阅读说明：本技术 一种靶向成纤维细胞活化蛋白的治疗药物及其制备方法 (Therapeutic drug targeting fibroblast activation protein and preparation method thereof ) 是由 陈小元 徐鹏飞 郭志德 吴晓明 文雪君 于 2021-08-10 设计创作，主要内容包括：本发明提供一种靶向成纤维细胞活化蛋白的治疗药物前体化合物,其结构如下式(I)所示。本发明还提供一种靶向成纤维细胞活化蛋白的治疗药物,是以式(I)化合物为配体,经治疗性核素标记得到的配合物,其结构如式(II)所示。本发明还提供所述前体化合物和所述治疗药物的制备方法,及其在制备FAP蛋白高表达肿瘤的治疗药物中的应用。本发明所述的靶向成纤维细胞活化蛋白的治疗药物利用截短型伊文思蓝的修饰的成纤维活化蛋白抑制剂能够显著延长其循环半衰期,并且能够增强肿瘤摄取富集和保留时间。(The invention provides a therapeutic drug precursor compound targeting fibroblast activation protein, which has a structure shown in the following formula (I). The invention also provides a therapeutic drug of the targeted fibroblast activation protein, which is a complex obtained by using the compound of the formula (I) as a ligand and labeling a therapeutic nuclide, and the structure of the complex is shown as the formula (II). The invention also providesThe precursor compound, the preparation method of the therapeutic drug and the application of the precursor compound in preparing the therapeutic drug for FAP protein high expression tumor. The therapeutic drug targeting the fibroblast activation protein can remarkably prolong the circulation half-life of the therapeutic drug by utilizing the modified fibroblast activation protein inhibitor of the truncated Evans blue, and can enhance the uptake, enrichment and retention time of tumors.)

1. A therapeutic prodrug compound targeting fibroblast activation protein has a structure shown in the following formula (I):

2. a process for preparing a compound of claim 1 comprising the steps of:

reacting the tEB derivative with polyethylene glycol-2 COOH under the action of a condensation reagent HATU; then carrying out condensation reaction with FAPI small molecular inhibitor under the action of HATU; then removing Boc protection by TFA; finally reacting with DOTA-NHS to obtain a compound shown as a formula (I), which is recorded as 'DOTA-tEB-FAPI 02'; the synthetic route is as follows:

3. a therapeutic drug targeting fibroblast activation protein, which is a radiolabeled complex of DOTA-tEB-FAPI02, namely the compound DOTA-tEB-FAPI02 of the formula (I) in claim 1 is used as a ligand, and the complex is labeled by a therapeutic nuclide and can be used as a radionuclide therapeutic probe.

4. The therapeutic agent of claim 3, wherein: the therapeutic nuclide is selected from¹⁷⁷Lu、⁹⁰Y、²¹³Bi、²¹¹At、²²³Ra or²²⁵Ac, any one of them.

5. A therapeutic agent targeting a fibroblast activation protein, characterized by: therapeutic nuclides using the compound of formula (I) DOTA-tEB-FAPI02 as defined in claim 1 as ligand¹⁷⁷Lu is marked to obtain a complex, and the structure of the complex is shown as a formula (II):

6. a process for preparing a therapeutic agent according to claim 3, comprising the steps of: comprising dissolving a compound of formula (I) according to claim 1 in a buffer solution or deionized water; adding radionuclide solution into the obtained solution, and sealing for 5-40min to obtain radionuclide-labeled complex.

7. A process for preparing a therapeutic agent according to claim 3, comprising the steps of: 50 μ g of a compound of formula (I) according to claim 1 in 50 μ l of 0.1M buffer (pH 4) and acetonitrile 300uL, 3 μ l of 10mM AlCl is added₃The solution, and 50. mu.l of it was added¹⁸F^-Fully mixing and heating the aqueous solution (10-30mCi) to 105-; taking a C18 separation small column, slowly leaching with 10mL of absolute ethyl alcohol, and leaching with 10mL of water; the cooled labeling solution is diluted with 10mL of water and transferred to a separation column, the unlabelled 18F ions are removed with 10mL of water and the 50% strength ethanol is usedLeaching the product with normal saline; diluting the leacheate with normal saline, and carrying out sterile filtration to obtain the therapeutic drug injection shown in the formula (II).

