WO2022028421A1 - 可荧光示踪的氨基酸衍生物及其制备方法和应用 - Google Patents

可荧光示踪的氨基酸衍生物及其制备方法和应用 Download PDF

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WO2022028421A1
WO2022028421A1 PCT/CN2021/110331 CN2021110331W WO2022028421A1 WO 2022028421 A1 WO2022028421 A1 WO 2022028421A1 CN 2021110331 W CN2021110331 W CN 2021110331W WO 2022028421 A1 WO2022028421 A1 WO 2022028421A1
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amino acid
traceable
reaction
acid derivative
formula
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French (fr)
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王玉记
王彦明
朱迪
卢玉
刘波涛
曲爱娟
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首都医科大学
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Definitions

  • the invention relates to the technical field of biomedicine, in particular to a fluorescent traceable amino acid derivative and a preparation method and application thereof.
  • PTMs Post-translational modifications
  • PTMs Post-translational modifications
  • Peptidyl arginine deiminase 4 (PAD4) is one such target because it acts similarly to histone deacetylases on gene expression. PAD4 acts as a transcriptional co-regulator that catalyzes the calcium-dependent conversion of specific arginine residues in proteins to citrulline.
  • PAD4 is not only a transcriptional co-repressor of the tumor suppressor protein p53, but also involved in mediating the formation of malignant tumors. Due to its regulatory role in cell signaling pathways and disease pathogenesis, PAD4 has emerged as a potential therapeutic target for a variety of diseases. Based on the small molecule substrate N-benzoyl-L-arginine amide (BAA) of PAD4, researchers have developed a variety of effective and irreversible haloacetamidine PAD4 inhibitors, but there are still unsatisfactory biological activities, etc. problem.
  • BAA N-benzoyl-L-arginine amide
  • the purpose of the present invention is to provide a fluorescent traceable amino acid derivative, a preparation method and application thereof, and the fluorescent traceable amino acid derivative provided by the present invention has good biological activity.
  • the present invention provides a fluorescent traceable amino acid derivative, which has the structure shown in formula I:
  • R is any one of the groups represented by formulas 1 to 10:
  • the present invention provides the preparation method of the fluorescently traceable amino acid derivative according to the above technical solution, wherein,
  • the first reaction raw material is Tos ⁇ Arg(NO 2 )-OBzl, HCl ⁇ LV-OBzl or N-( 2-aminoethyl)methanesulfonamide;
  • the preparation method of the fluorescently traceable amino acid derivative comprises the following steps:
  • the structural formula of the second reaction raw material is as follows:
  • the third reaction raw material is dissolved in tetrahydrofuran, the obtained mixed solution is mixed with 1-hydroxybenzotriazole and dicyclohexylcarbodiimide, and then activated, then the obtained activation system is mixed with benzylamine, and N-methyl
  • the pH value of the obtained mixed solution is adjusted to 8-9 by morpholine, and a condensation reaction is carried out to obtain the third intermediate product;
  • the fourth intermediate product and 4-chloro-7-nitro-2,1,3-benzoxaoxadiazole are dissolved in methanol, and the resulting mixed solution is treated with N,N-diisopropylethylamine The pH value is adjusted to 9.5 ⁇ 10.5, and the substitution reaction is carried out to obtain the fifth intermediate product;
  • the structural formula of the third reaction raw material is as follows:
  • the fourth reaction raw material, the fifth reaction raw material and methanol are mixed, and the pH value of the obtained mixed solution is adjusted to 9.5-10.5 by using N,N-diisopropylethylamine, and a substitution reaction is carried out to obtain an amino acid derivative that can be traced by fluorescence. thing;
  • the fifth reaction raw material is ethyl 2-chloroacetimidate or ethyl 2-fluoroacetimidate, and the structural formula of the fourth reaction raw material is as follows:
  • n 3 or 4.
  • the temperature of the substitution reaction in (i) is 15-35°C.
  • the temperature of the substitution reaction in (ii) is 15-35° C.
  • the hydrolysis reaction is carried out under ice bath conditions
  • the ammonia substitution reaction is carried out under ice bath conditions.
  • the temperatures of the condensation reaction, hydrogenolysis reaction and substitution reaction in (iii) are independently 15-35° C., and the hydrolysis reaction is carried out under ice bath conditions.
  • the temperature of the substitution reaction in (iv) is 15-35°C.
  • the present invention provides the application of the fluorescently traceable amino acid derivatives described in the above technical solution in the preparation of antitumor drugs.
  • the tumor comprises lung cancer, colon cancer, osteosarcoma or breast cancer.
  • the present invention provides a fluorescent traceable amino acid derivative having the structure shown in formula I.
  • the amino acid derivative provided by the present invention mainly uses a fluorescent traceable functional group to modify the amino acid skeleton, NBD-Cl(4 -Chloro-7-nitro-2,1,3-benzoxaoxadiazole) has the characteristics of low polarity and strong fluorescence, which can be used for subcellular imaging by modifying the N-terminus of the amino acid backbone. It is small in size, lacks reaction orthogonality, and has little interference with the biochemical reaction of the organism itself.
  • the results of the examples show that the fluorescently traceable amino acid derivatives provided by the present invention have good biological activity and can be fluorescently traced in vitro and in vivo.
  • Fig. 1 is the TEM image, SEM image, hydrated particle size change curve and Zeta potential change curve of compounds ZD-B and ZD-E-1;
  • Figure 2 is a graph showing the antitumor activity of different test compounds in the mouse S180 sarcoma model
  • Figure 3 is a graph showing the antitumor activity of the dominant test compounds in the mouse S180 sarcoma model
  • Figure 4 is a graph showing the growth trend of tumors in mice injected with compounds ZD-E-1 and ZD-F-1;
  • Figure 5 is a graph showing the visceral body ratio of mice injected with compounds ZD-E-1 and ZD-F-1;
  • Figure 6 is a graph showing the fluorescence co-localization of compounds ZD-B and ZD-E-1 in U2Os cells.
  • the present invention provides a fluorescent traceable amino acid derivative, which has the structure shown in formula I:
  • R is in formula I, and R is any one of the groups represented by formulas 1 to 10:
  • the fluorescently traceable amino acid derivative is specifically any one of the compounds represented by formulas I-1 to I-10:
  • the present invention provides a method for preparing the fluorescently traceable amino acid derivative.
  • the present invention preferably adopts different preparation methods according to the type of R in the fluorescently traceable amino acid derivative, which will be described in detail below.
  • the raw materials used are commercially available commodities well known to those skilled in the art.
  • the preparation method of the fluorescently traceable amino acid derivative comprises the following steps:
  • the first reaction raw material is Tos ⁇ Arg(NO 2 )-OBzl, HCl ⁇ LV-OBzl or N-( 2-aminoethyl)methanesulfonamide;
  • the reaction route is as follows:
  • the molar ratio of the 4-chloro-7-nitro-2,1,3-benzoxaoxadiazole (NBD-Cl) to the first reaction raw material is preferably 5:6; the The dosage ratio of NBD-Cl to methanol is preferably 5 mmol: 90-110 mL, more preferably 5 mmol: 100 mL.
  • NBD-Cl is preferably dissolved in methanol, and the first reaction raw material is added to the obtained NBD-Cl methanol solution under the stirring condition of an ice bath (0° C.), and then N,N-diisopropylethylamine ( DIPEA) to adjust the pH value of the obtained mixed solution to 9.5-10.5, remove the ice bath, and carry out the substitution reaction in the dark.
  • DIPEA N,N-diisopropylethylamine
  • the pH value of the obtained mixed solution is preferably adjusted to 10 by using N,N-diisopropylethylamine (DIPEA).
  • the temperature of the substitution reaction is preferably 15-35°C, more preferably 20-30°C; the present invention preferably performs the substitution reaction at room temperature, that is, no additional heating or cooling is required; In the embodiment, the substitution reaction is specifically carried out under the condition of 25°C.
  • the present invention preferably separates and purifies the obtained system through a medium-pressure preparation column, the used purification reagent is a mixture of EA and PE, and the volume fraction of EA is 35-50%, which is dissolved in methanol, sonicated, and left to stand for precipitation.
  • the solid is filtered under reduced pressure, and the obtained solid material is the amino acid derivative that can be traced by fluorescence.
  • the source of the first reaction raw material is not particularly limited in the present invention, and it can be prepared by using a commercially available commodity or a well-known method well known to those skilled in the art.
  • the Tos ⁇ Arg(NO 2 )-OBzl and N-(2-aminoethyl) methanesulfonamide are specifically commercially available products, and the HCl ⁇ LV-OBzl is preferably based on the methods well known to those skilled in the art.
  • the method is prepared, and the reaction scheme is as follows (an example of a specific preparation method is described in detail in Example 2 of the present invention):
  • the preparation method of the fluorescently traceable amino acid derivative comprises the following steps:
  • the structural formula of the second reaction raw material is as follows:
  • the reaction route is as follows:
  • the present invention 4-chloro-7-nitro-2,1,3-benzoxaoxadiazole, methanol and the second reaction raw material are mixed, and N,N-diisopropylethylamine is used to make the obtained mixed solution
  • the pH value is adjusted to 9.5-10.5, and the substitution reaction is carried out in the dark condition to obtain the first intermediate product.
  • the molar ratio of the NBD-Cl to the second reaction raw material (HCl ⁇ Orn(Boc)-OBzl) is preferably 5:6, and the dosage ratio of the NBD-Cl to methanol is preferably 5mmol:90 ⁇ 110 mL, more preferably 5 mmol: 100 mL.
  • NBD-Cl is preferably dissolved in methanol, and the second reaction raw material is added to the obtained NBD-Cl methanol solution under the condition of stirring in an ice bath, and then the pH value of the obtained mixed solution is adjusted to 9.5-10.5 by DIPEA, and then removed.
  • the substitution reaction was carried out in an ice bath and protected from light.
  • the pH value of the obtained mixed solution is preferably adjusted to 10 by using DIPEA.
  • the temperature of the substitution reaction is preferably 15-35° C., more preferably 20-30° C.; in the present invention, the substitution reaction is preferably carried out at room temperature.
  • the present invention preferably separates and purifies the obtained system through a medium pressure preparation column, and the used purification reagent is a mixture of EA and PE, and the volume fraction of EA is 35% to obtain the first intermediate product (NBD-Orn (Boc). )-OBzl).
  • the present invention dissolves the first intermediate product in ethyl acetate, mixes the obtained mixed solution with an ethyl acetate solution of HCl, and then performs a hydrolysis reaction to obtain a second intermediate product.
  • the concentration of the first intermediate product in the obtained mixed solution is preferably 0.35-0.45 mol/L, more preferably 0.4 mol/L; the acetic acid of the HCl
  • the concentration of HCl in the ethyl ester solution is preferably 3.5 to 4.5 mol/L, more preferably 4 mol/L; the volume ratio of the mixed solution to the ethyl acetate solution of HCl (denoted as HCl/EA solution) is preferably 1:2 .
  • the first intermediate product is preferably dissolved in ethyl acetate, and then HCl/EA solution is added to the obtained mixed solution in a fume hood under the condition of stirring in an ice bath, and the hydrolysis reaction is carried out under the condition of stirring in an ice bath.
  • the present invention preferably under the condition of stirring in a warm water bath at 37 ° C, the obtained system is vacuumed and drained, the residue is redissolved with dry EA and then drained again, and this operation is continued for 3 times until there is no obvious acid. Gas remains, and a second intermediate product is obtained.
  • the present invention dissolves the second intermediate product in methanol, uses triethylamine to adjust the pH value of the obtained mixed solution to 9.5-10.5, and performs ammonia substitution reaction under light-shielding conditions to obtain fluorescent light. Traced amino acid derivatives.
  • the second intermediate product is preferably dissolved in methanol, and the pH value is adjusted to 9.5-10.5 (preferably 10) with triethylamine under the stirring condition of ice bath, and the ammonia substitution reaction is carried out under the condition of avoiding light.
  • the present invention preferably separates and purifies the obtained system through a medium pressure preparative column.
  • the purification reagent used is a mixture of EA and PE, and the volume fraction of EA is 32% to obtain fluorescently traceable amino acid derivatives.
