Technetium-99m labeled folic acid derivative containing D-proline modification, preparation method and application

Abstract

The present invention relates to the fields of radiopharmaceutical chemistry and clinical nuclear medicine technology, and specifically to a D-proline-modified folate derivative and its use. The radioactive preparation obtained by labeling the D-proline-modified folate derivative with a radionuclide can be prepared by kitting. It has high radiochemical purity and good stability. It exhibits significant uptake at the tumor site in tumor-bearing mice, is significantly inhibited by inhibitors, has a good target / non-target ratio, and exhibits low renal uptake. It can be used as a novel tumor imaging agent targeting folate receptors.

Description

Technical Field

[0001] The invention belongs to the technical field of radiopharmaceuticals and clinical nuclear medicine, and particularly relates to a technetium-99m-labeled D-proline-modified folic acid derivative, a preparation method and an application thereof. Background Art

[0002] The folate receptor (FR) is a glycoprotein with a molecular weight of approximately 38-40 kDa, attached to the cell membrane via glycosylphosphatidylinositol. It primarily comprises three isoforms: α, β, and γ. The FR-α is expressed in a limited number of healthy tissues, such as the renal proximal tubules and epithelial cells of the intestine, lung, and choroid plexus. In other healthy tissues, FRs are located on the apical surface of polarized epithelial cells, making it difficult for them to bind externally absorbed folate. Consequently, FR-α expression is highly conserved in healthy tissues other than the kidney. However, studies have found that FR-α is highly expressed in many epithelial malignancies, such as ovarian, breast, endometrial, lung, and nasopharyngeal carcinomas. The expression level correlates with the degree of tissue differentiation, with higher FR-α expression observed in less differentiated or advanced malignancies. Therefore, FR-α isoforms have become a hot topic for research in the diagnosis and treatment of tumors with high FR-α expression using radioactively targeted molecular probes.

[0003] 99m Tc is the most widely used single-photon radionuclide in clinical practice. It has a half-life of 6.02 h, suitable gamma-ray energy (140 keV) and coordination chemical diversity, and can be obtained by elution from a molybdenum-technetium generator. It is cheap and easy to obtain. Therefore, the development of a new type of FR targeting radionuclide is a promising method. 99m Tc tumor radiopharmaceuticals have important practical significance. 99m Although most of the Tc-labeled radioactive complexes targeting FR have high uptake and good target / non-target ratios at tumor sites that positively express FR, these complexes have high radioactivity accumulation in the mouse kidneys, causing certain radiation damage to the kidneys. In addition, many drugs need to be separated and purified using high-performance liquid chromatography before injection, and the preparation steps are complicated, which is not convenient for clinical promotion. Therefore, it is necessary to develop new drugs that are easy to promote and have suitable kidney uptake. 99m Tc-labeled tumor imaging agent targeting FR.

[0004] The linker connects the targeting group and the chelating group connected to the radionuclide, and plays an important role in regulating the efficacy and pharmacokinetics of radiopharmaceuticals. The present invention uses D-proline as a linker to improve the pharmacokinetic properties of the complex, on the one hand, to maintain high uptake of the complex in tumors, and on the other hand, to reduce the uptake of the complex in the kidneys, thereby reducing radiation damage to the kidneys. 99m Based on the above background, the present invention synthesizes folic acid derivatives containing D-proline and hydrazino nicotinamide, and with the participation of other co-ligands, it is subjected to the 99m The exploration of new tumor radiopharmaceuticals that specifically target FR by Tc labeling has important scientific significance and broad clinical application prospects, and is also an important task facing this field. Summary of the Invention

[0005] The purpose of the present invention is to provide a technetium-99m labeled D-proline modified folic acid derivative which is simple to prepare, has high radiochemical purity, specifically targets FR and has suitable kidney uptake, and also to provide a preparation method thereof.

