CN110551119B - 6-Aminoamido-n-hexanoylcarbolinecarboxylic acid benzyl ester, its preparation and application - Google Patents

Abstract

The invention discloses (3S)-N-(6-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate benzyl ester with the following structure (where AA is selected as Residues from L-Glu(OBzl), Residues of L-Ile, Residues of L-Lys, Residues of L-Leu, Residues of L-Met, Residues of L-Pro, Residues of L-Ser Residues). Their preparation methods and their anti-tumor metastasis effects are disclosed, as well as their anti-inflammatory effects, and their applications in the preparation of anti-tumor metastasis drugs and anti-inflammatory drugs are clarified.

Description

6-Aminoamido-n-hexanoylcarbolinecarboxylic acid benzyl ester, its preparation and application

technical field

The present invention relates to (3S)-N-(6-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester. The invention relates to its preparation method, its in vivo anti-tumor metastasis effect, and their anti-inflammatory effect. Therefore, the present invention relates to its application in the preparation of anti-tumor metastases and anti-inflammatory drugs. The invention belongs to the field of biomedicine.

technical background

Malignant tumors seriously threaten human health. Lung cancer is one of the most aggressive human cancers. For patients with advanced lung cancer, usually 10%-15% only survive 5 years. This difficult situation has not improved significantly over the past 30 years. In many clinical cases, lung cancer has metastasized to surrounding tissues before being diagnosed. Tumor metastases, especially lung metastases, are the greatest risk of death in cancer patients. So far, there are still no antitumor drugs that can prevent tumor metastasis in clinical use. Inflammation further worsens the prognosis of tumors and patients with tumor metastasis. So far, there are no anti-tumor drugs that can prevent inflammation and tumor metastasis in clinical use. The invention of drugs with triple effects of anti-tumor, anti-tumor metastasis and anti-inflammatory is the frontier of anti-tumor drug research. The inventor's previous invention (Patent Application Publication No. CN 106349148A, Application No. CN201510409682.6) once disclosed that the amino acid benzyl ester-substituted bisindole acetic alcohol has triple effects of anti-tumor, anti-tumor metastasis and anti-inflammatory at a dose of 0.2 μmol/kg. function (left). The inventors are not satisfied that the minimum effective dose of this type of bisindoleethanol amino acid benzyl ester to exert anti-tumor metastasis and anti-inflammatory effects is 0.2 μmol/kg. The dose is too high. For the past two years, the inventors have been looking for compounds with anti-metastatic effects at a minimum effective dose lower than 0.2 μmol/kg. Finally, the inventors found that aminoacyl-6-aminocaproic acid-modified benzyl tetrahydrocarboline carboxylate has anti-tumor metastasis and anti-inflammatory effects at a dose of 0.02 μmol/kg. Because the toxic and side effects of the drug can disappear with the dose reduction, the 10-fold reduction in the effective dose shows that this structural modification has outstanding technical effects. Thus, the inventors have come up with the present invention.

SUMMARY OF THE INVENTION

The first aspect of the present invention is to provide (3S)-N-(6-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester of the following structure (where AA is selected from the residues of L-Glu(OBzl), the residues of L-Ile, the residues of L-Lys, the residues of L-Leu, the residues of L-Met, the residues of L-Pro , residues of L-Ser).

The second content of the present invention is to provide a preparation method of (3S)-N-(6-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester , the method includes:

(1) Under the catalysis of dilute sulfuric acid, L-tryptophan benzyl ester and formaldehyde undergo Pictet-Spengler reaction to generate benzyl (3S)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate ester;

(2) in aqueous sodium hydroxide solution (2M), in water and dioxane mixed solution, 6-aminocaproic acid and di-tert-butyl dicarbonate (Boc) ₂ O react to generate 6-Boc-aminocaproic acid;

(3) In the presence of N,N-dicyclohexylcarbodiimide and N-hydroxybenzotriazole in anhydrous tetrahydrofuran, 6-Boc-aminocaproic acid was combined with (3S)-2,3,4 , 9-Tetrahydro-β-carboline-3-carboxylate benzyl ester was reacted to give (3S)-N-(6-Boc-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboyl Benzyl-3-carboxylate;

