Preparation method of a dimer MR probe targeting fibroblast activation protein

Abstract

The present invention relates to a preparation method of a dimeric MR probe targeting fibroblast activation protein, and the method comprises the following steps: (1) Dissolve succinic acid-lysine-FAPI-04 dimer in dimethylformamide, then add N,N-diisopropylethylamine and tetraazacyclododecane tetraacetic acid-N-hydroxysuccinimide ester, and react at room temperature to obtain a crude product; the crude product is concentrated to obtain tetraazacyclododecane tetraacetic acid-succinic acid-lysine-FAPI-04 dimer, denoted as DOTA-Suc-Lys-(FAPI-04)2; (2) Dissolve the DOTA-Suc-Lys-(FAPI-04)2 in H2O, then add GdCl3, and reflux at 100 °C for 1 hour to obtain gadolinium-tetraazacyclododecane tetraacetic acid-succinic acid-lysine-FAPI-04 dimer, denoted as Gd-DOTA-Suc-Lys(FAPI 04)2, abbreviated as the dimeric MR probe. The synthesis of the present invention has a high yield, the method is simple and easy to implement, and the obtained dimeric MR probe has better tumor targeting property, which is beneficial to MR tumor targeted imaging, so as to be expected to achieve the purpose of accurate MR diagnosis of early tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of imaging tumor targeting probes, and particularly to a preparation method of a dimer MR probe targeting fibroblast activation protein. Background Art

[0002] Malignant tumors are the main killers threatening human health, mainly due to the inability to diagnose early; poor drug targeting, drug resistance, and poor efficacy for metastasis, etc.

[0003] Molecular Imaging is a new discipline resulting from the combination of modern imaging techniques and molecular biology. It uses specific molecules in the body as target objects, and can display the functions and metabolisms at the molecular level in vivo through modern imaging equipment outside the body, and directly display the physiological and pathological processes at the cellular or molecular level through images. More importantly, molecular imaging can detect diseases at the molecular level, improve the level of early disease diagnosis, and truly achieve early diagnosis and early treatment of diseases.

[0004] Magnetic resonance imaging (MRI) is a non-invasive imaging method, with advantages such as high resolution, no radioactive radiation, and long-term imaging. It is used in clinical medicine to evaluate the anatomical structure of the human body and the structure and function of tissues or organs, so as to diagnose diseases. The difference in relaxation time between tissues and between tissues and lesions is the basis for magnetic resonance imaging diagnosis. However, MRI is not very sensitive to the lesions of tiny tissues, molecular activities, or cell activities. Therefore, in MRI enhanced examination, by intravenously administering a contrast agent to artificially change the TIWI or T2WI signal intensity contrast between tissues and lesions, the sensitivity of disease diagnosis can be improved.

[0005] Currently, the commonly used contrast agent for nuclear magnetic resonance (MR) is a paramagnetic chelate containing gadolinium (Gd). It mainly shortens the T1 value, increases the signal intensity contrast between lesions and normal tissues on T1WI images, and helps localize and qualitatively analyze the lesions through different enhancement methods and types of lesions. For example, the traditional MR gadolinium chelate gadoterate meglumine (Gd-DOTA) belongs to an ionic non-specific extracellular fluid contrast agent. By using paramagnetic small molecule contrast agents, etc., it can help improve the sensitivity and contrast of imaging. Since it mainly diagnoses diseases through the lesion enhancement mode, that is, the blood supply situation, it lacks specificity in the diagnosis of tumors and is prone to missed diagnosis and misdiagnosis.

[0006] Tumor tissues are composed of parenchymal cells and stromal cells. Currently, most new MR probes target tumor parenchymal cells. However, studies have shown that stromal cells account for a larger proportion. Cancer-associated fibroblasts (CAFs) are the main stromal cells in the tumor cell growth microenvironment and an important part of the tumor stroma. In tumor lesions, the tumor stroma is very abundant. In some tumor lesions, its proportion can be as high as 90% of the entire tumor tissue, significantly higher than that of tumor parenchymal cells. There are obvious differences between cancer-associated fibroblasts and normal fibrosis: the former characteristically highly expresses fibroblast activation protein (FAP), while the latter expresses very little of this protein. More than 90% of malignant tumors show selective high expression of fibroblast activation protein, especially in breast, colon, and pancreatic tumors characterized by desmoplastic reactions, and it is hardly expressed in normal tissues and benign tumors. Currently, FAP has become an important target for tumor diagnosis and treatment. Fibroblast activation protein inhibitors (FAPIs) can specifically bind to FAP and selectively target FAP-positive tissues, thereby enabling imaging of the tumor stroma.