[0006] Specifically, the present invention provides the following technical solution: a technetium-99m labeled folic acid derivative containing D-proline modification, the structure of which is as follows (I):

[0007]

[0008] The corresponding 99m Tc complexes specifically bind to FR, with high tumor uptake and low non-target uptake, an excellent target-to-non-target ratio, and low kidney uptake, which can achieve satisfactory results in the diagnosis and treatment of tumors with high FR expression.

[0009] The present invention also provides a radioactive preparation, which comprises the above-mentioned D-proline-modified folic acid derivative labeled with a radioactive nuclide.

[0010] Preferably, in the above radioactive preparation, the radioactive nuclide portion is a metal radionuclide.

[0011] Preferably, in the above radioactive preparation, the metal radionuclide is 99m Tc, 99 Tc, 94m Tc, 94 Tc, 52 Mn, 186 Re or 188 Re.

[0012] Most preferably, in the above radioactive preparation, the radionuclide is99m Tc, the structural formula of the radioactive agent is (II):

[0013]

[0014] Where: L is 99m Tc forms a stable 99m The co-ligand components of Tc complexes are N-tris(hydroxymethyl)methylglycine (Tricine) and sodium triphenylphosphine tris-metasulfonate (TPPTS), N-tris(hydroxymethyl)methylglycine (Tricine) and sodium diphenylphosphine benzene-3-sulfonate (TPPMS), 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), etc.

[0015] The present invention also provides the use of the radioactive preparation in the diagnosis and / or treatment of FR-highly expressed tumors.

[0016] The beneficial effects of the present invention are as follows: the present invention provides a D-proline-modified derivative, a preparation method and an application thereof; the radioactive preparation obtained by labeling the derivative with a radionuclide has high uptake in tumors with high folate receptor expression, a good tumor / non-target ratio and appropriate kidney uptake, and is a safe, effective and popularizable new tumor radiopharmaceutical. DETAILED DESCRIPTION

[0017] The present invention provides a preparation method and application of a technetium-99m labeled folic acid derivative containing D-proline modification. In a preferred embodiment, the present invention provides a folic acid derivative having a general structural formula of 99m Radioactive preparations of Tc-DPFA-L:

[0018]

[0019] Where: L is 99m Tc forms a stable 99mThe co-ligand components of Tc complexes are N-tris(hydroxymethyl)methylglycine (Tricine) and sodium triphenylphosphine tris-metasulfonate (TPPTS), N-tris(hydroxymethyl)methylglycine (Tricine) and sodium diphenylphosphine benzene-3-sulfonate (TPPMS), 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), etc.

[0020] The preparation steps are as follows:

[0021] a: Synthesis of ligand DPFA:

[0022] Weigh an appropriate amount of compound 1 into a 25 mL round-bottom three-necked flask and dissolve it in dimethyl sulfoxide (DMSO). Then, add an appropriate amount of triethylamine. Under nitrogen, add an appropriate amount of compound 2. Allow to react overnight at room temperature in the dark. After the reaction, wash repeatedly with cold ether and dichloromethane, then dry under vacuum to obtain the ligand DPFA.

[0023] The specific synthetic route is:

[0024]

[0025] b: 99m Preparation of Tc-DPFA-L complex:

[0026] Dissolve DPFA and Tricine in normal saline, add TPPTS or TPPMS or NIC or ISONIC or PDA or PSA, add SnCl2·2H2O, adjust the solution pH to 5.0, and then add freshly washed Na 99m TcO4 solution was reacted at 100°C for 30 minutes to obtain the 99m Tc-DPFA-L complex.

[0027] Prepared by the above method 99m The radiochemical purity of the Tc-DPFA-L complex is greater than 90%, it is a hydrophilic substance, and has good in vitro stability. 99m Tc-DPFA-L has high tumor uptake and can be significantly inhibited. It has a high target to non-target ratio and low kidney uptake. SPECT / CT imaging results show obvious radioactive concentration in the tumor, which can also be significantly inhibited. Therefore, it is worthy of promotion and application as a new type of FR-targeted tumor imaging agent.