  • the preparation method of the fluorescently traceable amino acid derivative comprises the following steps:
  • the third reaction raw material is dissolved in tetrahydrofuran, the obtained mixed solution is mixed with 1-hydroxybenzotriazole and dicyclohexylcarbodiimide, and then activated, then the obtained activation system is mixed with benzylamine, and N-methyl
  • the pH value of the obtained mixed solution is adjusted to 8-9 by morpholine, and a condensation reaction is carried out to obtain the third intermediate product;
  • the fourth intermediate product and 4-chloro-7-nitro-2,1,3-benzoxaoxadiazole are dissolved in methanol, and the resulting mixed solution is treated with N,N-diisopropylethylamine The pH value is adjusted to 9.5 ⁇ 10.5, and the substitution reaction is carried out to obtain the fifth intermediate product;
  • the structural formula of the third reaction raw material is as follows:
  • the fluorescently traceable amino acid derivative when n is preferably 3, has the structure shown in formula I-5, and when n is preferably 4, the fluorescently traceable amino acid derivative has the formula Structure shown in I-7.
  • the third reaction raw material is dissolved in tetrahydrofuran, the obtained mixed solution is mixed with 1-hydroxybenzotriazole and dicyclohexylcarbodiimide, and then activated, and then the obtained activation system is mixed with benzylamine, and N- The pH value of the obtained mixed solution was adjusted to 8-9 by methylmorpholine, and a condensation reaction was carried out to obtain the third intermediate product.
  • the dosage ratio of the third reaction raw material and tetrahydrofuran (THF) is preferably 1 mmol: (9-11) mL, more preferably 1 mmol: 10 mL;
  • the molar ratio of oxazole (HOBt) and dicyclohexylcarbodiimide (DCC) is preferably 10:(11-13):(11-13), more preferably 10:12:12;
  • the dosage ratio of benzylamine is preferably 10 mmol: (1.6-5.6) mL.
  • the third reaction raw material is preferably dissolved in tetrahydrofuran, and 1-hydroxybenzotriazole and dicyclohexylcarbodiimide are added to the obtained mixed solution under the condition of stirring in an ice bath, and activated for 8 to 12 minutes, and a white solid is formed.
  • the activation is preferably 10min
  • benzylamine is added to the obtained activation system, the pH value of the obtained mixed solution is adjusted to 8-9 by N-methylmorpholine (NMM), and the ice bath is removed. , the condensation reaction is carried out.
  • the temperature of the condensation reaction is preferably 15-35°C, more preferably 20-30°C; the present invention preferably performs the condensation reaction at room temperature.
  • the third intermediate product is dissolved in methanol, and in the presence of palladium carbon, a hydrogenolysis reaction is carried out in a hydrogen atmosphere to obtain a fourth intermediate product.
  • the dosage ratio of the third intermediate product to methanol is preferably (5-10) mmol: (40-50) mL; the palladium-carbon is used as a catalyst, and the dosage can ensure that the hydrogenolysis reaction proceeds smoothly.
  • the temperature of the hydrogenolysis reaction is preferably 15-35°C, more preferably 20-30°C; in the present invention, the hydrogenolysis reaction is preferably carried out at room temperature.
  • the obtained system is preferably filtered under normal pressure, the palladium carbon is removed, and the solvent in the filtrate is removed to obtain the fourth intermediate product.
  • the fourth intermediate product and 4-chloro-7-nitro-2,1,3-benzoxaoxadiazole are dissolved in methanol, and N,N-di
  • the pH value of the obtained mixed solution is adjusted to 9.5-10.5 by isopropylethylamine, and the substitution reaction is carried out to obtain the fifth intermediate product.
  • the molar ratio of the NBD-Cl and the fourth intermediate product is preferably 5:6; the dosage ratio of the NBD-Cl and methanol is preferably 5 mmol:(40-100) mL.
  • NBD-Cl and the fourth intermediate product are preferably dissolved in methanol, and under the condition of stirring in an ice bath, DIPEA is used to adjust the pH value of the system to 9.5-10.5; the ice bath is removed, and a substitution reaction is performed.
  • DIPEA is used
  • the pH of the resulting mixed solution is preferably adjusted to 10.
  • the temperature of the substitution reaction is preferably 15-35° C., more preferably 20-30° C.; in the present invention, the substitution reaction is preferably carried out at room temperature.
  • the present invention preferably spins the obtained system under reduced pressure, and the obtained residue is separated and purified by a medium pressure preparation column.
  • the purification reagent used is a mixture of EA and PE, and the volume fraction of EA is 35%. Five intermediate products.
  • the present invention dissolves the fifth intermediate product in ethyl acetate, mixes the obtained mixed solution with an ethyl acetate solution of HCl, and then performs a hydrolysis reaction to obtain an amino acid derivative that can be fluorescently traced .
  • the concentration of the fifth intermediate product in the obtained mixed solution is preferably 0.5-0.6 mol/L; the concentration of HCl in the HCl ethyl acetate solution is preferably is 2-4 mol/L; the volume ratio of the mixed solution to HCl in ethyl acetate solution (referred to as HCl/EA solution) is preferably 1: (2-3).
  • the fifth intermediate product is preferably dissolved in ethyl acetate, and then HCl/EA solution is added to the obtained mixed solution in a fume hood under the condition of stirring in an ice bath, and the hydrolysis reaction is carried out under the condition of stirring in an ice bath.
  • the present invention preferably performs suction filtration on the obtained system to remove the solvent, and sequentially uses ethyl acetate and diethyl ether to carry out suction filtration and washing on the obtained residue to obtain a fluorescent traceable amino acid derivative (in the form of hydrochloride) .
  • the preparation method of the fluorescently traceable amino acid derivative comprises the following steps:
  • the fourth reaction raw material, the fifth reaction raw material and methanol are mixed, and the pH value of the obtained mixed solution is adjusted to 9.5-10.5 by using N,N-diisopropylethylamine, and a substitution reaction is carried out to obtain an amino acid derivative that can be traced by fluorescence. thing;
  • the fifth reaction raw material is ethyl 2-chloroacetimidate or ethyl 2-fluoroacetimidate, and the structural formula of the fourth reaction raw material is as follows:
  • n 3 or 4;
  • the reaction route is as follows:
  • the fluorescently traceable amino acid derivative when n is preferably 3, has the structure shown in formula I-6 or formula I-9; when n is preferably 4, the fluorescently traceable amino acid derivative has the structure shown in formula I-6 or formula I-9.
  • the amino acid derivative has the structure shown in formula I-8 or I-10.
  • the fifth reaction raw material is ethyl 2-chloroacetylimide; when R is a group represented by formula 9 or formula 10 In the case of a group, the fifth reaction raw material is 2-fluoroacetylimide ethyl ester.
  • the molar ratio of the fourth reaction raw material and the fifth reaction raw material is preferably 1:(1 ⁇ 2), and the dosage ratio of the fourth reaction raw material to methanol is preferably (1 ⁇ 3) mmol:( 40 ⁇ 50)mL.
  • the fourth reaction raw material is preferably dissolved in methanol
  • the fifth reaction raw material is added to the obtained mixed solution under the condition of stirring in an ice bath
  • the pH value of the system is adjusted by DIPEA to 9.5-10.5
  • the ice bath is removed
  • the substitution reaction is carried out.
  • the pH value of the obtained mixed solution is preferably adjusted to 10 by using DIPEA.
  • the temperature of the substitution reaction is preferably 15-35° C., more preferably 20-30° C.; in the present invention, the substitution reaction is preferably carried out at room temperature.
  • the present invention preferably spins the obtained system to dry the solvent, the residue is reconstituted with methanol aqueous solution with a volume fraction of 5%, and is separated and purified by C18 silica gel column chromatography, and the used reagent is a volume fraction of 40-60%.
  • the methanol aqueous solution spin off the methanol, freeze-dry to remove the water, and obtain the amino acid derivatives that can be traced by fluorescence.
  • the present invention provides the application of the fluorescently traceable amino acid derivatives described in the above technical solution in the preparation of antitumor drugs.
  • the tumor preferably includes lung cancer, colon cancer, osteosarcoma or breast cancer.
  • the antitumor drug comprises the fluorescently traceable amino acid derivative and a pharmaceutically acceptable excipient; the content of the fluorescently traceable amino acid derivative in the antitumor drug is preferably 20-80wt %, the pharmaceutically acceptable adjuvant preferably includes one or more of cyclodextrin, ⁇ -hydroxycyclodextrin and mannitol; the dosage form of the antitumor drug preferably includes lyophilized powder, nanoemulsion or Liposomes.
  • the target product ZD-A is a yellow solid powder with bright green fluorescence in organic solvent.
  • the reaction was judged to be complete.
  • the obtained system was separated and purified by a medium pressure preparative column (the purification reagent used was a mixture of EA and PE, and the volume fraction of EA was 50%), dissolved in 2 mL of methanol, sonicated, and left to stand to precipitate a solid, and filtered under reduced pressure to obtain 750 mg (32.1%)
  • the target product ZD-B is a yellow solid powder with bright green fluorescence in organic solvent.
  • the obtained system was separated and purified by a medium pressure preparative column (the used purification reagent was a mixture of EA and PE, and the volume fraction of EA was 35%), and was dissolved in 2 mL of methanol and then sonicated. (46.3%)
  • the target product ZD-C is orange-red solid powder with bright green fluorescence in organic solvent.
  • Weight loss method Weigh 0.998g (5mmol) of NBD-Cl into an eggplant flask, dissolve with 100mL of anhydrous methanol to obtain a yellow clear and transparent solution, add a stirrer; under ice bath stirring, add 2.15g (6mmol) of HCl ⁇ Orn (Boc)-OBzl, and adjust the pH value to 10 with 2.6 mL DIPEA; remove the ice bath, and react in the dark for 8 h at room temperature. It was observed that the color of the reaction solution gradually deepened to dark green, accompanied by green fluorescence.
  • the obtained system was separated and purified by a medium pressure preparative column (the used purification reagent was a mixture of EA and PE, and the volume fraction of EA was 35%) to obtain 990 mg (41.2%) of the target product NBD-Orn(Boc)-OBzl, which was Orange-red solid with bright green fluorescence in organic solvents.
  • the solvent was spin-dried, and the residue was reconstituted with methanol aqueous solution with a volume fraction of 5%, separated and purified by C18 silica gel column chromatography (the reagent used was a methanol aqueous solution with a volume fraction of 60%), and the methanol was spin-off and then lyophilized for removal. water to obtain 210 mg (50.0%) of the target product ZD-E-1 as an orange-red solid powder.
  • Weight loss method Accurately weigh 0.998g (5mmol) of 4-chloro-7-nitro-2,1,3-benzoxaoxadiazole (NBD-Cl), put it in an eggplant bottle, and dissolve it with 100mL of anhydrous methanol.
  • the obtained system was separated and purified by a medium pressure preparative column (the used purification reagent was a mixture of EA and PE, and the volume fraction of EA was 35%) to obtain 900 g (36.2%) of the target product NBD-Lys(Boc)-NBzl, which was Orange-red solid with bright green fluorescence in organic solvents.
  • the eggplant bottle is connected with a plug, and the reaction solution is decompressed and drained with a vacuum circulating water pump. The residue is reconstituted with 20 mL of dry EA and then drained again.
  • the reaction was judged to be complete.
  • the solvent was spin-dried, the residue was reconstituted with methanol aqueous solution with a volume fraction of 5%, the obtained system was separated and purified by C18 silica gel column chromatography (the reagent used was a methanol aqueous solution with a volume fraction of 40%), and the methanol was removed by concentration under reduced pressure. After that, the water was removed by lyophilization in a vacuum freeze dryer to obtain 520 g (36.9%) of the target product ZD-F-1, which was an orange-red solid powder.
  • A is the TEM image of the compound ZD-B
  • B is the TEM image of the compound ZD-E-1
  • C is the SEM image of the compound ZD-B
  • D is the SEM image of the compound ZD-E-1
  • E is the SEM image of the compound ZD-E-1.
  • the change curve of the hydrated particle size of compound ZD-B in solutions of different pH values within 72h (the test time is 0.5h, 24h, 48h and 72h, respectively), and F is the change of compound ZD-E-1 in solutions of different pH values within 72h.
  • the hydration particle size change curve, G is the Zeta potential change curve of compound ZD-B in different pH value solutions within 72h
  • H is the Zeta potential change curve of compound ZD-E-1 in different pH value solutions within 72h. It can be seen from Fig. 1 that ZD-B is elongated, and ZD-E-1 is spherical.
  • the tested cell lines S180 (mouse ascites tumor cells), LLC (mouse lung cancer cells), A549 (human non-small cell lung cancer cells), HCT116 (human colon cancer cells), U2Os (human osteosarcoma cells) and MCF-7 ( Human breast cancer cells) were purchased from Nanjing KGI Company.
  • test compounds When preparing the test samples, the test compounds were prepared into a sample solution with a concentration of 500 ⁇ M (final concentration of 100 ⁇ M) in PBS buffer containing 0.5% DMSO, which was used for the preliminary screening of the in vitro anti-cell proliferation experiment.
