in 20 ml of ethanol, and add cyclo(ylidene)isopropyl malonate (Merfur's acid) (1.5 mmol) and 5 drops of piperidine. The reaction was stirred overnight at room temperature. The reaction mixture was filtered under reduced pressure and washed with ethanol and methanol to obtain pure product (7). Yield 48%.

6). Synthesis of 1-(2-amino)ethyl-3-(3-(4-methoxy)phenyl)propenylindole-2-one hydrochloride:

Compound (7) (1mmol) was dissolved in 30ml of dichloromethane, 4ml of glacial acetic acid was added, and sodium borohydride (4.0mmol) was added at 0oC. After reacting for 1 hour, the reaction solution was added to 20 ml of dichloromethane, and then washed with brine. The organic phase was dried with anhydrous Na ₂ SO ₄ , and the solvent was evaporated to obtain compound (8);

Compound (8) (1 mmol) was dissolved in tetrahydrofuran, and phenylsilane (3 mmol) and triethylamine (1.5 mmol) were added respectively. The reaction solution was stirred at room temperature for 15 minutes, then 10 ml of water was added to the reaction solution, and stirred for 15 min; the reaction solution was added ether (50 ml), washed with water (2×50 ml), brine (50 ml), separated the organic phase, and used Washed with brine, and then purified by flash column chromatography (n-hexane/ethyl acetate=10/1) to obtain a colorless oil (9);

Compound (9) (2.0 mmol) and piperidine (0.4 mmol) were added to a suspension of compound (5) (1 mmol) in ethanol (15 ml). Heat to 90oC to react for 1.0 hour, cool the reaction solution to room temperature, and filter to obtain a precipitate, which is compound (10);

The Boc-protected compound (10) (1 mmol) was dissolved in ethyl acetate (3 mL), then dioxane-HCl (1 mmol) was added, and the mixture was stirred for 2 hours. A suspension appeared, filtered and washed with ethyl acetate and ether to give 1-(2-amino)ethyl-3-(3-(4-methoxy)phenyl)propenylindole-2-one hydrochloride , The yield is 33%.

Example 4

Inhibition of cancer cell proliferation experiment:

(1) Experimental method: Cells were seeded in 96-well plates at 5×104 (U937)/well or 1×104 (PC-3, DU145, M12, HT29)/well, and prepared by adding different concentrations of Example 1 of the present invention LD12, after 24 hours of incubation at 37°C (5% CO2), add 20 μL CellTiter to each well Aqueous One Solution Reagent (Promega, Madison, WI). One hour later, cell viability was determined by measuring the absorbance at 490 nm with a microplate reader.

(2) Experimental results: the half-inhibitory concentrations of LD12 on different types of cancer cells are shown in Table 1,

Table 1. Inhibitory effect of LD12 on cancer cell proliferation*

^* indicates that the cells were treated with different concentrations of the compounds to be tested for 24 hours, and then the cell viability was measured by the MTT method and the IC50 was calculated.

The results showed that the compound LD12 had inhibitory effect on the proliferation of various cancer cells, and its IC50 was at single-digit μmol concentration level, but its sensitivity to HT29 cells was slightly lower.

Example 5

In vitro Kinase Selective Screening Screening assay:

(1) Experimental method: By measuring the activities of different kinases, the LD12 prepared in Example 1 of the present invention was used to screen a group of kinases at a concentration of 10 μM.

(2) Experimental results: the inhibition rates of LD12 on different types of kinases are shown in Table 2;

Table 2. In vitro kinase selectivity screening results of LD12

Kinase Inhibition rate(%) Kinase Inhibition rate(%) ABL 6 LCK 4 AKT1 3 LYN 0 AKT2 8 MAPKAPK2 3 AMPK 76 MARK1 0 AurA -4 MEK1 69 BTK 12 MEK2 27 CAMK2 60 MET -1 CAMk4 71 MSK1 6 CDK2 -2 MST2 -3 CHK1 11 p38a -13 CHK2 8 p70S6K 5 Ck1d -5 PAK2 10 c-Raf -1 PDK1 -2 c-TAK1 6 PI3Ka 45 DYRK1a -5 PIM2 38 Erk1 twenty one PKA -3 Erk2 11 PKCb2 16 FGFR1 3 PPML -8 FLT3 38 PKD2 0 FYN 0 PKGa -2 GSK3b 0 PRAK 4 HGK -1 ROCK2 -1 IGF1R -1 RSK1 2 INSR -2 SGK1 14 IRAK4 -1 SRC 2 KDR -28 SYK 9

It can be seen from the results that LD12 can significantly inhibit MEK1, CAMK2, CAMK4 and AMPK at this concentration. At the same time, LD12 can moderately inhibit PI3Ka. In basic cell research, it was found that LD12 inhibited the phosphorylation of Akt, the downstream substrate of PI3K, but the inhibitory activity was lower than that of p-MEK and p-ERK. In conclusion, these results can definitely prove that LD12 can simultaneously inhibit multiple signaling pathways in the process of cancer development, showing that LD12 and its derivatives have multi-channel inhibitory activity, and can be used as new anticancer agents for development and research.

