A hynic-modified 4-(2-aminoethyl)benzenesulfonamide derivative and use thereof

Abstract

The present application relates to the field of radiopharmaceutical chemistry and clinical nuclear medicine technology, and particularly relates to a HYNIC modified 4-(2-aminoethyl) phenyl sulfonamide derivative and application thereof. The radioactive preparation obtained by labeling the HYNIC modified 4-(2-aminoethyl) phenyl sulfonamide derivative with a radionuclide can be prepared through kit, has high radiochemical purity and good stability, has a high uptake and target-to-non-target ratio at the tumor site of a HT-29 tumor-bearing mouse, and is specifically combined with carbonic anhydrase IX, so the radioactive preparation is a new type of tumor radioactive drug with clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of radiopharmaceutical chemistry and clinical nuclear medicine, and in particular to a radiolabeled HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative and its application. Background Art

[0002] Hypoxia is a common feature of solid tumors. Under hypoxic conditions, cancer cell metabolism favors glucose degradation, leading to the accumulation of large amounts of metabolic waste products such as lactate and carbon dioxide, resulting in acidification. Under these conditions, tumor cells express an enzyme called carbonic anhydrase IX (CA IX, CA9) to regulate intracellular and extracellular pH and combat acidosis, thereby promoting tumor cell proliferation, metastasis, and invasion. CA IX is a 459-amino acid metallotransmembrane protein divided into four domains: an N-terminal proteoglycan-like domain (PG), a catalytic domain, a transmembrane domain, and an intracellular domain. It regulates intracellular and extracellular pH by catalyzing the simple reaction of carbon dioxide hydration to bicarbonate, thereby achieving the physiological conditions required for tumor cell survival and proliferation. Because CA IX is expressed only in a very small number of healthy tissues, it can be used as a tumor-associated biomarker and is a promising target for tumor imaging and treatment. For most patients with clear cell renal cell carcinoma (ccRCC), CAIX is highly expressed even under normoxic conditions due to the deletion of the tumor suppressor protein (von Hippel-Lindau protein, pVHL) gene. Therefore, CAIX is a biomarker strongly associated with clear cell renal cell carcinoma.

[0003] At present, a large number of literatures have reported a variety of radionuclides (such as: 99m Tc, 68 Ga, 18 Compounds labeled with F, etc. are used for imaging and diagnosis of CAIX-related diseases, but most of them have the disadvantages of low tumor uptake, high kidney uptake, and low target-to-non-target ratio. Therefore, it is urgent to develop a radioactive probe with high tumor uptake and good imaging effect for the diagnosis of clinically relevant diseases. 99m Tc has ideal nuclide properties: it emits gamma rays with appropriate energy (140keV), has a suitable half-life (T 1 / 2 =6.02h), can be easily obtained by 99 Mo- 99m Tc generator was obtained. Therefore, it was developed 99mTc-labeled radioactive probes targeting CA IX have important clinical significance and practical value. Hydrazinyl nicotinamide (HYNIC) is a commonly used bifunctional linker, which can be used with co-ligands such as N-tris(hydroxymethyl)methylglycine (tricine) and triphenylphosphine tris(sulfonate) sodium (TPPTS) to prepare stable 99m Based on the above facts, the present invention is to prepare a HYNIC modified 4-(2-aminoethyl)benzenesulfonamide derivative, which is combined with the co-ligand to form a HYNIC complex. 99m Tc radiolabeling was used to prepare the corresponding radioactive probe to develop a new tumor imaging agent targeting CA IX, which has important scientific significance and broad clinical application prospects. Summary of the Invention

[0004] The purpose of the present invention is to provide a novel CAIX-targeting 99m The invention discloses a Tc-labeled HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative and a preparation method thereof.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] A HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative and its application, wherein the structural formula is (I):

[0007]

[0008] The derivatives prepared 99m The Tc complex specifically binds to CA IX, has high tumor uptake, and has an excellent target-to-non-target ratio, achieving satisfactory results in the diagnosis and treatment of tumors with high CA IX expression.

[0009] The present invention also provides a radioactive preparation, which comprises the HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative labeled with a radioactive nuclide and its application.