  • the sample solution For compounds whose half-inhibitory concentration on the tested cell lines, that is, the IC 50 value is lower than 100 ⁇ M, the sample solution was diluted to 50 ⁇ M, 25 ⁇ M, 12.5 ⁇ M, 6.25 ⁇ M, and 3.125 ⁇ M, and the MTT method was used again to detect its effect on the tested cell line.
  • the IC 50 value of the test cell line was determined, and the determination was repeated at least three times under the same experimental conditions until the obtained IC 50 value was stable and reliable.
  • Positive control group adriamycin, prepared with PBS buffer containing 0.5% DMSO to the required concentration; negative control group: PBS buffer containing 0.5% DMSO; Blank group: PBS buffer containing 0.01% DMSO.
  • Seeding cells Dilute the cells in the logarithmic growth phase in good growth condition to a cell concentration of 3-5 ⁇ 10 4 cells/mL with medium, and evenly seed them in a 96-well plate, with 100 ⁇ L per well (each peripheral well). Sealed with 100 ⁇ L of PBS buffer), the inoculated 96-well plate was placed in a cell culture incubator at 37° C., 5% CO 2 for 8 h.
  • Post-processing add 25 ⁇ L of pre-prepared MTT solution to each well, continue to incubate for 4 hours, and then take out; discard the supernatant (suspended cells need to be centrifuged at 3000 rpm for 10 min), add 150 ⁇ L DMSO to each well, and shake on a cell shaker for 15 minutes , after the formazan is fully dissolved, the microplate reader measures the OD value of each well at a wavelength of 570 nm (the ideal range is between 0.3 and 1.4).
  • the inhibition rate of the anti-cell proliferation activity of the test compounds was calculated according to formula a, each experiment was repeated at least three times, and the IC 50 values of the test compounds were calculated in prism, and the results are shown in Table 1.
  • Inhibition rate [(average OD value of negative control group-average OD value of test compound group)/average OD value of negative control group-average OD value of Blank group] ⁇ 100% Formula a.
  • mice used in the experiment were SPF-grade male ICR mice with a body weight of 20 ⁇ 2 g, purchased from Beijing Weitong Lihua Animal Experiment Technology Co., Ltd., and raised in the animal barrier of the Laboratory Animal Department of Capital Medical University.
  • the specific method was as follows: the tumor-forming mice were administered for 7 consecutive days by group, the tumor length was measured every other day, and the tumor volume was calculated. 24h after the last administration, the mice were taken out and weighed, and the eyeballs were taken out after anesthesia with ether to collect blood. The mice were sacrificed by severed neck, and the tumor mass was excised and each organ was weighed (it should be ensured that the operation is performed as soon as possible and the exfoliation is complete and clean). The tumor inhibition rate was calculated according to formula b, and the results are shown in Table 2 and Figure 2; and by calculating the viscera-to-body ratio of each organ, it was analyzed whether the test compounds had obvious physiological effects on the organs of mice.
  • Tumor inhibition rate (average tumor weight in Vehicle group-average tumor weight in administration group)/average tumor weight in Vehicle group ⁇ 100% Formula b.
  • n 10;
  • ip intraperitoneal injection
  • iv tail vein injection
  • n 10.
  • ZD-A, ZD-B, ZD-E-1 and ZD-E-1 were screened out.
  • Structure (ZD-F-1) three doses of 10 ⁇ mol/kg, 5 ⁇ mol/kg and 2 ⁇ mol/kg were set to evaluate their ability to inhibit tumor growth in mice. Due to the poor water solubility of ZD-A and ZD-B, intraperitoneal injection can only be used, while ZD-E-1 and ZD-F-1 have no such concerns and can choose tail vein injection.
  • Tested cell line U2Os (human osteosarcoma cells): purchased from Nanjing KGI Company.
  • Seeding cells PBS buffer, McCoy's 5A medium containing 10% fetal bovine serum, and trypsin-EDTA digestion solution were placed in a 37° C. water bath to preheat for 15 minutes. Select U2Os cells with good growth conditions and in the logarithmic growth phase, discard the original medium in the bottle, rinse with 1 mL of PBS buffer twice, add 1 mL of trypsin-EDTA digestion solution, and incubate in a cell incubator for 2 min ( Suspension cells do not require digestion). Observe under the microscope that the cell shape becomes round and can fall off and dissociate from the bottle wall.

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Abstract

本发明提供了可荧光示踪的氨基酸衍生物及其制备方法和应用，属于生物医药技术领域。本发明提供的氨基酸衍生物主要采用一种可荧光示踪的官能团对氨基酸骨架进行修饰，NBD-Cl(4-氯-7-硝基-2,1,3-苯并氧杂噁二唑)具有低极性、强荧光的特点，以其修饰氨基酸骨架的N端，可用于亚细胞成像，且其相对体积较小，缺乏反应正交性，对生物体自身生化反应干扰小。实施例的结果显示，本发明提供的可荧光示踪的氨基酸衍生物生物活性好，可在体内外进行荧光示踪。

Description

可荧光示踪的氨基酸衍生物及其制备方法和应用
本申请要求于2020年08月03日提交中国专利局、申请号为202010766644.7、发明名称为“可荧光示踪的氨基酸衍生物及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药技术领域，尤其涉及可荧光示踪的氨基酸衍生物及其制备方法和应用。
背景技术
近些年来，人们在与癌症的斗争中取得了显著的进步，但它仍然是人们目前面临的极其严峻的公共卫生挑战。
组蛋白的翻译后修饰(PTM)是表观遗传调控的标志。研究表明，表观遗传修饰剂对肿瘤抑制基因的沉默，是肿瘤发生过程中的早期事件。在过去的几十年间，针对组蛋白脱乙酰基酶和DNA甲基转移酶的抗癌药物的批准，突出了表观遗传学在人类疾病中的重要作用，并表明控制基因表达的因素可作为新型药物靶标。肽基精氨酸脱亚氨酶4(PAD4)就是这样一种靶标，因为它对基因表达的作用与组蛋白脱乙酰基酶类似。PAD4作为一种转录共调节剂，可催化蛋白质中特定精氨酸残基向瓜氨酸的钙依赖性转化。在癌症中，PAD4不仅是抑癌蛋白p53的转录共抑制因子，还参与介导恶性肿瘤的形成。由于其在细胞信号传导途径和疾病发病机理中的调控作用，PAD4已成为多种疾病的潜在治疗靶标。基于PAD4的小分子底物N-苯甲酰-L-精氨酰胺(BAA)，研究者们已经开发出多种有效且不可逆的卤乙脒类PAD4抑制剂，但仍存在生物活性不理想等问题。
发明内容
本发明的目的在于提供可荧光示踪的氨基酸衍生物及其制备方法和应用，本发明提供的可荧光示踪的氨基酸衍生物生物活性好。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种可荧光示踪的氨基酸衍生物，具有式I所示结构：
Figure PCTCN2021110331-appb-000001
式I中，R为式1～10所示基团中的任一种：
Figure PCTCN2021110331-appb-000002
本发明提供了上述技术方案所述可荧光示踪的氨基酸衍生物的制备方法，其中，
(i)当R为式1、式2或式3所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括以下步骤：