8. Use of the precursor compound of claim 1 or the therapeutic agent of claim 3 or a pharmaceutically acceptable salt thereof for the preparation of a therapeutic agent for tumors with high expression of FAP protein.

9. The use of claim 8, wherein: the therapeutic drug of claim 3 is prepared into an injection which can be administered by intravenous injection and is used for the radiotherapy of patients with tumors with high expression of FAP protein.

10. The use of claim 9, wherein: the FAP protein high-expression tumor comprises but is not limited to breast cancer, colorectal cancer, ovarian cancer, lung cancer, gastric cancer or pancreatic cancer.

Technical Field

The invention relates to the field of nuclear medicine and molecular imaging, in particular to a therapeutic drug targeting fibroblast activation protein and a preparation marker thereof.

Background

Fibroblast Activation Protein (FAP) is a membrane serine peptidase expressed on the surface of tumor stroma-activated fibroblasts. Studies have shown that high expression of fibroblast activation protein is detected on the stromal fibroblast cell surface of more than 90% of epithelial malignancies. Therefore, FAP has become an important target for tumor imaging and therapy.

At present, radionuclide-labeled inhibitors of Fibroblast Activation Protein (FAPI) represented by quinolinic acid derivatives have made important progress in the field of precise imaging of tumors. For example, metal species⁶⁸Ga-labeled FAPI has achieved over 30 different types of tumor-specific PET imaging. Due to the fact that⁶⁸Ga³⁺The generator generates low primary output and short half-life (only 68min), so that mass production and distribution cannot be realized, and the diagnosis and application of the generator are greatly limited to clinical popularization. In addition, the FAPI is rapidly metabolized and eluted, so that the effective dose of a tumor part is low, the retention time is too short, and the requirement of therapeutic application cannot be met. Therefore, there is a need to develop new therapeutic drugs for FAPI with appropriate metabolic kinetics, higher tumor uptake doses and longer tumor retention times to meet the nuclide therapy needs.

Disclosure of Invention

Based on the above background, the primary object of the present invention is to develop a therapeutic nuclide drug targeting fibroblast activation protein.

To achieve the above object, the present invention was developed based on the following concept: by adopting an in-vivo in-situ albumin combination strategy and modifying FAPI (FAPI) through truncated Evans blue, albumin is used as a FAPI delivery carrier, so that the half-life period of the albumin in peripheral blood is prolonged, and the uptake enrichment and retention time of the albumin in tumors is prolonged.

The invention firstly provides a therapeutic drug precursor compound of a targeted fibroblast activation protein, which has the structure shown as the following formula (I):

on this basis, the present invention further provides a process for preparing a precursor compound of formula (I), comprising the steps of:

reacting the tEB derivative with polyethylene glycol-2 COOH under the action of a condensation reagent HATU; then carrying out condensation reaction with FAPI small molecular inhibitor under the action of HATU; then removing Boc protection by TFA; finally reacting with DOTA-NHS to obtain the compound shown in the formula (I), which is marked as 'DOTA-tEB-FAPI 02'.

The synthetic route of the steps is as follows:

the invention further provides a therapeutic drug targeting fibroblast activation protein, wherein the therapeutic drug is a radiolabeled complex of DOTA-tEB-FAPI02, namely the compound DOTA-tEB-FAPI02 shown in the formula (I) is used as a ligand, and the complex is obtained by labeling a therapeutic nuclide and can be used as a radionuclide therapeutic probe.

The therapeutic nuclide can be selected¹⁷⁷Lu、⁹⁰Y、²¹³Bi、²¹¹At、²²³Ra or²²⁵Ac, any one of them.

The preferred therapeutic drug of the invention is a therapeutic nuclide with the compound DOTA-tEB-FAPI02 of the formula (I) as a ligand¹⁷⁷Lu is marked to obtain a complex, and the structure of the complex is shown as a formula (II):

the invention also provides a preparation method of the therapeutic drug, which comprises the steps of dissolving the precursor compound in a buffer solution or deionized water; adding radionuclide solution into the obtained solution, and sealing for 5-40min to obtain radionuclide-labeled complex.