[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 is 99m 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 in the Tc complex are triphenylphosphine tris-metasulfonic acid sodium (TPPTS), diphenylphosphine benzene-3-sulfonic acid sodium (TPPMS), 3,3'-(phenylphosphine diyl) di(benzene-1-sulfonic acid) disodium (TPPDS), nicotinic acid (NIC), isonicotinic acid (ISONIC), 3,5-pyridinedicarboxylic acid (PDA), 3-pyridinesulfonic acid (PSA), etc.

[0015] The present invention also provides the use of the radioactive preparation in the diagnosis or treatment of tumors with high CA IX expression.

[0016] The beneficial effects of the present invention are as follows: the present invention provides a HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative and its application, and the radioactive preparation obtained by labeling it with a radionuclide has high uptake in tumors with high CA IX expression and a high tumor / non-target ratio, and is a safe, effective and popularizable new tumor radiopharmaceutical. DETAILED DESCRIPTION

[0017] The present invention provides a HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative and its application. In a preferred embodiment, the present invention provides a HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative having a general structural formula of [ 99m The radioactive preparation of Tc]Tc-HYNIC-AEBS (tricine / L) has the following structural formula:

[0018]

[0019] Where: L is 99m Tc forms a stable 99m The co-ligand components in the Tc complex are triphenylphosphine tris-metasulfonic acid sodium (TPPTS), diphenylphosphine benzene-3-sulfonic acid sodium (TPPMS), 3,3'-(phenylphosphine diyl) di(benzene-1-sulfonic acid) disodium (TPPDS), nicotinic acid (NIC), isonicotinic acid (ISONIC), 3,5-pyridinedicarboxylic acid (PDA), 3-pyridinesulfonic acid (PSA), etc.

[0020] The preparation steps are as follows:

[0021] a. Synthesis of ligand HYNIC-AEBS

[0022] Compound AEBS was weighed into a round-bottom flask, and an appropriate amount of DMF was added to dissolve it. Then, triethylamine and compound 1 were added and reacted at 60°C for 6 h.

[0023] After the reaction, the solvent was removed by rotary evaporation, and the ligand HYNIC-AEBS was obtained by column chromatography separation and purification.

[0024]

[0025] b. Complexes 99m Preparation of Tc]Tc-HYNIC-AEBS(tricine / L)

[0026] Dissolve 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, then add ligand HYNIC-AEBS, add freshly washed [ 99m Tc]NaTcO4 solution, react at 100℃ for 30min to obtain the [ 99m Tc]Tc-HYNIC-AEBS(tricine / L) complex.

[0027] The [ 99m The radiochemical purity of the [Tc]Tc-HYNIC-AEBS (tricine / L) complex is greater than 95%, it is hydrophilic, and exhibits excellent in vitro stability. It exhibits high tumor uptake in HT-29 tumor-bearing mice. Injection of the inhibitor acetazolamide (AZA) significantly reduces tumor uptake, demonstrating that its tumor uptake is specific for CA IX. Imaging results demonstrate significant tumor accumulation and that tumor uptake can be significantly inhibited by the AZA inhibitor. This makes it a novel, high-performance SPECT molecular probe for tumor imaging.

[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] The present invention is described in detail below by way of examples: A HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative and its application, the general structural formula of which is [ 99m Tc]Tc-HYNIC-AEBS(tricine / L)

[0030]

[0031] Where: L is 99m Tc forms a stable 99mThe co-ligand components in the Tc complex are triphenylphosphine tris-metasulfonic acid sodium (TPPTS), diphenylphosphine benzene-3-sulfonic acid sodium (TPPMS), 3,3'-(phenylphosphine diyl) di(benzene-1-sulfonic acid) disodium (TPPDS), nicotinic acid (NIC), isonicotinic acid (ISONIC), 3,5-pyridinedicarboxylic acid (PDA), 3-pyridinesulfonic acid (PSA), etc.