将4-氯-7-硝基-2,1,3-苯并氧杂噁二唑、甲醇和第一反应原料混合，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，避光条件下进行取代反应，得到可荧光示踪的氨基酸衍生物；所述第一反应原料为Tos·Arg(NO 2)-OBzl、HCl·LV-OBzl或N-(2-氨基乙基)甲磺酰胺；
(ii)当R为式4所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括以下步骤：
将4-氯-7-硝基-2,1,3-苯并氧杂噁二唑、甲醇和第二反应原料混合，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，避光条件下进行取代反应，得到第一中间产物；
将所述第一中间产物溶解于乙酸乙酯中，将所得混合液与HCl的乙酸乙酯溶液混合，之后进行水解反应，得到第二中间产物；
将所述第二中间产物溶解于甲醇中，采用三乙胺将所得混合液的pH值调节至9.5～10.5，避光条件下进行氨取代反应，得到可荧光示踪的氨基酸衍生物；
所述第二反应原料的结构式如下：
Figure PCTCN2021110331-appb-000003
所述第一中间产物的结构式如下：
Figure PCTCN2021110331-appb-000004
所述第二中间产物的结构式如下：
Figure PCTCN2021110331-appb-000005
(iii)当R为式5或式7所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括以下步骤：
将第三反应原料溶解于四氢呋喃中，将所得混合液与1-羟基苯并三唑、二环己基碳二亚胺混合后进行活化，之后将所得活化体系与苄胺混合，采用N-甲基吗啡啉将所得混合液的pH值调节至8～9，进行缩合反应，得到第三中间产物；
将所述第三中间产物溶解于甲醇中，在钯碳存在条件下，于氢气氛围中进行氢解反应，得到第四中间产物；
将所述第四中间产物和4-氯-7-硝基-2,1,3-苯并氧杂噁二唑溶解于甲醇中，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，进行取代反应，得到第五中间产物；
将所述第五中间产物溶解于乙酸乙酯中，将所得混合液与HCl的乙酸乙酯溶液混合，之后进行水解反应，得到可荧光示踪的氨基酸衍生物；
所述第三反应原料的结构式如下：
Figure PCTCN2021110331-appb-000006
所述第三中间产物的结构式如下：
Figure PCTCN2021110331-appb-000007
所述第四中间产物的结构式如下：
Figure PCTCN2021110331-appb-000008
所述第五中间产物的结构式依次如下：
Figure PCTCN2021110331-appb-000009
(iv)当R为式6、式8、式9或式10所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括以下步骤：
将第四反应原料、第五反应原料和甲醇混合，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，进行取代反应，得到可荧光示踪的氨基酸衍生物；
所述第五反应原料为2-氯乙酰亚氨酸乙酯或2-氟乙酰亚氨酸乙酯，所述第四反应原料的结构式如下：
Figure PCTCN2021110331-appb-000010
其中，所述第三反应原料、第三中间产物、第四中间产物、第五中间产物和第四反应原料的结构式中，n＝3或4。
优选地，所述(i)中取代反应的温度为15～35℃。
优选地，所述(ii)中取代反应的温度为15～35℃，水解反应在冰浴条件下进行，氨取代反应在冰浴条件下进行。
优选地，所述(iii)中缩合反应、氢解反应和取代反应的温度独立为15～35℃，水解反应在冰浴条件下进行。
优选地，所述(iv)中取代反应的温度为15～35℃。
本发明提供了上述技术方案所述可荧光示踪的氨基酸衍生物在制备抗肿瘤药物中的应用。
优选地，所述肿瘤包括肺癌、结肠癌、骨肉瘤或乳腺癌。
本发明提供了一种具有式I所示结构的可荧光示踪的氨基酸衍生物，本发明提供的氨基酸衍生物主要采用一种可荧光示踪的官能团对氨基酸骨架进行修饰，NBD-Cl(4-氯-7-硝基-2,1,3-苯并氧杂噁二唑)具有低极性、强荧光的特点，以其修饰氨基酸骨架的N端，可用于亚细胞成像，且其相对体积较小，缺乏反应正交性，对生物体自身生化反应干扰小。实施例的结果显示，本发明提供的可荧光示踪的氨基酸衍生物生物活性好，可在体内外进行荧光示踪。
说明书附图
图1为化合物ZD-B和ZD-E-1的TEM图、SEM图、水合粒径变化曲线以及Zeta电位变化曲线；
图2为不同受试化合物在小鼠S180肉瘤模型中的抗肿瘤活性图；
图3为优势受试化合物在小鼠S180肉瘤模型中的抗肿瘤活性图；
图4为注射化合物ZD-E-1和ZD-F-1的小鼠肿瘤生长趋势图；
图5为注射化合物ZD-E-1和ZD-F-1的小鼠脏体比统计图；
图6为化合物ZD-B和ZD-E-1在U2Os细胞内的荧光共定位图。
具体实施方式
本发明提供了一种可荧光示踪的氨基酸衍生物，具有式I所示结构：
Figure PCTCN2021110331-appb-000011
式I中，R为式I中，R为式1～10所示基团中的任一种：
Figure PCTCN2021110331-appb-000012
在本发明中，所述可荧光示踪的氨基酸衍生物具体为式I-1～I-10所示化合物中的任一种：
Figure PCTCN2021110331-appb-000013
(记为ZD-A)，
Figure PCTCN2021110331-appb-000014
(记为ZD-B)，
Figure PCTCN2021110331-appb-000015
(记为ZD-C)，
Figure PCTCN2021110331-appb-000016
(记为ZD-D)，
Figure PCTCN2021110331-appb-000017
Figure PCTCN2021110331-appb-000018
(记为ZD-E)，
Figure PCTCN2021110331-appb-000019
(记为ZD-E-1)，
Figure PCTCN2021110331-appb-000020
(记为ZD-F)，
Figure PCTCN2021110331-appb-000021
(记为ZD-F-1)，
Figure PCTCN2021110331-appb-000022
(记为ZD-E-2)，
Figure PCTCN2021110331-appb-000023
(记为ZD-F-2)。
本发明提供了所述可荧光示踪的氨基酸衍生物的制备方法，本发明优选根据可荧光示踪的氨基酸衍生物中R的种类采用不同的制备方法，下面详细说明。
在本发明中，若无特殊说明，所用原料为本领域技术人员熟知的市售商品。
在本发明中，当R为式1、式2或式3所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括 以下步骤：
将4-氯-7-硝基-2,1,3-苯并氧杂噁二唑、甲醇和第一反应原料混合，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，避光条件下进行取代反应，得到可荧光示踪的氨基酸衍生物；所述第一反应原料为Tos·Arg(NO 2)-OBzl、HCl·LV-OBzl或N-(2-氨基乙基)甲磺酰胺；
反应路线如下所示：
Figure PCTCN2021110331-appb-000024
在本发明中，所述4-氯-7-硝基-2,1,3-苯并氧杂噁二唑(NBD-Cl)和第一反应原料的摩尔比优选为5：6；所述NBD-Cl与甲醇的用量比优选为5mmol：90～110mL，更优选为5mmol：100mL。本发明优选将NBD-Cl溶解于甲醇中，在冰浴(0℃)搅拌条件下，向所得NBD-Cl甲醇溶液中加入第一反应原料，然后采用N,N-二异丙基乙胺(DIPEA)将所得混合液的pH值调节至9.5～10.5，撤去冰浴，避光条件下进行取代反应。在本发明中，采用N,N-二异丙基乙胺(DIPEA)将所得混合液的pH值优选调节至10。
在本发明中，所述取代反应的温度优选为15～35℃，更优选为20～30℃；本发明优选在室温条件下进行取代反应，即不需要额外的加热或降温；在本发明的实施例中，具体是在25℃条件下进行取代反应。本发明优选采用TLC监测反应进程，观察到所述NBD-Cl原料点消失后，判断反应完全；其中，按体积比计，TLC监测用展开剂优选为石油醚(PE)：乙酸乙酯(EA)＝(1～2)：1。
所述取代反应结束后，本发明优选将所得体系经中压制备柱分离纯化，所用纯化试剂为EA和PE的混合物，EA的体积分数为35～50％，并用甲醇溶解后超声，静置析出固体，经减压过滤，所得固体物料即为可荧光示踪的氨基酸衍生物。
本发明对所述第一反应原料的来源没有特殊限定，采用本领域技术人员熟知的市售商品或熟知的方法制备得到均可。在本发明中，所述Tos·Arg(NO 2)-OBzl和N-(2-氨基乙基)甲磺酰胺具体采用市售商品，所述HCl·LV-OBzl优选按照本领域技术人员熟知的方法制备得到，反应路线如下(具体制备方法的一个示例在本发明实施例2中详细说明)：
Figure PCTCN2021110331-appb-000025
在本发明中，当R为式4所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括以下步骤：
将4-氯-7-硝基-2,1,3-苯并氧杂噁二唑、甲醇和第二反应原料混合，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，避光条件下进行取代反应，得到第一中间产物；
将所述第一中间产物溶解于乙酸乙酯中，将所得混合液与HCl的乙酸乙酯溶液混合，之后进行水解反应，得到第二中间产物；
将所述第二中间产物溶解于甲醇中，采用三乙胺将所得混合液的pH值调节至9.5～10.5，避光条件下进行氨取代反应，得到可荧光示踪的氨基酸衍生物；
所述第二反应原料的结构式如下：
Figure PCTCN2021110331-appb-000026
所述第一中间产物的结构式如下：
Figure PCTCN2021110331-appb-000027
所述第二中间产物的结构式如下：
Figure PCTCN2021110331-appb-000028
反应路线如下所示：
Figure PCTCN2021110331-appb-000029
本发明将4-氯-7-硝基-2,1,3-苯并氧杂噁二唑、甲醇和第二反应原料混合，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，避光条件下进行取代反应，得到第一中间产物。在本发明中，所述NBD-Cl与第二反应原料(HCl·Orn(Boc)-OBzl)的摩尔比优选为5：6，所述NBD-Cl与甲醇的用量比优选为5mmol：90～110mL，更优选为5mmol：100mL。本发明优选将NBD-Cl溶解于甲醇中，在冰浴搅拌条件下，向所得NBD-Cl甲醇溶液中加入第二反应原料，然后采用DIPEA将所得混合液的pH值调节至9.5～10.5，撤去冰浴，避光条件下进行取代反应。在本发明中，采用DIPEA将所得混合液的pH值优选调节至10。
在本发明中，所述取代反应的温度优选为15～35℃，更优选为20～30℃；本发明优选在室温条件下进行取代反应。本发明优选采用TLC监测反应进程，观察到所述NBD-Cl原料点消失后，判断反应完全；其中，按体积比计，TLC监测用展开剂优选为PE：EA＝2：1。
所述取代反应结束后，本发明优选将所得体系经中压制备柱分离纯化，所用纯化试剂为EA和PE的混合物，EA的体积分数为35％，得到第一中间产物(NBD-Orn(Boc)-OBzl)。
得到第一中间产物后，本发明将所述第一中间产物溶解于乙酸乙酯中，将所得混合液与HCl的乙酸乙酯溶液混合，之后进行水解反应，得到第二中间产物。在本发明中，所述第一中间产物溶于乙酸乙酯后，所得混合液中第一中间产物的浓度优选为0.35～0.45mol/L，更优选为0.4mol/L；所述HCl的乙酸乙酯溶液中HCl的浓度优选为3.5～4.5mol/L，更优选为4mol/L；所述混合液与HCl的乙酸乙酯溶液(记为HCl/EA溶液)的体积比优选为1：2。
本发明优选将第一中间产物溶解于乙酸乙酯中，之后在通风橱中，冰浴搅拌条件下，向所得混合液中加入HCl/EA溶液，冰浴搅拌条件下进行水解反应。本发明优选采用TLC监测反应进程，观察到所述第一中间产物的原料点消失后，判断反应完全；其中，按体积比计，TLC监测用展开剂优选为EA：H 2O：冰醋酸(HAc)＝6：1：1。
所述水解反应结束后，本发明优选在37℃温水浴搅拌条件下，将所得体系减压抽干，残留物用 干燥EA复溶后再次抽干，继续重复该操作3次，至无明显酸气残留，得到第二中间产物。
