CN105273043A - Amino-acid benzyl ester modified beta-carboline, activity, nanometer structure, synthesis and application - Google Patents

Abstract

The invention discloses 1-[ethyl-Asp(Ala-OBzl)Ala-OBzl]-beta-carboline-3-carboxylic acid benzyl ester, discloses a preparation method for the compound, discloses the nanometer structure of the compound, discloses the activity of the compound on inhibiting tumor cell proliferation, and discloses application of the compound to prepare antitumor medicines.

Description

Amino acid benzyl ester modified β-carboline, activity, nanostructure, synthesis and application

technical field

The present invention relates to 1-[ethyl-Asp(Ala-OBzl)Ala-OBzl]-beta-carboline-3-carboxylic acid benzyl ester, relates to its preparation method, relates to its activity of inhibiting tumor cell proliferation, relates to its effect on The inhibition of tumor growth in S180-bearing mice further involves the cutting effect between them and tumor cell DNA. Therefore, the present invention relates to its application in the preparation of antitumor drugs. The invention belongs to the field of biomedicine.

Background technique

In recent years, cancer has become a world problem given the dramatic increase in cancer incidence and mortality worldwide. The number of cancer patients in my country ranks among the top in the world, and the number of cancer patients is increasing, and the demand for anti-tumor drugs is also increasing. There are many limitations in the current clinical application of anti-tumor drugs. With the advancement of science and technology and the development of science, finding safe and effective new anti-tumor drugs is one of the hotspots in drug research.

Cancer is a general term for a group of diseases that can affect any part of the body. Other terms used are malignancy and neoplasm. According to the World Health Organization, cancer is one of the leading causes of death in the world, especially in developing countries. The number of cancer deaths worldwide is expected to continue to rise, exceeding 13.1 million by 2030. Therefore, the development of new anti-tumor drugs with high efficiency, low toxicity and small side effects has always been one of the important topics of new drug research.

With the understanding of the characteristics of tumors and the nature of pathogenesis, anti-tumor drugs have been transitioning from traditional cytotoxic drugs to non-cytotoxic drugs in recent years. β-carbolines are cytotoxic antineoplastic compounds of natural origin. The inventors realized that the essence of the anti-tumor cytotoxicity of β-carboline is intercalation with tumor cell DNA. This form of cutting can cause cytotoxicity. The inventors have found that β-carboline can be inserted between the bases of the double helix between the DNA of tumor cells. In further studies, the inventors realized that the antitumor activity of β-carbolines comes from intercalation. The inventors also realized that introducing a dipeptide at the 1-position of β-carboline to generate benzyl β-carboline-3-carboxylate and derivatives can enhance the effect of β-carboline on tumor cells and enhance the anti-tumor activity. Based on these findings, the inventors proposed benzyl 1-[ethyl-Asp(Ala-OBzl)Ala-OBzl]-β-carboline-3-carboxylate. Compared with the inventor's previous invention of 1-(ethyl-AA-OBzl)-beta-carboline-3-carboxylate benzyl ester, the outstanding creativity of the present invention lies in that the link between β-carboline and tumor cell DNA is retained. The intercalation effect, showing high antitumor activity at low dose. Thus, benzyl 1-[ethyl-Asp(Ala-OBzl)Ala-OBzl]-β-carboline-3-carboxylate showed good tumor cell proliferation inhibitory effect and antitumor activity.

Contents of the invention

The first object of the present invention is to provide 1-[ethyl-Asp(Ala-OBzl)Ala-OBzl]-β-carboline-3-carboxylic acid benzyl ester of the following formula.

The second content of the present invention is to provide the method for preparing 1-[ethyl-Asp(Ala-OBzl)Ala-OBzl]-β-carboline-3-carboxylic acid benzyl ester, the method is made up of the following steps:

1) In the presence of trifluoroacetic acid (TFA), Trp-OBzl undergoes Pictet-Spengler condensation with 1,1,3,3-tetramethoxypropane to generate 3S-1-(2,2-dimethoxyethyl )-2,3,49-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid benzyl ester;

2) In a mixed solution of tetrahydrofuran (THF) and water, 3S-1-(2,2-dimethoxyethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4 -b]benzyl indole-3-carboxylate is oxidized by potassium permanganate to 1-(2,2-dimethoxyethyl)-1H-pyrido-3,4-b]indole-3- Benzyl carboxylate;

