CN109081801B

CN109081801B - Acidic amino acid modified indoloethanol derivative, and synthesis, activity and application thereof

Info

Publication number: CN109081801B
Application number: CN201710442553.6A
Authority: CN
Inventors: 赵明; 彭师奇; 王玉记; 吴建辉; 吴晨
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2017-06-13
Filing date: 2017-06-13
Publication date: 2021-03-30
Anticipated expiration: 2037-06-13
Also published as: CN109081801A

Abstract

The invention discloses 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole (wherein AA is L-Asp (OBzl) residue and L-Glu (OBzl) residue), discloses a preparation method thereof, discloses the antitumor activity thereof, discloses the antitumor metastasis activity thereof and discloses the anti-inflammatory activity thereof, thus the invention discloses the application thereof in preparing antitumor drugs, antitumor metastasis drugs and anti-inflammatory drugs.

Description

Acidic amino acid modified indoloethanol derivative, and synthesis, activity and application thereof

Technical Field

The invention relates to 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole, a preparation method thereof, antitumor activity thereof, antitumor metastasis activity thereof and anti-inflammatory activity thereof, and thus relates to application thereof in preparing antitumor drugs, antitumor metastasis drugs and anti-inflammatory drugs. The invention belongs to the field of biological medicine.

Background

Malignant tumors seriously threaten human health. Among them lung cancer is one of the most aggressive human cancers. For patients with advanced lung cancer, 10% -15% of the patients typically survive for only 5 years. The situation of this difficulty has not improved significantly over the last 30 years. In many clinical cases, lung cancer has metastasized to surrounding tissues before it is diagnosed. Tumor metastasis, especially tumor lung metastasis, is the greatest risk of death in tumor patients. To date, there is no antitumor drug that can prevent tumor metastasis for clinical use. Inflammation further worsens the prognosis of patients with tumors and tumor metastases. To date, no antitumor drug that can prevent inflammation and tumor metastasis has been used clinically. Hair-like deviceThe medicine with the triple effects of resisting tumor, resisting tumor metastasis and resisting inflammation is the leading edge of research on the anti-tumor medicine. The inventor's prior invention (patent application publication No. CN 106349148A, application No. CN 201510409682.6) has disclosed that amino acid benzyl ester substituted bisindole acetic acid alcohol has triple effects of anti-tumor, anti-tumor metastasis and anti-inflammatory at 0.2 mu mol/kg (left formula). The inventors have had two unsatisfactory points with respect to this type of benzyl bisindoloethanol amino acid. The first dissatisfaction was that the MTT models showed that their antitumor activity was derived from cytotoxic effects (IC of all compounds except 1 inhibiting proliferation of 5 tumor cells)₅₀8.2-62.2. mu.M). Clinical application shows that cytotoxic drugs have large toxic and side effects. That is, such benzyl bisindoloethanol amino acid ester is at great risk of toxic side effects. The second point of dissatisfaction is that their minimum effective dose for exerting the triple effects of anti-tumor, anti-tumor metastasis and anti-inflammatory is 0.2. mu. mol/kg, which is higher. Over the past two years, the inventors have been searching for non-cytotoxic compounds with triple effects of anti-tumor, anti-tumor metastasis and anti-inflammatory, with a minimum effective dose of less than 0.2 μmol/kg. Finally, the inventor finds that the acidic amino acid benzyl ester (Glu (OBzl) -OBzl and Asp (OBzl) -OBzl) modified indoleethanol (right formula) has triple effects of anti-tumor, anti-tumor metastasis and anti-inflammatory at the dose of 0.02 mu mol/kg. Because the toxic and side effects of the medicine can disappear along with the reduction of the dosage, the reduction of the effective dosage by 10 times shows that the structure modification has outstanding technical effect. In addition, MTT models show their IC for tumor cell proliferation₅₀Are all larger than 200. mu.M. That is, the acidic amino acid benzyl ester (Glu (OBzl) -Obzl and Asp (OBzl) -OBzl) modified indoleethanol has no toxic side effects of cytotoxic compounds. The inventor proposes the invention according to two advantages.

Disclosure of Invention

In a first aspect of the invention there is provided 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole of the formula (AA being an L-Asp (OBzl) residue and an L-Glu (OBzl) residue).