得到第二中间产物后，本发明将所述第二中间产物溶解于甲醇中，采用三乙胺将所得混合液的pH值调节至9.5～10.5，避光条件下进行氨取代反应，得到可荧光示踪的氨基酸衍生物。本发明优选将所述第二中间产物溶解于甲醇中，在冰浴搅拌条件下，用三乙胺调节pH值至9.5～10.5(优选为10)，避光条件下进行氨取代反应。本发明优选采用TLC监测反应进程，观察到所述第二中间产物的原料点消失后，判断反应完全；其中，按体积比计，TLC监测用展开剂优选为PE：EA＝2：1。
所述氨取代反应结束后，本发明优选将所得体系经中压制备柱分离纯化，所用纯化试剂为EA和PE的混合物，EA的体积分数为32％，得到可荧光示踪的氨基酸衍生物。
在本发明中，当R为式5或式7所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括以下步骤：
将第三反应原料溶解于四氢呋喃中，将所得混合液与1-羟基苯并三唑、二环己基碳二亚胺混合后进行活化，之后将所得活化体系与苄胺混合，采用N-甲基吗啡啉将所得混合液的pH值调节至8～9，进行缩合反应，得到第三中间产物；
将所述第三中间产物溶解于甲醇中，在钯碳存在条件下，于氢气氛围中进行氢解反应，得到第四中间产物；
将所述第四中间产物和4-氯-7-硝基-2,1,3-苯并氧杂噁二唑溶解于甲醇中，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，进行取代反应，得到第五中间产物；
将所述第五中间产物溶解于乙酸乙酯中，将所得混合液与HCl的乙酸乙酯溶液混合，之后进行水解反应，得到可荧光示踪的氨基酸衍生物；
所述第三反应原料的结构式如下所示：
Figure PCTCN2021110331-appb-000030
所述第三中间产物的结构式如下所示：
Figure PCTCN2021110331-appb-000031
所述第四中间产物的结构式如下所示：
Figure PCTCN2021110331-appb-000032
所述第五中间产物的结构式依次如下所示：
Figure PCTCN2021110331-appb-000033
其中，所述第三反应原料、第三中间产物、第四中间产物和第五中间产物的结构式中，n＝3或4；
反应路线如下所示(产物以盐酸盐形式体现)：
Figure PCTCN2021110331-appb-000034
在本发明中，当n优选为3时，所述可荧光示踪的氨基酸衍生物具有式I-5所示结构，当n优选为4时，所述可荧光示踪的氨基酸衍生物具有式I-7所示结构。
本发明将第三反应原料溶解于四氢呋喃中，将所得混合液与1-羟基苯并三唑、二环己基碳二亚胺 混合后进行活化，之后将所得活化体系与苄胺混合，采用N-甲基吗啡啉将所得混合液的pH值调节至8～9，进行缩合反应，得到第三中间产物。在本发明中，所述第三反应原料与四氢呋喃(THF)的用量比优选为1mmol：(9～11)mL，更优选为1mmol：10mL；所述第三反应原料与1-羟基苯并三唑(HOBt)、二环己基碳二亚胺(DCC)的摩尔比优选为10：(11～13)：(11～13)，更优选为10：12：12；所述第三反应原料与苄胺的用量比优选为10mmol：(1.6～5.6)mL。
本发明优选将第三反应原料溶解于四氢呋喃中，在冰浴搅拌条件下，向所得混合液中加入1-羟基苯并三唑和二环己基碳二亚胺，活化8～12min，有白色固体析出；在本发明中，所述活化优选为10min，之后向所得活化体系中加入苄胺，采用N-甲基吗啡啉(NMM)将所得混合液的pH值调节至8～9，撤去冰浴，进行缩合反应。在本发明中，所述缩合反应的温度优选为15～35℃，更优选为20～30℃；本发明优选在室温条件下进行缩合反应。本发明优选采用TLC监测反应进程，观察到所述第三反应原料的原料点消失后，判断反应完全；其中，按体积比计，TLC监测用展开剂优选为二氯甲烷：甲醇＝30：1。
所述缩合反应结束后，本发明优选将所得体系进行减压过滤，去除滤液中THF，残余物加入乙酸乙酯，之后依次采用饱和NaHCO 3溶液、饱和NaCl溶液、5wt％KHSO 4溶液、饱和NaCl溶液、5wt％NaHCO 3溶液、饱和NaCl溶液洗涤，洗涤后加入无水Na 2SO 4干燥；干燥后常压过滤除去Na 2SO 4，滤液减压浓缩得无色油状物；经中压制备柱分离纯化，按体积比计，收集产物所用试剂为二氯甲烷：甲醇＝95：5，得到第三中间产物。
得到第三中间产物后，本发明将所述第三中间产物溶解于甲醇中，在钯碳存在条件下，于氢气氛围中进行氢解反应，得到第四中间产物。在本发明中，所述第三中间产物与甲醇的用量比优选为(5～10)mmol：(40～50)mL；所述钯碳作为催化剂，其用量保证氢解反应顺利进行即可。在本发明中，所述氢解反应的温度优选为15～35℃，更优选为20～30℃；本发明优选在室温条件下进行氢解反应。本发明优选采用TLC监测反应进程，观察到所述第三中间产物的原料点消失后，判断反应完全；其中，按体积比计，TLC监测用展开剂优选为二氯甲烷：甲醇＝10：1。
所述氢解反应结束后，本发明优选将所得体系进行常压过滤，将钯碳除去，去除滤液中溶剂，得到第四中间产物。
得到第四中间产物后，本发明将所述第四中间产物和4-氯-7-硝基-2,1,3-苯并氧杂噁二唑溶解于甲醇中，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，进行取代反应，得到第五中间产物。在本发明中，所述NBD-Cl和第四中间产物的摩尔比优选为5：6；所述NBD-Cl和甲醇的用量比优选为5mmol：(40～100)mL。
本发明优选将NBD-Cl和第四中间产物溶解于甲醇中，在冰浴搅拌条件下，采用DIPEA调节体系pH值至9.5～10.5；撤去冰浴，进行取代反应，在本发明中，采用DIPEA将所得混合液的pH值优选调节至10。在本发明中，所述取代反应的温度优选为15～35℃，更优选为20～30℃；本发明优选在室温条件下进行取代反应。本发明优选采用TLC监测反应进程，观察到所述NBD-Cl的原料点消失后，判断反应完全；其中，按体积比计，TLC监测用展开剂优选为石油醚：乙酸乙酯＝2：1。
所述取代反应结束后，本发明优选将所得体系进行减压旋干，所得残余物经中压制备柱分离纯化，所用纯化试剂为EA和PE的混合物，EA的体积分数为35％，得到第五中间产物。
得到第五中间产物后，本发明将所述第五中间产物溶解于乙酸乙酯中，将所得混合液与HCl的乙酸乙酯溶液混合，之后进行水解反应，得到可荧光示踪的氨基酸衍生物。在本发明中，所述第五中间产物溶于乙酸乙酯后，所得混合液中第五中间产物的浓度优选为0.5～0.6mol/L；所述HCl的乙酸乙酯溶液中HCl的浓度优选为2～4mol/L；所述混合液与HCl的乙酸乙酯溶液(记为HCl/EA溶液)的体积比优选为1：(2～3)。
本发明优选将第五中间产物溶解于乙酸乙酯中，之后在通风橱中，冰浴搅拌条件下，向所得混合液中加入HCl/EA溶液，冰浴搅拌条件下进行水解反应。本发明优选采用TLC监测反应进程，观察到所述第五中间产物原料点消失后，判断反应完全；其中，按体积比计，TLC监测用展开剂优选为EA：H 2O：HAc＝6：1：1。
所述水解反应结束后，本发明优选将所得体系进行抽滤去除溶剂，依次采用乙酸乙酯和乙醚对所得剩余物进行抽滤洗涤，得到可荧光示踪的氨基酸衍生物(盐酸盐形式)。
在本发明中，当R为式6、式8、式9或式10所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括以下步骤：
将第四反应原料、第五反应原料和甲醇混合，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，进行取代反应，得到可荧光示踪的氨基酸衍生物；
所述第五反应原料为2-氯乙酰亚氨酸乙酯或2-氟乙酰亚氨酸乙酯，所述第四反应原料的结构式如下：
Figure PCTCN2021110331-appb-000035
其中，所述第四反应原料的结构式中，n＝3或4；
反应路线如下所示：
Figure PCTCN2021110331-appb-000036
在本发明中，当n优选为3时，所述可荧光示踪的氨基酸衍生物具有式I-6或式I-9所示结构，当n优选为4时，所述可荧光示踪的氨基酸衍生物具有式I-8或I-10所示结构。
在本发明中，具体的，当R为式6或式8所示基团时，所述第五反应原料为2-氯乙酰亚氨酸乙酯；当R为式9或式10所示基团时，所述第五反应原料为2-氟乙酰亚氨酸乙酯。
在本发明中，所述第四反应原料和第五反应原料的摩尔比优选为1：(1～2)，所述第四反应原料与甲醇的用量比优选为(1～3)mmol：(40～50)mL。本发明优选将第四反应原料溶解于甲醇中，在冰浴搅拌条件下向所得混合液中加入第五反应原料，采用DIPEA调节体系的pH值至9.5～10.5，撤去冰浴，进行取代反应。在本发明中，采用DIPEA将所得混合液的pH值优选调节至10。
在本发明中，所述取代反应的温度优选为15～35℃，更优选为20～30℃；本发明优选在室温条件下进行取代反应。本发明优选采用TLC监测反应进程，观察到所述第四反应原料点消失后，判断反应完全；其中，按体积比计，TLC监测用展开剂优选为EA：H 2O：HAc＝6：1：1。
所述取代反应结束后，本发明优选将所得体系旋干溶剂，残余物用体积分数为5％的甲醇水溶液复溶，用C18硅胶柱层析分离纯化，所用试剂为体积分数为40～60％的甲醇水溶液，旋除甲醇后冻干去除水分，得到可荧光示踪的氨基酸衍生物。
本发明提供了上述技术方案所述可荧光示踪的氨基酸衍生物在制备抗肿瘤药物中的应用。在本发明中，所述肿瘤优选包括肺癌、结肠癌、骨肉瘤或乳腺癌。在本发明中，所述抗肿瘤药物包括所述可荧光示踪的氨基酸衍生物和药学上可接受的辅料；所述抗肿瘤药物中可荧光示踪的氨基酸衍生物的含量优选为20～80wt.％，所述药学上可接受的辅料优选包括环糊精、β-羟基环糊精和甘露醇中的一种或几种；所述抗肿瘤药物的剂型优选包括冻干粉、纳米乳或脂质体。
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
NBD-Arg(NO 2)-OBzl(ZD-A)的合成
减重法称取0.998g(5mmol)4-氯-7-硝基-2,1,3-苯并氧杂噁二唑(NBD-Cl)于茄瓶中，用100mL无水甲醇溶解，得到黄色澄清透明溶液，加入搅拌子；冰浴搅拌条件下，加入1.85g(6mmol)Tos·Arg(NO 2)-OBzl，并用2.6mLN,N-二异丙基乙胺(DIPEA)调节pH值至10；撤冰浴，室温(25℃)条件下避光反应8h，观察到反应液颜色逐渐加深至墨绿色，伴有绿色荧光产生，TLC(按体积比计，展开剂为PE：EA＝1：1，Rf＝0.25)监测反应进程，观察到NBD-Cl原料点消失后，判断反应完全。反应结束后所得体系经中压制备柱分离纯化(所用纯化试剂为EA和PE的混合物，EA的体积分数为50％)，并用2mL甲醇溶解后超声，静置析出固体，减压过滤，得到610mg(26.8％)目标产物ZD-A，为黄色固体粉末，有机溶剂中具有明亮的绿色荧光。M.p.:166.0～168.8℃；
Figure PCTCN2021110331-appb-000037
(C＝0.1,CH 3OH)；ESI-MS(m/z):471.3[M-H] -； 1H-NMR(300MHz,DMSO-d 6):δ(ppm)＝9.50(d,J＝6.7Hz,1H),8.53(s,1H),8.50(s,1H),8.00(s,1H),7.85(s,1H),7.32(s,5H),6.41(s,1H),5.18(s,2H),4.76(s,1H),3.19(dt,J＝6.8Hz,6.2Hz,2H),2.04(m,2H),1.65(m,2H)； 13C-NMR(75MHz,DMSO-d 6):δ/ppm＝171.0,159.8,144.6,137.8,136.0,128.9,128.6,128.4,100.7,67.1,56.4,28.4,25.3。
实施例2
Boc-LV-OBzl的合成
减重法称取2.31g(10mmol)Boc-Leu(叔丁氧羰基亮氨酸)于茄瓶中，用150mL无水四氢呋喃(THF)溶解，得到无色澄清透明溶液，加入搅拌子；冰浴搅拌条件下，依次加入1.62g(12mmol)1-羟基苯并三唑(HOBt)和2.47g(12mmol)二环己基碳二亚胺(DCC)，活化30min后，有白色固体析出；向茄瓶中加入2.92g(11mmol)HCl·Val-OBzl，并用N-甲基吗啡啉(NMM)调节pH值至8；撤冰浴，室温条件下搅拌反应8h，用TLC(按体积比计，展开剂为CH 2Cl 2：CH 3OH＝30：1，R f＝0.38)监测反应进程，观察到Boc-Leu的原料点消失，判断反应完全。反应结束后用真空循环水泵减压过滤，并用EA润洗滤饼，滤液减压浓缩至干，除去THF，残留物用150mL EA溶解，再次抽滤除去固体物料，并将滤液转移至250mL分液漏斗中，依次用饱和NaHCO 3溶液萃洗3次(30mL/次)、饱和NaCl溶液萃洗3次(30mL/次)、5wt％KHSO 4溶液萃洗3次(30mL/次)、饱和NaCl溶液萃洗3次(30mL/次)、饱和NaHCO 3溶液萃洗3次(30mL/次)、饱和NaCl溶液萃洗3次(30mL/次)，观察到EA层在萃洗过程中颜色变淡至无色；将EA层用无水Na 2SO 4干燥2h后，过滤除去Na 2SO 4，滤液减压浓缩得无色油状物，经中压制备柱分离纯化(所用纯化试剂为甲醇和二氯甲烷的混合物，甲醇的体积分数为5％)，得到3.68g(87.6％)目标产物Boc-LV-OBzl，为白色固体粉末。ESI-MS(m/z):421[M+H] +。
