CN107459550B

CN107459550B - Carboline formyl-AA-fluorouracil-acetyl-Orn-NHCH2C6H5Their synthesis, activity and use

Info

Publication number: CN107459550B
Application number: CN201610391393.2A
Authority: CN
Inventors: 赵明; 彭师奇; 王玉记; 吴建辉; 吴建民
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2016-06-03
Filing date: 2016-06-03
Publication date: 2020-03-17
Anticipated expiration: 2036-06-03
Also published as: CN107459550A

Abstract

The invention discloses 4N of the formula^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅The invention discloses a preparation method of the compounds, discloses the antitumor activity of the compounds and the anti-inflammatory activity of the compounds, and thus discloses the application of the compounds as antitumor drugs and anti-inflammatory drugs.

Description

Carboline formyl-AA-fluorouracil-acetyl-Orn-NHCH2C6H5Their synthesis, activity and use

Technical Field

The invention relates to 4N^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅To a process for their preparation, to their inhibitory effect on the growth of tumour cells, to their anti-tumour effect and to their anti-inflammatory effect. The invention belongs to the field of biological medicine.

Background

β -carboline is an important natural product with broad pharmacological effects such as antithrombotic and antitumor effects the inventors have disclosed a series of β -carboline-3-formyl-amino acids, β -carboline-3-formyl-amino acid benzyl esters, β -carboline-3-formyl-oligopeptides with antithrombotic and antitumor activity which are not satisfactory at this level of activity for intraperitoneal administration at a dose of 8.9-10 μmol/kg. the inventors are thus dissatisfied with experimental exploration of low dose oral objectives the inventors have found that the introduction of a salicylic-like substituent at position 1 of carboline is unsatisfactory and indeed leads to a substantial reduction of the dose in the sense that, of the 1- (salicylic acid derivative) - β -carboline-3-formyl amino acid, 1- (salicylic acid derivative) - β -carboline-3-formyl-carboline benzyl ester and 1- (salicylic acid derivative) - β -carboline-3-formyl-oligopeptides represented by the following formulae (left, R is amino acid benzyl ester group or polypeptide group), some of the water-soluble compounds are potent at doses of benzyl ester 3-formyl-1- (salicylic acid derivative) - β -carboline-formyl amino acid ester groups which are not effective at the dose which leads to the instability of wldv-1-phagy-1-counter-hydroxy-benzoyl ester group which is not expected by the inventors at the usual dose of the ww group (ww-3-phagy).

Then, the inventors have found that the use of Orn-AA-NHCH has been attempted after the experimental investigation of β -carboline-3-formyl derivatives having no substituent at the 1-position₂C₆H₅The β -carboline-3-formyl-Orn-AA-NHCH is obtained by replacing AA-OBzl₂-C₆H₅Not only has definite anti-tumor effect under the dosage of 1nmol/kg, but also has definite therapeutic effect on the complication inflammation of the tumor under the dosage of 0.1 mu mol/kg. Clearly, this is an unexpected technical effect. Based on this finding, the inventors have proposed the present invention.

Disclosure of Invention

The first content of the invention is to provide N^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅[ wherein AA is selected from L-Met, L-Cys (Bzl), L-Arg (NO)₂) L-Lys (5-fluorouracil-1-acetyl) residue]。

A second aspect of the present invention is to provide N^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅[ wherein AA is selected from L-Met, L-Cys (Bzl), L-Arg (NO)₂) L-Lys (5-fluorouracil-1-acetyl) residue]The method of (2), the methodThe method comprises the following steps:

(1) carrying out Pictet-Spengler condensation on L-tryptophan and formaldehyde under the catalysis of dilute sulfuric acid to generate (3s) - β -tetrahydrocarboline-3-carboxylic acid;

(2) (3s) - β -tetrahydrocarboline-3-carboxylic acid to (3s) - β -tetrahydrocarboline-3-carboxylic acid methyl ester;

(3) oxidizing (3s) - β -tetrahydro carboline-3-carboxylic acid methyl ester into β -carboline-3-carboxylic acid methyl ester by potassium permanganate;

(4) β -carboline-3-carboxylic acid methyl ester is hydrolyzed in 2N NaOH solution to generate β -carboline-3-carboxylic acid;

(5) β -carboline-3-carboxylic acid is coupled with L-AA-OBzl (AA in the formula is defined as above) to obtain β -carboline-3-formyl-AA-OBzl;

(6) 5-Fluorouracil with Br-CH in 6N KOH solution₂COOH coupling to give 5-fluorouracil-1-ylacetic acid

(5-Fu-Ac)；

(7) β -carboline-3-formyl-AA-OBzl (AA in the formula is defined as above) is hydrolyzed in 2N NaOH solution to generate β -carboline-3-formyl-AA;

(8) β -carboline-3-formyl-AA (AA is as defined above) and Orn (Boc) -OBzl to obtain N^α- (β -carboline-3-formyl-AA) -orn (boc) -OBzl;