3) In a mixed solution of acetic acid, water and hydrochloric acid, 1-(2,2-dimethoxyethyl)-1H-pyrido[3,4-b]indole-3-carboxylic acid benzyl ester is hydrolyzed Benzyl 1-acetaldehyde-β-carboline-3-carboxylate was obtained;

4) In the presence of dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBt), Boc-Asp was condensed with Ala-OBzl in anhydrous tetrahydrofuran (THF) to form Boc-Asp (Ala- OBzl) Ala-OBzl;

5) removing Boc from Boc-Asp(Ala-OBzl)Ala-OBzl in an ethyl acetate solution of hydrogen chloride to obtain Asp(Ala-OBzl)-Ala-OBzl;

6) In THF solution, benzyl 1-acetaldehyde-β-carboline-3-carboxylate and Asp(Ala-OBzl)-Ala-OBzl were ammoniated and reduced to 1-[ethyl -Asp(Ala-OBzl)Ala-OBzl]-β-carboline-3-carboxylic acid benzyl ester.

The third content of the present invention is to determine the nanostructure of 1-[ethyl-Asp(Ala-OBzl)Ala-OBzl]-β-carboline-3-carboxylic acid benzyl ester.

The fourth content of the present invention is to evaluate the effect of benzyl 1-[ethyl-Asp(Ala-OBzl)Ala-OBzl]-β-carboline-3-carboxylate on inhibiting tumor cell proliferation.

The fifth content of the present invention is to clarify the inhibitory effect of benzyl 1-[ethyl-Asp(Ala-OBzl)Ala-OBzl]-β-carboline-3-carboxylate on tumor growth in S180-bearing mice.

The sixth content of the present invention is to evaluate the cutting action between the compound 1-[ethyl-Asp(Ala-OBzl)Ala-OBzl]-β-carboline-3-carboxylic acid benzyl ester and DNA.

Description of drawings

Figure 1 Synthetic route of compound 4. .i) thionyl chloride, benzyl alcohol; ii) trifluoroacetic acid, 1,1,3,3-tetramethoxypropane, dichloromethane; iii) potassium permanganate, tetrahydrofuran ice bath; iv) concentrated hydrochloric acid , glacial acetic acid, water; v) sodium cyanoborohydride, N-methylmorpholine, Asp(Ala-OBzl)Ala-OBzl.

Figure 2 Transmission electron micrographs of compound 4 in pure water at concentrations of 1×10 ^-5 M, 1×10 ^-7 M, and 1×10 ^-9 M.

Fig. 3 UV spectra of compound 4 and CT-DNA cuttings.

Fig. 4 Fluorescence spectra of compound 4 and CT-DNA cuttings.

Fig. 5 The viscosity curve of compound 4 and CT-DNA cuttings.

detailed description

In order to further illustrate the present invention, a series of examples are given below. These examples are entirely illustrative, and 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-1-(2,2-dimethoxyethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid Benzyl esters (1)

Pour 5g (15.2mmol) tryptophan benzyl ester into a 250mL round bottom flask, dissolve it in 50mL of dichloromethane, add 5mL of 1,1,3,3-tetramethoxypropane and 5mL of trifluoro Acetic acid, reaction at room temperature, reaction TLC (petroleum ether/acetone, 4/1) monitoring, 48h after the raw material point disappears, adjust pH=7 with concentrated ammonia water, concentrate to dryness under reduced pressure, dissolve the residue with ethyl acetate, ethyl acetate The solution was washed with saturated sodium bicarbonate solution, saturated sodium chloride solution, and saturated potassium bisulfate solution, then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure, and the residue was separated by column chromatography (petroleum ether/acetone , 4:1) afforded 2.8 g (47%) of the title compound as a pale yellow oil. ESI-MS (m/e): 395 [M+H] ⁺ .