HCl·LV-OBzl的合成
将1.26g(3mmol)Boc-LV-OBzl置于茄瓶中，加入5mL干燥EA使其溶解。在通风橱中，冰浴搅拌条件下，继续向溶液中滴加10mL HCl的乙酸乙酯溶液(记为HCl/EA溶液，HCl的浓度为4mo/L)，瓶口接上干燥管，冰浴搅拌条件下反应3h，观察到有橙红色固体逐渐沿瓶壁析出，TLC(按体积比计，展开剂为CH 2Cl 2：CH 3OH＝10：1，R f＝0.30)监测反应进程，观察到Boc-LV-OBzl原料点消失后，判断反应完全。反应结束后，在37℃温水浴搅拌体积下，茄瓶接具塞单通，用真空循环水泵将反应液减压抽干，残留物用20mL干燥EA复溶后再次抽干，重复该操作3次，至无明显酸气残留，最后加入无水乙醚磨洗抽干，得到990mg(92.5％)目标产物HCl·LV-OBzl，为白色固体粉末。ESI-MS (m/z):3201[M+H] +。
NBD-LV-OBzl(ZD-B)的合成
减重法称取0.998g(5mmol)4-氯-7-硝基-2,1,3-苯并氧杂噁二唑(NBD-Cl)于茄瓶中，用100mL无水甲醇溶解，得到黄色澄清透明溶液，加入搅拌子；冰浴搅拌条件下，加入2.14g(6mmol)HCl·LV-OBzl，并用2.6mL DIPEA调节pH值至10；撤冰浴，室温条件下避光反应8h，观察到反应液颜色逐渐加深至墨绿色，伴有绿色荧光产生，TLC(按体积比计，展开剂为PE：EA＝1：1，R f＝0.28)监测反应进程，观察到NBD-Cl原料点消失后，判断反应完全。反应结束后所得体系经中压制备柱分离纯化(所用纯化试剂为EA和PE的混合物，EA的体积分数为50％)，并用2mL甲醇溶解后超声，静置析出固体，减压过滤，得到750mg(32.1％)目标产物ZD-B，为黄色固体粉末，有机溶剂中具有明亮的绿色荧光。M.p.:147.4～149.1℃；
Figure PCTCN2021110331-appb-000038
(C＝0.1,CH 3OH)；ESI-MS(m/z):482.5[M-H] -； 1H-NMR(300MHz,DMSO-d 6):δ(ppm)＝9.38(s,1H),8.58(s,1H),8.52(d,J＝8.9Hz,1H),7.34(s,5H),6.42(s,1H),5.12(d,J＝4.1Hz,2H),4.52(s,1H),4.27(t,J＝6.4Hz,1H),2.11(m,1H),1.91(s,1H),1.67(d,J＝32.0Hz,2H),0.91(t,J＝5.7Hz,6H),0.86(d,J＝6.3Hz,6H)； 13C-NMR(75MHz,DMSO-d 6):δ/ppm＝171.4,144.7,137.9,136.2,128.8,128.6,128.5,122.3,100.1,66.5,58.0,56.2,30.3,24.8,23.3,22.0,19.4,18.6。
实施例3
NBD-N-(2-aminoethyl)methanesulfonamide(ZD-C)的合成
减重法称取0.998g(5mmol)NBD-Cl于茄瓶中，用100mL无水甲醇溶解，得到黄色澄清透明溶液，加入搅拌子；冰浴搅拌条件下，加入834mg(6mmol)N-(2-氨基乙基)甲磺酰胺(N-(2-aminoethyl)methanesulfonamide)，并用2.6mL DIPEA调节pH值至10；撤冰浴，室温条件下避光反应8h，观察到反应液颜色逐渐加深至墨绿色，伴有绿色荧光产生，TLC(按体积比计，展开剂为PE：EA＝2：1，R f＝0.30)监测反应进程，观察到NBD-Cl原料点消失后，判断反应完全。反应结束后所得体系经中压制备柱分离纯化(所用纯化试剂为EA和PE的混合物，EA的体积分数为35％)，并用2mL甲醇溶解后超声，静置析出固体，减压过滤，得到697mg(46.3％)目标产物ZD-C，为橙红色固体粉末，有机溶剂中具有明亮的绿色荧光。M.p.:198.8～203.5℃；
Figure PCTCN2021110331-appb-000039
(C＝0.1,CH 3OH)；ESI-MS(m/z):300.1[M-H] -； 1H-NMR(300MHz,DMSO-d 6):δ(ppm)＝9.40(s,1H),8.55(d,J＝8.8Hz,1H),7.26(t,J＝5.7Hz,1H),6.46(d,J＝8.9Hz,1H),3.62(s,2H),3.29(q,J＝6.1Hz,2H),2.94(s,3H)。
实施例4
NBD-Orn(Boc)-OBzl的合成
减重法称取0.998g(5mmol)NBD-Cl于茄瓶中，用100mL无水甲醇溶解，得到黄色澄清透明溶液，加入搅拌子；冰浴搅拌条件下，加入2.15g(6mmol)HCl·Orn(Boc)-OBzl，并用2.6mLDIPEA调节pH值至10；撤冰浴，室温条件下避光反应8h，观察到反应液颜色逐渐加深至墨绿色，伴有绿色荧光产生，TLC(按体积比计，展开剂为PE：EA＝2：1，R f＝0.30)监测反应进程，观察到NBD-Cl原料点消失后，判断反应完全。反应结束后所得体系经中压制备柱分离纯化(所用纯化试剂为EA和PE的混合物，EA的体积分数为35％)，得到990mg(41.2％)目标产物NBD-Orn(Boc)-OBzl，为橙红色固体，有机溶剂中具有明亮的绿色荧光。ESI-MS(m/z):484.5[M-H] -； 1H-NMR(300MHz,DMSO-d 6):δ(ppm)＝9.51(s,1H),8.52(d,J＝8.9Hz,1H),7.32(s,5H),6.84(s,1H),6.41(s,1H),5.17(s,2H),3.34(s,1H),2.94(q,J＝6.1Hz,2H),1.99(m,2H),1.54(m,2H),1.34(s,9H)。
NBD-Orn(cycto)(ZD-D)的合成
将970mg(2mmol)NBD-Orn(Boc)-OBzl置于茄瓶中，加入5mL干燥EA使其溶解，在通风橱 中，冰浴搅拌条件下，继续向溶液中滴加10mLHCl/EA溶液(HCl的浓度为4mo/L)，瓶口接上干燥管，冰浴搅拌条件下反应3h，观察到有橙红色固体逐渐沿瓶壁析出，TLC(按体积比计，展开剂为EA：H 2O：乙酸(HAc)＝6：1：1，R f＝0.35)监测反应进程，观察到NBD-Orn(Boc)-OBzl原料点消失后，判断反应完全。反应结束后，在37℃温水浴搅拌条件下，茄瓶接具塞单通，用真空循环水泵将反应液减压抽干，残留物用20mL干燥EA复溶后再次抽干，重复该操作3次，至无明显酸气残留，将残留物用20mL甲醇溶解，冰浴搅拌条件下，用三乙胺调节pH值至10，避光反应6h，TLC(按体积比计，展开剂为PE：EA＝2：1，R f＝0.35)监测反应进程，观察到原料点消失后，判断反应完全。反应结束后，所得体系经中压制备柱分离纯化(所用纯化试剂为EA和PE的混合物，EA的体积分数为32％)，得到560mg(76.3％)目标产物ZD-D，为橙红色固体粉末。M.p.:218.1～220.7℃；
Figure PCTCN2021110331-appb-000040
(C＝0.1,CH 3OH)；ESI-MS(m/z):300.3[M+Na] +； 1H-NMR(300MHz,DMSO-d 6):δ(ppm)＝9.35(s,1H),8.52(d,J＝9.0Hz,1H),7.86(s,1H),6.54(d,J＝9.1Hz,1H),4.64(s,1H),3.21(m,2H),2.16(m,2H),1.90(m,2H)。
实施例5
N-苄氧羰基-N-ε-叔丁氧羰基-L-鸟氨酰苄胺(Z-Orn(Boc)-NBzl)的合成
准确称取3.660g(10mmol)N-苄氧羰基-N-ε-叔丁氧羰基-L-鸟氨酸于茄瓶中，用100mL无水THF溶解，得到无色澄清透明溶液，并加入搅拌子；在冰浴搅拌条件下，加入1.620g(12mmol)HOBt和2.472g(12mmol)DCC，活化10min，有白色固体析出；向反应体系中加入1.6mL苄胺，并用NMM调节反应液pH值至8；撤去冰浴，室温条件下反应12h，用TLC监测反应进程，按体积比计，展开剂为二氯甲烷：甲醇＝30：1，N-苄氧羰基-N-ε-叔丁氧羰基-L-鸟氨酸的原料点消失后，判断反应完全。反应结束后减压过滤，旋蒸旋干THF，得到黄色油状物质；加入60mL乙酸乙酯，之后依次采用饱和NaHCO 3溶液(25mL×3)、饱和NaCl溶液(25mL×3)、5wt％KHSO 4溶液(25mL×3)、饱和NaCl溶液(25mL×3)、5wt％NaHCO 3溶液(25mL×3)、饱和NaCl溶液(25mL×3)洗涤，洗涤过程中溶液逐渐变浅至无色，洗涤后加入无水Na 2SO 4干燥2h；常压过滤除去Na 2SO 4，滤液减压浓缩得无色油状物；经中压制备柱分离纯化(按体积比计，收集产物所用纯化试剂为二氯甲烷：甲醇＝95：5)，得到4.490g(98.7％)目标产物Z-Orn(Boc)-NBzl，为白色固体。ESI-MS(m/e):455[M+H] +； 1H-NMR(300MHz,DMSO-d 6):δ(ppm)＝8.39(m,1H),7.42(d,J＝8.0Hz,1H),7.34(m,5H),7.27(m,5H),6.79(m,1H),5.03(s,2H),4.28(d,J＝5.3Hz,2H),3.99(m,1H),2.89(t,J＝5.8Hz,2H),1.60(m,2H),1.51(m,2H),1.37(s,9H)。
N-ε-叔丁氧羰基-L-鸟氨酰苄胺(Orn(Boc)-NBzl)的合成
将4.490g(9.9mmol)Z-Orn(Boc)-NBzl置于茄瓶中，加入40mL甲醇溶解，得到澄清透明溶液；称取0.449g钯碳(Pd/C)于茄瓶中，装好三通，同时开启搅拌；将三通竖直一侧接氢气袋，侧面通口接水泵，将茄瓶中空气抽尽，换成氢气，反复换气三次后，将反应装置移到氢解反应通风橱中，室温条件下进行反应2h，用TLC监测反应进度，按体积比计，展开剂为二氯甲烷：甲醇＝10：1，Z-Orn(Boc)-NBzl原料点消失后，判断反应完全。反应结束后常压过滤，将钯碳除去，滤液旋干得到3.988g(88.8％)目标产物Orn(Boc)-NBzl，为白色固体粉末。ESI-MS(m/e):321[M+H] +。
N-(7-硝基-2,1,3-苯并氧杂噁二唑-4-基)-N-ε-叔丁氧羰基-L-鸟氨酰苄胺(NBD-Orn(Boc)-NBzl)的合成
准确称取0.998g(5mmol)4-氯-7-硝基-2,1,3-苯并氧杂噁二唑(NBD-Cl)和1.942g(6mmol)Orn(Boc)-NBzl于茄瓶中，用40mL无水甲醇溶解，得到无色澄清透明溶液，并加入搅拌子；冰浴搅拌条件下，用2.6mLDIPEA调节体系pH值至10；撤去冰浴，室温条件下反应5h，用TLC监测反应进度，按体积比计，展开剂为石油醚：乙酸乙酯＝2：1，NBD-Cl原料点消失后，判断反应完全。 反应结束后减压旋干，所得残余物经中压制备柱分离纯化(所用纯化试剂为EA和PE的混合物，EA的体积分数为35％)，得到2.028g(83.8％)目标产物NBD-Orn(Boc)-NBzl，为橙红色固体，有机溶剂中具有明亮的绿色荧光。ESI-MS(m/e):483.4[M-H] -。
(S)-5氨基-N-苄基-2-((7-硝基-2,1,3-苯并氧杂噁二唑-4-基)氨基)戊烷酰胺(NBD-Orn(HCl)-NBzl，ZD-E)的合成
将2.028g(5.5mmol)NBD-Orn(Boc)-NBzl置于茄瓶中，用10mL干燥乙酸乙酯溶解，得到无色澄清透明溶液，并加入搅拌子；冰浴搅拌条件下，于通风橱中，加入20mLHCl/EA溶液(HCl的浓度为2mo/L)，瓶口接上干燥管；冰浴搅拌反应2h，用TLC监测反应进度，按体积比计，展开体系为乙酸乙酯：水：冰醋酸＝6：1：1，NBD-Orn(Boc)-NBzl原料点消失后，判断反应完全。反应结束后抽滤去除溶剂，向剩余物中加入干燥乙酸乙酯15mL×3次，乙醚15mL×1次，抽滤去除溶剂，得到1.762g(96.1％)目标产物(盐酸盐形式)，即(S)-5-氨基-N-苄基-2-((7-硝基-2,1,3-苯并氧杂噁二唑-4-基)氨基)戊烷酰胺盐酸盐，为红色固体。ESI-MS(m/e):384[M+H] +。
实施例6
(S)-N-苄基-5-(2-氯乙酰亚胺基)-2-((7-硝基-2,1,3-苯并氧杂噁二唑-4-基)氨基)戊烷酰胺(NBD-Orn(Cl)-NBzl，ZD-E-1)的合成
准确称取420mg(1mmol)(S)-5-氨基-N-苄基-2-((7-硝基-2,1,3-苯并氧杂噁二唑-4-基)氨基)戊烷酰胺盐酸盐置于茄瓶中，用40mL无水甲醇溶解，得到澄清透明溶液，加入搅拌子；冰浴搅拌条件下，加入187mg(1.2mmol)2-氯乙酰亚氨酸乙酯盐酸盐，并用DIPEA调节体系的pH值至10；撤去冰浴，室温条件下反应6h，用TLC监测反应进度，按体积比计，展开剂为乙酸乙酯：水：冰醋酸＝6：1：1，(S)-5-氨基-N-苄基-2-((7-硝基-2,1,3-苯并氧杂噁二唑-4-基)氨基)戊烷酰胺盐酸盐原料点消失后，判断反应完全。反应结束后旋干溶剂，残余物用体积分数为5％的甲醇水溶液复溶，用C18硅胶柱层析分离纯化(所用试剂为体积分数为60％的甲醇水溶液)，旋除甲醇后冻干去除水分，得到210mg(50.0％)目标产物ZD-E-1，为橙红色固体粉末。纯度：97.34％；M.p.:88.2～90.0℃；