[0007] Clemens Kratochwil et al. from Heidelberg University in Germany screened out two more optimal fibroblast activation protein effective inhibitors (FAPIs): FAPI-02 and FAPI-04 molecules from more than 30 alternative compounds, and prepared positron imaging agents [68Ga]-FAPI-04 and [68Ga]-FAPI-02 by labeling with the radioactive positron nuclide 68 gallium [68Ga]. Studies have shown that FAPI-04 has better chemical properties. [68Ga]-DOTA-FAPI-04 is a hot positron emission tomography (PET) imaging agent currently studied at home and abroad. It has now entered the clinical trial stage, and its sensitivity and specificity in diagnosing tumors have been widely recognized. It shows good sensitivity in the detection of 28 primary solid tumors and metastases. Some studies have shown that the sensitivity of [68Ga]Ga-DOTA-FAPI PET / CT for primary tumors is 98.2%, and the sensitivity for lymph node diagnosis is 86.4%. However, PET equipment is high-end and the examination cost is expensive. It must be labeled with the positron nuclide 68 Ga for imaging, and it is radioactive. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a preparation method of a dimer MR probe targeting fibroblast activation protein, which is simple in method and easy to implement.

[0009] To solve the above problems, a preparation method of a dimer MR probe targeting fibroblast activation protein according to the present invention includes the following steps:

[0010] ⑴ Dissolve succinic acid-lysine-FAPI-04 dimer (Suc-Lys-(FAPI-04)2) in dimethylformamide (DMF), then add N,N-diisopropylethylamine (DIPEA) and tetraazacyclododecane tetraacetic acid succinimide ester (DOTA-NHS), and react at room temperature for 120 min to obtain a crude product; the crude product is concentrated to obtain tetraazacyclododecane tetraacetic acid-succinic acid-lysine-FAPI-04 dimer, denoted as DOTA-Suc-Lys-(FAPI-04)2;

[0011] ⑵ Dissolve the DOTA-Suc-Lys-(FAPI-04)2 in H2O, then add GdCl3, and reflux at 100 °C for 1 hour to obtain gadolinium-tetraazacyclododecane tetraacetic acid-succinic acid-lysine-FAPI-04 dimer, denoted as Gd-DOTA-Suc-Lys(FAPI 04)2, abbreviated as dimer MR probe.

[0012] In the step ⑴, the molar ratio of succinic acid-lysine-FAPI-04 dimer, N,N-diisopropylethylamine, and tetraazacyclododecane tetraacetic acid succinimide ester is 1:3-4:2.5-3.5; the mass-volume ratio of succinic acid-lysine-FAPI-04 dimer to dimethylformamide is 55-65 mg:1 ml.

[0013] In the step ⑵, the molar ratio of DOTA-Suc-Lys-(FAPI-04)2 to GdCl3 is 1:4.5-5.5; the mass-volume ratio of DOTA-Suc-Lys-(FAPI-04)2 to H2O is 4.5-5.5 mg:1 ml.

[0014] The present invention has the following advantages compared with the prior art:

[0015] 1. The present invention constructs a dimer MR molecular probe Gd-DOTA-Suc-Lys-(FAPI 04)2 targeting fibroblast activation protein for the first time. Compared with the monomer MR molecular probe, the dimer MR molecular probe has a higher affinity for binding to the target, which is beneficial for imaging for a longer time.

[0016] 2. The present invention has modified the structure of gadoteric acid meglumine, a commonly used MR imaging agent in clinics, and taken fibroblast activation protein (FAP), which is highly expressed in the stroma of malignant tumors but hardly expressed in benign or normal tissues, as the imaging target. A dimer of fibroblast activation protein inhibitors (FAPIs) FAPI-04 molecules is linked to the Gd-DOTA molecule to prepare a specific MR molecular probe Gd-DOTA-Suc-Lys-(FAPI 04)2 targeting the tumor stroma. This probe can recognize and bind to FAP-positive tissue cells highly expressed in tumors, endowing the MR probe with tumor targeting properties, facilitating MR tumor targeting imaging, and thus expected to achieve the purpose of precise early tumor MR diagnosis.

[0017] 3. The present invention combines the superior soft tissue resolution ability and high-definition imaging efficiency of MR with the high target specificity of Gd-DOTA-Suc-Lys-(FAPI 04)2 molecular imaging. Its clinical transformation will improve the precise diagnosis level of early tumors in the field of magnetic resonance at home and abroad, providing a diagnostic basis for the early treatment of patients.

[0018] 4. The present invention has a high synthesis yield, and the method is simple and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following further elaborates on the specific implementation manners of the present invention in conjunction with the drawings.

[0020] Figure 1 It is a synthetic technical scheme diagram of Gd-DOTA-Suc-Lys-(FAPI 04)2 of the present invention.