[0032] The preparation method is as follows, but is not limited to the complexes illustrated:

[0033] a. Synthesis of HYNIC-AEBS

[0034] AEBS (0.100 g, 0.5 mmol) was weighed into a round-bottom flask and dissolved in DMF. Then, 0.6 mL of triethylamine and compound 1 (0.264 g, 0.6 mmol) were added and reacted at 60°C for 6 h. After completion of the reaction, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (dichloromethane:methanol = 10:1, v / v) to obtain the ligand HYNIC-AEBS (0.177 g, 67%). 1 H NMR (400MHz, DMSO-d6) δ11.36(s,1H),9.02(s,1H),8.59(d,J=2.4Hz,1H),8.48(t,J=5.1Hz,1H),8.02(d,J=8.5Hz,2H),7.80 –7.72(m,3H),7.44(d,J=8.3Hz,2H),7.36(t,J=7.5Hz,1H),7.32–7.23(m,3H),3.50(q,J=6.7Hz,2H),2.93(t,J=7.3Hz,2H).; 13 C NMR(101MHz,DMSO-d6)δ165.25,159.28,147.80,145.54,144.56,142.44,141.31,138.40,132.28,129.92 ,129.78,128.64,127.46,126.50,126.27,125.93,121.80,106.71,49.22,40.93,35.35.;HR-MS(ESI)for C 21 H 20 N5O6S2Na[M+H] + :found 526.0825,calcd 526.0753.

[0035] b. Complexes 99m Preparation of Tc]Tc-HYNIC-AEBS (tricine / TPPTS)

[0036] 1 mg of tricine and 2 mg of TPPTS were weighed and dissolved in 0.5 mL of normal saline. Citrate buffer with a pH of 5.0 was added to adjust the solution to pH 5.0. 2 μg of ligand HYNIC-AEBS and 0.5 mL of freshly washed [ 99m Tc]NaTcO4 (about 370MBq), heated in boiling water bath for 30min to obtain the [ 99m Tc]Tc-HYNIC-AEBS (tricine / TPPTS) complex.

[0037] The present invention 99m Performance determination of Tc]Tc-HYNIC-AEBS (tricine / TPPTS) complex:

[0038] 1. Identification of Complexes

[0039] a. High performance liquid chromatography (HPLC) identification

[0040] In HPLC, a C18 reverse phase column and an SCL-10AVP high pressure liquid chromatograph were used. Phase A was water (containing 0.1% trifluoroacetic acid), and phase B was methanol (containing 0.1% trifluoroacetic acid). The gradient was: 0-2 min: 10% phase B, 2-10 min: 10% to 90% phase B, 10-15 min: 90% phase B, 15-20 min: 90% to 10% phase B, and 20-30 min: 10% phase B. The injection volume was 20 μL, and the flow rate was 1 mL / min. 99m Tc]Tc-HYNIC-AEBS(tricine / TPPTS) retention time (R t ) is: 11.44min.

[0041] b. Thin layer chromatography (TLC) identification

[0042] TLC identification was performed using polyamide and rapid chromatography paper as stationary phases, acetonitrile and acetone / raw salt (1:1) as developing solvents. 99m Tc]R of Tc-HYNIC-AEBS(tricine / TPPPTS) f The value is 0-0.1, and the complex [ 99m Tc]R of Tc-HYNIC-AEBS(tricine / TPPPTS) f The value is 0.7-1.0.

[0043] Table 1. Chromatographic results of each component

[0044]

[0045] The radiochemical purity of the markers determined by the above two methods was greater than 95%.

[0046] 2. Determination of lipid-water partition coefficient of complex

[0047] Take 0.9 mL of pH = 7.4 phosphate buffer (0.025 mol / L) in a 5 mL centrifuge tube, add 1 mL of n-octanol and 0.1 mL of 99m Tc]Tc-HYNIC-AEBS (tricine / TPPTS) solution, cover with a stopper, vortex to make it uniform, and centrifuge for 5 minutes (5000r / min). Then, take 3×0.1mL from the organic phase and the aqueous phase respectively, measure the radioactivity counts of the two phases, and calculate their distribution coefficient D (D=radioactivity of organic phase / radioactivity of aqueous phase). Repeat three times. 99m The lipid-water partition coefficient (log D 7.4 ) is -2.90±0.03, indicating that it is a hydrophilic substance.

