Technetium-99m-labeled FAPI derivatives containing dual pharmacophore groups, preparation method and application

Abstract

The present invention discloses a technetium-99m labeled FAPI derivative containing dual pharmacophores with a structural general formula of 99m Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS), and a preparation method and application thereof. Through two steps of the synthesis of the ligand HYNIC-PEG4-2F46 and 99m the preparation of Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS), the 99m Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex is obtained. The complex is easy to prepare, has high radiochemical purity and good stability, has high uptake and a high target-to-non-target ratio at the tumor site of U87MG tumor-bearing mice, and has specific binding to FAP in tumors, and is a novel tumor radioactive drug with clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of radiopharmaceuticals, and particularly relates to a technetium-99m labeled FAPI derivative containing dual pharmacophore groups, a preparation method thereof, and an application thereof. Background Art

[0002] The tumor microenvironment plays an extremely important role in the occurrence and development of tumors. Tumor-associated fibroblasts are an important component of the tumor microenvironment and play important roles in the proliferation, migration, immune escape, radio- and chemo-resistance, and energy metabolism of tumor cells. Fibroblast activation protein (FAP) is an important molecular marker on the surface of tumor-associated fibroblasts. Because it is selectively expressed on the cell membranes of fibroblasts in the stroma of most solid tumors, it has become an important target for tumor detection and treatment.

[0003] FAP small molecule inhibitors (FAPI) based on (4-quinolinoyl)-glycyl-2-cyanopyrrolidine have high affinity and high selectivity. Through continuous modification and improvement of the structure, a series of 68 Ga-labeled FAPI complexes have been reported and used for tumor imaging in primary and metastatic lesions. Among them, 68 68 Ga]Ga-FAPI-04 shows good biodistribution, rapid uptake in tumors, and good image contrast. Studies in cancer patients have shown that it has high uptake in primary occult carcinoma, sarcoma, cholangiocarcinoma, esophageal cancer, breast cancer, and lung cancer. Based on the structural improvement of FAPI-04, 68 68 Ga]Ga-FAPI-46 shows higher tumor uptake and retention than 68

[0004] 68 68 Ga]Ga-FAPI-46, and 68 68 ​​​​​​The tumor uptake of Ga]Ga-DOTA-2P(FAPI)2 was higher than that of [ 68 This indicates that the molecular probe containing two pharmacophores can effectively improve tumor-specific uptake, reduce non-target uptake, and improve the target-to-non-target ratio.

[0005] In the current reports on molecular probes targeting FAP, 68 Ga-labeled complexes account for the largest proportion, about 92%. 18 F-labeled FAPI molecular probe [ 18 F]AlF-NOTA-FAPI, [ 18 F]F-FAPI-74 and [ 18 F]AlF-P-FAPI, etc., have high specific tumor uptake and excellent imaging effect. However, the above probes are all PET (positron emission tomography) imaging agents, which are expensive and have certain limitations on clinical application and promotion. 99m Tc's SPECT (Single-Photon Emission Computed Tomography) diagnostic technology has the following advantages: 99m Tc is a 99 Mo / 99m SPECT nuclides prepared by Tc generators are convenient and easy to obtain. Due to the large number of SPECT devices in the world, about 27,000 units, they can be used in a wider market. The production process of medicine boxes is simpler and more controllable. The low price and the inclusion of medical insurance can benefit more patients. In recent years, the development of SPECT / CT and software based on cadmium zinc telluride (CZT) technology, spiral quantitative SPECT / CT production, further improve the imaging quality and shorten the imaging time, SPECT / CT imaging has ushered in the dawn again.

[0006] at present 99m There are few reports on Tc-labeled SPECT-based FAP-targeted molecular probes, among which [ 99m Tc]Tc-FAPI-34 and [ 99m Tc][Tc-(CN-PEG4-FAPI)6] + It has high affinity and high tumor uptake, but the high uptake of non-target organs in the abdomen restricts its application to a certain extent. In order to develop an excellent SPECT tumor molecular probe targeting FAP, in 2021, we developed a 99mTechnetium-labeled FAPI derivatives (Patent No.: ZL2021115355016), which are composed of FAPI derivatives modified with D-proline containing the HYNIC group (HYNIC-DP-FAPI), the co-ligand tricine, and sodium triphenylphosphine trisulfonate (TPPTS), and 99m coordinate with technetium to form a stable 99m Tc(HYNIC-DP-FAPI)(Tricine / TPPTS). This complex has good in vitro and in vivo stability and FAP affinity. Biodistribution shows high uptake in the tumor sites of tumor-bearing mice and low uptake in non-target organs such as the heart, liver, and muscle. However, due to high blood uptake, the tumor / blood ratio needs to be improved.