In a preferred embodiment of the present invention, the preparation method specifically comprises the following steps: 50 μ g of the compound of formula (I) was dissolved in 20 μ L DMSO, 200 μ L buffer (pH 5.5) was added, the mixture was shaken to dissolve completely, and about 5mCi was added¹⁷⁷LuCl₃. The mixture is shaken up and then heated to 95 ℃ for reaction for 30 min. After the reaction was completed, the reaction mixture was cooled to room temperature. A C18 separation cartridge was loaded and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water¹⁷⁷Lu ion, then 0.3mL of 10mM HCl in ethanol solution to obtain the product. Diluting the leacheate with normal saline, and carrying out sterile filtration to obtain the therapeutic drug injection shown in the formula (II).

The buffer solution is a substance for stabilizing the pH value of the reaction solution, and can be acetate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate, phosphate, a mixture thereof and the like.

Other chemicals used in the above synthesis steps are commercially available.

In still another aspect, the invention also provides application of the compound shown in the formula (I) or (II) or pharmaceutically acceptable salt thereof in preparing a therapeutic drug for tumors with high expression of FAP protein.

In the preferable application of the invention, the complex of the formula (II) is prepared into an injection, is administered by intravenous injection and is used for patients with tumors with high FAP protein expression.

In the application, the FAP protein high-expression tumor comprises but is not limited to breast cancer, colorectal cancer, ovarian cancer, lung cancer, gastric cancer or pancreatic cancer.

In the nuclide treatment drug structure of the targeted fibroblast activation protein, the modified fibroblast activation protein inhibitor of the truncated Evans blue can be used for obviously prolonging the circulation half-life period of the drug, and enhancing the uptake, enrichment and retention time of tumors. In addition, the therapeutic medicine of the targeted fibroblast activation protein is matched with certain diagnostic medicines of the targeted fibroblast activation protein, so that the diagnosis and treatment effects of the fibroblast activation protein positive tumor are expected to be improved. The diagnostic drug targeting fibroblast activation protein may be a radiolabeled complex represented by the following formula (III):

drawings

FIG. 1 is a mass spectrum of Compound 3 of example 1 of the present invention.

FIG. 2 is a mass spectrum of Compound 5 of example 1 of the present invention.

FIG. 3 is a mass spectrum of DOTA-tEB-FAPI02, a compound of example 1 of the present invention.

FIG. 4 is a schematic diagram showing the HPLC quality control result in example 2 of the present invention.

FIG. 5 prepared in example 2 of the invention¹⁷⁷Lu-DOTA-tEB-FAPI02 distributes experimental results in normal mice.

FIG. 6 prepared in example 2 of the invention¹⁷⁷SPECT imaging of Lu-DOTA-tEB-FAPI02 at different time points in U87 tumor-bearing mice.

Detailed Description

The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.

Example 1: preparation of compound DOTA-tEB-FAPI02

Synthesis of Compound 4:

to compound 3(1.0mmol) in N, N-dimethylformamide was added COOH-PEG, respectively₂-COOH(1.50mmol)、HATU(1.0mmol)And N, N-diisopropylethylamine (3.0mmol), stirred at room temperature overnight and the reaction monitored by HPLC for completion. The solvent was distilled off under reduced pressure to obtain a crude product. The crude product was reverse phase pillared and lyophilized to give pure compound 4 in 67% yield. Wherein, the mass spectrum of the compound 3 is shown in figure 1.