(9)N^α- (β -carboline-3-formyl-AA) -Orn (Boc) -OBzl (wherein AA is as defined above) in 2N NaOH solution to produce N^α- (β -carboline-3-formyl-AA) -orn (boc);

(10)N^α- (β -carboline-3-formyl-AA) -Orn (Boc) (AA has the same definition as above) and benzylamine to obtain N^α- (β -carboline-3-formyl-AA) -Orn (Boc) -NHCH₂C₆H₅；

(11)N^α- (β -carboline-3-formyl-AA) -Orn (Boc) -NHCH₂C₆H₅(wherein AA is as defined above) in 4N HCl-ethyl acetate solution at 0 deg.C to remove Boc and convert to N^α- (β -carboline-3-formyl-AA) -Orn-NHCH₂C₆H₅；

(12)N^α- (β -carboline-3-formyl-AA) -Orn-NHCH₂C₆H₅(wherein AA is as defined above) is coupled to 5-Fu-Ac,to obtain N^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅。

The third aspect of the present invention is the evaluation N^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅(AA in the formula is as defined above) are disclosed.

The fourth aspect of the present invention is the evaluation N^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅(wherein AA is as defined above) in the formula.

Drawings

FIG. 1.N^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅I) Formaldehyde, concentrated H₂SO₄(ii) a ii) methanol, SOCl₂；iii)KMnO₄Acetone; iv)2N NaOH, methanol, ice bath; v)1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), N, N-Dimethylformamide (DMF); vi)4N HCl/EA, ice bath; vii)60 ℃, KOH, Br-CH₂COOH; viii) O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), anhydrous DMF, NMM. AA ═ L-Met in 6a-14 a; AA ═ L-cys (bzl) in 6b-14 b; AA ═ L-Arg (NO) in 6c-14c₂) (ii) a In 6d-14d, AA is L-Lys (5-fluorouracil-1-acetyl).

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 β -Tetrahydrocarboline-3-carboxylic acid (2)

Placing 400mL of distilled water into a 500mL eggplant bottle, slowly adding 0.2mL of concentrated sulfuric acid, uniformly stirring for 10 minutes, continuously adding 5g (25.0mmol) of tryptophan into the obtained dilute sulfuric acid, and completely dissolving the tryptophan by ultrasonic oscillation; 10mL of 40% formaldehyde solution was added to the reaction mixture at room temperatureThe reaction was carried out for 6 hours. Slowly dropwise adding concentrated ammonia water into the reaction solution at the temperature of 0 ℃ in ice bath, adjusting the pH value to 6, leading the reaction bottle to be turbid, standing for 2 hours, and filtering out white precipitate under reduced pressure. The filtered off colourless solid was spread on a petri dish and dried in a fume hood to give 4.8g (yield 90.7%) of product as a milky powder, ESI-MS (M/z):205[ M + H]⁺。

EXAMPLE 2 preparation β -Tetrahydrocarboline-3-carboxylic acid methyl ester (3)

Adding 36mL of methanol into a 100mL eggplant bottle, dropwise adding 2.4mL of thionyl chloride into the eggplant bottle by using a constant-pressure funnel at-10 ℃, stirring for 30 minutes, adding 2.16g (10.0mmol) of β -tetrahydrocarboline-3-carboxylic acid, removing the ice salt bath, gradually returning to room temperature, continuing the reaction for 24 hours, detecting the disappearance of the reaction raw material by TLC (dichloromethane: methanol 20:1), removing the solvent under reduced pressure, adding 15mL of methanol into the residue, removing the solvent under reduced pressure, repeating the steps for 3 times, adding 10mL of anhydrous ether, removing the solvent under reduced pressure to obtain yellow powder, dissolving the yellow powder by using 50mL of ethyl acetate, transferring the yellow powder to a 100mL separating funnel, adding 20mL of saturated sodium bicarbonate solution, extracting and washing for 3 times, then extracting and washing for 3 times by using 10mL of saturated sodium chloride solution, drying the organic phase with anhydrous sodium sulfate overnight, filtering, concentrating the filtrate under reduced pressure, and separating the residue by using a silica gel column chromatography to obtain 1.023g of yellow powdery solid (yield of 44.5%), ESI-MS (M/e): 231M + H]⁺。

EXAMPLE 3 preparation β -carboline-3-carboxylic acid methyl ester (4)

Dissolving 2.3g (10.0mmol) of β -tetrahydrocarboline-3-carboxylic acid methyl ester with 60mL of acetone, slowly adding 2.21g (14.0mmol) of potassium permanganate at the temperature of 0 ℃ of an ice bath, stirring and mixing uniformly, removing the ice bath, reacting at room temperature for 6 hours, monitoring the reaction by a TLC plate (dichloromethane: methanol 20:1), filtering the reaction solution by a Buchner funnel after the reaction is finished, transferring the filter cake to a 100mL eggplant bottle, adding 50mL of acetone, stirring for 30 minutes, filtering by the Buchner funnel, repeating the step for 3 times until TLC monitoring shows that no product is extracted from the filtrate, combining the filtrates, concentrating under reduced pressure, and carrying out silica gel column chromatography separation and purification on the residue to obtain 750mg of a light yellow solid (the yield is 33.2%), ESI-MS (M/e):227[ M + H ], (M + H)]⁺。