Example 2 Preparation of 3S-1-(2,2-dimethoxyethyl)-pyrido[3,4-b]indole-3-carboxylic acid benzyl ester (2)

3.5 g (8.9 mmol) of 1-(2,2-dimethoxyethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxy Pour benzyl ester into a 250ml round bottom flask, dissolve in 100mL tetrahydrofuran, add 2.8g (17.77mmol) potassium permanganate under ice cooling, react for 4h, TLC (petroleum ether/acetone, 4/1) detects that the ultraviolet point disappears After the fluorescence point appeared, filter the reaction solution, add THF to rinse the filter cake, concentrate the filtrate under reduced pressure to remove THF, then extract the residual water phase with 70 mL of dichloromethane for a total of 3 extractions, combine the ester phases, and wash 3 times with saturated saline , dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/acetone, 3/1) to obtain 1.8 mg (51.9%) of the title compound as a pale yellow solid. ESI-MS (m/e): 391 [M+H] ⁺ .

Embodiment 3 prepares 1-acetaldehyde group-beta-carboline-3-carboxylate benzyl ester (3)

In a 100ml round bottom flask, add 1g (2.56mmol) 3S-1-(2,2-dimethoxyethyl)-pyrido[3,4-b]indole-3-carboxylic acid benzyl ester, 10ml Dissolve it with acetic acid, then add 1.25ml of water and 1.25ml of hydrochloric acid, stir at room temperature for 4 hours, TLC monitoring (petroleum ether/acetone, 3/1) the raw material spots disappear, add 100ml of ice-water mixture to the round bottom flask, and stir under ice bath Filter after 10 minutes, wash the filter cake with water and 5% aqueous sodium bicarbonate solution, dry it in the air, and directly apply it to the next reaction.

Example 4 Preparation of 1-[ethyl-Asp(Ala-OBzl)Ala-OBzl]-β-carboline-3-carboxylic acid benzyl ester (4)

Add 1690mg (2.25mmol) HCl·Asp(Ala-OBzl)Ala-OBzl, 20mL tetrahydrofuran into a round bottom flask, stir under ice bath, add 2mL N-methylmorpholine dropwise, adjust the pH to 8, dropwise dissolve 500mg (1.45mmol) tetrahydrofuran solution of benzyl 1-acetaldehyde-β-carboline-3-carboxylate, add an appropriate amount of anhydrous magnesium sulfate, stir at room temperature for 30min, add 45mg (0.714mmol) sodium cyanoborohydride, room temperature After stirring for 24 hours, TLC plate (dichloromethane/methanol, 30/1) monitored the disappearance of raw material spots, and stopped the reaction. The reaction solution was filtered, the filtrate was concentrated to dryness under reduced pressure, and the residue was dissolved in 150 mL of dichloromethane, and the obtained solution was washed three times with saturated aqueous sodium bicarbonate solution, saturated aqueous sodium chloride solution, and saturated aqueous potassium hydrogensulfate solution, and the dichloromethane layer was washed three times. Dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain a syrupy substance that is purified by silica gel column chromatography (dichloromethane/methanol, 50/1), and recrystallized from methanol/ether to obtain 783 mg (66.2%) of the title compound , a colorless solid. Mp165-166°C; ESI-MS (m/e): 784[M+H] ⁺ ; IR (KBr): 3286.7, 3201.8, 3178.7, 3089.9, 3062.9, 3032.1, 2700.3, 1732.1, 1651.1, 1550.8, 1500.6, 1454.3, 1373.3, 1346.3, 1303.9, 1249.9, 1199.7, 1134.1, 1111.0, 1060.9, 1010.7, 736.8, ^698.2.1 HNMR (300MHz, DMSO-d ₆ ) δ/ppm=12.64(s, 1H), 9.72(s, 1H), 9.27(d, 1H, J=6.3Hz), 8.86(d, 2H, J=6.6Hz), 8.40(d, 2H, J=8.1Hz), 7.70(d, 1H, J=8.4Hz), 7.61(t, 1H, J=7.5Hz), 7.52(d, 2H, J=6.9Hz), 7.32(m, 12H), 5.42(s, 2H), 5.08(m, 4H), 4.35(m, 3H), 3.67(m, 2H), 3.56 (s, 2H), 3.38 (s, 1H), 2.92 (s, 2H), 1.30 (dd, 6H, J ₁ =7.5 Hz, J ₂ =11.4 Hz).

