HYNIC-modified 4-(2-aminoethyl) benzenesulfonamide derivative and application thereof

Abstract

The invention relates to the technical field of radiopharmaceutical chemistry and clinical nuclear medicine, in particular to an HYNIC modified 4-(2-aminoethyl) benzenesulfonamide derivative and application thereof. The radioactive preparation obtained by labeling the HYNIC-modified 4-(2-aminoethyl) benzenesulfonamide derivative with radionuclide can be prepared by a medicine box, has high radiochemical purity and good stability, has high uptake and target-to-non-target ratio at the tumor site of an HT-29 tumor-bearing mouse, can be specifically combined with carbonic anhydrase IX, and can be used for detecting the tumor site of the HT-29 tumor-bearing mouse, so that the HYNIC-modified 4-(2-aminoethyl) benzenesulfonamide derivative can be used for detecting the tumor site of the HT-29 tumor-bearing mouse. The compound is a novel tumor radiopharmaceutical 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 radioactively labeled 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 sugar degradation, which leads to the accumulation of a large amount of metabolic waste such as lactic acid and carbon dioxide in the cell and acidification. Under this condition, tumor cells express an enzyme called carbonic anhydrase IX (CA IX, CA9) to regulate the pH inside and outside the cell and resist acidosis, thereby promoting tumor cell proliferation, metastasis and invasion. CA IX is a metal transmembrane protein composed of 459 amino acids, which can be divided into four domains: N-terminal proteoglycan-like domain (proteoglycan like PG), catalytic domain, transmembrane domain, and intracellular domain. It regulates the pH inside and outside the tumor cell by catalyzing the simple reaction of carbon dioxide hydration to bicarbonate to achieve the physiological conditions required for tumor cell survival and reproduction. Since CAIX is only expressed in very few healthy tissues, CAIX can be used as a tumor-related marker and is a very potential tumor imaging and treatment target. For most patients with clear cell renal cell carcinoma (ccRCC), CAIX is highly expressed even under normoxic conditions due to the loss 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 related diseases. 99m Tc has ideal nuclide properties: it emits gamma rays with appropriate energy (140 keV) and has a suitable half-life (T 1 / 2 =6.02h), can be easily obtained by 99 Mo- 99m Tc generator is obtained. Therefore, it is developed 99mTc-labeled radioactive probes targeting CA IX have important clinical significance and practical value. Hydrazinyl nicotinamide (HYNIC) is a commonly used bifunctional linker that can be used with coligands such as N-tris(hydroxymethyl)methylglycine (tricine) and triphenylphosphine tris(sulfonate) (TPPTS) to prepare stable 99m Based on the above facts, the present invention prepares a HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative and combines it with a co-ligand 99m Tc radioactive labeling 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 cytotoxic drug that is simple to prepare, has high radiochemical purity, and specifically targets CAIX. 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 binds specifically to CA IX, has high tumor uptake, and has an excellent target to non-target ratio, and has 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 above 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 radioactive nuclide.

[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 of Tc complexes are triphenylphosphine tris-sulfonate sodium (TPPTS), diphenylphosphine benzene-3-sulfonate 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 above radioactive preparation in the field of 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 the derivative 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 radioactive drug. 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 of Tc complexes are triphenylphosphine tris-sulfonate sodium (TPPTS), diphenylphosphine benzene-3-sulfonate 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] Weigh compound AEBS into a round-bottom flask, add an appropriate amount of DMF to dissolve it, then add triethylamine and compound 1, and react at 60°C for 6 hours.

[0023] After the reaction was completed, 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 saline, add TPPTS or TPPMS or NIC or ISONIC or PDA or PSA, add SnCl2·2H2O, adjust the solution pH to 5.0, 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%, and it is a hydrophilic substance with good in vitro stability. It has a high uptake in the tumor site of HT-29 tumor-bearing mice. After injection of the acetazolamide (AZA) inhibitor, the tumor uptake is significantly reduced, indicating that its uptake in the tumor is specific to CA IX. The imaging results show that it is significantly concentrated in the tumor site, and its uptake in the tumor can be significantly inhibited by the AZA inhibitor. It is a new SPECT molecular probe with excellent performance that can be used 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. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used.

[0029] The present invention is described in detail by way of examples below: 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 of Tc complexes are triphenylphosphine tris-sulfonate sodium (TPPTS), diphenylphosphine benzene-3-sulfonate 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] The compound AEBS (0.100 g, 0.5 mmol) was weighed into a round-bottom flask, and an appropriate amount of DMF was added to dissolve it, and then 0.6 mL of triethylamine and compound 1 (0.264 g, 0.6 mmol) were added, and the mixture was reacted at 60° C. for 6 h. After the reaction, the solvent was removed by rotary evaporation, and the ligand HYNIC-AEBS (0.177 g, 67%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 10:1, V / V). 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] Weigh 1 mg tricine and 2 mg TPPTS and dissolve them in 0.5 mL normal saline. Add pH 5.0 citrate buffer to adjust the solution to pH 5.0. Then add 2 μg ligand HYNIC-AEBS and 0.5 mL freshly washed [ 99m Tc]NaTcO4 (about 370MBq), heated in a 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 a SCL-10AVP high pressure liquid chromatograph were used. Phase A was water (containing 0.1% trifluoroacetic acid), phase B was methanol (containing 0.1% trifluoroacetic acid), and the gradient was 0-2 min B phase was 10%, 2-10 min B phase changed from 10% to 90%, 10-15 min B phase was 90%, 15-20 min B phase changed from 90% to 10%, and 20-30 min B phase was 10%. The injection volume was 20 μL and the flow rate was 1 mL / min. Determination [ 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. In the fast chromatography paper-acetone / raw salt (1:1), 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 is 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 out 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 the organic phase / radioactivity of the aqueous phase), and 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 after it was placed in mouse serum at room temperature for 6 hours and at 37°C 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 hours and in mouse serum at 37°C for 4 hours, 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), and 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 the tumor-bearing mice were killed 120 minutes later. The heart, liver, lung, kidney, spleen, stomach, bone, meat, small intestine, large intestine, blood, tumor and other related tissues and organs were taken, wiped clean and weighed, and the radioactivity count was measured on a γ-Counter to calculate the percentage of injected dose per gram (%ID / g) of each tissue. The number of mice in each time item was 3. 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 the tumor, while the uptake of the tumor in the inhibition group was significantly reduced, further indicating that its uptake in the tumor was specific and that it could be used as a new type of tumor molecular probe.

[0056] 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, the derivatives obtained by structural modification using HYNIC as a chelating agent and the radioactive preparations obtained by radionuclide labeling of co-ligands such as Tricine and 3,3'-(phenylphosphinediyl)di(benzene-1-sulfonic acid) disodium (TPPDS), Tricine and glucoheptonate, Tricine and glucosamine, Tricine and mannitol, Tricine and diphenylphosphinobenzoic acid also fall within the scope of the present invention.

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 radioactive agent comprises the HYNIC-modified 4-(2-aminoethyl)benzenesulfonamide derivative of 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 (II): Where: L is 99m Tc forms a stable 99m The co-ligand components of Tc complexes are triphenylphosphine tris-sulfonate sodium (TPPTS), diphenylphosphine benzene-3-sulfonate 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.

5. Use of the radioactive preparation according to any one of claims 2 to 4 in the preparation of tumor radioactive drugs.

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