EXAMPLE 4 preparation β -carboline-3-carboxylic acid (5)

Dissolving 2.26g (10.0mmol) of β -carboline-3-carboxylic acid methyl ester with methanol and tetrahydrofuran, adjusting pH to 12 with 2N NaOH solution, reacting at room temperature for 4 hours, adjusting pH to 7 with saturated potassium hydrogen sulfate solution, removing methanol and tetrahydrofuran under reduced pressure, adjusting pH of water phase to 2 with saturated potassium hydrogen sulfate solution, precipitating a large amount of solid, filtering, rinsing the filter cake with 20mL of distilled water to obtain 1.89g (yield 89.2%) of light yellow solid, ESI-MS (M/e):213[ M + H ], [ M]⁺。

EXAMPLE 5 preparation of 5-Fluorouracil-1-Ylacetic acid (13)

2.56g (45.7mmol) of KOH was dissolved in 8mL of distilled water, and 1.56g (12.0mmol) of 5-fluorouracil was added thereto, followed by stirring at 60 ℃ for 30 minutes. 2.78g (20.0mmol) of Br-CH₂COOH was dissolved in 8mL of distilled water, and the solution was slowly dropped into the reaction mixture. The reaction was carried out at 60 ℃ for 6 hours. The reaction was monitored by TLC (ethyl acetate: water: glacial acetic acid ═ 5:1: 2). After the reaction is completed, the heating is stopped, the pH value is adjusted to 5.5 by concentrated hydrochloric acid under ice bath, and the mixture is kept stand for 2 hours, so that no solid is separated out. Then the pH value is adjusted to 2 by concentrated hydrochloric acid in ice bath, and the mixture is kept still for 12 hours, and colorless solid is separated out. Filtering, collecting filter cake, and recrystallizing with cold and hot water to obtain colorless needle-like solid 1.14g (yield 50.7%), ESI-MS (M/e):187[ M-H]^-。

EXAMPLE 6 preparation β -carboline-3-formyl-Met-OBzl (6a)

Dissolving 212mg (1.0mmol) of β -carboline-3-carboxylic acid in 10mL of anhydrous DMF, adding 149mg (1.1mmol) of HOBt at 0 ℃ in an ice bath for activation for 5 minutes, then adding 296mg (1.3mmol) of EDC, activating for 30 minutes, then adding 263mg (1.1mmol) of HCl & Met-OBzl, adjusting the pH value to 8 by NMM, removing the ice bath, reacting at room temperature for 6 hours, monitoring the reaction by TLC (dichloromethane: methanol ═ 20:1), adding a saturated sodium chloride solution to the reaction solution after the reaction is finished, generating yellow turbidity, adding ethyl acetate for extraction and washing, extracting and washing the water layer for three times (10mL × 3), detecting no product point in the water layer by TLC, collecting the ethyl acetate layer, sequentially extracting and washing three times (10mL × 3) by saturated sodium carbonate, saturated sodium chloride, 5% potassium sulfate solution, saturated sodium chloride, saturated sodium carbonate and saturated sodium chloride solution in turn, adding anhydrous sodium sulfate for drying overnight, filtering off the sodium sulfate, and filtering off the filtrateThe solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 297mg (yield: 68.5%) of a colorless solid, ESI-MS (M/z):434[ M + H]⁺。

EXAMPLE 7 preparation β -carboline-3-formyl-Met (7a)

Placing 433mg (1.0mmol) of β -carboline-3-formyl-Met-OBzl in a 100mL eggplant bottle, adding 50mL of methanol, dissolving, stirring at 0 deg.C for 15min in ice bath, adjusting pH to 12 with 2N NaOH solution, reacting in ice bath, monitoring by TLC (dichloromethane: methanol 20:1), adjusting pH to 7 with 5% potassium hydrogen sulfate solution in ice bath after reaction, removing methanol under reduced pressure, adjusting pH to 2 with saturated potassium hydrogen sulfate solution in ice bath, precipitating yellow solid, filtering to obtain 292mg of light yellow solid (yield 85.1%), ESI-MS (M/z):342[ M-H-M/z): 342]^-。

EXAMPLE 8 preparation of N^α- (β -carboline-3-formyl-Met) -Orn (Boc) -OBzl (8a)