Synthesis of Compound 5:

in a 50mL flask, compound 4(0.1mmol), HATU (0.1mmol) and N, N-diisopropylethylamine (0.3mmol), FAPI small molecule inhibitor compound 1(0.1mmol) and 5mL of N, N-dimethylformamide were charged, reacted at room temperature and the completion of the reaction was monitored by HPLC. The solvent was distilled off under reduced pressure to obtain a crude product. The crude product was reverse phase pillared and lyophilized to give pure compound 5 in 54% yield. FIG. 2 is a mass spectrum of Compound 5. The structure of the FAPI small molecule inhibitor compound 1 is shown as the following formula (IV):

synthesis of compound DOTA-tEB-FAPI 02:

in a 25mL flask, compound 5(0.1mmol) was dissolved in TFA/dichloromethane (1: 9 by volume) to remove Boc protection, the progress of deprotection was monitored by HPLC until the reaction was complete, the solvent was blown dry with nitrogen, and DOTA-NHS (0.05g, 0.1mmol) and N, N-diisopropylethylamine (0.04g, 0.3mmol) were added sequentially to 5mL of N, N-dimethylformamide. The reaction system is stirred for reaction at room temperature, the reaction is monitored by HPLC until the reaction is finished, and the solvent is removed by reduced pressure distillation to obtain a crude product. The crude product was reverse phase pillared and lyophilized to give the pure compound DOTA-tieb-FAPI 02 in 55% yield. FIG. 3 is a mass spectrum of compound DOTA-tEB-FAPI 02.

The synthetic route of the steps is as follows:

example 2.177 preparation of the Lu-DOTA-tEB-FAPI02 Complex:

50 micrograms of example 1The compound DOTA-tieb-FAPI 02 prepared by the method is dissolved in 20 microliters DMSO, 200 mul of buffer solution (pH 5.5) is added, the mixture is shaken to be completely dissolved, and about 5mCi is added¹⁷⁷LuCl₃. The mixture is shaken up and then heated to 95 ℃ for reaction for 30 min. After the reaction was completed, the reaction mixture was cooled to room temperature. A C18 separation cartridge was loaded and rinsed slowly with 10mL of absolute ethanol and then 10mL of water. The labeling solution was diluted with 10mL of water and applied to a column, and the unlabeled fraction was removed with 10mL of water¹⁷⁷Lu ion, then 0.3mL of 10mM HCl in ethanol solution to obtain the product. Diluting the eluate with normal saline, and sterile filtering to obtain the final product¹⁷⁷Lu-DOTA-tEB-FAPI02 injection. The HPLC quality control results are shown in FIG. 4.

Experimental examples, analysis and application effects

1. HPLC analysis identification

The HPLC system is as follows: Shimadzulc-20A; a C18 column (YMC, 3 μm, 4.6X 150mm) was used for the analysis. Detection wavelength 254nm, flow rate 1mL/min, elution gradient: 0-3 minutes: 10% acetonitrile (0.1% TFA) and 90% water (0.1% TFA) remained unchanged; 3-16 minutes: increase to 90% acetonitrile (0.1% TFA) and 10% water (0.1% TFA); 16-18 min: reduced to 10% (0.1% TFA) and 90% water (0.1% TFA); 18-20 min: 10% acetonitrile (0.1% TFA) and 90% water (0.1% TFA) were maintained.

2、¹⁷⁷Experiments with Lu-DOTA-tEB-FAPI02 complex in normal mice and U87 tumor-bearing model mice.

Prepared according to the method of example 2 and with a purity of more than 95%¹⁷⁷Lu-DOTA-tEB-FAPI02, injected by tail vein with 1.3MBq in normal mice and U87 tumor bearing model mice¹⁷⁷Lu-tEB-FAPI. Pharmacokinetics were tested at different time points after injection and SPECT imaging was performed, and the results are shown in fig. 5 and 6. The results show that it is possible to display,¹⁷⁷Lu-DOTA-tEB-FAPI02 has good pharmacokinetics in normal mice and can be continuously taken up by tumor tissues and maintained for more than 48 hours in U87 tumor-bearing model mice, which indicates that¹⁷⁷Lu-DOTA-tEB-FAPI02 has significantly enhanced tumor uptake and retention time and can be used as a tumor therapeutic.

In conclusion, in the scheme provided by the invention, the modified fibroblast activation protein inhibitor utilizing the truncated Evans blue can obviously prolong the circulation half-life period of the inhibitor, and can enhance the uptake enrichment and retention time of tumors.

In addition, the therapeutic drug of the targeted fibroblast activation protein is matched with some diagnostic drugs of the targeted fibroblast activation protein for use, so that the diagnosis and treatment effect of the fibroblast activation protein positive tumor is expected to be improved.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.