(4) In an ethyl acetate solution of hydrogen chloride (4M) under ice bath, (3S)-N-(6-Boc-amino-n-hexanoyl)-2,3,4,9-tetrahydrocarboline-3-carboxyl Acid benzyl ester removes Boc to obtain (3S)-N-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester;

(5) (3S)-N-(6-amino-n-hexanoyl)-2,3 in anhydrous tetrahydrofuran in the presence of N,N-dicyclohexylcarbodiimide and N-hydroxybenzotriazole , 4,9-tetrahydro-β-carboline-3-carboxylate benzyl ester and Boc-AA (where AA is selected from the residues of L-Glu(OBzl), the residues of L-Ile, the residues of L-Lys residues, residues of L-Leu, residues of L-Met, residues of L-Pro, residues of L-Ser) react to give (3S)-N-(6-Boc-AA-amino-n-hexanoyl) )-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester;

(6) In ethyl acetate solution of hydrogen chloride (4M) under ice bath, (3S)-N-(6-Boc-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboyl Removal of Boc from benzyl phospholine-3-carboxylate to give (3S)-N-(6-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate benzyl ester .

The third aspect of the present invention is to evaluate the inhibition of C57BL/ 6 Mice lung cancer metastasis activity.

The fourth aspect of the present invention is to evaluate (3S)-N-(6-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate benzyl ester with low ICR Inhibition of inflammation in mice.

Description of drawings

Figure 1. Synthetic route of (3S)-N-(6-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester. i)HCHO,H ₂ O,H ₂ SO ₄ ; ii) N,N-dicyclohexylcarbodiimide (DCC), N-hydroxybenzotriazole (HOBt), N-methylmorpholine, tetrahydrofuran; iii) dioxygen Hexacyclic, aqueous sodium hydroxide, di-tert-butyl dicarbonate (Boc) _2O ; iv) hydrogen chloride in ethyl acetate (4M). AA=L-glutamic acid residue in 5a,6a; AA=L-isoleucine residue in 5b,6b; AA=L-lysine residue in 5c,6c; AA=L in 5d,6d - Leucine residue; AA = L-methionine residue in 5e, 6e; AA = L-proline residue in 5f, 6f; AA = L-serine residue in 5g, 6g.

Detailed ways

In order to further illustrate the present invention, a series of examples are given below. These examples are purely illustrative, they are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

Example 1 Preparation of (3S)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (1)

1.5 mL of concentrated H ₂ SO ₄ was slowly added to 300 mL of water under ice bath, and stirred for 3 min. Then 5.00 g (15.1 mmol) L-tryptophan benzyl ester hydrochloride and 6 mL formaldehyde solution (40%) were added. After stirring at room temperature for 72h, TLC showed the disappearance of L-tryptophan benzyl ester hydrochloride. The reaction solution was adjusted to pH 7 with concentrated ammonia water under ice bath, and filtered. The filter cake was dissolved with 100 mL of ethyl acetate, and the obtained solution was washed with saturated aqueous sodium chloride solution (40 mL×3), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol=90:1) to obtain 1.9 g (41%) of the title compound as a yellow oily solid. ESI-MS (m/e): 307 [M+H] ⁺ .

Example 2 Preparation of 6-tert-butoxycarbonylaminocaproic acid (2)

5.00 g (38.2 mmol) of 6-aminocaproic acid (EACA) were suspended in 50 mL of distilled water. The resulting suspension was added with 10 mL of aqueous sodium hydroxide solution (2M) under an ice bath and stirred until dissolved. To the resulting solution, a solution of 9.2 g (42.2 mmol) (Boc) ₂ O and 20 mL of dioxane was added and the pH was adjusted to 9 with aqueous sodium hydroxide solution (2 M) and stirred for 30 min. The ice bath was removed and stirred at room temperature until TLC showed the disappearance of 6-aminocaproic acid. Under ice bath, the pH of the reaction solution was adjusted to 7 with saturated aqueous KHSO ₄ solution, and the dioxane was removed by concentration under reduced pressure. The reaction solution was adjusted to pH 2 with saturated aqueous KHSO ₄ solution under ice bath, and extracted with ethyl acetate (50 mL×3). The ethyl acetate layer was washed with saturated aqueous NaCl solution (40 mL×3), and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain 8.0 g (91%) of the title compound as a colorless oil. ESI-MS (m/e): 232 [M+H] ⁺ .