Dissolving 343mg (1.0mmol) of β -carboline-3-formyl-Met in 10mL of anhydrous DMF, adding 149mg (1.1mmol) of HOBt at 0 ℃ in ice bath for activation for 5 minutes, then adding 296mg (1.3mmol) of HCl & EDC, activating for 30 minutes, then adding 354mg (1.1mmol) of HCl & Orn (Boc) -OBzl, adjusting the pH value to 8 with NMM, removing the ice bath, reacting at room temperature for 6 hours, monitoring the reaction by TLC (dichloromethane: methanol ═ 20:1), and after the reaction is finished, purifying by silica gel column chromatography to obtain 446mg of light yellow solid (yield is 68.9%), ESI-MS (M/z):648[ M + H + 1 ]: 648]⁺。

Example 9 preparation of N^α- (β -carboline-3-formyl-Met) -Orn (Boc) (9a)

647mg (1.0mmol) N^α- (β -carboline-3-formyl-Met) -Orn (Boc) -OBzl was placed in a 50mL eggplant flask, 20mL methanol was added and dissolved, the mixture was stirred for 15 minutes at 0 ℃ in ice bath, the pH was adjusted to 12 with 2N NaOH solution, the reaction was carried out in ice bath, the reaction was monitored by TLC (dichloromethane: 20:1), after completion of the reaction, the pH was adjusted to 7 with 5% potassium hydrogensulfate solution in ice bath, methanol was removed under reduced pressure, the pH was adjusted to 2 with saturated potassium hydrogensulfate solution in ice bath, a yellow solid precipitated, and the mixture was filtered to obtain 467mg (yield 83.8%) of a yellow solid, ESI-MS (M/z):556[ M-H ], (M-H): 556]^-。

Examples10 preparation of N^α- (β -carboline-3-formyl-Met) -Orn (Boc) -NHCH₂C₆H₅(10a)

557mg (1.0mmol) of N^α- (β -carboline-3-formyl-Met) -Orn (Boc) was placed in a 50mL eggplant flask, 10mL anhydrous DMF was added, after dissolution, 149mg (1.1mmol) of HOBt was added at 0 ℃ for activation for 5 minutes, 296mg (1.3mmol) of HCl & EDC was added, after activation for 30 minutes, 118mg (1.1mmol) of C₇H₉And N, adjusting the pH value to be 8 by using NMM. The ice bath was removed and the reaction was allowed to proceed at room temperature for 6 hours. The reaction was monitored by TLC (dichloromethane: methanol ═ 20: 1). After the reaction is finished, the compound is treated as shown in the formula 6' a. Purification by silica gel column chromatography gave 424mg (65.6% yield) of an ESI-MS (M/z) pale yellow solid 647[ M + H ]]⁺。

Example 11 preparation of N^α- (β -carboline-3-formyl-Met) -Orn-NHCH₂C₆H₅(11a)

646mg (1.0mmol) of N^α- (β -carboline-3-formyl-Met) -Orn (Boc) -NHCH₂C₆H₅Placed in a 50mL eggplant bottle, added with 2mL of anhydrous ethyl acetate under ice-bath, stirred for 10 minutes, added with 10mL of 4N HCl/EA, added with a drying tube, and reacted under ice-bath. TLC monitoring (dichloromethane: methanol 20: 1). After the reaction is completed, pumping the solvent of the reaction solution by using a diaphragm pump, after the solvent is pumped to be dry, adding 5mL of anhydrous ethyl acetate into a reaction bottle, pumping the solvent by using the diaphragm pump so as to pump off the residual acid gas of the reaction solution, repeating the pumping for three times, finally adding 10mL of anhydrous ethyl ether, soaking for 10 minutes, pumping the solvent, repeating the pumping for three times to obtain 520mg of yellow solid (the yield is 95.3%), and ESI-MS (M/z):547[ M + H ], (M/z):547]⁺。

Example 12 preparation of N^α- (β -carboline-3-formyl-Met) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅(14a)

273mg (0.5mmol) of N^α- (β -carboline-3-formyl-Met) -Orn-NHCH₂C₆H₅The mixture was placed in a 50mL Erlenmeyer flask, 10mL of anhydrous DMF was added, and after dissolution, 209mg (0.55mmol) of HBTU and 104mg (0.55mmol) of 5-Fu-Ac were added at 0 ℃ in an ice bath, and the pH was adjusted to 8 with NMM. The ice bath was removed and the reaction was allowed to react at room temperature for 6 hours and monitored by TLC (dichloromethane: methanol: glacial acetic acid 10:1:5 drops). And after the reaction is finished, pouring the reaction liquid into a watch glass, and drying the watch glass in a fume hood. The residue was purified by column chromatography on silica gel to give 42mg (yield: 11.7%) of a pale yellow solid; ESI-MS (M/z):717[ M + H]⁺；mp:167-168℃；