[0007] In order to effectively solve the problem that the tumor uptake of the technetium-labeled FAP tumor molecular probe and the tumor / blood ratio need to be improved, this patent modifies the structure based on the pharmacophore of FAPI-46 to obtain a ligand containing two FAPI-46 pharmacophores (HYNIC-PEG4-2F46), and then coordinates with the co-ligand Tricine / TPPTS and 99m technetium to form a stable tumor molecular probe 99m Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) with high stability, strong targeting, good target-to-non-target ratio, and easy to promote, 99m which is used for early tumor diagnosis, staging, and efficacy evaluation, and will lay a good foundation for the realization of translational medicine and precision medicine. Summary of the Invention

[0008] The object of the present invention is to provide a technetium-99m-labeled FAPI derivative containing dual pharmacophores, its preparation method, and application.

[0009] To achieve the above object, the technetium-99m-labeled FAPI derivative containing dual pharmacophores provided by the present invention 99m Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) has a structure shown in formula (I):

[0010]

[0011] In this structural formula: the nitrogen atom on the hydrazino group in the HYNIC-PEG4-2F46 molecule, the phosphorus atom in the co-ligand TPPTS, and the oxygen and nitrogen atoms in tricine coordinate with 99m technetium to obtain 99m Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex.

[0012] Preparation method of technetium-99m labeled FAPI derivative containing dual pharmacophore groups, and the preparation steps are as follows:

[0013] a: Synthesis of ligand HYNIC-PEG4-2F46:

[0014] Dissolve compound 1 and compound 2 in an appropriate amount of N,N-dimethylformamide (DMF), then add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N-ethyldiisopropylamine (DIPEA), stir and react at room temperature. After the reaction is completed, spin-dry the reaction solution, add 25% triethylamine / tetrahydrofuran (TEA / THF), react at room temperature for 3 h, and separate and purify by reverse-phase preparative liquid chromatography to obtain compound 3; dissolve compound 4 in DMF, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt), react at room temperature, then add compound 3 and N-methylmorpholine (NMM), react at room temperature, spin-dry the solvent, dissolve it in trifluoroacetic acid (TFA) again, react at room temperature for 30 min, then add the reaction solution to ice-cold diethyl ether, a large amount of solid precipitates, centrifuge, and purify the obtained solid by reverse-phase preparative liquid chromatography to obtain compound 5; weigh an appropriate amount of compound 5 in a round-bottom flask, dissolve it in an appropriate amount of DMF, then successively add DIPEA and compound 6, react at room temperature for 6 h, spin-dry the solvent, and separate and purify by reverse-phase preparative liquid chromatography to obtain HYNIC-PEG4-2F46. The specific synthesis route is as follows:

[0015]

[0016] b: 99m Preparation of [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex:

[0017] Weigh an appropriate amount of Tricine, TPPTS, and HYNIC-PEG4-2F46 ligand and dissolve them in physiological saline, adjust the pH of the solution to 5.0, add an appropriate amount of freshly eluted Na 99m TcO4, and heat in a boiling water bath for 30 min to obtain the described 99m [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex.