In a second aspect, the present invention provides a process for the preparation of 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole (AA is L-asp (OBzl) residue and L-glu (OBzl) residue) comprising the steps of:

1) indole ethanol and ethyl bromoacetate react in tetrahydrofuran at 80 ℃ under the catalysis of sodium hydride to generate 1-ethoxycarbonylmethyl-3-ethoxycarbonylmethoxyethyl-indole (1);

2) saponifying the compound 1 in a 2N NaOH aqueous solution to obtain 1-carboxymethyl-3-carboxymethoxyethyl indole (2);

3) and reacting the compound 2 with L-amino acid benzyl ester in anhydrous tetrahydrofuran in the presence of dicyclohexylcarbodiimide and N-hydroxybenzotriazole to generate 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole (AA is L-Asp (OBzl) residue and L-Glu (OBzl) residue).

In a third aspect of the invention, 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole (AA: L-Asp (OBzl) residue and L-Glu (OBzl) residue) was evaluated for its anti-metastatic activity.

A fourth aspect of the present invention is to evaluate the antitumor activity of 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole (AA is the L-Asp (OBzl) residue and the L-Glu (OBzl) residue).

A fifth aspect of the present invention is to evaluate the anti-inflammatory activity of 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole (AA is the L-Asp (OBzl) residue and the L-Glu (OBzl) residue).

Drawings

FIG. 1. scheme for the synthesis of 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole i) ethyl bromoacetate, NaH, 80 ℃; ii) 2N NaOH in water; iii) dicyclohexylcarbodiimide, N-hydroxybenzotriazole, N-methylmorpholine; AA ═ asp (obzl) residue in 3a, AA ═ L-glu (obzl) residue in 3 b.

Detailed Description

To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.

EXAMPLE 1 preparation of 1-ethoxycarbonylmethyl-3-ethoxycarbonylmethoxyethyl-indole (1)

To a solution of 5.00g (31mmol) of indoloethanol and 50mL of anhydrous Tetrahydrofuran (THF) was slowly added 2.98g (4mmol, 60%) of NaH at room temperature, and after stirring for 30 minutes 17.22mL (5mmol) of ethyl bromoacetate was slowly added dropwise, and the mixture was heated at 80 ℃ for 48 hours. TLC (petroleum ether/ethyl acetate, 3/1) showed the reaction was complete. The heating was stopped and the reaction mixture was cooled to room temperature. The solid was filtered off and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 3/1) to give 1.54g (15%) of the title compound as a yellow syrup. ESI-MS (M/e) 334[ M + H]⁺。

EXAMPLE 2 preparation of 1-carboxymethyl-3-carboxymethoxyethyl indole (2)

1.31g (3.9mmol) of 1-ethoxycarbonylmethyl-3-ethoxycarbonylmethoxyethyl-indole (1) are dissolved in 10mL of methanol in ice. To the resulting solution was added dropwise a 2N aqueous NaOH solution to adjust the pH of the solution to 12, and after stirring for 5 hours TLC (petroleum ether/ethyl acetate, 3/1) indicated that the reaction was complete. The reaction mixture was saturated with KHSO₄Adjusting pH of the aqueous solution to 7, concentrating under reduced pressure, extracting the residue with 15mL ethyl acetate for 3 times, and collecting the water layer with saturated KHSO₄The aqueous solution was adjusted to pH 4 and extracted 3 times with 15mL of ethyl acetate. The separated aqueous layer was continuously saturated with KHSO₄Adjusting pH to 2 with water solution, extracting with 15mL ethyl acetate for 3 times, combining the separated ethyl acetate layers, washing with 15mL saturated aqueous NaCl solution for 3 times, and adding anhydrous Na₂SO₄Drying for 12 hours. Filtration and concentration of the filtrate under reduced pressure gave 0.68g (62%) of the title compound as a yellow syrup. ESI-MS (M/e):276[ M-H]^-；Mp 97-100℃；

(C ═ 0.1, ethanol); IR (KBr, cm)^-1):3196, 3051,2892,1651,1469,1435,1175,1124,840,901,724；¹H NMR(300MHz,DMSO-d₆)δ/ppm ＝12.731(s,2H),7.549(d,J＝7.8Hz,1H),7.324(d,J＝6.9Hz,1H),7.172(s,1H),7.114(t,J ＝6.9Hz,1H),7.021(t,J＝6.9Hz,1H),4.937(s,2H),4.053(s,2H),3.725(t,J＝7.2Hz,2H), 2.947(t,J＝7.2Hz,2H)。

