NEAR-INFRARED FLUORESCENT MOLECULE ACTIVE TARGETING FOLATE RECEPTOR AND PREPARATION METHOD THEREOF

Abstract

The present disclosure relates to the fields of near-infrared surgical navigation fluorescent molecules, cell marker imaging and so on, and in particular discloses an active targeting near-infrared fluorescent small molecule structure and a preparation method thereof. The present disclosure prepares the active targeting near-infrared fluorescent small molecule with pemetrexed disodium and derivatives thereof as active targeting groups by utilizing an organic total synthesis method. Such active targeting near-infrared fluorescent molecule has the advantages of high active targeting property, strong specificity, good water solubility, high fluorescence quantum yield and so on.

Claims

1. A near-infrared fluorescent molecule active targeting a folate receptor, with a structural formula as follows: ##STR00018## wherein M is independently selected from the group consisting of H, Na, and K.

2. A method for preparing a near-infrared fluorescent molecule active targeting a folate receptor, comprising following steps: S1, synthesizing S0456, comprising following step: S11, mixing and heating 4-hydrazinophenylsulfonic acid, 3-methyl-2-butane, and glacial acetic acid to 110-130 ° C., refluxing a resultant for 16-20 h in nitrogen atmosphere, precipitating in ethyl acetate, then filtering and collecting a crude product in a form of pink solid, dissolving an obtained product in methanol, dropwise adding a dissolving solution into a solution of potassium hydroxide and isopropanol under mild conditions, and filtering and washing a crude mixture to obtain a product 1, in brown solid; S12, adding the product 1 and 1,4-butyl sultone into a toluene solution in nitrogen atmosphere to be heated at 100-120 ° C. for 40-55 h, cooling a mixed material to room temperature, precipitating a crude product, adding a crude mixture into methanol to be stirred for 30 minutes; followed by precipitating, filtering, and collecting, dissolving a resultant again in a mixture of water and methanol at 2:1, and slowly adding a mixed solution into acetonitrile with a dropping funnel; and filtering a precipitate and collecting a pink solid product 2; and S13, refluxing and heating aminobenzene, cyclohexanone, the product 2, Vilsmeier-Haack reagent, and anhydrous sodium acetate in anhydrous ethanol for 6-8 h in nitrogen atmosphere, cooling a reaction mixture to room temperature, followed by filtering, washing with ethanol and methanol, and collecting a green solid product 3; S2, synthesizing a pemetrexed targeted drug and preparing Pemetrexed-Tyr: the synthesizing a pemetrexed targeted drug: dissolving a pemetrexed hydrolytic acid in DMF, sequentially adding HATU, O-tert-butyl-L-tyrosine tert-butyl ester hydrochloride, and DIEA into a flask, stirring until a resultant is dissolved completely, reacting at room temperature for 25-35 min under nitrogen protection, dropwise adding a reacted solution into an HCI solution to generate a light yellow precipitate, and drying the light yellow precipitate to obtain a solid product 4, that is, the pemetrexed targeted drug; and the preparing Pemetrexed-Tyr: putting the product 4 into a round-bottom flask, adding trifluoroacetic acid, stirring a mixture for two hours, adding a resultant into methyl tert-butyl ether, followed by precipitating and filtering, and drying in vacuum to obtain a product 5, that is, the Pemetrexed-Tyr; and S3, reacting Pemetrexed-Tyr and S0456 to generate Pemetrexed-Tyr-S0456, comprising following steps: S31, to an aqueous solution of S0456, dropwise adding a Pemetrexed-Tyr trianion solution with pH of 10-12 at 18-28 ° C.; S32, raising a temperature of a reaction mixture to 85-95 ° C., stirring for 40-55 minutes, and monitoring formation of product by TLC; S33, after a product is formed, cooling the reaction mixture to room temperature, and transferring the reaction mixture into stirred acetone as a stable flow through a cannula, so as to obtain a green precipitate; S34, filtering the precipitate on a sintering funnel under vacuum by an air pump, and washing a resultant with acetone; and S35, drying a green powder-like solid to obtain Pemetrexed-Tyr-S0456, wherein a structural formula of the product 1 is as follows: ##STR00019## a structural formula of the product 2 is as follows: ##STR00020## a structural formula of the product 3, that is, the S0456 is as follows: ##STR00021## a structural formula of the product 4, that is, the pemetrexed targeted drug is as follows: ##STR00022## a structural formula of the product 5, that is, the Pemetrexed-Tyr is as follows: ##STR00023## and a structural formula of the Pemetrexed-Tyr-S0456, that is, the near-infrared fluorescent molecule active targeting a folate receptor is as follows: ##STR00024##