[0018] Prepared by the above method 99mThe radiochemical purity of the [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex is greater than 95%, it is a hydrophilic substance, and has good in vitro stability. It has high uptake and good retention at the tumor site in tumor-bearing mice (1 h after injection: 21.07 ± 1.70% ID / g; 4 h: 15.09 ± 2.13% ID / g). After injection of the FAP inhibitor for inhibition, the tumor uptake was significantly reduced (from 21.07 ± 1.70% ID / g to 4.91 ± 0.48% ID / g), indicating that its uptake in tumors is specific to FAP. Compared with the technetium-99m-labeled FAPI derivative containing the HYNIC group ( 99m Tc(HYNIC-DP-FAPI)(Tricine / TPPTS)), the uptake of this drug in U87MG tumors was significantly increased 1 hour after injection (from 16.26 ± 2.71% ID / g to 21.07 ± 1.70% ID / g), and the target-to-non-target ratio was significantly increased (tumor / muscle ratio increased from 6.20 to 8.18; tumor / blood ratio increased from 2.42 to 3.10), achieving the expected effect. The imaging results show that it has obvious accumulation at the tumor site, low uptake in non-target tissues, and the uptake in tumors can be significantly inhibited by the FAP inhibitor. It is a new type of SPECT molecular probe with excellent performance for tumor imaging. Detailed implementation mode

[0019] The present invention is described in detail below by examples: A technetium-99m-labeled FAPI derivative containing dual pharmacophores labeled with technetium-99m, with the structural general formula 99m [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS), and its structural formula is as follows:

[0020]

[0021] In this structural formula: The nitrogen atom on the hydrazino group in the HYNIC-PEG4-2F46 molecule, the phosphorus atom in the co-ligand TPPTS, and the oxygen and nitrogen atoms in Tricine coordinate with 99m [Tc to obtain 99m [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex.

[0022] 99m The preparation method of the [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex, and its preparation steps are as follows:

[0023] a. Synthesis of HYNIC-PEG4-2F46 ​

[0024] Compound 1 (200 mg) and Compound 2 (195 mg) were dissolved in 10 mL of DMF. Then, HATU (182 mg) and DIPEA (215 μL) were added, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC until completion. The reaction solution was evaporated to dryness by rotary evaporation, and then 10 mL of 25% TEA / THF was added. The reaction was carried out at room temperature for 3 h and then evaporated to dryness. The product was separated and purified by reverse-phase preparative liquid chromatography to obtain Compound 3 (215 mg, 72%);

[0025] Compound 4 (32 mg) was dissolved in 5 mL of DMF. Then, EDCI (30 mg) and HOBt (21 mg) were added, and the reaction was carried out at room temperature for 10 min. Then, Compound 3 (215 mg) and NMM (42 μL) were added, and the reaction was carried out at room temperature for 2 h. The solvent was evaporated to dryness, and the residue was dissolved in 5 mL of TFA. The reaction was carried out at room temperature for 30 min. Then, the reaction solution was added to 50 mL of ice-cold diethyl ether, and a large amount of solid precipitated. The solid was centrifuged, and the obtained solid was purified by reverse-phase preparative liquid chromatography to obtain Compound 5 (131 mg, 63%);

[0026] Compound 5 (131 mg) was dissolved in 5 mL of DMF. Then, DIPEA (44 μL) was added, and finally Compound 6 (44 mg) was added. The reaction was carried out at room temperature for 6 h. The solvent was evaporated to dryness, and the product was separated and purified by reverse-phase preparative liquid chromatography to obtain HYNIC-PEG4-2F46 (76 mg, 46%). 1 H NMR (400 MHz, Methanol-d4) δ 9.02 (s, 1H), 8.63 (d, J = 5.1 Hz, 3H), 8.37 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.08 (s, 1H), 7.98 (d, J = 9.5 Hz, 3H), 7.89 (d, J = 7.5 Hz, 1H), 7.66 (dd, J = 8.1, 5.3 Hz, 6H), 7.61 (s, 3H), 7.53 - 7.38 (m, 3H), 7.17 (s, 1H), 5.16 - 5.12 (m, 3H), 4.39 - 4.20 (m, 12H), 4.11 (dt, J = 20.3, 10.1 Hz, 4H), 3.83 - 3.76 (m, 5H), 3.67 (q, J = 6.5 Hz, 12H), 3.54 - 3.51 (m, 34H), 3.15 (d, J = 2.5 Hz, 11H), 2.62 (s, 6H), 2.48 - 2.40 (m, 4H), 2.27 (s, 3H), 2.14 (s, 6H). MS: M (Formula: C 95 H 124 F4N 21 NaO 21S; MW: 2027.21), [M - Na + 4H] / 3: found 669.3, calcd 669.3。

[0027] The synthetic route is as follows:

[0028]

[0029] b. 99m Preparation of [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) Complex

[0030] Weigh 1 mg of Tricine and 2 mg of TPPTS and dissolve them in 0.5 mL of physiological saline. Add succinate buffer with a pH of 5.0 to adjust the solution pH to 5.0. Then, sequentially add 20 μg of the ligand HYNIC-PEG4-2F46 and 0.5 mL of freshly rinsed Na 99m TcO4 (about 370 MBq), and heat it in a boiling water bath for 30 min to obtain the 99m [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex.