EXAMPLE 3 preparation of 1- (acetyl-Asp (OBzl) -3- (ethoxyacetyl-Asp (OBzl) -OBzl) indole (3a)

A solution of 0.55g (2.0mmol) of 1-carboxymethyl-3-carboxymethoxyethyl-indole (2), 0.54g (4.0mmol) of N-hydroxybenzotriazole and 10mL of anhydrous THF was stirred for 30 minutes to obtain a reaction solution A. 0.82g (4.0mmol) of dicyclohexylcarbodiimide was dissolved in 5mL of anhydrous THF to obtain a reaction solution B. The reaction solution B was slowly added dropwise to the reaction solution A under ice-cooling, and stirred for 30 minutes. A solution of 2.79g (8.0mmol) of HCl. Asp (OBzl) -OBzl and 15mL of anhydrous THF was then added. The reaction mixture was adjusted to pH 9 with N-methylmorpholine and stirred at room temperature for 10 hours. TLC (CH)₂Cl₂/CH₃OH, 30/1, plus 3 drops of acetic acid) showed the reaction was complete. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in 30mL of ethyl acetate. The resulting solution was sequentially diluted with 20mL of saturated NaHCO₃Washing with water solution 3 times, washing with 20mL saturated NaCl water solution 3 times, and washing with 20mL 5% KHSO₄Washing with aqueous solution 3 times, washing with 20mL saturated NaCl aqueous solution 3 times, and washing with 20mL saturated NaHCO aqueous solution₃3 washes with aqueous solution and 3 washes with 20mL of saturated aqueous NaCl solution. Adding anhydrous Na into the ethyl acetate layer₂SO₄Drying for 12 hr, filtering, concentrating the filtrate under reduced pressure, and subjecting the residue to silica gel column Chromatography (CH)₂Cl₂/CH₃OH, 30/1, plus 3 drops of acetic acid) to yield 0.50g (29%) of the title compound as a colorless syrup. ESI-MS (M/e):868[ M + H]⁺；

(C ═ 0.1, ethanol); IR (KBr, cm)^-1):3407,3033,2929,1732,1679,1517,1170,967, 738,696；¹H NMR(300MHz,DMSO-d₆)δ/ppm＝8.793(d,J＝8.1Hz,1H),8.183(d,J＝8.4Hz, 1H),7.537(d,J＝1.5Hz,1H),7.339(m,20H),7.231(d,J＝4.8Hz,1H),7.051(s,1H),7.023 (m,2H),5.089(m,8H),4.821(m,4H),3.941(s,2H),3.669(t,J＝6.3Hz,2H),2.926(m,6 H)。

EXAMPLE 4 preparation of 1- (acetyl-Glu (OBzl) -3- (ethoxyacetyl-Glu (OBzl) -OBzl) indole (3b)

From 0.55g (2.0mmol) of 1-carboxymethyl-3-carboxymethoxyethyl-indole (2) and 2.91g (8.0mmol) of HCl. Glu (OBzl) -OBzl, 0.53g (30%) of the title compound is obtained as colorless syrup by the method of example 3. ESI-MS (M/e):896 [ M + H]⁺；

(C ═ 0.1, ethanol); IR (KBr, cm)^-1):3361,3033,2934,1732,1678,1518, 1454,1164,738,696；¹H NMR(300MHz,DMSO-d₆)δ/ppm＝8.726(d,J＝7.8Hz,1H),8.067 (d,J＝7.8Hz,1H),7.545(d,J＝7.5Hz,1H),7.340(m,20H),7.247(d,J＝7.2Hz,1H),7.116 (s,1H),7.004(m,2H),5.081(m,8H),4.803(m,2H),4.398(m,2H),3.947(s,2H),3.678(m, 2H),2.957(t,J＝6.9Hz,2H),2.439(m,4H),2.112(m,2H),1.903(m,2H)。