Claims (7)

1. the dual pathways inhibitor taking Raf/MEK/ERK and PI3K/Akt as target spot, chemical name is 1-(2-amino) ethyl-3-(3-(4-methoxyl group) phenyl) acrylic indol-2-one hydrochlorate, has the compound of structure formula I:

2. claimed in claim 1 a kind ofly it is characterized in that taking Raf/MEK/ERK and PI3K/Akt as the preparation method of the dual pathways inhibitor of target spot, comprise the following steps:

1) N-phenylethylenediamine (2) is synthetic:

Taking toluene as solvent, 2-bromine ethylamine hydrobromide and aniline (1) reflux 14-18h, cooling, through extraction, washing, purification, obtain compound (2) N-phenylethylenediamine; The mol ratio of 2-bromine ethylamine hydrobromide and aniline (1) is 1:(1.5～2.5);

2) N-tertbutyloxycarbonyl-N '-phenylethylenediamine (3) is synthetic:

Taking methanol as solvent, by step 1) the N-phenylethylenediamine (2) that makes and di-t-butyl carbonic ester mix, and reaction 3-5h, obtains compound (3) N-tertbutyloxycarbonyl-N '-phenylethylenediamine; The mol ratio of N-phenylethylenediamine (2) and di-t-butyl carbonic ester is 1:(1～1.5);

3) N-(2-(N '-chloracetyl) phenylamino) ethyl carbamic acid tert-butyl alcohol ester (4) synthetic:

By step 2) N-tertbutyloxycarbonyl-N '-phenylethylenediamine of making is dissolved in dichloromethane, adds triethylamine and is cooled to 0oC, under stirring, is added dropwise to chloracetyl chloride, reaction 2-4h, pours reactant liquor in ethyl acetate into, washing, dry, obtain compound (4); The mol ratio of N-tertbutyloxycarbonyl-N '-phenylethylenediamine (3), triethylamine, chloracetyl chloride is (1～1.5): (1～1.5): 1;

4) N-(2-oxindole-1-yl) ethyl carbamic acid tert-butyl alcohol ester (5) is synthetic:

Under nitrogen protection, to palladium, 2-(di-t-butyl phosphino-) biphenyl, step 3) in the compound (4) that makes, add anhydrous triethylamine and dry toluene, 70-90oC reacts 14-18h, cooling, dilutes by ethyl acetate, filter, concentrated, purification, obtains compound (5) N-(2-oxindole-1-yl) ethyl carbamic acid tert-butyl alcohol ester; The mol ratio of palladium, 2-(di-t-butyl phosphino-) biphenyl, compound (4), anhydrous triethylamine, dry toluene is 2:1:(1～2): (2～4): (1～3);

5) 5-(4-methoxyl group) phenylmethylene-2,2-dimethyl-1,3-bis-oxazolidine-4,6-diketone (7) synthetic:

Taking ethanol as solvent, P-methoxybenzal-dehyde (6), malonic acid ring (Asia) isopropyl ester, piperidines mix, and under room temperature, stirring reaction spends the night, and filters, and washing, obtains compound (7); The mol ratio of P-methoxybenzal-dehyde (6), malonic acid ring (Asia) isopropyl ester is (1～1.5): (1～1.5);

6) 1-(2-amino) ethyl-3-(3-(4-methoxyl group) phenyl) acrylic indol-2-one hydrochlorate is synthetic:

Taking dichloromethane as solvent, add compound (7), under sour existence, drip sodium borohydride in 0oC, reaction obtains compound (8); Compound (7) is 1:(3～4 with the mol ratio of sodium borohydride);

Taking oxolane as solvent, add compound (8), phenyl silane, triethylamine, reaction 15-30min, washing, purification, obtains compound (9); The mol ratio of compound (8), phenyl silane, triethylamine is 1:(2～4): (1.5～3);

Compound (9) and piperidines are joined in the alcohol suspension of compound (5), be heated to 70-90oC reaction 1-2h, reactant liquor is cooled to room temperature, filter to obtain compound (10); The mol ratio of compound (9), piperidines, compound (5) is (1～2): (0.2～0.4): 1;

The compound (10) of Boc protection is dissolved in to ethyl acetate, then adds dioxane-HCl,, there is float in reaction 1-3h, filters, and washing, to obtain final product; Compound (10) is (1～2) with the mol ratio of dioxane-HCl: (1～2).

3. a kind of preparation method taking Raf/MEK/ERK and PI3K/Akt as the dual pathways inhibitor of target spot as claimed in claim 2, it is characterized in that, in described step (1), the operation of extraction, washing, purification, be specially: add the KOH aqueous solution of water and 50%, stratification; With saturated NaCl washing water and with dichloromethane extraction for several times; After merging organic facies, wash anhydrous Na with saturated NaCl ₂sO ₄dry, evaporate to dryness after filtering, column chromatography carries out purification.

4. a kind of preparation method taking Raf/MEK/ERK and PI3K/Akt as the dual pathways inhibitor of target spot as claimed in claim 2, it is characterized in that, in described step (3), the mode that adds of chloracetyl chloride is: first drip 80% of chloracetyl chloride addition, react after 1.5-3 hour, drip again the chloracetyl chloride of residue 20%, reaction 30-60 minute.

5. a kind of preparation method taking Raf/MEK/ERK and PI3K/Akt as the dual pathways inhibitor of target spot as claimed in claim 2, it is characterized in that, in described step (4), filtration, concrete operations concentrated, purification are: adopt diatomite filtration, filtrate is spin-dried for concentrated, obtain product crude product, crude product carries out purification through silica gel chromatography.

6. as claimed in claim 2 a kind ofly it is characterized in that taking Raf/MEK/ERK and PI3K/Akt as the preparation method of the dual pathways inhibitor of target spot, in described step (6), the preparation of compound (8) acid used is glacial acetic acid.

Described in claim 1 a kind of dual pathways inhibitor taking Raf/MEK/ERK and PI3K/Akt as target spot in the application of preparing in antitumor drug.