[0021] Figure 2 It is a purity detection diagram of the synthesis of the product Gd-DOTA-Suc-Lys-(FAPI 04)2 of the present invention.

[0022] Figure 3 It is an LC-MS diagram of the product Gd-DOTA-Suc-Lys-(FAPI 04)2 of the present invention.

[0023] Figure 4 It is an in vitro longitudinal relaxation rate test diagram (7.0T MR) of Gd-DOTA-Suc-Lys-(FAPI 04)2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] Such as Figure 1As shown, the present invention starts from intermediate 7, and the main purpose is to synthesize the end product 8 Gd-DOTA-Suc-Lys-(FAPI 04)2.

[0025] A preparation method of a dimeric MR probe targeting fibroblast activation protein, comprising the following steps:

[0026] ⑴ Dissolve succinic acid-lysine-FAPI-04 dimer (Suc-Lys-(FAPI-04)2, compound 5) in dimethylformamide (DMF), then add N,N-diisopropylethylamine (DIPEA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid N-hydroxysuccinimide ester (DOTA-NHS, compound 6), and react at room temperature for 120 min to obtain a crude product; the crude product is concentrated to obtain 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-succinic acid-lysine-FAPI-04 dimer (compound 7), denoted as DOTA-Suc-Lys-(FAPI-04)2.

[0027] Wherein: the molar ratio of succinic acid-lysine-FAPI-04 dimer, N,N-diisopropylethylamine, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid N-hydroxysuccinimide ester is 1:3-4:2.5-3.5; the mass-volume ratio of succinic acid-lysine-FAPI-04 dimer to dimethylformamide is 55-65 mg:1 ml.

[0028] The molecular weight of Suc-Lys-(FAPI-04)2 is 1182.29; the molecular weight of DOTA-NHS is 501.49; the molecular weight of DIPEA is 129; the molecular weight of DOTA-Suc-Lys-(FAPI-04)2 is 1568.70.

[0029]

[0030]

[0031] ⑵ Dissolve DOTA-Suc-Lys-(FAPI-04)2 in H2O, then add GdCl3, and reflux at 100 °C for 1 hour to obtain gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-succinic acid-lysine-FAPI-04 dimer, denoted as Gd-DOTA-Suc-Lys(FAPI-04)2, simply referred to as FAPI-04 dimeric MR probe.

[0032] Wherein: the molar ratio of DOTA-Suc-Lys-(FAPI-04)2 to GdCl3 is 1:4.5-5.5; the mass-volume ratio of DOTA-Suc-Lys-(FAPI-04)2 to H2O is 4.5-5.5 mg:1 ml.

[0033] The molecular weight of Gd-DOTA-Suc-Lys(FAPI 04)2 is 1723.91.

[0034]

[0035]

[0036]

Working principle

[0037] The recognition and binding of the labeled compound to the target are the basis of molecular imaging, belonging to the category of receptor imaging, and having the advantages of high sensitivity and strong specificity in disease diagnosis. More than 90% of malignant tumors have a high selective expression of fibroblast activation protein (FAP), while it is hardly expressed in normal tissues and benign tumors. Therefore, FAP has become an important target for tumor imaging. Fibroblast activation protein inhibitors (FAPIs) can specifically bind to FAP and selectively target tissues positive for FAP, based on which specific imaging of the tumor stroma can be performed.

[0038] FAPI-04 is an effective inhibitor of fibroblast activation protein, which can specifically bind to the target fibroblast activation protein (FAP). The dimer (FAPI-04)2 can further improve the ability to bind to the target compared with the monomer and prolong the interaction time between the target and the receptor.

[0039] In order to construct the dimer (FAPI-04)2 molecule into an MR probe molecule, the present invention introduces Gd-DOTA to construct a novel MR molecular probe Gd-DOTA-Suc-Lys-(FAPI 04)2, which not only targets the tumor stroma but also improves the interaction time between the receptor and the target, facilitating imaging.

[0040] The label [68Ga] or gadolinium (Gd) only plays a tracer role. The former requires a PET device to detect the signal, and the latter requires an MR device to detect the corresponding signal. Whether imaging can be performed depends on the properties of the labeled compound and the imaging device. According to the principle of identity, since [68Ga]-FAPI-04 has tumor stroma targeting, the novel MR probe Gd-DOTA-Suc-Lys-(FAPI 04)2 has better tumor targeting.