(c ═ 0.1, methanol); IR (cm)^-1):3272,3063,2980,2956,2872, 1697,1639,1596,1497,1461,1377,1339,1242,729,695；¹H-NMR(300MHz,DMSO-d₆): δ/ppm＝12.05(s,1H),11.98(s,1H),8.94(s,1H),8.86(s,1H),8.81(d,J＝8.4Hz,1H),8.52(t,J ＝5.7Hz,1H),8.43(d,J＝7.5Hz,1H),8.40(d,J＝7.5Hz,1H),8.21(t,J＝5.7Hz,1H),8.01(d, J＝6.9Hz,1H),7.67(d,J＝8.1Hz,1H),7.61(t,J＝7.8Hz,1H),7.35-7.22(m,6H),4.73(m,1H),4.40-4.32(m,2H),4.27-4.20(m,3H),3.09-3.05(m,2H),2.09-2.04(m,5H),1.70(m,1H),1.58 (m,1H),1.46(m,2H)。

EXAMPLE 13 preparation β -carboline-3-formyl-Cys (Bzl) -OBzl (6b)

From 212mg (1.0mmol) β -carboline-3-carboxylic acid and 331mg (1.1mmol) HCl.Cys (Bzl) -OBzl as in example 6, 375mg (75.5% yield) of the title compound were obtained as ESI-MS (M/z):496[ M + H ] as a light yellow oil]⁺。

EXAMPLE 14 preparation β -carboline-3-formyl-Cys (Bzl) (7b)

From 495mg (1.0mmol) β -carboline-3-formyl-Cys (Bzl) -OBzl 324mg (80.1% yield) of the title compound as yellow solid were obtained according to example 7 ESI-MS (M/z):404[ M-H]^-。

Example 15 preparation of N^α- [ β -carboline-3-formyl-Cys (Bzl)]-Orn(Boc)-OBzl(8b)

From 405mg (1.0mmol) β -carboline-3-formyl-Cys (Bzl) and 354mg (1.1mmol) HCl. Orn (Boc) -OBzl as per example 8, 537mg (75.7% yield) of the title compound was obtained as light yellow solid ESI-MS (M/z):710[ M + H ], (M/z)]⁺。

Example 16 preparation of N^α- [ β -carboline-3-formyl-Cys (Bzl)]-Orn(Boc)(9b)

From 709mg (1.0mmol) of N according to example 9^α- [ β -carboline-3-formyl-Cys (Bzl)]-Orn (Boc) -OBzlTo 471mg (76.1% yield) of the title compound as a yellow solid, ESI-MS (M/z):618[ M-H]^-。

Example 17 preparation of N^α- [ β -carboline-3-formyl-Cys (Bzl)]-Orn(Boc)-NHCH₂C₆H₅(10b)

From 619mg (1.0mmol) N according to example 10^α- [ β -carboline-3-formyl-Cys (Bzl)]Orn (Boc) and 118mg (1.1mmol) C₇H₉N yield 479mg (67.6% yield) of the title compound as a pale yellow solid, ESI-MS (M/z):709[ M + H]⁺。

EXAMPLE 18 preparation of N^α- [ β -carboline-3-formyl-Cys (Bzl)]-Orn-NHCH₂C₆H₅(11b)

From 708mg (1.0mmol) N according to example 11^α- [ β -carboline-3-formyl-Cys (Bzl)]-Orn(Boc)-NHCH₂C₆H₅587mg (96.5% yield) of the title compound are obtained as a yellow solid in ESI-MS (M/z):575[ M + H]⁺。

Example 19 preparation of N^α- [ β -carboline-3-formyl-Cys (Bzl)]-N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅(14b)

From 304mg (0.5mmol) N according to example 12^α- [ β -carboline-3-formyl-Cys (Bzl)]-Orn-NHCH₂C₆H₅And 104mg (0.55mmol) of 5-Fu-Ac gave 41mg (yield 10.5%) of the title compound as a pale yellow solid; ESI-MS (M/z):779[ M + H]⁺；mp:159-160℃；[α]_D ²⁵-50.0 (c-0.1, methanol); IR (cm)^-1):3278,3062,3015, 2966,2887,2824,1692,1639,1597,1496,1460,1376,1241,729,692；¹H-NMR(300MHz, DMSO-d6):δ/ppm＝12.45(s,1H),8.97(s,1H),8.88(s,1H),8.86(d,J＝10.8Hz,1H),8.75(d,J ＝7.8Hz,1H),8.58(t,J＝6.0Hz,1H),8.41(d,J＝7.8Hz,1H),8.24(t,J＝5.7Hz,1H),7.89(d,J ＝6.9Hz,1H),7.61(d,J＝7.5Hz,1H),7.35-7.18(m,11H),4.93(m,1H),4.42-4.26(m,5H),3.77 (m,2H),3.12-3.06(m,2H),2.96(m,1H),2.88(m,1H),1.67(m,1H),1.59(m,1H),1.49(m, 2H)。

EXAMPLE 20 preparation β -carboline-3-formyl-Arg (NO)₂)-OBzl(6c)

From 212mg (1.0mmol) β -carboline-3-carboxylic acid and 340mg (1.1mmol) HCl Arg (NO) according to example 6₂) OBzl gave 338mg (67.1% yield) of the title compound as a pale yellow oil, ESI-MS (M/z):504[ M + H]⁺。

EXAMPLE 21 preparation β -carboline-3-formyl-Arg (NO)₂)(7c)

