Fluorescently-traceable amino acid derivative and preparation methods and use thereof
The present disclosure provides a fluorescently-traceable amino acid derivative and a preparation method and use thereof, and belongs to the technical field of biomedicine. In the present disclosure, an amino acid skeleton of the amino acid derivative is modified mainly by a fluorescently-traceable functional group. 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) has low polarity and strong fluorescence, and can be used to modify an N-terminal of the amino acid skeleton to conduct subcellular imaging. Moreover, the NBD-Cl has a relatively small volume, lacks reaction orthogonality, and causes little interference with biochemical reactions of the organism itself. The results of examples show that the fluorescently-traceable amino acid derivative has a desirable biological activity and can be fluorescently traced in vivo and in vitro.
1 . A fluorescently-traceable amino acid derivative, having a structure shown in formula I:
wherein, in formula I, R is any one of the groups shown in formula 1 to formula 10:
2 . The fluorescently-traceable amino acid derivative according to claim 1 , wherein the fluorescently-traceable amino acid derivative is any one selected from the group consisting of compounds shown in formula I-1 to formula I-10:
3 . The fluorescently-traceable amino acid derivative according to claim 1 , wherein when the fluorescently-traceable amino acid derivative is the compound shown in formula I-5, formula I-7, or formula I-8, the fluorescently-traceable amino acid derivative is a hydrochloride of the amino acid derivative.
4 . A preparation method of the fluorescently-traceable amino acid derivative according to claim 1 , wherein
(i) when R is the group shown in formula 1, formula 2, or formula 3, the preparation method of the fluorescently-traceable amino acid derivative comprises the following steps:
mixing 4-chloro-7-nitro-2,1,3-benzoxadiazole, methanol, and a first reaction raw material, adjusting a resulting mixed solution to a pH value of 9.5 to 10.5 with N,N-diisopropylethylamine, and conducting substitution in the dark to obtain the fluorescently-traceable amino acid derivative; wherein the first reaction raw material is selected from the group consisting of Tos·Arg(NO 2 )-OBzl, HCl·LV-OBzl, and N-(2-aminoethyl) methanesulfonamide;
(ii) when R is the group shown in formula 4, the preparation method of the fluorescently-traceable amino acid derivative comprises the following steps:
mixing 4-chloro-7-nitro-2,1,3-benzoxadiazole, methanol, and a second reaction raw material, adjusting a resulting mixed solution to a pH value of 9.5 to 10.5 with N,N-diisopropylethylamine, and conducting substitution in the dark to obtain a first intermediate product;
dissolving the first intermediate product in ethyl acetate, mixing a resulting mixed solution with an ethyl acetate solution of HCl, and conducting hydrolysis to obtain a second intermediate product; and
dissolving the second intermediate product in methanol, adjusting a resulting mixed solution to a pH value of 9.5 to 10.5 with triethylamine, and conducting ammonia substitution in the dark to obtain the fluorescently-traceable amino acid derivative; wherein
the second reaction raw material has a structural formula as follows:
the first intermediate product has a structural formula as follows:
the second intermediate product has a structural formula as follows:
(iii) when R is the group shown in formula 5 or formula 7, the preparation method of the fluorescently-traceable amino acid derivative comprises the following steps:
dissolving a third reaction raw material in tetrahydrofuran, mixing a resulting mixed solution I with 1-hydroxybenzotriazole and dicyclohexylcarbodiimide to conduct activation, mixing a resulting activation system with benzylamine, adjusting an obtained mixed solution II to a pH value of 8 to 9 with N-methylmorpholine, and conducting condensation to obtain a third intermediate product;
dissolving the third intermediate product in methanol, and conducting hydrogenolysis in a hydrogen atmosphere in the presence of palladium on carbon to obtain a fourth intermediate product;
dissolving the fourth intermediate product and 4-chloro-7-nitro-2,1,3-benzoxadiazole in methanol, adjusting a resulting mixed solution to a pH value of 9.5 to 10.5 with N,N-diisopropylethylamine, and conducting substitution to obtain a fifth intermediate product; and
dissolving the fifth intermediate product in ethyl acetate, mixing a resulting mixed solution with an ethyl acetate solution of HCl, and conducting hydrolysis to obtain the fluorescently-traceable amino acid derivative; wherein
the third reaction raw material has a structural formula as follows:
the third intermediate product has a structural formula as follows:
the fourth intermediate product has a structural formula as follows:
the fifth intermediate product has a structural formula as follows:
and
(iv) when R is the group shown in formula 6, formula 8, formula 9, or formula 10, the preparation method of the fluorescently-traceable amino acid derivative comprises the following steps:
mixing the fourth reaction raw material, the fifth reaction raw material, and methanol, adjusting a resulting mixed solution to a pH value of 9.5 to 10.5 with N,N-diisopropylethylamine, and conducting substitution to obtain the fluorescently-traceable amino acid derivative; wherein
the fifth reaction raw material is selected from the group consisting of ethyl 2-chloroacetimidate and ethyl 2-fluoroacetimidate, and the fourth reaction raw material has a structural formula as follows:
and
in the structural formulas of the third reaction raw material, the third intermediate product, the fourth intermediate product, the fifth intermediate product, and the fourth reaction raw material, n is 3 or 4.