[0028] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or the product instructions were used.

[0029] Example 1

[0030] This embodiment provides a 99m Tc-labeled folic acid derivative containing D-proline modification, referred to as 99m Tc-DPFA-L, the structural formula is as follows:

[0031]

[0032] Where: L is 99m Tc forms a stable 99m The co-ligand components of Tc complexes are N-tris(hydroxymethyl)methylglycine (Tricine) and sodium triphenylphosphine tris-metasulfonate (TPPTS), N-tris(hydroxymethyl)methylglycine (Tricine) and sodium diphenylphosphine benzene-3-sulfonate (TPPMS), 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), etc.

[0033] The preparation method is as follows, but is not limited to the complexes illustrated:

[0034] a. Synthesis of ligand DPFA

[0035] 73 mg of compound 1 (0.15 mmol) was weighed into a 25 mL round-bottom three-necked flask and dissolved in 3 mL of dimethyl sulfoxide (DMSO). 75 mg of triethylamine (0.75 mmol) was then added. Under nitrogen, 96 mg of compound 2 (0.18 mmol) was added and allowed to react overnight at room temperature in the dark. After completion of the reaction, the mixture was repeatedly washed with cold ether and dichloromethane and dried under vacuum to yield 86 mg of the ligand DPFA (a 63.7% yield). 1H NMR (600MHz, DMSO-d6) δ (ppm): 8.98 (s, 1H), 8.60 (s, 1H), 8.34 (d, J = 23.3Hz, 1H), 7.97 (d, J = 7.5Hz, 2H), 7 .83(s,2H),7.73(d,J=7.8Hz,1H),7.63(d,J=7.9Hz,1H),7.31(s,1H),7.25(d,J=6.0Hz,1H),7.20(s,1H) ,6.88(s,1H),6.59(d,J=8.0Hz,1H),4.44(s,2H),4.30(s,1H),3.62(s,1H),3.51(d,J=17.5Hz,2H),3.08 (s,2H),2.95(s,1H),2.17(d,J=47.8Hz,2H),1.79(d,J=36.9Hz,6H),1.09(dd,J=14.6,7.2Hz,1H).HR-MS for C 39 H 40 N 13 O 10 S[M-Na] - :found 882.2753,calcd 882.2747.

[0036] b. 99m Preparation of Tc-DPFA-TPPTS Complex

[0037] 5 μg of ligand DPFA, 2 mg of N-tris(hydroxymethyl)methylglycine (Tricine), and 2 mg of triphenylphosphine tris(sulfonate) (TPPTS) were dissolved in an appropriate amount of physiological saline, and then 30 μg of SnCl2·2H2O and 0.1 mL of succinate buffer (pH = 5.0) were added. Freshly washed Na 99m TcO4, react at 100℃ for 30min to obtain the target complex 99m The radiochemical purity of Tc-DPFA-TPPTS was greater than 90% as determined by TLC and HPLC.

[0038]

[0039] c. 99m Preparation of Tc-DPFA-TPPMS Complex

[0040] 5 μg of ligand DPFA, 2 mg of N-tris(hydroxymethyl)methylglycine (Tricine), and 2 mg of diphenylphosphine-3-phenylsulfonate sodium (TPPMS) were dissolved in an appropriate amount of physiological saline, and then 30 μg of SnCl2·2H2O and 0.1 mL of succinate buffer (pH = 5.0) were added. Then, freshly washed Na 99m TcO4, react at 100℃ for 30min to obtain the target complex 99m The radiochemical purity of Tc-DPFA-TPPMS was greater than 90% as determined by TLC and HPLC.

[0041]

[0042] d. 99m Preparation of Tc-DPFA-NIC complex

[0043] Take 5 μg of ligand DPFA, 2 mg of N-tris(hydroxymethyl)methylglycine (Tricine), and 2 mg of nicotinic acid (NIC), dissolve them in an appropriate amount of normal saline, add 30 μg of SnCl2·2H2O and 0.1 mL of succinate buffer (pH = 5.0), and then add freshly washed Na 99m TcO4, react at 100℃ for 30min to obtain the target complex 99m The radiochemical purity of Tc-DPFA-NIC was greater than 90% as determined by TLC and HPLC.