Figure PCTCN2021110331-appb-000041
(C＝0.1,CH 3OH)；ESI-MS(m/z):458.7[M-H] -； 1H-NMR(300MHz,DMSO-d 6):δ(ppm)＝9.94(s,1H),9.52(s,1H),9.35(s,1H),9.09(s,1H),8.79(s,1H),8.56(d,J＝8.7Hz,1H),7.26(m,5H),6.36(s,1H),4.40(m,1H),4.36(s,2H),4.31(m,2H),3.29(m,2H),2.03(m,2H),1.68(m,2H)； 13C-NMR(75MHz,DMSO-d 6):δ/ppm＝170.2,167.3,162.8,158.9,158.4,139.4,128.7,127.7,127.3,118.2,114.4,58.6,42.8,42.2,41.4,24.7,24.3。
实施例7
Z-Lys(Boc)-NBzl的合成
减重法准确称取3.8g(10mmol)Z-Lys(Boc)-OH于茄瓶中，用100mL无水THF溶解，得到无色澄清透明溶液，加入搅拌子；冰浴搅拌下，依次加入1.62g(12mmol)HOBt和2.47g(12mmol)DCC，活化10min后，有白色固体析出；向茄瓶中滴加入5.6mL苄胺，并用NMM调节pH至8；撤冰浴，室温条件下搅拌反应8h，用TLC(按体积比计，展开剂为CH 2Cl 2：CH 3OH＝30：1，R f＝0.38)监测反应进程，观察到Z-Lys(Boc)-OH原料点消失，判断反应完全。反应结束后用真空循环水泵减压过滤，并用EA润洗滤饼；滤液减压浓缩至干，除去THF，残留物用150mL EA溶解，再次抽滤除去固体物料，并将滤液转移至250mL分液漏斗中，依次用饱和NaHCO 3溶液萃洗3次(30mL/次)、饱和NaCl溶液萃洗3次(30mL/次)、5wt％KHSO 4溶液萃洗3次(30mL/次)、饱和NaCl溶液萃洗3次(30mL/次)、饱和NaHCO 3溶液萃洗3次(30mL/次)、饱和NaCl溶液萃洗3次(30mL/次)，观察到EA层在萃洗过程中颜色变淡至无色；将EA层用无水Na 2SO 4干燥2h后，过滤除去Na 2SO 4，滤液减压浓缩得无色油状物，经中压制备柱分离纯化(所用纯化试剂为甲醇和二氯甲烷的混 合物，甲醇的体积分数为5％)，得到3.85g(82.3％)目标产物Z-Lys(Boc)-NBzl，为白色固体粉末。ESI-MS(m/z):469[M+H] +。
Lys(Boc)-NBzl的合成
将2.34g(5mmol)Z-Lys(Boc)-NBzl置于茄瓶中，用50mL甲醇溶解，得到无色澄清透明溶液；搅拌条件下，向茄瓶中加入234mg Pd/C，接上三通和氢气袋，用真空循环水泵抽尽反应瓶内空气，通入氢气，重复操作3次，保持茄瓶与氢气袋相通，于通风橱中室温搅拌反应3h，TLC(按体积比计，展开剂为CH 2Cl 2：CH 3OH＝10：1，R f＝0.25)监测反应进程，Z-Lys(Boc)-NBzl原料点消失后，判断反应完全。反应结束后常压过滤除去Pd/C，滤液减压浓缩至干，得到1.59g(95.2％)目标产物Lys(Boc)-NBzl，为白色固体粉末。ESI-MS(m/z):335[M+H] +。
NBD-Lys(Boc)-NBzl的合成
减重法准确称取0.998g(5mmol)4-氯-7-硝基-2,1,3-苯并氧杂噁二唑(NBD-Cl)置于茄瓶中，用100mL无水甲醇溶解，得到黄色澄清透明溶液，加入搅拌子；冰浴搅拌条件下，加入2.0g(6mmol)Lys(Boc)-NBzl，并用2.6mL DIPEA调节pH值至10；撤冰浴，室温条件下避光反应8h，观察到反应液颜色逐渐加深至墨绿色，伴有绿色荧光产生，TLC(按体积比计，展开剂为PE：EA＝2：1，R f＝0.30)监测反应进程，NBD-Cl原料点消失后，判断反应完全。反应结束后所得体系经中压制备柱分离纯化(所用纯化试剂为EA和PE的混合物，EA的体积分数为35％)，得到900g(36.2％)目标产物NBD-Lys(Boc)-NBzl，为橙红色固体，有机溶剂中具有明亮的绿色荧光。ESI-MS(m/z):497.5[M-H] -； 1H-NMR(300MHz,DMSO-d 6):δ(ppm)＝9.36(s,1H),8.73(s,1H),8.53(d,J＝8.8Hz,1H),7.28(m,5H),6.77(t,J＝5.1Hz,1H),6.34(s,1H),4.36(m,1H),4.30(s,2H),2.89(s,2H),1.95(s,2H),1.40(m,4H),1.33(m,9H)。
NBD-Lys(HCl)-NBzl(ZD-F)的合成
将1.49g(3mmol)NBD-Lys(Boc)-NBzl置于茄瓶中，加入5mL干燥的乙酸乙酯使其溶解，在通风橱中，冰浴搅拌条件下，继续向溶液中滴加15mL HCl/EA溶液(HCl的浓度为4mo/L)，瓶口接上干燥管，冰浴搅拌条件下反应3h，观察到有橙红色固体逐渐沿瓶壁析出，TLC(按体积比计，展开剂为EA：H 2O：HAc＝6：1：1，R f＝0.35)监测反应进程，NBD-Lys(Boc)-NBzl原料点消失后，判断反应完全。反应结束后再37℃温水浴搅拌条件下，茄瓶接具塞单通，用真空循环水泵将反应液减压抽干，残留物用20mL干燥EA复溶后再次抽干，重复该操作3次，至无明显酸气残留，最后加入无水乙醚磨洗抽干，得到1.24g(95.4％)目标产物ZD-F(具体为盐酸盐形式)，为橙红色固体粉末。M.p.:120.2～123.5℃；
Figure PCTCN2021110331-appb-000042
(C＝0.1,CH 3OH)；ESI-MS(m/z):434[M+H] +。
实施例8
NBD-Lys(Cl)-NBzl(ZD-F-1)的合成
将1.3g(3mmol)NBD-Lys(HCl)-NBzl置于茄瓶中，用50mL无水甲醇溶解，得到橙红色澄清透明溶液，加入搅拌子；冰浴搅拌条件下，加入0.499g(3.2mmol)2-氯乙酰亚氨酸乙酯盐酸盐，并用3.6mLDIPEA调节pH值至10；撤冰浴，室温条件下避光反应8h，TLC(按体积比计，展开剂为EA：H 2O：HAc＝6：1：1，R f＝0.38)监测反应进程，并用茚三酮显色，观察到NBD-Lys(HCl)-NBzl原料点消失后，判断反应完全。反应结束后旋干溶剂，残余物用体积分数为5％的甲醇水溶液复溶，所得体系经C18硅胶柱层析分离纯化(所用试剂为体积分数为40％的甲醇水溶液)，减压浓缩除去甲醇后，经真空冷冻干燥机冻干除水，得到520g(36.9％)目标产物ZD-F-1，为橙红色固体粉末。M.p.:118.1～120.4℃；
Figure PCTCN2021110331-appb-000043
(C＝0.1,CH 3OH)；ESI-MS(m/z):472.4[M-H] -； 1H-NMR(300MHz,DMSO-d 6):δ(ppm)＝10.26(s,1H),9.61(s,1H),9.22(s,1H),8.96(t,J＝5.3Hz,1H),8.52(d,J＝8.8Hz,1H),7.26(m,5H),6.41(s,1H),4.47(m,1H),4.43(s,2H),4.31(d,J＝5.1Hz,2H),3.27(t,J＝5.9Hz,2H), 2.00(m,2H),1.60(m,2H),1.47(m,2H)； 13C-NMR(75MHz,DMSO-d 6):δ/ppm＝170.5,162.6139.5,128.7,127.7,127.3,49.0,42.8,42.4,39.6,27.0,23.6。
测试例1 纳米形貌表征及稳定性研究
利用透射电子显微镜和扫描电子显微镜研究了化合物ZD-B和ZD-E-1的纳米形态，并用纳米粒度仪研究了二者在溶液中的水合粒径和Zeta电位，结果如图1所示，图1中，A为化合物ZD-B的TEM图，B为化合物ZD-E-1的TEM图，C为化合物ZD-B的SEM图，D为化合物ZD-E-1的SEM图，E为化合物ZD-B在不同pH值溶液中72h内(测试时间分别为0.5h、24h、48h和72h)的水合粒径变化曲线，F为化合物ZD-E-1在不同pH值溶液中72h内的水合粒径变化曲线，G为化合物ZD-B在不同pH值溶液中72h内的Zeta电位变化曲线，H为化合物ZD-E-1在不同pH值溶液中72h内的Zeta电位变化曲线。由图1可知，ZD-B呈长条状，ZD-E-1呈球状。在纳米粒度仪连续72h测定的粒径变化曲线中，pH＝7.0时，ZD-B和ZD-E-1都处于粒径最小；ZD-B在pH＝2.0和5.5情况下，粒径大小有很大变化；ZD-E-1在pH＝2.0时，粒径几乎没有变化，pH＝5.5时，粒径先增大后减小。在连续72h测定的Zeta电位变化曲线中，ZD-B和ZD-E-1都未出现较大的变化。
测试例2 评价可荧光示踪的氨基酸衍生物的体外抗肿瘤细胞增殖活性(MTT法)
受试细胞株S180(小鼠腹水瘤细胞)、LLC(小鼠肺癌细胞)、A549(人非小细胞肺癌细胞)、HCT116(人结肠癌细胞)、U2Os(人骨肉瘤细胞)和MCF-7(人乳腺癌细胞)均购自南京凯基公司。
制备受试样品时，将受试化合物用含0.5％DMSO的PBS缓冲液配成浓度为500μM(终浓度100μM)的样品溶液，用于体外抗细胞增殖实验的初筛。对于对受试细胞株的半数抑制浓度，即IC 50值低于100μM的化合物，将样品溶液逐级稀释为50μM、25μM、12.5μM、6.25μM、3.125μM后，再次用MTT法检测其对受试细胞株的IC 50值，并在相同的实验条件下重复测定至少三次以上，直至所得IC 50值稳定可靠。
阳性对照组：阿霉素，用含0.5％DMSO的PBS缓冲液配制成所需浓度；阴性对照组：为含0.5％DMSO的PBS缓冲液；Blank组：为含0.01％DMSO的PBS缓冲液。
具体方法为：
1)接种细胞：将生长状况良好，处于对数生长期的细胞用培养基稀释至3～5×10 4个/mL的细胞浓度，均匀接种于96孔板中，每孔100μL(外围各孔用100μLPBS缓冲液液封)，将接种后的96孔板置于37℃、5％CO 2的细胞培养箱中孵育8h。
2)给药：观察细胞生长及贴壁情况，当贴壁率达50％以上时，按照预设复孔给25μL不同化合物或不同浓度的受试样品溶液，每板均设置阴性和阳性对照组以及Blank组；轻轻拍打使其样品液分散均匀，置于细胞培养箱中孵育48h。
3)后处理：每孔加入事先配制好的MTT溶液25μL，继续孵育4h后取出；弃去上清液(悬浮细胞需离心3000rpm，10min)后，每孔加入150μL DMSO，于细胞摇床上震荡15min，使甲臜充分溶解后，酶标仪测定各孔在570nm波长下的OD值(理想范围在0.3～1.4之间)。
按照式a计算受试化合物的抗细胞增殖活性的抑制率，每个实验至少重复三次，在prism中计算受试化合物的IC 50值，结果如表1所示。
抑制率＝[(阴性对照组的平均OD值-受试化合物组的平均OD值)/阴性对照组的平均OD值-Blank组的平均OD值]×100％    式a。
表1 受试化合物对各细胞株的体外抗细胞增殖活性的IC 50值(Mean±SDμM)
Figure PCTCN2021110331-appb-000044
Figure PCTCN2021110331-appb-000045
测试例3 评价可荧光示踪的氨基酸衍生物体内抑制肿瘤生长实验(小鼠S180纤维肉瘤模型)
实验所用小鼠为SPF级雄性ICR小鼠，体重20±2g，购买于北京维通利华动物实验技术有限公司，在首都医科大学实验动物部动物屏障内进行饲养。
具体方法为：按组别对成瘤小鼠进行连续7天的给药，每隔一天测量肿瘤长短并计算肿瘤体积。末次给药24h后，取出称重，并用乙醚麻醉后摘眼球取血，所得全血在1000rpm条件下离心10min，吸取血清备用。将小鼠断颈处死，并剥取瘤块及各脏器称重(应保证尽快操作且剥取完整干净)。按式b计算抑瘤率，结果如表2和图2所示；并通过计算各脏器的脏体比，分析受试化合物对小鼠脏器有无明显的生理影响。
抑瘤率＝(Vehicle组平均瘤重-给药组平均瘤重)/Vehicle组平均瘤重×100％    式b。
1)不同受试化合物之间的比较
表2 受试化合物的体内给药分组及结果统计表
Figure PCTCN2021110331-appb-000046
注：表2中“YW3-56”化学名称为N-(1-(苄基氨基)-5-(2-氯乙二酰亚胺基)-1-氧戊烷-2-基)-6-(二甲基氨基)-2-萘酰胺，采用US20120108562(Gong Chen,Yanming Wang,Pingxin Li,Jing Hu,Shu Wang,Yuji Wang.Therapeutic compositions and methods)专利公开的方法制备得到；
ip，腹腔注射；n＝10；
经单因素方差分析，*代表与Vehicle组相比，P<0.05，有差异；**代表与Vehicle组相比，P<0.01，有显著性差异；a代表与YW3-56组相比，P>0.05，无差异(图2)。