EXAMPLE 5 determination of the anti-tumor metastasis Activity of Compounds 3a, b

The assay model was inoculated with Lewis mouse lung carcinoma cells (LLC, purchased from ATCC) in DMEM medium (containing 10% inactivated fetal bovine serum, 1X 10)⁵U/L penicillin and 100mg/L streptomycin), and the cells are enriched by passage every two days according to an adherent cell culture method. Digesting the cells when the cells are in good growth state and in logarithmic growth phase, and adjusting the cell density to 1 × 10 with physiological saline⁷one/mL. Staining with placental blue to count viable cells>95 percent. Inbred C57BL/6 male mice (SPF grade, body weight 20. + -.2 g) were taken and left-handed mice fixed. The right anterior limb axillary skin of the mouse was disinfected with 75% ethanol. The LLC tumor cell suspension is injected subcutaneously into the axilla of a mouse with a 1mL sterile syringe held in the right hand, and 0.2mL is injected into each mouse. After the mice are inoculated for 10 days, tumors with the diameter of about 4-5mm grow out, namely the tumor source. The Lewis lung cancer tumor-bearing mice are inoculated for 10 days and anesthetized by ether, and then the cervical vertebrae are removed for killing. Soaking in 75% ethanol for 10min, sterilizing, and removing tumor on clean bench. Well-grown tumor tissue was selected, minced in a sterile plate, and placed in a tissue homogenizer made of glass. According to the ratio of tumor mass to weightThe cell suspension was prepared by adding physiological saline at a temperature of 4 ℃ to a volume of 1 to 3(g to mL) of saline and gently grinding. The cell suspension is screened by 200-mesh cells to prepare single cell suspension. Adjusting the cell density of the single cell suspension to 1.5X 10 with physiological saline⁷one/mL. Staining with placental blue to count viable cells>95 percent. Left-handed inbred C57BL/6 male mice were fixed and their right anterior limb axillary skin was disinfected with 75% ethanol. The tumor cell suspension was injected subcutaneously into the mouse axilla with a 1mL sterile syringe in the right hand, 0.2mL each. 10 days after inoculation, the mice developed tumors of 4-5mm in diameter, and the inoculated mice were randomly grouped by the measured tumor volume. Each group had 12 mice. Mice on day 11 of tumor inoculation were dosed 1 time daily for 12 consecutive days with tumor volumes measured and recorded every two days, with an oral administration of either a 20 μmol/kg/day normal saline solution of the putative anti-tumor metastasis peptide Arg-Gly-Asp-ser (rgds) or a 0.02 μmol/kg/day normal saline solution of compound 3a, b or a 2 μmol/kg/day normal saline solution of compound 2 or an oral administration of 10 mL/kg/day normal saline. The next day of the last administration, tumor volume was measured, cervical spine was removed by ether anesthesia and sacrificed, tumor of the mice was weighed, lung of the mice was taken and tumor nodules transferred from the lung of the tumor were counted. Statistical analysis of the data was performed using the t-test. The results are shown in Table 1. Not only was compound 3a, b effective in inhibiting tumor lung metastasis at a dose of 0.02 μmol/kg, but there was no significant difference in activity from compound 2 at 1000 times higher RGDS and 100 times higher doses than them. These data indicate that the present invention has significant technical effects.