[0031] The present invention 99m Performance determination of [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex:

[0032] 1. Identification of the complex

[0033] 99m [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) is identified by high performance liquid chromatography (HPLC): using a C18 reverse column, an SCL-10AVP type high pressure liquid chromatograph, phase A is water (containing 0.1% trifluoroacetic acid), phase B is acetonitrile (containing 0.1% trifluoroacetic acid), the gradient is that phase B is 10% from 0 - 2 min, phase B changes from 10% to 90% from 2 - 10 min, phase B is 90% from 10 - 15 min, phase B changes from 90% to 10% from 15 - 20 min, and phase B is 10% from 20 - 30 min. The injection volume is 20 μL and the flow rate is 1 mL / min. Measure 99m [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) retention time (R t ) is: 10.27 min.

[0034] 2. Determination of the lipophilic-hydrophilic partition coefficient of the complex

[0035] ​Take 0.9 mL of phosphate buffer (0.025 mol / L) with pH 7.4 in a 5 mL centrifuge tube. Add 1 mL of n-octanol and 0.1 mL of 99m Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) solution to the centrifuge tube, cover with a stopper, shake well, and centrifuge for 5 min (5000 r / min). Then, take out 3×0.1 mL from the organic phase and the aqueous phase respectively, measure the radioactivity counts of the two phases, and calculate their partition coefficient D (D = radioactivity of the organic phase / radioactivity of the aqueous phase). Repeat five groups. The measured 99m lipid-water partition coefficient (logD) of the Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex is -3.10 ± 0.06, indicating that it is a hydrophilic substance.

[0036] 3. Determination of the stability of the complex

[0037] Measure the radiochemical purity of the complex after it is placed at room temperature and in mouse serum at 37 °C for 4 hours. The results show that 99m the radiochemical purity of the Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex is greater than 90% after being placed at room temperature and in mouse serum at 37 °C for 4 hours, indicating good in vitro stability.

[0038] 4. Biodistribution experiment of the complex in tumor-bearing mice

[0039] Inject 0.10 mL of 99m Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) labeling solution (about 3.7×10 5 Bq) into the tail vein of Balb / c model mice bearing U87MG tumors. Sacrifice the mice under anesthesia 1 h and 4 h after injection. In addition, perform an in vivo inhibition experiment on 99m Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) using FAPI as follows: Inject 100 μL of physiological saline solution containing 100 μg of FAPI into the tail vein of mice. Inject 0.10 mL of 99m Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) labeling solution (about 3.7×10 5(Bq). The mice were sacrificed 1 h after anesthesia. Relevant tissues and organs such as the heart, liver, lung, kidney, spleen, bone, small intestine, stomach, muscle, blood, and tumor were taken, wiped clean, weighed, and their radioactivity counts were measured on a γ-Counter, and the percentage injected dose per gram (%ID / g) of each tissue was calculated. The number of mice in each time point was 4. The results are shown in Table 1.

[0040] Table 1 99m Biodistribution of [99mTc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) in U87MG tumor-bearing Balb / c nude mice (%ID / g, n = 4)

[0041]

[0042] 5. SPECT imaging of the complex in tumor-bearing mice

[0043] Inject 0.2 mL (about 37 MBq) of [99mTc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) solution via the tail vein of the Balb / c model mice bearing U87MG tumors. One hour later, anesthesia was induced using isoflurane gas. In the inhibition group, 100 μL of a physiological saline solution containing 100 μg of FAPI was injected 30 min in advance, and then 99m 0.2 mL (about 37 MBq) of the [99mTc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex was injected. One hour later, anesthesia was induced using isoflurane gas. The mice were fixed prone, and imaging was performed using SPECT / CT. The SPECT imaging results showed that in the experimental group 99m [99mTc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) accumulated significantly in the tumor, while the uptake in the tumor in the inhibition group was significantly reduced, further indicating its specific uptake in the tumor, suggesting that it can be used as a novel SPECT molecular probe with excellent tumor affinity. 99m