Example 3 Preparation of (3S)-N-(6-Boc-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (3)

4.15g (18mmol) Boc-6-aminocaproic acid was dissolved in 80mL anhydrous tetrahydrofuran, 2.46g (18.2mmol) N-hydroxybenzotriazole (HOBt) and 4.04g (19.6mmol) N were added under ice bath, N-dicyclohexylcarbodiimide (DCC), stirred for 60 minutes. Then, a solution of 5.06 g (16.5 mmol) of (3S)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester and 80 mL of anhydrous tetrahydrofuran was added to the obtained reaction solution. The reaction mixture was adjusted to pH 9 with N-methylmorpholine (NMM) in an ice bath and stirred at room temperature for 24 hours. TLC showed disappearance of (3S)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The solution was washed successively with saturated aqueous NaHCO ₃ solution (40 mL×3), saturated aqueous NaCl solution (40 mL×3), saturated aqueous KHSO ₄ solution (40 mL×3), saturated aqueous NaCl solution (40 mL×3), and saturated aqueous NaHCO ₃ solution (40 mL×3), washed with saturated aqueous NaCl solution (40 mL×3). The ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ , filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol=160:1) to obtain 5.43 g (64%) of the title compound, which was Yellow oily solid. ESI-MS (m/e): 520 [M+H] ⁺ .

Example 4 Preparation of (3S)-N-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (4)

Under ice bath, 3.4g (6.6mmol) of (3S)-N-(6-Boc-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester was added to 40mL Hydrogen chloride in ethyl acetate (4M) was dissolved, stirred for 3 hours, TLC showed (3S)-N-(6-Boc-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3 - Disappearance of benzyl carboxylate. The reaction mixture was concentrated to dryness under reduced pressure with stirring. The residue was dissolved in anhydrous ethyl acetate, and the solution was concentrated to dryness under reduced pressure. This operation is repeated three times. The residue was thoroughly washed with anhydrous ether to obtain 2.8 g (94%) of the title compound as a tan solid. ESI-MS (m/e): 420 [M+H] ⁺ ; Mp 223-226°C. ¹ HNMR (300MHz, DMSO-d6:δ/ppm=10.963(d,J=11.4Hz,1H), 7.894(s,3H), 7.458(d,J=7.5Hz,1H), 7.327(t,J= 7.5Hz, 1H), 7.204(dd, J ₁ =1.5Hz, J ₂ =7.2Hz, 1H), 7.158-6.972(m, 6H), 5.607(d, J=4.7Hz, 1H), 5.059(s, 2H), 4767(d, J＝15.6Hz, 1H), 4.610(d, J＝17.4Hz, 1H), 3.426(m, 1H), 3.021(m, 1H), 2.754(m, 2H), 2.613( m, 1H), 2.389 (m, 1H), 1.568-1.546 (m, 4H), 1.399-1.323 (m, 2H).

Example 5 Preparation of (3S)-N-(6-Boc-Glu(OBzl)-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (5a)

Dissolve 2.44g (7.2mmol) Boc-Glu (OBzl) and 0.97g (7.2mmol) N-hydroxybenzotriazole (HOBt) with 30mL anhydrous tetrahydrofuran, add 1.48g (7.2mmol) N under ice bath, N-dicyclohexylcarbodiimide (DCC), stirred for 40 minutes. Then, 3.00 g (6.6 mmol) of (3S)-N-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester was added to the obtained reaction solution It was mixed with 30 mL of anhydrous tetrahydrofuran solution, adjusted to pH 8 with N-methylmorpholine under ice bath, and stirred at room temperature for 12 hours. TLC showed that (3S)-N-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester disappeared, the reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the residue The mixture was dissolved in 100 mL of ethyl acetate. The solution was washed successively with saturated aqueous NaHCO ₃ solution (40 mL×3), saturated aqueous NaCl solution (40 mL×3), saturated aqueous KHSO ₄ solution (40 mL×3), saturated aqueous NaCl solution (40 mL×3), and saturated aqueous NaHCO ₃ solution (40 mL×3), washed with saturated aqueous NaCl solution (40 mL×3). The ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ , filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol=60:1) to obtain 1.52g (31%) of the title compound, which was Pale yellow solid. ESI-MS (m/z): 739 [M+H] ⁺ .