Experimental example 1 evaluates the effect of compound 4 on inhibiting tumor cell proliferation

Compound 4 was prepared to the desired concentration with 1640 medium containing 0.4% DMSO. A549, MCF-7, HL60, S180, and SF-295 cells in good growth state and in logarithmic growth phase were seeded in 96-well plates at a density of 4×10 ⁴ cells/mL, 100 μL per well. Cultivate in a 5% _CO2 incubator at 37°C for 4 hours, add the sterilized solution of compound 4 according to the preset concentration gradient of 100 μM, 50 μM, 25 μM and 12.5 μM, and add an equal volume of dissolved samples containing 0.4 μM to the control group. 1640 medium in % DMSO. After continuing to cultivate for 48 hours, add 25 μL of MTT solution with a concentration of 5 mg/mL to each well and incubate at 37 °C for 4 hours. After carefully removing the supernatant, add 100 μL DMSO to each well and shake for about 15 minutes to dissolve the precipitate. Immediately measure the OD (absorbance) value on a microplate reader at a wavelength of 570 nm. The inhibition rate was calculated according to inhibition rate=[(OD average value of 1640 medium group containing 0.4% DMSO-OD average value of compound 4 group)/OD value of 1640 medium group containing 0.4% DMSO]×100%". The experiment was repeated 3 times in parallel, and the inhibition rate was plotted against the concentration of compound 4 to calculate the IC ₅₀ (half effective inhibitory concentration) value.

Compound 4 was prepared to the desired concentration with DMEM medium containing 0.4% DMSO. U2OS and HaCaT cells in good growth state and in the logarithmic growth phase were inoculated in 96-well plates at a density of 4×10 ⁴ cells/mL, 100 μL per well. Cultivate in a 37°C, 5% _CO2 incubator for 4 hours, add the sterilized solution of compound 4 according to the preset concentration gradient of 100 μM, 50 μM, 25 μM and 12.5 μM, and add an equal volume of dissolved samples containing 0.4 μM to the control group. %DMSO in DMEM medium. After continuing to cultivate for 48 hours, add 25 μL of MTT solution with a concentration of 5 mg/mL to each well and incubate at 37 °C for 4 hours. After carefully removing the supernatant, add 100 μL DMSO to each well and shake for about 15 minutes to dissolve the precipitate. Immediately measure the OD (absorbance) value on a microplate reader at a wavelength of 570 nm. The inhibition rate was calculated as inhibition rate=[(OD average value of DMEM medium group containing 0.4% DMSO-OD average value of compound 4 group)/OD average value of DMEM medium group containing 0.4% DMSO]×100%. The experiment was repeated 3 times in parallel, and the inhibition rate was plotted against the concentration of compound 4 to calculate the IC ₅₀ (half effective inhibitory concentration) value of the compound of the present invention.

The results are listed in Table 1 and Table 2. The results showed that compound 4 generally had an inhibitory effect on tumor cells, with an average IC ₅₀ of 20 μM, and did not harm normal skin cells HaCaT, with an IC ₅₀ greater than 100.

Table 1 Compound 4 inhibits tumor cell proliferation activity (IC ₅₀ , )

n=18

Table 2 Compound 4 inhibits tumor cell proliferation activity (IC ₅₀ , )

n=18

Experimental Example 2 Evaluation of the activity of compound 4 in inhibiting tumor growth in S180 mice

Compound 4 was added with DMSO (dimethyl sulfoxide) to help dissolve it before measurement, and suspended with 0.5% CMC-Na. Doxorubicin was dissolved in saline. Under sterile conditions, take the S180 sarcoma inoculated in ICR mice for 7-10 days, add appropriate amount of normal saline to prepare tumor cell suspension, the number of cells is 1.4× ¹⁰⁷ cells/mL, and inoculate under the skin of healthy male ICR mice forelimb axils , each mouse was injected with 0.2 mL. 24 hours after tumor inoculation, mice in compound 4 treatment group were orally administered 0.2 mL of compound 4 in CMC-Na suspension daily for 7 consecutive days at a dose of 1 μmol/kg. Mice in the blank group were orally administered 0.2 mL of 0.5% sodium carboxymethyl cellulose. Doxorubicin (dose 2 μmol/kg) was used as positive control. On the 8th day of the experiment, the mice were weighed. After ether anesthesia, the tumors, hearts, liver, spleen, kidneys and brains of the mice in each group were dissected and weighed. The tumor inhibition rate and viscera body ratio of the animals in each group were calculated. The curative effect of solid tumors is expressed by tumor weight and inhibition percentage. Inhibition rate of tumor weight %=[1-(tumor weight of compound 4 group/tumor weight of blank group)]×100%.