From 503mg (1.0mmol) β -carboline-3-formyl-Arg (NO) according to example 7₂) OBzl gave 335mg (yield 81.2%) of the title compound as a yellow solid, ESI-MS (M/z):412[ M-H]^-。

EXAMPLE 22 preparation of N^α- [ β -carboline-3-formyl-Arg (NO)₂)]-Orn(Boc)-OBzl(8c)

From 413mg (1.0mmol) β -carboline-3-formyl-Arg (NO) according to example 8₂) And 354mg (1.1mmol) of HCl. Orn (Boc) -OBzl to give 471mg (yield 65.7%) of the title compound as a pale yellow solid, ESI-MS (M/z):718[ M + H: (M/z)]⁺。

Example 23 preparation of N^α- [ β -carboline-3-formyl-Arg (NO)₂)]-Orn(Boc)(9c)

From 717mg (1.0mmol) of N according to example 9^α- [ β -carboline-3-formyl-Arg (NO)₂)]Orn (Boc) -OBzl to yield 485mg (77.4% yield) of the title compound as a yellow solid, ESI-MS (M/z):626[ M-H]^-。

Example 24 preparation of N^α- [ β -carboline-3-formyl-Arg (NO)₂)]-Orn(Boc)-NHCH₂C₆H₅(10c)

From 627mg (1.0mmol) N according to example 10^α- [ β -carboline-3-formyl-Arg (NO)₂)]Orn (Boc) to give 441mg (61.6% yield) of the title compound as a pale yellow solid, ESI-MS (M/z):717[ M + H]⁺。

EXAMPLE 25 preparation of N^α- [ β -carboline-3-formyl-Arg (NO)₂)]-Orn-NHCH₂C₆H₅(11c)

From 716mg (1.0mmol) of N according to example 11^α- [ β -carboline-3-formyl-Arg (NO)₂)]-Orn(Boc)-NHCH₂C₆H₅To yield 582mg (yield 94)5%) the title compound as a yellow solid, ESI-MS (M/z):617[ M + H]⁺。

Example 26 preparation of N^α- [ β -carboline-3-formyl-Arg (NO)₂)]-N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅(14c)

From 308mg (0.5mmol) N according to example 12^α- [ β -carboline-3-formyl-Arg (NO)₂)]-Orn-NHCH₂C₆H₅And 104mg (0.55mmol) of 5-Fu-Ac gave 33mg (8.3% yield) of the title compound as a pale yellow solid; ESI-MS (M/z):787[ M + H]⁺；mp:178-179℃；

(c ═ 0.1, methanol); IR (cm)^-1):3280,3063,2965,2923,2886,1692,1657,1649,1596,1497,1460,1339,1245,1145,730,691；¹H-NMR(300MHz,DMSO-d₆):δ/ppm＝12.12(s,1H),11.88(s,1H),8.94(s,1H),8.85(s,1H),8.73(d,J＝7.8Hz, 1H),8.61-8.55(m,2H),8.41(d,J＝7.8Hz,1H),8.26(t,J＝5.4Hz,1H),8.02(d,J＝6.9Hz,1H), 7.67(d,J＝8.1Hz,1H),7.60(t,J＝7.5Hz,1H),7.33-7.21(m,6H),4.71(m,1H),4.36-4.26(m, 5H),3.25-3.15(m,2H),3.10-3.06(m,2H),1.89-1.71(m,3H),1.61-1.42(m,5H)。

EXAMPLE 27 preparation β -carboline-3-formyl-Lys (Boc) -OBzl (6d)

From 212mg (1.0mmol) β -carboline-3-carboxylic acid and 370mg (1.1mmol) HCl Lys (Boc) -OBzl367mg (69.3% yield) as in example 6 the title compound was obtained as a colourless solid, ESI-MS (M/z):531[ M + H ] 531]⁺。

EXAMPLE 28 preparation β -carboline-3-formyl-Lys (Boc) (7d)

From 530mg (1.0mmol) β -carboline-3-formyl-Lys (Boc) -OBzl 374mg (yield 85.1%) of the title compound as yellow solid were obtained according to example 7, ESI-MS (M/z):439[ M-H]^-。

EXAMPLE 29 preparation of N^α- [ β -carboline-3-formyl-Lys (Boc)]-Orn(Boc)-OBzl(8d)

From 440mg (1.0mmol) β -carboline-3-formyl-Lys (Boc) and 354mg (1.1mmol) HCl. Orn (Boc) -OBzl 45 mg were obtained according to example 86mg (61.4% yield) of the title compound as a pale yellow solid, ESI-MS (M/z):744[ M + H]⁺。

Example 30 preparation of N^α- [ β -carboline-3-formyl-Lys (Boc)]-Orn(Boc)(9d)

From 743mg (1.0mmol) N as in example 9^α- [ β -carboline-3-formyl-Lys (Boc)]Orn (Boc) -OBzl gave 480 mg (73.5% yield) of the title compound as a yellow solid, ESI-MS (M/z):652[ M-H]^-。