Example 6 Preparation of (3S)-N-(6-Boc-Ile-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (5b)

According to the method of Example 5, from 2.60 g (5.7 mmol) (3S)-N-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester and 1.45 g (6.3 mmol) of Boc-Ile to give 1.19 g (33%) of the title compound as a pale yellow solid. ESI-MS (m/z): 633 [M+H] ⁺ .

Example 7 Preparation of (3S)-N-(6-Boc-Lys(Boc)-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (5c)

From 0.86 g (1.87 mmol) Boc-Lys(Boc) and 0.42 g (1.37 mmol) (3S)-N-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro according to the method of Example 5 -β-Carboline-3-carboxylate benzyl ester gave 0.54 g (39%) of the title compound as a pale yellow solid. ESI-MS (m/z): 748 [M+H] ⁺ .

Example 8 Preparation of (3S)-N-(6-Boc-Leu-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (5d)

According to the method of Example 5, from 2.10 g (4.6 mmol) of (3S)-N-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester and 1.28 g (5.5 mmol) of Boc-Leu to give 1.62 g (56%) of the title compound as a pale yellow solid. ESI-MS (m/z): 633 [M+H] ⁺ .

Example 9 Preparation of (3S)-N-(6-Boc-Met-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (5e)

According to the method of Example 5, from 1.80 g (4.0 mmol) of (3S)-N-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester and 1.28 g (5.1 mmol) of Boc-Met to give 0.35 g (14%) of the title compound as a pale yellow solid. ESI-MS (m/z): 651 [M+H] ⁺ .

Example 10 Preparation of (3S)-N-(6-Boc-Pro-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (5f)

According to the method of Example 5, from 2.00 g (4.4 mmol) (3S)-N-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester and 1.04 g (4.8 mmol) of Boc-Pro to give 1.20 g (44%) of the title compound as a pale yellow solid. ESI-MS (m/z): 617 [M+H] ⁺ .

Example 11 Preparation of (3S)-N-(6-Boc-Ser-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (5g)

According to the method of Example 5, from 1.5 g (3.3 mmol) of (3S)-N-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester and 0.74 g (3.6 mmol) of Boc-Ser to give 0.30 g (15%) of the title compound as a pale yellow solid. ESI-MS (m/z): 607 [M+H] ⁺ .

Example 12 Preparation of (3S)-N-(6-Glu(OBzl)-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (6a)

1000 mg of (3S)-N-(6-Boc-Glu(OBzl)-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester was added under ice bath 15mL of hydrogen chloride in ethyl acetate (4M) and stirred for 2.5h, TLC (ethyl acetate:water:glacial acetic acid=6:1:1.2) showed (3S)-N-(6-Boc-Glu(OBzl)-amino n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester disappeared. The reaction solution was concentrated to dryness under reduced pressure. The residue was dissolved in anhydrous ethyl acetate, and then concentrated to dryness under reduced pressure. This operation is repeated three times. The residue was thoroughly washed with anhydrous ether, then purified by C18 column chromatography (methanol:water=40:60) and lyophilized to obtain 490 mg (54%) of the title compound as a pale yellow solid. ESI-MS(m/e):639[M+H]+.Mp:103-105℃; ^1H NMR(300MHz,DMSO-d6:δ/ppm=10.963(d,J=10.5Hz,1H), 8.608(m, 1H), 8.328(s, 3H), 7.455(d, J=7.5Hz, 1H), 7.395-7.305(m, 6H), 7.236-7.101(m, 7H), 5.685(d, J= 4.5Hz, 1H), 5.096(s, 2H), 5.053(s, 2H), 4761(d, J=15.6Hz, 1H), 4.629(d, J=17.1Hz, 1H), 3.803(dd, J ₁ =5.7Hz, J ₂ =12.0Hz 1H), 3.419(m, 1H), 3.145-2.965(m, 3H), 2.574(m, 1H), 2.457-2.389(m, 3H), 2.039-1.985(m, 2H), 1.582-1.508 (m, 2H), 1.482-1.421 (m, 2H), 1.374-1.300 (m, 2H).