The tumor weights are listed in Table 3. The results in Table 3 showed that the tumor weight of the mice in the compound 4 group was significantly lower than that of the mice in the blank group, indicating that the compound 4 had good antitumor activity. This effective dose of compound 4 (1 μmol/kg) is 8.9 times lower than the effective dose of 1-(ethyl-AA-OBzl)-β-carboline-3-carboxylic acid benzyl ester (8.9 μmol/kg) disclosed by the inventors .

Table 34 Effects on tumor growth in S180 tumor-bearing mice

n=15; a) Compared with 0.5% sodium carboxymethylcellulose group, p<0.01.

The transmission electron microscope of experimental example 3 compound 4

Compound 4 was prepared into 10 ^-5 M, 10 ^-7 M and 10 ^-9 M solutions with triple-distilled water, and a small amount (about 10 μL) was dropped on the surface of the copper grid, and the copper grid was lined with filter paper, and dried naturally. Observe its morphology and particle size under an electron microscope (JEOL, JEM-1230), and record it with photos. The electron micrograph of compound 4 is shown in Figure 2. It can be seen that compound 4 self-assembles into nanospheres with a diameter of 70-210 nm in aqueous solution.

Experimental example 4 Determination of the cutting effect of compound 4 and CT-DNA

In order to confirm the cutting between compound 4 and tumor cell DNA, the cutting effect of compound 4 and CT-DNA was described by ultraviolet spectrum and fluorescence spectrum.

1) Using UV spectrum to describe the cutting effect of compound 4 and CT-DNA

2 mL of compound 4 with a concentration of 10 ^-5 M was placed in a cuvette, and its ultraviolet spectrum was measured. Then use a micro-syringe to add CT-DNA solution with a concentration of 4.70×10 ^-4 M to the sample cup and the blank cup in turn (20 μL each time, so it is considered to keep the volume of the test system constant, that is, the system concentration remains constant) for 5 minutes Afterwards, the ultraviolet spectrum of the mixture was measured (the final concentrations of CT-DNA were 0, 4.7, 9.4, 14.1, 18.8, and 23.5 μM), and the changes of the ultraviolet spectrum before and after the DNA was combined with the compound were investigated. The resulting UV spectrum is shown in Figure 3. The results showed that when no CT-DNA was added, the maximum absorption wavelength of compound 4 in the wavelength range of 230-400nm was 282nm, and the absorption intensity was 0.416. After adding CT-DNA, as the concentration of CT-DNA increases, the π→π* transition of compound 4 has a certain degree of color reduction effect in the wavelength range of 230-400nm, and an obvious red shift phenomenon occurs. When the concentration of CT-DNA was 23.5μM, its maximum absorption wavelength was 288.5nm, red-shifted by 6.5nm, and its maximum absorption was 0.302, which decreased by 0.114 and decreased by 27.4%, indicating that compound 4 and CT-DNA had cuttage.

2) Using fluorescence spectrum to describe the cutting effect of 4 and CT-DNA

Pipette 2 mL of compound 4 with a concentration of 10 ^-5 M into a quartz cuvette, and measure the fluorescence spectrum. Add 20 μL of CT-DNA solution with a concentration of 4.70×10 ^-4 M to the cuvette successively (20 μL is added each time, so it is considered that the volume of the test system remains constant, that is, the system concentration remains constant), and after 5 minutes, measure the fluorescence quenching Spectra (the final concentrations of CT-DNA were 0, 4.7, 9.4, 14.1, 18.8, 23.5 μM, respectively). The fluorescence emission and excitation slits are 5nm and 2.5nm respectively, the excitation wavelength is fixed at 280nm, the scanning speed is 1500nm/min, the emission spectrum ranges from 320 to 500nm, and is measured at 37°C. The resulting fluorescence spectrum is shown in Figure 4. The results showed that compound 4 was a fluorescent substance with a maximum emission wavelength of 382nm and a fluorescence intensity of 3904. With the continuous addition of CT-DNA, the fluorescence intensity gradually decreased. When the concentration of CT-DNA was 23.5 μM, the fluorescence intensity was 3052, which decreased by 852, which was 21.8%. The maximum absorption peak is red-shifted. It can be seen that DNA has a fluorescence quenching effect on compound 4, indicating that compound 4 and CT-DNA have been intertwined.