[0044]

[0045] e. 99m Preparation of Tc-DPFA-ISONIC Complex

[0046] Take 5 μg of ligand DPFA, 2 mg of N-tris(hydroxymethyl)methylglycine (Tricine), 2 mg of isonicotinic acid (ISONIC), dissolve in an appropriate amount of physiological saline, then add 30 μg of SnCl2·2H2O and 0.1 mL of succinate buffer (pH = 5.0), and then add freshly washed Na 99m TcO4, react at 100℃ for 30min to obtain the target complex 99m The radiochemical purity of Tc-DPFA-ISONIC was greater than 90% as determined by TLC and HPLC.

[0047]

[0048] f. 99m Preparation of Tc-DPFA-PDA Complex

[0049] Take 5 μg of ligand DPFA, 2 mg of N-tris(hydroxymethyl)methylglycine (Tricine), and 2 mg of 3,5-pyridinedicarboxylic acid (PDA), dissolve them in an appropriate amount of physiological saline, then add 30 μg of SnCl2·2H2O and 0.1 mL of succinate buffer (pH = 5.0), and then add freshly washed Na 99m TcO4, react at 100℃ for 30min to obtain the target complex 99m The radiochemical purity of Tc-DPFA-PDA was greater than 90% as determined by TLC and HPLC.

[0050]

[0051] g. 99m Preparation of Tc-DPFA-PSA Complex

[0052] Take 5 μg of ligand DPFA, 2 mg of N-tris(hydroxymethyl)methylglycine (Tricine), 2 mg of 3-pyridinesulfonic acid (PSA), dissolve in an appropriate amount of physiological saline, then add 30 μg of SnCl2·2H2O and 0.1 mL of succinate buffer (pH = 5.0), and then add freshly washed Na 99m TcO4, react at 100℃ for 30min to obtain the target complex 99m The radiochemical purity of Tc-DPFA-PSA was greater than 90% as determined by TLC and HPLC.

[0053]

[0054] Experiments show that the complex 99m The properties of Tc-DPFA-L are as follows:

[0055] 1. Identification of Complexes

[0056] (1) TLC method

[0057] Thin layer chromatography (TLC) was used to determine the radiochemical yield and radiochemical purity of the labeled compound. The development system 1 used was Whatman filter paper-raw salt, and the development system 2 was polyamide film-acetonitrile. Under this system, the R f The values are shown in Table 1.

[0058] Table 1 R of radioactive components in various systems f value

[0059]

[0060]

[0061] The chromatographic identification results were99m The radiochemical yield and radiochemical purity of the Tc-DPFA-L complex were both greater than 90%, and it was used in subsequent experiments without further purification.

[0062] (2) HPLC method

[0063] High performance liquid chromatography (HPLC) was used to identify the radiochemical purity of the labeled substance: pure water containing 0.1% trifluoroacetic acid (phase A) and acetonitrile containing 0.1% trifluoroacetic acid (phase B) were used as the mobile phases, and the elution gradients were shown in Table 2 below.

[0064] Table 2. HPLC elution gradient

[0065]

[0066] The HPLC identification results showed that 99m The retention time of the Tc-DPFA-L complex is between 9.0 and 10.0 min.