Example 13 Preparation of (3S)-N-(6-Ile-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (6b)

Following the method of Example 12, obtained from 840 mg of (3S)-N-(6-Boc-Ile-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester 560 mg (79%) of the title compound as a pale yellow solid. ESI-MS (m/e): 533[M+H] ⁺ .Mp121-122°C; ¹ H NMR (300MHz, DMSO-d6: δ/ppm=10.997 (d, J=13.2Hz, 1H), 8.541 ( m, 1H), 8.240 (s, 3H), 7.454 (d, J=7.5Hz, 1H), 7.329 (t, J=7.8Hz, 1H), 7.205 (dd, J ₁ =2.1Hz, J ₂ =5.7 Hz,1H),7.162-7.104(m,6H),5.602(d,J＝4.8Hz,1H),5.058(s,2H),4769(d,J＝15.6Hz,1H),4.637(d,J =17.4Hz, 1H), 3.586(m, 1H), 3.422(m, 1H), 3.196(m, 1H), 3.028(m, 1H), 2.566(m, 1H), 2.417(m, 1H), 1.815 (m, 1H), 1.592-1.310 (m, 6H), 1.113 (m, 2H), 0.911-0.840 (m, 6H).

Example 14 Preparation of (3S)-N-(6-Lys-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (6c)

According to the method of Example 12, from 800 mg of (3S)-N-(6-Boc-Lys(Boc)-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid The benzyl ester gave 430 mg (75%) of the title compound as a pale yellow solid. ESI-MS (m/e): 548.3[M+H] ⁺ .Mp: 118.3-120.1°C; ¹ HNMR (300MHz, DMSO-d6: δ/ppm=10.990 (d, J=15.3Hz, 1H), 8.642 (m,1H),8.292(s,3H),8.055(s,3H),7.454(d,J=7.8Hz,1H),7.331(dd,J1 ₌ 7.8Hz,J2= _15.6Hz ,1H) ,7.205(dd,J1 ₌ 1.8Hz,J2=7.5Hz,1H), _7.162-7.086 (m,6H),5.596(d,J=4.8Hz,1H),5.061(s,2H),4775( d, J＝15.6Hz, 1H), 4.634(d, J＝17.4Hz, 1H), 3.744(m, 1H), 3.451(m, 1H), 3.109-2.937(m, 3H), 2.746(m, 2H) ), 2.584(m, 1H), 2.430(m, 1H), 1.742(m, 2H), 1.621-1.313(m, 10H).

Example 15 Preparation of (3S)-N-(6-Leu-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (6d)

According to the method of Example 12, from 1000 mg of (3S)-N-(6-Boc-Leu-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester 605 mg (72%) of the title compound as a pale yellow solid. ESI-MS (m/e): 533[M+H] ⁺ .Mp111-113°C; ¹ H NMR (300MHz, DMSO-d6: δ/ppm=10.998 (d, J=13.2Hz, 1H), 8.635 ( dd, J ₁ =5.1Hz, J ₂ =10.5Hz 1H), 8.281(s, 3H), 7.455(d, J=7.5Hz, 1H), 7.328(t, J=7.8Hz, 1H), 7.200(dd , J ₁ =2.1Hz, J ₂ =7.5Hz,1H),7.158-7.072(m,6H),5.613(d,J=4.5Hz,1H),5.057(s,2H),4769(d,J= 15.6Hz, 1H), 4.630(d, J=17.4Hz, 1H), 3.707(m, 1H), 3.447(m, 1H), 3.174-3.030(m, 3H), 2.569(m, 1H), 2.419( m, 1H), 1.645-1.306 (m, 9H), 0.908 (d, J=6.0 Hz, 3H), 0.888 (d, J=6.0 Hz, 3H).