以上实验选择同一给药剂量，并采用腹腔注射的方式，以阿霉素为阳性对照组，对先导化合物YW3-56及含不同氨基酸主体化合物进行初步评价。通过单因素方差分析，ZD-A和ZD-C两组化合物的平均瘤重与Vehicle组相比有差异，而ZD-B和ZD-E-1的平均瘤重与Vehicle组相比有显著性差异，且所有受试化合物的平均瘤重与YW3-56组相比，均未表现出差异。表2以及图2中结果表明，这几种受试化合物在10μmol/kg的剂量下，均表现出一定的抗肿瘤生长活性，其中又以ZD-B和ZD-E-1的活性最为突出。
2)对优势化合物的量效分析
基于以上结果，得到了几个具有体内抗肿瘤生长活性和可修饰位点的化合物结构，对其进行进一步的量效分析。结果见表3和图3。
表3 优势氨基酸主体化合物的体内给药分组及结果统计表
Figure PCTCN2021110331-appb-000047
Figure PCTCN2021110331-appb-000048
注：ip，腹腔注射；iv，尾静脉注射；n＝10。
经单因素方差分析，*代表与Vehicle组相比，P<0.05，有差异；**表示与Vehicle组相比，P<0.01，有显著性差异；a表示与YW3-56组相比，P>0.05，无差异(图3)。
在对不同氨基酸主体化合物的体内抗肿瘤生长活性有了初步的探讨后，从中筛选出ZD-A、ZD-B、ZD-E-1以及ZD-E-1的侧链亚甲基单元增加的结构(ZD-F-1)，设置10μmol/kg、5μmol/kg、2μmol/kg三个给药剂量，评价它们对小鼠体内肿瘤生长的抑制能力。由于ZD-A和ZD-B的水溶性较差，因此只能采用腹腔注射的方式，而ZD-E-1和ZD-F-1则无此顾虑，可以选择尾静脉注射的方式。经单因素方差分析，发现这四种化合物在2μmol/kg的剂量下，其平均瘤重仍与Vehicle组相比有差异。其中又以ZD-E-1活性最为显著，在三种剂量下均与Vehicle组相比有显著性差异，且与YW3-56组相比无差异。实验结果表明，化合物ZD-E-1在三种剂量下，均表现出良好且稳定的抗肿瘤生长活性，优于其类似物ZD-F-1。之后对这两种化合物的小鼠肿瘤生长趋势和小鼠脏体比进行分析，比较这两种化合物的抗肿瘤生长能力和毒性强弱。结果见图4和图5，其中，经单因素方差分析，*代表与Vehicle组相比，P<0.05，有差异；**代表与Vehicle组相比，P<0.01，有显著性差异。
由图4可以看出，化合物ZD-E-1和ZD-F-1的小鼠肿瘤生长趋势大致相同，但在第3天后，其大小逐渐产生差异，可用以佐证其瘤重结果。由图5可知，通过比较两种化合物的小鼠脏体比，发现ZD-E-1在高剂量(10μmol/kg和5μmol/kg)下，对肝脏重量的影响，及在三种剂量下对脑重影响，与Vehicle组相比有差异，而ZD-F-1对肝重和脑重影响无差异。实验结果表明，尽管ZD-E-1对肿瘤生长的抑制能力要优于ZD-F-1，但其毒副作用也更明显，可能会引起小鼠肝脏的衰竭和脑部病变。
测试例4 评价化合物ZD-B和ZD-E-1细胞内分布(激光共聚焦)
受试细胞株：U2Os(人骨肉瘤细胞)：购自南京凯基公司。
具体方法为：
1)接种细胞：将PBS缓冲液、含10％胎牛血清的McCoy’s 5A培养基、胰蛋白酶-EDTA消化液置于37℃水浴锅中预热15min。选取生长状况良好，处于对数生长期的U2Os细胞，弃去瓶内原有培养基后，用1mLPBS缓冲液润洗2次后，加入1mL胰蛋白酶-EDTA消化液，于细胞培养箱中孵育2min(悬浮细胞无需消化)。镜下观察细胞形态变圆且能从瓶壁上脱落游离，加入1mL培养基终止消化，并用滴管轻轻吹打使细胞完全脱落并分散，转移至15mL离心管中，3000rpm、3min条件下离心弃上清，重新加入3mL培养基，并用吸管轻轻吹打使其分散均匀。于2mLEP管中稀释10 倍后，吸取10μL细胞悬液于细胞计数板上镜下计数，细胞总数＝(四大格内细胞总数/4)×10000×稀释倍数。最后将细胞悬液用培养基稀释至1×10 5个/mL的细胞浓度，并均匀接种于共聚焦小皿中，每皿1mL。将接种后的共聚焦小皿置于37℃、5％CO 2的细胞培养箱中孵育8h。
2)给药：观察细胞生长及贴壁情况，当贴壁率达50％以上时，按照预设组别给予250μL终浓度为10μM和5μM的受试样品溶液。轻轻拍打使其分散均匀，继续置于细胞培养箱中孵育8h和24h。
3)固定：按不同时间点取出共聚焦小皿，小心吸出培养基，用PBS缓冲液润洗3遍，加入1mL4％多聚甲醛固定液，室温下固定30min；
4)染色：将固定液吸出后，用PBS缓冲液润洗3遍，加入1mL现配的0.5μg/mL(1:10000)DAPI溶液，染色3～5min，并用PBS缓冲液润洗3遍；
5)观察：加入含有抗荧光淬灭剂的PBS缓冲液(5μL+995μLPBS)，荧光显微镜或者激光共聚焦下观察。
结果如图6所示(DAPI对细胞核进行染色，呈蓝色；化合物荧光为绿色(Ex＝480nm；Em＝520nm)；Merge为二者叠加图)，图6中，A为ZD-B，B为ZD-E-1。由图6可知，化合物ZD-B并没有进入细胞核，而是分散在细胞质中，说明其作用靶点不是核内PAD4，其对癌细胞增殖的抑制作用可能涉及其他的作用机制；而ZD-E-1则在细胞核和胞质中均有分布，并且随浓度的升高和时间的延长，进入胞内的量也越大。说明了ZD-E-1通过作用于PAD4通路，来实现对癌细胞增殖的抑制。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (19)

  1. 一种可荧光示踪的氨基酸衍生物，其特征在于，具有式I所示结构：
    Figure PCTCN2021110331-appb-100001
    式I中，R为式1～10所示基团中的任一种：
    Figure PCTCN2021110331-appb-100002
  2. 根据权利要求1所述可荧光示踪的氨基酸衍生物，其特征在于，所述可荧光示踪的氨基酸衍生物为式I-1～I-10所示化合物中的任一种：
    Figure PCTCN2021110331-appb-100003
  3. 根据权利要求1所述可荧光示踪的氨基酸衍生物，其特征在于，当可荧光示踪的氨基酸衍生物为I-5、I-7或I-8时，所述可荧光示踪的氨基酸衍生物为氨基酸衍生物的盐酸盐。
  4. 权利要求1～3所述可荧光示踪的氨基酸衍生物的制备方法，其特征在于，
    (i)当R为式1、式2或式3所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括以下步骤：
    将4-氯-7-硝基-2,1,3-苯并氧杂噁二唑、甲醇和第一反应原料混合，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，避光条件下进行取代反应，得到可荧光示踪的氨基酸衍生物；所 述第一反应原料为Tos·Arg(NO 2)-OBzl、HCl·LV-OBzl或N-(2-氨基乙基)甲磺酰胺；
    (ii)当R为式4所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括以下步骤：
    将4-氯-7-硝基-2,1,3-苯并氧杂噁二唑、甲醇和第二反应原料混合，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，避光条件下进行取代反应，得到第一中间产物；
    将所述第一中间产物溶解于乙酸乙酯中，将所得混合液与HCl的乙酸乙酯溶液混合，之后进行水解反应，得到第二中间产物；
    将所述第二中间产物溶解于甲醇中，采用三乙胺将所得混合液的pH值调节至9.5～10.5，避光条件下进行氨取代反应，得到可荧光示踪的氨基酸衍生物；
    所述第二反应原料的结构式如下：
    Figure PCTCN2021110331-appb-100004
    所述第一中间产物的结构式如下：
    Figure PCTCN2021110331-appb-100005
    所述第二中间产物的结构式如下：
    Figure PCTCN2021110331-appb-100006
    (iii)当R为式5或式7所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括以下步骤：
    将第三反应原料溶解于四氢呋喃中，将所得混合液与1-羟基苯并三唑、二环己基碳二亚胺混合后进行活化，之后将所得活化体系与苄胺混合，采用N-甲基吗啡啉将所得混合液的pH值调节至8～9，进行缩合反应，得到第三中间产物；
    将所述第三中间产物溶解于甲醇中，在钯碳存在条件下，于氢气氛围中进行氢解反应，得到第四中间产物；
    将所述第四中间产物和4-氯-7-硝基-2,1,3-苯并氧杂噁二唑溶解于甲醇中，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，进行取代反应，得到第五中间产物；
    将所述第五中间产物溶解于乙酸乙酯中，将所得混合液与HCl的乙酸乙酯溶液混合，之后进行水解反应，得到可荧光示踪的氨基酸衍生物；
    所述第三反应原料的结构式如下
    Figure PCTCN2021110331-appb-100007
    所述第三中间产物的结构式如下：
    Figure PCTCN2021110331-appb-100008
    所述第四中间产物的结构式如下：
    Figure PCTCN2021110331-appb-100009
    所述第五中间产物的结构式依次如下：
    Figure PCTCN2021110331-appb-100010
    (iv)当R为式6、式8、式9或式10所示基团时，可荧光示踪的氨基酸衍生物的制备方法包括以下步骤：
    将第四反应原料、第五反应原料和甲醇混合，采用N,N-二异丙基乙胺将所得混合液的pH值调节至9.5～10.5，进行取代反应，得到可荧光示踪的氨基酸衍生物；
    所述第五反应原料为2-氯乙酰亚氨酸乙酯或2-氟乙酰亚氨酸乙酯，所述第四反应原料的结构式如下：
    Figure PCTCN2021110331-appb-100011
    其中，所述第三反应原料、第三中间产物、第四中间产物、第五中间产物和第四反应原料的结构式中，n＝3或4。
  5. 根据权利要求4所述的制备方法，其特征在于，所述(i)中取代反应的温度为15～35℃。
  6. 根据权利要求4所述的制备方法，其特征在于，所述(ii)中取代反应的温度为15～35℃，水解反应在冰浴条件下进行，氨取代反应在冰浴条件下进行。
  7. 根据权利要求4所述的制备方法，其特征在于，所述(iii)中缩合反应、氢解反应和取代反应的温度独立为15～35℃，水解反应在冰浴条件下进行。
  8. 根据权利要求4所述的制备方法，其特征在于，所述(iv)中取代反应的温度为15～35℃。
  9. 权利要求1～3所述可荧光示踪的氨基酸衍生物在制备抗肿瘤药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述肿瘤包括肺癌、结肠癌、骨肉瘤或乳腺癌。
  11. 一种抗肿瘤药物，其特征在于，包括权利要求1～3所述可荧光示踪的氨基酸衍生物和药学上可接受的辅料。
  12. 根据权利要求11所述的抗肿瘤药物，其特征在于，所述可荧光示踪的氨基酸衍生物为氨基酸衍生物的盐酸盐。
  13. 根据权利要求11所述的抗肿瘤药物，其特征在于，所述抗肿瘤药物中可荧光示踪的氨基酸衍生物的含量为20～80wt.％。
  14. 根据权利要求11所述的抗肿瘤药物，其特征在于，所述药学上可接受的辅料包括环糊精、β-羟基环糊精和甘露醇中的一种或几种。
  15. 根据权利要求11所述的抗肿瘤药物，其特征在于，所述抗肿瘤药物的剂型包括冻干粉、纳米乳或脂质体。
  16. 权利要求1～3所述可荧光示踪的氨基酸衍生物在抗肿瘤中的应用。
  17. 根据权利要求16所述的应用，其特征在于，所述肿瘤包括肺癌、结肠癌、骨肉瘤或乳腺癌。
  18. 根据权利要求1～3任一项所述的可荧光示踪的氨基酸衍生物，其特征在于，当所述可荧光示踪的氨基酸衍生物为式I-1时，给药量为2μmol/kg、5μmol/kg或10μmol/kg；
    当所述可荧光示踪的氨基酸衍生物为式I-2时，给药量为2μmol/kg、5μmol/kg和10μmol/kg；
    当所述可荧光示踪的氨基酸衍生物为式I-3时，给药量为10μmol/kg；
    当所述可荧光示踪的氨基酸衍生物为式I-4时，给药量为10μmol/kg；
    当所述可荧光示踪的氨基酸衍生物为式I-5时，给药量为10μmol/kg；
    当所述可荧光示踪的氨基酸衍生物为式I-6时，给药量为2μmol/kg、5μmol/kg或10μmol/kg；
    当所述可荧光示踪的氨基酸衍生物为式I-8时，给药量为2μmol/kg、5μmol/kg或10μmol/kg；
  19. 根据权利要求1～3任一项所述的可荧光示踪的氨基酸衍生物，其特征在于，当所述可荧光示踪的氨基酸衍生物为式I-1或I-2时，采用腹腔注射；
    当所述可荧光示踪的氨基酸衍生物为式I-6或I-8时，采用腹腔注射或静脉注射。
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