5 . The preparation method according to claim 4 , wherein the fluorescently-traceable amino acid derivative is any one selected from the group consisting of compounds shown in formula I-1 to formula I-10:
6 . The preparation method according to claim 4 , wherein when the fluorescently-traceable amino acid derivative is the compound shown in formula I-5, formula I-7, or formula I-8, the fluorescently-traceable amino acid derivative is a hydrochloride of the amino acid derivative.
7 . The preparation method according to claim 4 , wherein in (i), the substitution is conducted at 15° C. to 35° C.
8 . The preparation method according to claim 4 , wherein in (ii), the substitution is conducted at 15° C. to 35° C., the hydrolysis is conducted in an ice bath, and the ammonia substitution is conducted in an ice bath.
9 . The preparation method according to claim 4 , wherein in (iii), the condensation, the hydrogenolysis, and the substitution each are conducted at 15° C. to 35° C., and the hydrolysis is conducted in an ice bath.
10 . The preparation method according to claim 4 , wherein in (iv), the substitution is conducted at 15° C. to 35° C.
11 . A method for treating a tumor, comprising administrating the fluorescently-traceable amino acid derivative according to claim 1 to a subject in need thereof.
12 . The method according to claim 11 , wherein the fluorescently-traceable amino acid derivative is any one selected from the group consisting of compounds shown in formula I-1 to formula I-10:
13 . The method according to claim 11 , wherein when the fluorescently-traceable amino acid derivative is the compound shown in formula I-5, formula I-7, or formula I-8, the fluorescently-traceable amino acid derivative is a hydrochloride of the amino acid derivative.
14 . The method according to claim 11 , wherein the tumor comprises lung cancer, colon cancer, osteosarcoma, or breast cancer.
15 . The fluorescently-traceable amino acid derivative according to claim 1 , wherein when the fluorescently-traceable amino acid derivative is the compound shown in formula I-1, a dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-2, the dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-3, the dosage is 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-4, the dosage is 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-5, the dosage is 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-6, the dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg; and
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-8, the dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg.
16 . The fluorescently-traceable amino acid derivative according to claim 2 , wherein when the fluorescently-traceable amino acid derivative is the compound shown in formula I-1, a dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-2, the dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-3, the dosage is 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-4, the dosage is 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-5, the dosage is 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-6, the dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg; and
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-8, the dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg.
17 . The fluorescently-traceable amino acid derivative according to claim 3 , wherein when the fluorescently-traceable amino acid derivative is the compound shown in formula I-1, a dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-2, the dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-3, the dosage is 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-4, the dosage is 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-5, the dosage is 10 μmol/kg;
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-6, the dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg; and
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-8, the dosage is 2 μmol/kg, 5 μmol/kg, or 10 μmol/kg.
18 . The fluorescently-traceable amino acid derivative according to claim 1 , wherein when the fluorescently-traceable amino acid derivative is the compound shown in formula I-1 or formula I-2, intraperitoneal injection is adopted; and
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-6 or formula I-8, the intraperitoneal injection or intravenous injection is adopted.
19 . The fluorescently-traceable amino acid derivative according to claim 2 , wherein when the fluorescently-traceable amino acid derivative is the compound shown in formula I-1 or formula I-2, intraperitoneal injection is adopted; and
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-6 or formula I-8, the intraperitoneal injection or intravenous injection is adopted.
20 . The fluorescently-traceable amino acid derivative according to claim 3 , wherein when the fluorescently-traceable amino acid derivative is the compound shown in formula I-1 or formula I-2, intraperitoneal injection is adopted; and
when the fluorescently-traceable amino acid derivative is the compound shown in formula I-6 or formula I-8, the intraperitoneal injection or intravenous injection is adopted.