Example 16 Preparation of (3S)-N-(6-Met-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (6e)

Following the procedure of Example 12, from 1100 mg of (3S)-N-(6-Boc-Met-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester 530 mg (57%) of the title compound as a pale yellow solid. ESI-MS (m/e): 551.2[M+H] ⁺ .Mp: 108.8-110.4°C; ¹ HNMR (300MHz, DMSO-d6: δ/ppm=10.962 (d, J=10.8Hz, 1H), 8.599 (q, J1 ₌ 5.4Hz, J2=10.5Hz, 1H), _8.319 (s, 3H), 7.456(d, J=7.5Hz, 1H), 7.329(t, J=7.5Hz, 1H), 7.206 (dd, J ₁ =2.1Hz, J ₂ =6.6Hz,1H),7.163-7.074(m,6H),5.604(d,J=4.8Hz,1H),5.059(s,2H),4769(d, J=15.6Hz, 1H), 4.631 (d, J=17.4Hz, 1H), 3.818 (dd, J1 ₌ 5.4Hz, J2=11.7Hz, 1H), _3.427 (m, 1H), 3.186-3.050( m,3H),2.568(m,1H),2.466-2.401(m,3H),2.059-1.962(m,5H),1.596-1.520(m,2H),1.495-1.433(m,2H),1.384- 1.308 (m, 2H).

Example 17 Preparation of (3S)-N-(6-Pro-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (6f)

According to the method of Example 12, from 950 mg of (3S)-N-(6-Boc-Pro-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester 598 mg (75%) of the title compound as a pale yellow solid. ESI-MS (m/e): 517.2 [M+H] ⁺ .Mp 116-117°C; ¹ H NMR (300 MHz, DMSO-d6: δ/ppm=10.975 (d, J=11.1 Hz, 1H), 9.188 (m, 2H), 8.610(dd, J ₁ =5.4Hz, J ₂ =11.1Hz, 1H), 7.456(d, J=7.8Hz, 1H), 7.328(t, J=7.5Hz, 1H), 7.202 (dd, J ₁ =2.1Hz, J ₂ =7.5Hz,1H),7.159-7.085(m,6H),5.605(d,J=4.5Hz,1H),5.058(s,2H),4767(d, J=15.6Hz, 1H), 4.636(d, J=17.7Hz, 1H), 4.21(m, 1H), 3.421(m, 1H), 3.224-3.099(m, 5H), 2.569(m, 1H), 2.438 (m, 1H), 2.273 (m, 1H), 1.889-1.786 (m, 3H), 1.552 (m, 2H), 1.457 (m, 2H), 1.322 (m, 2H).

Example 18 Preparation of (3S)-N-(6-Ser-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (6g)

Following the procedure of Example 12, from 825 mg of (3S)-N-(6-Boc-Ser-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester 448 mg (65%) of the title compound as a pale yellow solid. ESI-MS (m/e): 507.2[M+H] ⁺ .Mp 147-149°C; ¹ H NMR (300MHz, DMSO-d6: δ/ppm=10.990 (d, J=14.1Hz, 1H), 8.501 ( dd, J ₁ =5.1Hz, J ₂ =10.2Hz, 1H), 8.185(s, 3H), 7.454(d, J=7.8Hz, 1H), 7.331(t, J=7.8Hz, 1H), 7.243- 7.191(m, 1H), 7.162-7.085(m, 6H), 5.602(d, J=4.5Hz, 1H), 5.506(t, J=4.5Hz, 1H), 5.060(s, 2H), 4772(d , J=15.6Hz, 1H), 4.634(d, J=17.4Hz, 1H), 3.778-3.700(m, 3H), 3.426(m, 1H), 3.126-2.971(m, 3H), 2.574(m, 1H), 2.421 (m, 1H), 1.553 (m, 2H), 1.489-1.306 (m, 4H).