Description

BRIEF DESCRIPTION OF DRAWINGS

[0032] FIG. 1 is a structural diagram of an active targeting near-infrared fluorescent molecule of the present disclosure;

[0033] FIG. 2 is a synthetic flow diagram for preparation of the active targeting near-infrared fluorescent molecule;

[0034] FIG. 3 is a fluorescence performance diagram (fluorescence intensity) of the active targeting near-infrared fluorescent molecule prepared (Excitation (785 nm) and Emission (811 nm));

[0035] FIG. 4 is a fluorescence performance diagram (detection limit) of the active targeting near-infrared fluorescent molecule prepared (the fluorescence intensity was measured for samples of different concentrations, and this detection limit was determined by linear simulation);

[0036] FIG. 5 is a hydrogen spectrum of S0456 of the active targeting near-infrared fluorescent molecule prepared;

[0037] FIG. 6 is a mass spectrum of S0456 of the active targeting near-infrared fluorescent molecule prepared;

[0038] FIG. 7 is a mass spectrum of Pemetrexed-Tyr (OtBu)-OtBu prepared;

[0039] FIG. 8 is a mass spectrum of Pemetrexed-Tyr-OH prepared;

[0040] FIG. 9 is a mass spectrum of Pemetrexed-Tyr-S0456 prepared; and

[0041] FIG. 10 is a fluorescence microscopic image of the Pemetrexed-Tyr-S0456 uptake by KB cells.

DETAILED DESCRIPTION OF EMBODIMENTS

[0042] The technical solutions in the embodiments of the present disclosure will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present disclosure; apparently, only some but not all embodiments of the present disclosure are described. All of other embodiments, obtained by those ordinarily skilled in the art based on the embodiments of the present disclosure without using any creative efforts, shall fall into the scope of protection of the present disclosure.

[0043] As shown in FIG. 1, the present disclosure takes

##STR00010##

as a matrix, and S0456 and Pemetrexed are structurally linked together through tyrosine to finally form the active targeting near-infrared fluorescent molecule. In FIG. 1, moiety a is Pemetrexed structure, moiety b is tyrosine structure, moiety c is S0456 structure, and M is independently selected from H, Na, and K.

[0044] In the present embodiment, a following structural formula is taken as an example:

##STR00011##

[0045] A synthetic process thereof is as follows.

[0046] As shown in FIG. 2, the synthetic process of the active targeting near-infrared fluorescent molecule of the present disclosure includes following steps:

Synthesizing S0456:

[0047] (1) 4-hydrazinophenylsulfonic acid (1.6 g, 31.9 mmol), 3-methyl-2-butane (2.10 ml, 90 mmol), and glacial acetic acid (50 ml) were mixed and heated to 120 ° C. in nitrogen atmosphere for 18 h, After precipitation in ethyl acetate, a crude product was filtered and collected in a form of pink solid, and a resulting product (6.5 g, 25.4 mmol) was dissolved in methanol (50 mL). A dissolving solution was dropwise added into a solution of potassium hydroxide (1.7 g, 30 mmol) and isopropanol (20 ml) under mild conditions, and the crude mixture was filtered and washed to obtain a brown solid, with yield of 97%, and a structural formula of the brown solid is as follows:

##STR00012##

[0048] (2) Product 1 (2.3 g, 8.3 mmol) and 1,4-butyl sultone was added into a toluene solution in nitrogen atmosphere to be heated at 110 ° C. for 48 h. Mixed materials were cooled to room temperature and a solvent was precipitated. To a crude mixture methanol (10 ml) was added and the resultant was stirred for 30 minutes. Crude mixture was filtered; the resultant was collected, and dissolved in a mixture of water (10 ml) and methanol (50 ml) at 2:1 (v/v). The mixed solution was slowly added to acetonitrile (160 ml) with a dropping funnel. A precipitate was filtered and a pink solid was collected with yield of 40%, wherein a structural formula of the pink solid is as follows:

##STR00013##

[0049] (3) Product 2 (1.5 g, 2.79 mmol), Vilsmeier-Haack reagent (0.5 g, 1.39 mmol), and anhydrous sodium acetate (0.342 g, 4.17 mmol) were refluxed and heated in 20 mL of anhydrous ethanol for 6 h in nitrogen atmosphere. Reaction mixture was cooled to room temperature, and then filtered, washed with ethanol and methanol, and a brownish green solid (S0456) was collected with yield of 90%, wherein a structural formula of the product is as follows:

##STR00014##

[0050] FIG. 5 and FIG. 6 show hydrogen spectrum and mass spectrum of S0456, respectively.

[0051] Synthesizing a targeted drug: a pemetrexed hydrolytic acid (1.05 g, 3.52 mmol) was dissolved in DMF, and stirred until the resultant was dissolved, HATU (2.007 g, 5.28 mmol), O-tert-butyl-L-tyrosine tert-butyl ester hydrochloride (1.161 g, 3.52 mmol), and DIEA (1.364 g, 10.56 mmol) were sequentially added into a flask, and stirred until the resultant was dissolved completely, the reaction was carried out at room temperature for 30 min under the protection of nitrogen, and the reacted solution was dropwise added into 0.1 N aq. HCI (1.0 L, 0.14 M) to generate a light yellow precipitate, followed by suction filtration and vacuum drying to obtain (5) 2.04 g of a solid with yield of 95%, wherein a structural formula of the product is as follows:

##STR00015##

[0052] The mass spectrum of Pemetrexed-Tyr (OtBu)-OtBu is as shown in FIG. 7.

[0053] Solid 4 (2.04 g, 3.34 mmol) was placed in a round-bottom flask, TFA and H.sub.2O (95:5, 10 mL) were added, the resultant was stirred for two hours, methyl tert-butyl ether was added, followed by precipitation, filtration, and vacuum drying, to obtained 1.507 g of product with yield of 98%, wherein a structural formula of the product is as follows:

##STR00016##

[0054] The mass spectrum of Pemetrexed-Tyr-OH is as shown in FIG. 8.

[0055] Preparing Pemetrexed-Tyr-S0456: to an aqueous (18 mL) solution of S0456 (2.909 g, 3.276 mmol), a Pemetrexed-Tyr (1.507 g, 3.276 mmol) trianion solution with pH of 11 was added dropwise at 23 ° C. The temperature of reaction mixture was raised to 90 ° C., the reaction mixture was stirred at 90 ° C. for 45 minutes, and formation of 7 was monitored by TLC. After the formation of product was completed, the reaction mixture was cooled to room temperature, and transferred into stirred acetone (0.5 L) as a stable flow through a cannula, to obtain a green precipitate. The precipitate was filtered on a sintering funnel under the vacuum by an air pump, and washed with acetone (3×500 mL). The green powder-like solid was dried in high vacuum for 12 h, and 6 (4.34 g) was quantitatively obtained.

##STR00017##

[0056] Upon test, the fluorescence performance spectrum of the Pemetrexed-Tyr-S0456 product is shown in FIG. 3 and FIG. 4, and the mass spectrum is as shown in FIG. 9.

[0057] In order to verify effects of the present disclosure, the following verification experiment was performed.

[0058] KB cells were incubated with 1×10.sup.−6 mol/L Pemetrexed-Tyr-S0456 for 2 h, the cells were washed with PBS for 2-3 times, and then the KB cells were imaged with a laboratory self-made near-infrared fluorescent microscope to obtain white-light and fluorescent images, and images were fused with Image-J, as shown in FIG. 10. Bright KB cells indicate that small molecules target the folate receptor of KB cells, and fluorescence intensity was detected under excitation of excitation light.

[0059] The above-mentioned are merely for preferred specific embodiments of the present disclosure; but the scope of protection of the present disclosure is not limited thereto. Equivalent substitutions or changes made by any person familiar with the technical field, according to the technical solution of the present disclosure and improved concept thereof, within the technical scope disclosed in the present disclosure, should be covered within the scope of protection of the present disclosure.