[0044] ​Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention, by changing different linking agents such as amino acids, peptide chains, polyethylene glycol (PEG) chains, fatty chains, etc., or using co-ligands such as N-tris(hydroxymethyl)methylglycine (Tricine) and ethylenediamine-N,N'-diacetic acid (EDDA), N-tris(hydroxymethyl)methylglycine (Tricine) and sodium diphenylphosphinebenzenesulfonate (TPPMS), N-tris(hydroxymethyl)methylglycine (Tricine) and disodium 3,3'-(phenylphosphinediyl)bis(benzene-1-sulfonate) (TPPDS), N-tris(hydroxymethyl)methylglycine (Tricine) and nicotinic acid (NIC), N-tris(hydroxymethyl)methylglycine (Tricine) and isonicotinic acid (ISONIC), N-tris(hydroxymethyl)methylglycine (Tricine) and 3,5-pyridinedicarboxylic acid (PDA), N-tris(hydroxymethyl)methylglycine (Tricine) and 3-pyridinesulfonic acid (PSA), N-tris(hydroxymethyl)methylglycine (Tricine) and glucoheptonate, N-tris(hydroxymethyl)methylglycine (Tricine) and glucosamine, N-tris(hydroxymethyl)methylglycine (Tricine) and mannitol, N-tris(hydroxymethyl)methylglycine (Tricine) and diphenylphosphinebenzoic acid, etc., radiolabeled to obtain radioactive preparations all fall within the scope of protection required by the present invention.

Claims

1. A technetium-99m labeled FAPI derivative containing dual pharmacophore groups, with the general structural formula being 99m Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS), and its structure is shown as follows: In this structural formula: The nitrogen atom on the hydrazino group in the HYNIC-PEG4-2F46 molecule, the phosphorus atom in the co-ligand TPPTS, and the oxygen and nitrogen atoms in Tricine coordinate with 99m Tc to obtain 99m the [Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex.

2. A preparation method for obtaining the technetium-99m labeled FAPI derivative containing dual pharmacophore groups as described in claim 1, characterized in that, The process steps are as follows: a: Synthesis of ligand HYNIC-PEG4-2F46: Dissolve compound 1 and compound 2 in an appropriate amount of N,N-dimethylformamide (DMF), then add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N-ethyldiisopropylamine (DIPEA), stir the reaction at room temperature. After the reaction is completed, spin-dry the reaction solution, add 25% triethylamine / tetrahydrofuran (TEA / THF), react at room temperature for 3 h, and separate and purify by reverse-phase preparative liquid chromatography to obtain compound 3; dissolve compound 4 in DMF, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt), react at room temperature, then add compound 3 and N-methylmorpholine (NMM), react at room temperature, spin-dry the solvent, then dissolve in trifluoroacetic acid (TFA), react at room temperature for 30 min, then add the reaction solution to ice-cold diethyl ether, a large amount of solid precipitates, centrifuge, and purify the obtained solid by reverse-phase preparative liquid chromatography to obtain compound 5; weigh an appropriate amount of compound 5 in a round-bottom flask, dissolve it in an appropriate amount of DMF, then sequentially add DIPEA and compound 6, react at room temperature for 6 h, spin-dry the solvent, and separate and purify by reverse-phase preparative liquid chromatography to obtain HYNIC-PEG4-2F46; The specific synthesis route is as follows: b: 99m Preparation of Tc]Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex: Weigh an appropriate amount of N-tris(hydroxymethyl)methylglycine (Tricine), triphenylphosphine trisodium m-sulfonate (TPPTS), and HYNIC-PEG4-2F46 ligand and dissolve them in physiological saline. Adjust the pH of the solution to 5.0, and add an appropriate amount of freshly rinsed Na 99m TcO4. Heat it in a boiling water bath for 30 min to obtain the 99m Tc] Tc-(HYNIC-PEG4-2F46)(Tricine / TPPTS) complex.

3. Use of the technetium-99m-labeled FAPI derivative containing dual pharmacophore groups as claimed in claim 1 in the preparation of a tumor imaging agent.

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