Experimental Example 1 Evaluation of the anti-tumor metastatic activity of compounds 6a,b,c,d,e,f,g

1) Compounds 6a, b, c, d, e, f, g were dissolved in normal saline, RGDS tetrapeptide was dissolved in normal saline as a positive control, and normal saline was used as a negative control;

2) The oral dose of compound 6a,b,c,d,e,f,g is 0.02μmol/kg/day, the oral dose of compound 4 is 0.2μmol/kg/day, and the oral dose of normal saline is 0.2mL/20g/ day, the oral dose of RGDS tetrapeptide was 20 μmol/kg/day. Two weeks after tumor inoculation, tumors with a diameter of about 4-5 mm could be observed in the armpits of mice. The drug was administered continuously for 10 days and administered 10 times in total.

3) The experimental animals were C57BL/6 male mice (clean grade) with a body weight of 20±2g, 12 mice in each group.

4) Lewis mouse lung cancer cells (LLC): LLC cells were purchased from ATCC, adherent cells; DMEM medium.

5) The LLC cells were digested and diluted with physiological saline to 2×10 ⁷ cells/mL. The Tryanblue exclusion test showed that the number of viable cells was >95%. Take C57BL/6 male mice, male, fix the mouse with the left hand, sterilize the axillary skin of the right forelimb of the mouse with 75% ethanol, hold a 1 mL sterile syringe in the right hand and inject 0.2 mL/mouse of the tumor cell suspension into the axilla of the mice subcutaneously. The number of tumor cells was about 2×10 ⁶ /mL). Tumors with a diameter of about 1.5 to 2 cm can grow in mice 12 to 15 days after inoculation, which can be used as tumor sources for future use.

6) Take Lewis lung cancer tumor-bearing mice that have been inoculated well for 12 to 15 days, dislocate their cervical vertebrae, soak and disinfect with 75% ethanol for 10 minutes, bluntly dissect solid tumors in an ultra-clean bench, and select well-growing tumor tissues to wash away the floating blood , cut into pieces in a watch dish, put the cut tumor tissue into a glass tissue homogenizer, add 4°C pre-cooled normal saline to grind into a cell suspension, filter twice with a 200-mesh nylon mesh to make a single-cell suspension liquid. Dilute the cell suspension to 2×10 ⁷ cells/mL, stain with trypan blue, and calculate the number of viable cells >95%. Take C57BL/6 male mice, inoculate 0.2 mL of qualified cell suspension into the right armpit subcutaneously for each mouse, and observe. 10 to 12 days after inoculation, tumors with a diameter of about 4 to 5 mm could be observed in the armpits of the mice, and the tumor volume was measured, and the groups were averagely grouped according to the tumor volume, and the administration was started. The mice in the treatment group were given daily oral administration of compound 6a,b,c,d,e,f,g at a dose of 0.02 μmol/kg/day, and oral dose of compound 4 at 0.2 μmol/kg/day. The mice in the blank group were given 0.2 mL/20 g/day of normal saline by gavage every day. Mice in the positive control group were given 20 μmol/kg/day of RGDS tetrapeptide by gavage every day for ten consecutive days.

7) On the eleventh day, the mice were weighed, anesthetized with ether, and the mice were sacrificed by removing the cervical vertebrae. Then, the tumor growth site of the right axilla of the mice was fixed with forceps, the skin was cut, the tumor was exposed, bluntly dissected, and weighed; Lungs and the number of tumor nodules with lung metastasis were recorded. The experimental data were analyzed by t test and analysis of variance, and the number of metastatic tumor nodes was expressed as (mean ± SD g). The results are shown in Table 1. It can be seen from Table 1 that at an oral dose of 0.02 μmol/kg/day, compounds 6a, b, c, d, e, f, g can effectively inhibit the metastasis of lung tumors in mice. The present invention has significant technical effects.

Table 1 The number of lung tumor nodules in mice transferred by each group of compounds

a) Compared with normal saline, P<0.01, compared with RGDS, P>0.05; b) Compared with normal saline, P<0.05, compared with RGDS, P>0.05; c) Compared with normal saline, P<0.05; n =8.