[0041] The present invention converts the clinically validated tumor-targeting PET molecular probe [68Ga]Ga-DOTA-FAPI into an MR molecular probe Gd-DOTA-Suc-Lys-(FAPI04)2. This design introduces the essence of PET molecular imaging into the field of MR imaging, and is expected to combine the superior soft tissue resolution and high-definition imaging efficiency of MR with the high specificity targeting of molecular imaging. Through dimer modification, it promotes the long-term binding of the target to the receptor, resulting in a continuous increase in MRI signal in the tumor site, and improving the sensitivity and specificity diagnosis of early tumors.

[0042] Example A preparation method of a dimer MR probe targeting fibroblast activation protein, comprising the following steps:

[0043] ⑴ Dissolve Suc-Lys-(FAPI-04)2 (60.7 mg, 0.0513 mmol) in DMF (1 ml), then add DIPEA (33.11 μL, 0.205 mmol) and DOTA-NHS (77.18 mg, 0.154 mmol). After reacting at room temperature for 120 min, LC-MS shows that compound 5 is completely consumed, and a crude product is obtained. The crude product is concentrated to obtain 26.5 mg of DOTA-Suc-Lys-(FAPI-04)2. The yield is 32.9%.

[0044] ⑵ Dissolve DOTA-Suc-Lys-(FAPI-04)2 (25.6 mg, 0.016 mmol) in H2O (5 mL), then add GdCl3 (21.52 mL, 0.082 mmol), and reflux at 100 °C for 1 hour. LC-MS shows that compound 7 is completely consumed, and 23 mg of Gd-DOTA-Suc-Lys(FAPI 04)2 is obtained. The yield is 83.4%.

[0045]

Purity detection

[0046] The obtained Gd-DOTA-Suc-Lys(FAPI-04)2 is purified (A: 0.1% TFA 100% H2O; B: ACN), and the results are as Figure 2 shown. RT: 10.463 minutes, and its purity is 96.28%.

[0047]

LC-MS analysis

[0048] The obtained Gd-DOTA-Suc-Lys(FAPI-04)2 is analyzed by LC-MS, and the results are as Figure 3 shown: showing different molecular fragments, [M+3H] / 3, [M+4H] / 4, [M+2H] / 2, and the actual molecular weight is: 1723.62.

[0049]

In vitro experiments

[0050] The obtained Gd-DOTA-Suc-Lys(FAPI-04)2 was subjected to a longitudinal relaxation rate experiment: Gd-DOTA-Suc-Lys-(FAPI 04)2 with different concentration gradients was prepared in PBS for detection, as Figure 4 shown. The results showed that the longitudinal molar relaxation rate r1 of Gd-DOTA-Suc-Lys-(FAPI04)2 was 3.81 mM -1 s -1 .

Claims

1. Preparation method of a dimeric MR probe targeting fibroblast activation protein, comprising the following steps: ⑴ Dissolve succinic acid-lysine-FAPI-04 dimer in dimethylformamide, then add N,N-diisopropylethylamine and tetraazacyclododecane tetraacetic acid-N-hydroxysuccinimide ester, and react at room temperature for 120 min to obtain a crude product; the crude product is concentrated to obtain tetraazacyclododecane tetraacetic acid-succinic acid-lysine-FAPI-04 dimer, denoted as DOTA-Suc-Lys-(FAPI-04)2; The structural formula of the succinic acid-lysine-FAPI-04 dimer is: ； The structural formula of the tetraazacyclododecane tetraacetic acid-N-hydroxysuccinimide ester is: ； The structural formula of the DOTA-Suc-Lys-(FAPI-04)2 is: ； ⑵ Dissolve DOTA-Suc-Lys-(FAPI-04)2 in H2O, then add GdCl3, and reflux at 100 °C for 1 hour to obtain gadolinium-tetraazacyclododecane tetraacetic acid-succinic acid-lysine-FAPI-04 dimer, denoted as Gd-DOTA-Suc-Lys(FAPI04)2, abbreviated as the dimeric MR probe; the structural formula of the Gd-DOTA-Suc-Lys(FAPI 04)2 is: 。 2. The preparation method of a dimeric MR probe targeting fibroblast activation protein according to claim 1, characterized in that: In the step ⑴, the molar ratio of the succinic acid-lysine-FAPI-04 dimer, N,N-diisopropylethylamine, and tetraazacyclododecane tetraacetic acid-N-hydroxysuccinimide ester is 1:3-4:2.5-3.5; the mass-volume ratio of the succinic acid-lysine-FAPI-04 dimer to dimethylformamide is 55-65 mg:1 ml.

3. The preparation method of a dimeric MR probe targeting fibroblast activation protein according to claim 1, characterized in that: In the step ⑵, the molar ratio of DOTA-Suc-Lys-(FAPI-04)2 to GdCl3 is 1:4.5-5.5; the mass-volume ratio of DOTA-Suc-Lys-(FAPI-04)2 to H2O is 4.5-5.5 mg:1 ml.

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