3) Using the viscosity method to describe the cutting effect of compound 4 and CT-DNA

Put the Ubbeholde viscometer in a water bath at 37°C, measure the viscosity of PBS (three times in parallel), take it as T0, and then measure the viscosity of CT-DNA (100 μM) solution, the sample volume is 10 mL. Take 10 μL of compound 4 (concentration is 10 mM), that is, the final concentration of compound 4 in the sample is 10 μM. Pipette and mix the measurement three times to get the average value, and then add 10 μL to the sample solution successively, that is, the final concentration of compound 4 in the sample is 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. η=(tt ₀ )/t ₀ , where t ₀ is the time required for PBS to pass through the capillary, and t is the time required for the drug-containing CT-DNA solution to pass through the capillary. The binding ratio C ₅ /C _CT-DNA was plotted as (η/η ₀ ) ^1/3 . The viscosity change of CT-DNA is shown in Figure 5. The results showed that as the concentration of compound 4 increased, the viscosity of CT-DNA solution increased, and the increasing trend gradually decreased, indicating that compound 4 and CT-DNA had cuttings.

Claims (4)

1. compound 1-[ethyl-Asp (Ala-OBzl) Ala-OBzl]-β-carboline-3-benzyl carboxylate.

2. the preparation method of 1-[ethyl-Asp (Ala-OBzl) Ala-OBzl]-β-carboline-3-benzyl carboxylate of claim 1, the method is made up of following steps:

1) there is Pictet-Spengler condensation in Trp-OBzl and 1,1,3,3-tetramethoxy propane under trifluoracetic acid (TFA) exists, generate 3S-1-(2,2-dimethoxy-ethyl)-2,3,4,9-tetrahydrochysene-1H-pyrido [3,4-b] Indole-3-Carboxylic Acid benzyl ester;

2) in the mixing solutions of tetrahydrofuran (THF) (THF) with water, 3S-1-(2,2-dimethoxy-ethyl)-2,3,4,9-tetrahydrochysene-1H-pyrido [3,4-b] Indole-3-Carboxylic Acid benzyl ester is 1-(2 by potassium permanganate oxidation, 2-dimethoxy-ethyl)-1H-pyrido [3,4-b] Indole-3-Carboxylic Acid benzyl ester;

3) in the mixing solutions of acetic acid, water and hydrochloric acid, 1-(2,2-dimethoxy-ethyl)-1H-pyrido [3,4-b] Indole-3-Carboxylic Acid benzyl ester is hydrolyzed and obtains 1-aldehyde-base-β-carboline-3-benzyl carboxylate;

4) under dicyclohexylcarbodiimide (DCC) and N-hydroxy benzo triazole (HOBt) exist, Boc-Asp is Boc-Asp (Ala-OBzl) Ala-OBzl with Ala-OBzl condensation in anhydrous tetrahydro furan (THF);

5) in the ethyl acetate solution of hydrogenchloride, Boc-Asp (Ala-OBzl) Ala-OBzl is removed Boc and obtain Asp (Ala-OBzl)-Ala-OBzl;

6) in THF solution, 1-aldehyde-base-β-carboline-3-benzyl carboxylate and Asp (Ala-OBzl)-Ala-OBzl are reduced to 1-[ethyl-Asp (Ala-OBzl) Ala-OBzl]-β-carboline-3-benzyl carboxylate by sodium cyanoborohydride ammonification.

3. 1-[ethyl-Asp (Ala-OBzl) Ala-OBzl]-β-carboline-3-benzyl carboxylate nanostructure of claim 1.

4. 1-[ethyl-Asp (Ala-OBzl) Ala-OBzl]-β-carboline-3-benzyl carboxylate of claim 1 is preparing the application in antitumor drug.