[0048] 3. In vitro stability determination of the complex

[0049] The radiochemical purity of the complex was determined by placing it in mouse serum at room temperature for 6 hours and at 37℃ for 4 hours. The results showed that 99m The radiochemical purity of the complex was greater than 95% after being placed in room temperature for 6 h and in mouse serum at 37°C for 4 h, indicating good in vitro stability.

[0050] 4. Biodistribution of the complex in tumor-bearing mice

[0051] 0.1 mL of [ 99m Tc]Tc-HYNIC-AEBS (tricine / TPPTS) labeling solution (about 3.7×10 5 Bq), mice were killed at 30min and 120min after injection. 99m Tc]Tc-HYNIC-AEB S (tricine / TPPTS) was used to perform an in vivo inhibition experiment in mice. The method was as follows: 100 μL of saline solution containing 200 μg of AZA was injected into the mouse through the tail vein, and 60 minutes later, 0.1 mL of [ 99mTc]Tc-HYNIC-AEBS (tricine / TPPTS) labeling solution (about 3.7×10 5 Bq) and 120 minutes later, tumor-bearing mice were sacrificed. Heart, liver, lung, kidney, spleen, stomach, bone, flesh, small intestine, large intestine, blood, tumor, and other relevant tissues and organs were removed, cleaned, and weighed. Radioactivity was measured using a γ-counter, and the percent injected dose per gram (%ID / g) was calculated for each tissue. Three mice were included in each time period. The results are shown in Table 2.

[0052] Table 2. 99m Biodistribution of Tc]Tc-HYNIC-AEBS (tricine / TPPTS) in nude mice bearing HT-29 tumors

[0053]

[0054] 5. SPECT / CT Imaging of the Complex in Tumor-Bearing Mice

[0055] Injection of HT-29 tumor nu / nu model mice into the tail vein 99m Tc]Tc-HYNIC-AEBS (tricine / TPPTS) solution 0.2 mL (about 37 MBq), and after 120 minutes, isoflurane gas anesthesia was used. The inhibition group needed to be injected with 100 μL of saline solution containing 200 μg of acetazolamide 60 minutes in advance, and then injected with [ 99m Tc]Tc-HYNIC-AEBS (tricine / TPPTS) solution 0.2mL (about 37MBq), after 120 minutes, isoflurane gas anesthesia was used. The mice were fixed in prone position and imaged using SPECT / CT. SPECT / CT imaging results showed that in the experimental group [ 99m Tc]Tc-HYNIC-AEBS (tricine / TPPTS) was significantly concentrated in tumors, while the uptake of tumors in the inhibition group was significantly reduced, further demonstrating that its uptake in tumors was specific and that it could be used as a new type of tumor molecular probe.

[0056] Although the present application has been described in detail with general description and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present application. Therefore, these modifications or improvements made on the basis of the spirit of the present application, the derivatives obtained after structural modification with HYNIC as a chelating agent, and the radiopharmaceuticals obtained after radionuclide labeling of the co-ligands such as N-tris(hydroxymethyl)methylglycine (Tricine) and 3,3'-(phenylphosphine diyl) bis(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, N-tris(hydroxymethyl)methylglycine (Tricine) and diphenylphosphine benzoic acid, etc. also belong to the scope of the present application.

Claims

1. A HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative, characterized in that: The structural formula of the HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative is (I):

2. A radioactive preparation, characterized in that The structural formula of the radioactive agent is (II): Where: L is 99m Tc forms a stable 99m The co-ligand components of the Tc complex are triphenylphosphine tris-metasulfonic acid sodium (TPPTS), diphenylphosphine benzene-3-sulfonic acid sodium (TPPMS), 3,3'-(phenylphosphine diyl) di(benzene-1-sulfonic acid) disodium (TPPDS), nicotinic acid (NIC), isonicotinic acid (ISONIC), 3,5-pyridinedicarboxylic acid (PDA), and 3-pyridinesulfonic acid (PSA).

3. Use of the radioactive preparation according to claim 2 in the preparation of a tumor imaging agent.

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