[0067] 2. Determination of lipid-water partition coefficient of complex

[0068] Take 1.9mL of pH 7.4 phosphate buffer (0.025mol / L) in a 5mL centrifuge tube, add 2.0mL of n-octanol and 0.1mL of 99m The Tc-DPFA-L solution was stoppered, vortexed for 5 minutes, and centrifuged for 5 minutes (3000 rpm). Then, 3 × 0.1 mL of the solution was taken from each of the organic and aqueous phases. The radioactivity counts in the two phases were measured, and the partition coefficient D (D = radioactivity in the organic phase / radioactivity in the aqueous phase) was calculated. This was repeated three times. The lipid-water partition coefficient of the complex is shown in the following table:

[0069] Table 3 Results of lipid-water partition coefficients of complexes

[0070]

[0071]

[0072] The results of lipid-water partition coefficients showed that the complexes were all water-soluble substances.

[0073] 3. In vitro stability determination of the complex

[0074] The labeled complex 99m The radiochemical purity of Tc-DPFA-L was measured after it was placed in mouse serum at room temperature and at 37°C for 4 hours. The experimental results showed that the radiochemical purity of the complex was greater than 90% after it was placed in mouse serum at room temperature and at 37°C for 4 hours, indicating that it has good in vitro stability.

[0075] 4. Biodistribution Experiment of the Complex in Mice

[0076] In order to verify that a series of complexes are tumor imaging agents that specifically target FR, and the kidney, as an organ with high FR expression, can be used as one of the target organs to a certain extent, normal uptake and inhibition experiments were carried out in normal female Kunming mice using folic acid FA as an inhibitor. Each mouse was injected with 0.1 mL of complex solution (about 7.4×10 5 The control group received 100 μg of FA 30 minutes prior to administration. Two hours after administration, mice were sacrificed, and relevant tissues and organs, including heart, liver, lung, kidney, spleen, bone, intestine, stomach, muscle, and blood, were removed, cleaned, weighed, and radioactivity counted in a γ-counter. The percent injected dose per gram (%ID / g) was calculated for each tissue. Five mice were included in each phase. Biodistribution results are shown in Tables 4-9.

[0077] Table 4 99m Biodistribution results of Tc-DPFA-TPPTS in normal Kunming male mice (n=5)

[0078]

[0079] Table 5 99m Biodistribution results of Tc-DPFA-TPPMS in normal Kunming male mice (n=5)

[0080]

[0081]

[0082] Table 6 99m Biodistribution results of Tc-DPFA-NIC in normal Kunming male mice (n=5)

[0083]

[0084] Table 7 99m Biodistribution results of Tc-DPFA-ISONIC in normal Kunming male mice (n=5)

[0085]

[0086]

[0087] Table 8 99m Biodistribution results of Tc-DPFA-PDA in normal Kunming male mice (n=5)

[0088]

[0089] Table 9 99mBiodistribution results of Tc-DPFA-PSA in normal Kunming male mice (n=5)

[0090]

[0091] As shown in Tables 4-9, in the control group, the kidney, an organ with high PSMA expression, showed moderate renal uptake of all complexes at 2 hours, while uptake in blood and muscle was very low, with a high target-to-nontarget ratio and rapid clearance of nonspecific binding. Injection of the inhibitor FA 30 minutes beforehand significantly reduced renal uptake, demonstrating a robust inhibitory effect, demonstrating that these complexes specifically target FR.

[0092] In the above complexes, 99m Tc-DPFA-TPPTS is well taken up in the kidney where FR is highly expressed and can be significantly inhibited. It is also poorly taken up in non-target organs such as the intestine and blood. 99m Tc-DPFA-TPPTS was used as one of the representative compounds to conduct in-depth research in Balb / c female nude mice bearing KB tumors. The tumor-bearing mice were divided into a normal group and an inhibitory group. Each mouse was injected with 0.1 mL of the complex solution (approximately 7.4×10 5 The control group received 100 μg of FA 30 minutes prior to administration. Two hours after administration, mice were sacrificed, and relevant tissues and organs, including heart, liver, lung, kidney, spleen, bone, intestine, stomach, muscle, blood, and tumor, were removed, cleaned, weighed, and radioactivity counted in a γ-counter. The percent injected dose per gram (%ID / g) was calculated for each tissue. Biodistribution results in tumor-bearing mice are shown in Table 10.