TABLE 1 antitumor metastatic Activity of Compounds 3a, b

b) P <0.01 compared to saline, p >0.05 compared to RGDS and Compound 2; n-10

EXAMPLE 6 determination of the anti-tumor growth Activity of Compounds 3a, b

Dissolving adriamycin with physiological saline before measurement, and dissolving compound 2 and compounds 3a, b with physiological salineFor administration to S180 mice. Taking S180 ascites tumor liquid which is inoculated in a male ICR mouse and grows vigorously for 10 days in a sterile environment, diluting the S180 ascites tumor liquid into liquid (1:2) by using normal saline, fully mixing the liquid, dyeing the tumor cell suspension by using freshly prepared 0.2% trypan blue, uniformly mixing the liquid and the liquid, counting the liquid according to a white cell counting method, wherein the blue-dyed cell is a dead cell, and the non-dyed cell is a live cell. The cell concentration is 4-large-grid viable cell number/4 × 10⁴The cell density was calculated as x dilution factor ═ cell number/mL, and the cell survival rate was calculated as live cell number/(live cell number + dead cell number) × 100%. Homogenizing tumor solution with survival rate of more than 90% to density of 2.0 × 10⁷Cell suspension per mL. This cell suspension was inoculated subcutaneously (0.2 mL/mouse) in the right axilla of a mouse to prepare S180 tumor-bearing mice. 24h after inoculation, S180 tumor-bearing mice were intraperitoneally injected daily with a normal saline solution of doxorubicin (dose 2. mu. mol/kg/day g) or daily orally administered with a normal saline solution of Compound 2 (dose 5. mu. mol/kg/day) or daily orally administered with a normal saline solution of Compounds 3a, b (dose 0.02. mu. mol/kg/day). The administration is once daily for 12 days. The day after the last dose, tumor volume was measured, cervical spine was removed under ether anesthesia and sacrificed, then the right axillary tumor growth site of the mouse was fixed with forceps, and the skin was excised and the tumor was blunt-stripped and weighed. Efficacy was expressed as tumor weight (mean ± SD g), and data were analyzed by t-test and variance. The results are shown in Table 2. Not only was compound 3a, b effective in inhibiting tumor growth at a dose of 0.02 μmol/kg, but there was no significant difference in activity from compound 2 at doses 100-fold higher than them. These data indicate that the present invention has significant technical effects.

TABLE 2 Effect of Compounds 3a, 3b on tumor growth in S180 mice

a) P <0.05 compared to saline, p >0.05 compared to compound 2; n is 12.

EXAMPLE 7 determination of the anti-inflammatory Activity of Compounds 3a, b

Since xylene-induced ear swelling in mice is recognized as an acute inflammation model, the present invention measures the therapeutic effect of compounds 3a, b on a xylene-induced ear swelling model in mice. Because aspirin is a positive drug for treating acute inflammation, aspirin is selected as a positive control in the present invention. ICR male mice (body weight 20 ± 2g) were allowed to rest for 2 days at 22 ℃ with free access to water and food. Thereafter, the mice were randomly divided into a saline group (dose of 0.2 mL/mouse), an aspirin group (dose of 1.11mmol/kg), a Compound 2 group (dose of 2. mu. mol/kg) and a Compound 3a, b group (dose of 0.02. mu. mol/kg), and 12 mice per group. Mice were tested either orally with normal saline, orally with aspirin, orally with compound 2, or orally with compound 3a, b, as indicated. After 30min of administration, the left auricle of the mouse was evenly smeared with 30 μ L of xylene, and after 2h, the mouse was subjected to ether anesthesia, the neck was cut off, the left and right ears were cut off, round ears were taken at the same positions of the two ears by a 7mm punch, and the difference in swelling between the two ears was weighed and found to be the swelling degree. Namely swelling degree, namely the weight of the right ear disk to the weight of the left ear disk. The results are shown in Table 3. Not only did compounds 3a, b effectively inhibit xylene-induced ear swelling in mice at the 0.02 μmol/kg dose, but the activity was superior to compound 2, which is 100 times higher than the dose, with no significant difference from aspirin, which is 55500 times higher than the dose. These data indicate that the present invention has significant technical effects.

TABLE 3 Effect of Compounds 3a, b on xylene-induced ear swelling in mice

a) P <0.01 compared to saline and compound 2, p >0.05 compared to aspirin; n is 12.

Claims

1. 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole of the following formula,

wherein AA is a L-Asp (OBzl) residue or a L-Glu (OBzl) residue.

2. A process for preparing the 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole of claim 1, which comprises:

1) indole ethanol and ethyl bromoacetate react in tetrahydrofuran at 80 ℃ under the catalysis of sodium hydride to generate 1-ethoxycarbonylmethyl-3-ethoxycarbonylmethoxyethyl-indole (compound 1);

2) saponifying the compound 1 in a 2N NaOH aqueous solution to obtain 1-carboxymethyl-3-carboxymethoxyethyl indole (compound 2);

3) reacting the compound 2 with L-amino acid benzyl ester in anhydrous tetrahydrofuran in the presence of dicyclohexylcarbodiimide and N-hydroxybenzotriazole to generate 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole.

3. Use of the 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole according to claim 1 for the preparation of a medicament against tumor metastases.

4. Use of the 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole according to claim 1 for the preparation of a medicament against ascites tumors.

5. Use of the 1- (acetyl-AA-OBzl) -3- (ethoxyacetyl-AA-OBzl) indole according to claim 1 for the preparation of an anti-inflammatory medicament.