EXAMPLE 31 preparation of N^α- [ β -carboline-3-formyl-Lys (Boc)]-Orn(Boc)-NHCH₂C₆H₅(10d)

From 653mg (1.0mmol) N as in example 10^α- [ β -carboline-3-formyl-Lys (Boc)]Orn (Boc) and 118mg (1.1mmol) C₇H₉N gave 481mg (yield 64.8%) of the title compound as a pale yellow solid, ESI-MS (M/z):743 [ M + H]⁺。

Example 32 preparation of N^α- (β -carboline-3-formyl-Lys) -Orn-NHCH₂C₆H₅(11d)

From 742mg (1.0mmol) N as in example 11^α- [ β -carboline-3-formyl-Lys (Boc)]-Orn(Boc)-NHCH₂C₆H₅508mg (yield 93.7%) of the title compound are obtained as a yellow solid in ESI-MS (M/z):543[ M + H]⁺。

Example 33 preparation of N^α- [ β -carboline-3-formyl-Lys (5-fluorouracil-1-acetyl)]-N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅(14d)

From 271mg (0.5mmol) N according to example 12^α- (β -carboline-3-formyl-Lys) -Orn-NHCH₂C₆H₅And 210mg (1.1mmol) of 5-Fu-Ac gave 43mg (9.6% yield) of the title compound as a pale yellow solid; ESI-MS (M/z) 883 [ M + H]⁺；mp:177-178℃；

(c ═ 0.1, methanol); IR (cm)^-1):3277,3065,3008,2936, 2864,1692,1656,1597,1531,1497,1461,1242,727,696；¹H-NMR(300MHz,DMSO-d₆):δ/ppm＝12.32(s,1H),8.95(s,1H),8.84(s,1H),8.71(d,J＝8.4Hz,1H),8.63(t,J＝6.0Hz,1H), 8.39(d,J＝7.8Hz,1H),8.30(t,J＝5.1Hz,1H),8.22(t,J＝4.5Hz,1H),7.97(t,J＝6.0Hz,2H), 7.67(d,J＝7.5Hz,1H),7.60(t,J＝7.5Hz,1H),7.33-7.18(m,6H),4.65(m,1H),4.35-4.20(m, 7H),3.16-3.02(m,4H),1.68-1.58(m,3H),1.49-1.39(m,7H)。

EXAMPLE 34 evaluation of 14a-d antitumor cell proliferation Activity

The 14a-d of the present invention were all prepared to the desired concentration using medium containing 0.5% dimethyl sulfoxide (DMSO). 5 tumor cells of S180 (mouse sarcoma cell), HT-29 (human colon cancer cell), A549 (human lung cancer cell), MCF-7 (human breast cancer cell), and HL60 (human myeloblastosis cell) were used in combination.

The cells in good growth state and logarithmic growth phase are treated according to the 5 multiplied by 10⁴The cells were plated at a density of 100. mu.L/mL in 96-well plates. At 37 deg.C, 5% CO₂After 4 hours of incubation in an incubator, sterilized compounds of the invention were added at a predetermined concentration gradient of 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, using doxorubicin as a control. After an additional 48 hours of incubation, 25. mu.L of 5mg/mL thiazole blue solution was added to each well, incubated at 37 ℃ for 4 hours, the supernatant carefully removed (after centrifugation of the suspension cells and removal of the supernatant) and 100. mu.L DMSO was added to each well and the pellet was dissolved with shaking for about 15 min. O.d. (absorbance) values were immediately determined on a microplate reader at a wavelength of 570 nm. Calculating tumor inhibition rate and IC₅₀. The results are also shown in Table 1.

The results show that the 14a-d of the invention is sensitive to cell strains HT-29 and MCF-7 and has inhibition effect.

TABLE 114 a-d antitumor cell proliferation Activity (IC)₅₀±SDμM,n＝3)

EXAMPLE 35 evaluation of the antitumor Activity of 14a-d

Before measurement, 14a-d of the present invention was dissolved in 0.5% sodium carboxymethylcellulose (CMC-Na) with DMSO as an aid. Inoculating to ICR mice under aseptic condition for 7-10 days to obtain S180 meatAdding appropriate amount of normal saline to prepare into tumor cell suspension with cell number of 2 × 10⁷Perml, inoculated subcutaneously in the hind axilla of healthy male ICR mice, 0.2mL per mouse. 24h after tumor inoculation, the mice in the treatment group are orally administrated with 0.2ml by intragastric administration every day for 10 days, and the dose is 1 nmol/kg. The blank mice were gavaged orally with 0.2ml of 0.5% CMC-Na (containing 0.5% DMSO) daily. 5-fluorouracil (dose 150. mu. mol/kg) was used as a positive control, and 0.5% CMC-Na (containing 0.5% DMSO) was used as a negative control. The experiment is carried out until the 11 th day, the weight of the mice is weighed, the tumors of each group of mice are dissected and weighed, and finally, the tumor inhibition rate of each group of animals is counted. The therapeutic effect of solid tumors was expressed as a percentage of tumor weight inhibition, and was calculated as: (1-tumor weight in administration group/tumor weight in blank group) × 100%. The activity of the compounds is expressed as tumor weight or percent tumor inhibition and the data are shown in Table 2. The results show that 14a-d has good antitumor activity at a dose of 1nmol/kg, and show unexpected technical effects.