[0093] Table 10 99m Biodistribution results of Tc-DPFA-TPPTS in Balb / c female nude mice bearing KB tumors (n=4)

[0094]

[0095] KB tumors are positive tumors with high expression of FR. 99m The Tc-DPFA-TPPTS complex exhibited high tumor uptake in KB tumors and was significantly inhibited by the inhibitor FA, demonstrating that the complex specifically binds to FR. Similarly, renal uptake was low, at 5.94±0.48 ID% / g 2 hours after injection, suggesting that it avoids radiation damage to the kidneys.

[0096] 5. SPECT / CT imaging experiments of the complex in tumor-bearing mice

[0097] The imaging experiment was divided into a normal group and an inhibition group. 99mA Tc-DPFA-TPPTS complex solution (approximately 18.5 MBq) was administered. The inhibitory group received a 100 μg FA folic acid solution 30 minutes prior to administration. Two hours after administration, mice were anesthetized with 1.5% isoflurane. Scanning parameters were set for a 15-minute SPECT scan and a 4-minute CT scan. Images were acquired using HiSPECT and vivoquant 2.5 software. Mice were positioned in a prone position for SPECT / CT imaging.

[0098] SPECT imaging results showed that 99m Tc-DPFA-TPPTS was significantly concentrated in the tumor of the normal group mice. Except for a certain concentration in the kidney, the uptake in other non-target organs such as the liver and intestine was low. However, the uptake in the tumor and kidney of the inhibition group mice was significantly reduced, indicating that 99m Tc-DPFA-TPPTS can be used as a tumor molecular probe specifically targeting FR.

[0099] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention include, in addition to the D-proline-modified folic acid derivatives mentioned in the present invention, the L-proline-modified folic acid derivatives and the corresponding co-ligands M are N-tris(hydroxymethyl)methylglycine (Tricine) and ethylenediamine-N,N'-diacetic acid (EDDA), N-tris(hydroxymethyl)methylglycine (Tricine) and triphenylphosphine tris-metasulfonate (TPPTS), N-tris(hydroxymethyl)methylglycine (Tricine) and diphenylphosphine benzene-3-sulfonate (TPPMS), N-tris(hydroxymethyl)methyl The radioactive preparations obtained by labeling with radionuclides such as N-tris(hydroxymethyl)methylglycine (Tricine) and 2-(pyridin-4-yl)acetic acid (PA), 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), and N-tris(hydroxymethyl)methylglycine (Tricine) and 3-pyridinesulfonic acid (PSA) all fall within the scope of protection claimed in the present invention. In addition, radioactive preparations obtained by radionuclide labeling of folic acid derivatives modified with D-proline or L-proline and the co-ligand M being N-tris(hydroxymethyl)methylglycine (Tricine) and 3,3'-(phenylphosphinediyl)di(benzene-1-sulfonic acid) disodium (TPPDS), N-tris(hydroxymethyl)methylglycine (Tricine) and glucoheptonate, N-tris(hydroxymethyl)methylglycine (Tricine) and glucosamine, N-tris(hydroxymethyl)methylglycine (Tricine) and mannitol, or N-tris(hydroxymethyl)methylglycine (Tricine) and diphenylphosphinobenzoic acid also fall within the scope of protection claimed in the present invention.

Claims

1. A folic acid derivative modified with D-proline, characterized in that: Its structural formula is as follows:

2. A radioactive preparation, characterized in that The radioactive preparation comprises the D-proline-modified folic acid derivative according to claim 1 labeled with a radionuclide.

3. The radioactive preparation according to claim 2, characterized in that The radionuclide is 99m Tc, 99 Tc, 94m Tc, 94 Tc, 52 Mn, 186 Re or 188 Re.

4. The radioactive preparation according to claim 3, characterized in that The structural formula of the radioactive agent is as follows:

5. Use of the radioactive preparation according to any one of claims 2 to 4 in the preparation of a tumor imaging agent targeting folate receptors.

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