Experimental Example 2 Evaluation of the anti-inflammatory activity of compounds 6a,b,c,d,e,f,g

1) Compounds 6a, b, c, d, e, f, g were dissolved in normal saline, aspirin was dissolved in normal saline as a positive control, and normal saline was used as a negative control;

2) The oral dose of compound 6a,b,c,d,e,f,g is 0.02μmol/kg/day, the oral dose of compound 4 is 0.2μmol/kg/day, and the oral dose of normal saline is 0.2mL/20g/ day, the oral dose of aspirin was 1.11 mmol/kg/day.

3) The experimental animals were ICR male mice (clean grade), weighing 20±2g, randomly divided into groups, 12 mice in each group.

4) After 30 minutes of intragastric administration, 30 μL of xylene was evenly applied to the left auricle of the mouse. After 2 hours, the mouse was anesthetized with ether, killed by cervical dislocation, and the left and right ears were cut off. A round ear piece was taken from the position, weighed, and the difference of the swelling of the two ears was calculated as the swelling degree. That is, the degree of swelling = the weight of the left ear disc - the weight of the right ear disc. The experimental data were analyzed by t test and analysis of variance, and the ear swelling was expressed as (mean ± SD mg). The results are shown in Table 2. It can be seen from Table 2 that at an oral dose of 0.02 μmol/kg/day, compounds 6a, b, c, d, e, f, g can effectively inhibit the swelling of the ears of mice caused by xylene. The present invention has significant technical effects.

Table 2 Effects of each group of compounds on mouse ear swelling caused by xylene

a) Compared with normal saline, P<0.01, compared with Aspirin, P>0.05; b) Compared with normal saline, P<0.01;

c) Compared with normal saline, P<0.05; n=12.

Claims (3)

1. A benzyl ester of (3S)-N-(6-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate with the following structure in the preparation of antitumor application in transfer medicine,

In the formula, AA is selected from the residues of L-Glu(OBzl), the residues of L-Ile, the residues of L-Lys, the residues of L-Leu, the residues of L-Met, the residues of L-Pro, Residues of L-Ser. 2. The (3S)-N-(6-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate benzyl ester of claim 1 is used in the preparation of antitumor The application in the transfer medicine is characterized in that the compound is prepared by the following method: (1) Under the catalysis of dilute sulfuric acid, L-tryptophan benzyl ester and formaldehyde undergo Pictet-Spengler reaction to generate benzyl (3S)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate ester; (2) in 2M aqueous sodium hydroxide solution, in water and dioxane mixed solution, 6-aminocaproic acid and di-tert-butyl dicarbonate (Boc) ₂ O react to generate 6-Boc-aminocaproic acid; (3) In the presence of N,N-dicyclohexylcarbodiimide and N-hydroxybenzotriazole in anhydrous tetrahydrofuran, 6-Boc-aminocaproic acid was combined with (3S)-2,3,4 , 9-Tetrahydro-β-carboline-3-carboxylate benzyl ester was reacted to give (3S)-N-(6-Boc-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboyl Benzyl-3-carboxylate; (4) In 4M ethyl acetate solution of hydrogen chloride under ice bath, (3S)-N-(6-Boc-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3- Benzyl carboxylate removes Boc to obtain (3S)-N-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester; (5) In the presence of N,N-dicyclohexylcarbodiimide and N-hydroxybenzotriazole, in dry tetrahydrofuran, (3S)-N-(6-amino-n-hexanoyl)-2,3, 4,9-Tetrahydro-β-carboline-3-carboxylic acid benzyl ester reacts with Boc-AA to give (3S)-N-(6-Boc-AA-amino-n-hexanoyl)-2,3,4,9 - Benzyl tetrahydro-β-carboline-3-carboxylate; (6) (3S)-N-(6-Boc-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline- 3-Carboxylic acid benzyl ester removes Boc to obtain (3S)-N-(6-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester. 3. (3S)-N-(6-AA-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester of the following formula is used in the preparation of anti-inflammatory application in medicine,

In the formula, AA is selected from the residues of L-Glu(OBzl), the residues of L-Ile, the residues of L-Lys, the residues of L-Leu, the residues of L-Met, the residues of L-Pro, Residues of L-Ser.

Images