TABLE 214 influence of a-d on tumor regrowth in S180 sarcoma mice (mean. + -. SD g)

Note: n-10, a) p <0.05 compared to the CMC-Na group.

Example 36 evaluation of the effects of 14a-c on tumor regrowth in S180 sarcoma mice

Experimental methods As in example 35, when the compound was dosed at 1nmol/kg, 100nmol/kg and 1. mu. mol/kg, respectively, and a group of 5-fluorouracil was dosed at 1.5. mu. mol/kg, it was found that 5-fluorouracil was inactive at 1.5. mu. mol/kg, while 14a-c had good antitumor effect at 100nmol/kg, and thus the dose of 14a-c was reduced by 15 times as compared to 5-fluorouracil, and the data are shown in Table 3, and the results showed that 14a-c showed unexpected technical effects at 1 nmol/kg.

TABLE 314 a-c Effect on tumor regrowth in S180 sarcoma mice (mean. + -. SD g)

n 10, a) to 1nmol/kg dose group ratio p <0.05, b) to 100nmol/kg dose group ratio p <0.05, c) to CMC-Na ratio p > 0.05.

EXAMPLE 37 evaluation of the anti-inflammatory Activity of Compounds 14a-d

Male mice weighing 20. + -.2 g were orally administered 100nmol/kg of compound 14a-d or 1100. mu. mol/kg aspirin or 0.2mL/20g 0.5% CMC-Na (containing 0.5% DMSO) for 30 minutes, then mice were treated with xylene (0.03mL) on the left ear contour and 2 hours later the mice were sacrificed by cervical dislocation. The left and right ears of the mouse are cut off, round ear pieces are taken at the same positions of the two ears by a puncher with the diameter of 7mm, the round ear pieces are respectively weighed, and the weight difference of the two round ear pieces is calculated to be used as swelling degree. (degree of swelling-left auricle weight-right auricle weight) the activity of the compound is expressed in the degree of swelling. The experimental data statistics all adopt t test and variance analysis, and the swelling degree is (A)

mg) is given. The results show that the compounds of the invention have anti-inflammatory activity at a dose of 100nmol/kg, showing unexpected technical effects.

TABLE 414 influence of a-d on ear weight of mice with xylene-induced inflammation (mean. + -. SD mg)

Note: n-10, a) p <0.05 compared to the CMC-Na group.

Claims

1. 4N of the formula^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅,

Wherein AA is selected from L-Met, L-Cys (Bzl), L-Arg (NO)₂) L-Lys (5-fluorouracil-1-acetyl) residue.

2. Preparation of N according to claim 1^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅The method of (1), the method comprising:

(5) β -carboline-3-carboxylic acid is coupled with L-AA-OBzl to obtain β -carboline-3-formyl-AA-OBzl;

(6) 5-Fluorouracil (5-Fu) with Br-CH in 6N KOH solution₂COOH coupling to obtain 5-fluorouracil-1-yl acetic acid (5-Fu-Ac);

(7) β -carboline-3-formyl-AA-OBzl is hydrolyzed in 2N NaOH solution to generate β -carboline-3-formyl-AA;

(8) β -carboline-3-formyl-AA and Orn (Boc) -OBzl to obtain N^α- (β -carboline-3-formyl-AA) -orn (boc) -OBzl;

(9)N^α- (β -carboline-3-formyl-AA) -Orn (Boc) -OBzl hydrolyzing in 2N NaOH solution to generate N^α- (β -carboline-3-formyl-AA) -orn (boc);

(10)N^α- (β -carboline-3-formyl-AA) -Orn (Boc) coupling with benzylamine to give N^α- (β -carboline-3-formyl-AA) -Orn (Boc) -NHCH₂C₆H₅；

(11)N^α- (β -carboline-3-formyl-AA) -Orn (Boc) -NHCH₂C₆H₅In 4N hydrogen chloride-ethyl acetate solution, removing Boc at 0 deg.C, and converting into N^α- (β -carboline-3-formyl-AA) -Orn-NHCH₂C₆H₅；

(12)N^α- (β -carboline-3-formyl-AA) -Orn-NHCH₂C₆H₅(AA in the formula is as defined above) and 5-Fu-Ac to obtain N^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅。

3. N of claim 1^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅Application in preparing antitumor drugs.

4. N of claim 1^α- (β -carboline-3-formyl-AA) -N^ω(5-fluorouracil-1-acetyl) -Orn-NHCH₂C₆H₅Application in preparing anti-inflammatory drugs.