DUAL-MODE PROBE FOR DETECTING HYDROGEN SULFIDE AND USE THEREOF

Abstract

The present invention relates to a dual-mode probe for detecting of hydrogen sulfide and use thereof and, more specifically to a dual-mode probe that has excellent blood-brain barrier permeability and is capable of fluorescence and nuclear imaging and a use thereof for detecting hydrogen sulfide.

Claims

1. A complex compound into which the radioisotope Cu is introduced, defined by the following formula 1: ##STR00004## wherein R1 to R5 are each independently hydrogen or C1-C10 straight or branched alkyl, and Cu is .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, or .sup.67Cu; or a pharmaceutically acceptable salt thereof.

2. The complex compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R1 to R5 are each independently C1-C5 straight alkyl.

3. The complex compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the R1 to R3 are methyl.

4. The complex compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R1 to R3 are methyl, and R4 and R5 are hydrogen.

5. The complex compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the complex compound is a probe for detecting H.sub.2S.

6. The complex compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the lipophilicity log D.sub.7.4 of the complex compound is 1.5 to 3.0.

7. A contrast agent comprising the complex compound or pharmaceutically acceptable salt thereof according to claim 1.

8. The contrast agent according to claim 7, wherein the contrast agent is one or more selected from the group consisting of a contrast agent for positron emission tomography (PET), a contrast agent for fluorescence imaging, a contrast agent for gamma cameras, a contrast agent for single photon emission computed tomography (SPECT), a Cherenkov optical imaging contrast agent, and a contrast agent for charge-coupled device (CCD).

9. The contrast agent according to claim 7, wherein the contrast agent is a dual-modality contrast agent capable of simultaneous PET and fluorescence imaging.

10. The contrast agent according to claim 7, wherein the contrast agent is used to detect hydrogen sulfide H.sub.2S in the body of a subject.

11. The contrast agent according to claim 7, wherein the contrast agent is used to detect hydrogen sulfide H.sub.2S in the brain of a subject.

12. A composition for diagnosing inflammatory diseases, comprising the complex compound or pharmaceutically acceptable salt thereof according to claim 1.

13. The composition for diagnosing inflammatory diseases according to claim 12, wherein the inflammatory disease is one or more types selected from the group consisting of neuroinflammatory diseases, rheumatoid arthritis, non-rheumatic inflammatory arthritis, arthritis related to Lyme disease, inflammatory osteoarthritis, meningitis, osteomyelitis, inflammatory bowel disease, appendicitis, pancreatitis, sepsis, pyelitis, nephritis, and inflammation diseases due to bacterial infection.

14. The composition for diagnosing inflammatory diseases according to claim 13, wherein the neuroinflammatory disease is selected from the group consisting of Alzheimer's disease, vascular dementia, frontotemporal dementia, alcoholic dementia, Parkinson's disease, traumatic brain injury, Niemann-Pick disease, amyotrophic axial sclerosis, multiple sclerosis, Huntington's disease, Creutzfeldt-Jakob disease and stroke.

15. Use of the complex compound or pharmaceutically acceptable salt thereof according to claim 1 in preparing a composition for diagnosing inflammatory diseases.

16. A method for diagnosing inflammatory diseases, comprising: (a) administering the complex compound or pharmaceutically acceptable salt thereof according to claim 1 to a subject; (b) scanning the subject by using a fluorescence imaging device, a radioactive imaging device, or a combination thereof; and (c) analyzing the scanned image and diagnosing the subject as having an inflammatory disease when a fluorescence signal, a radiation signal, or a combination thereof is increased, as compared to the control group.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0069] FIG. 1 is a diagram showing the synthesis process of Cu-ASTM-FITC according to the present invention.

[0070] FIG. 2 shows .sup.1H NMR spectrum data of ATSM-FITC(4).

[0071] FIG. 3 shows .sup.13C NMR spectrum data of ATSM-FITC(4).

[0072] FIG. 4 shows HRMS spectrum data of ATSM-FITC(4).

[0073] FIG. 5 shows HRMS spectrum data of Cu-ATSM-FITC(5).

[0074] FIGS. 6A to 6D show experimental results on the sensitivity and selectivity of Cu-ATSM-FITC(5).

[0075] FIG. 6A Absorbance and emission spectra of Cu-ATSM-FITC(5) in PBS (10% DMSO, pH 7.4).

[0076] FIG. 6B Fluorescence spectrum of probe Cu-ATSM-FITC(5) (10 M) after reaction with various concentrations of H.sub.2S (0-100 M).

[0077] FIG. 6C Results of confirming the fluorescence intensity over time of Cu-ATSM-FITC(5) in the presence of NaHS, L-Cys, and GSH.

[0078] FIG. 6D Selectivity of Cu-ATSM-FITC(5) toward various biological competitors comprising H.sub.2S.

[0079] FIG. 7 is a diagram showing the mechanism by which Cu-ATSM-FITC(5) reacts with H.sub.2S to exhibit fluorescence activity.

[0080] FIG. 8 shows experimental results of confirming the detection limit of Cu-ATSM-FITC (5) in the presence of H.sub.2S.

[0081] FIG. 9 shows the results of calculating the quantum yield of ATSM-FITC(4) by comparison with the fluorescein standard value according to the equation.

[0082] FIG. 10 shows the results of confirming the change in fluorescence intensity of Cu-ATSM-FITC(5) in the presence or absence of H.sub.2S at various pH values.

[0083] FIGS. 11A to 11C show the results of in vitro evaluation of the toxicity of Cu(ATSM-FITC)(5) complex ((FIG. 11A) human cervical cancer HeLa cells; (FIG. 11B) human liver cancer cell line (HepG2); and (FIG. 11C) human embryonic kidney 293 (HEK293) cells).

[0084] FIGS. 12A to 12D show the results of in vitro MTT assay evaluation of the toxicity of CuS to various cell lines ((FIG. 12A) HeLa; (FIG. 12B) human embryonic kidney 293 (HEK293); (FIG. 12C) human liver cancer cells (HepG2), and (FIG. 12D) human brain glioblastoma cells (U87MG)).

[0085] FIG. 13 shows the results of histological H&E staining of the mouse brain cerebral cortex administered with a high dose of Cu(ATSM-FITC)(5).

[0086] FIG. 14 shows the results of treating live HeLa cells with Cu-ATSM-FITC(5) and detecting exogenous and endogenous H.sub.2S through fluorescence imaging.

[0087] FIGS. 15A to 15C show a schematic diagram showing the process in which .sup.64Cu-ATSM-FITC(6) radiolabeled with Cu-64 reacts with H.sub.2S to decomplex, and .sup.64CuS is precipitated (FIG. 15A); the UV-HPLC profile (black) of Cu-ATSM-FITC(5) and the radio-HPLC profile (red) of .sup.64Cu-ATSM-FITC(6) (FIG. 15B); and the decomplexation profile of .sup.64Cu-ATSM-FITC(6) reacted with various concentrations of NaHS (FIG. 15C).

[0088] FIG. 16 shows the results of evaluation of the stability of [64Cu][Cu(ATSM-FITC)](6) in PBS and FBS at 37 C.

[0089] FIGS. 17A to 17H are diagrams showing the results of in vivo detection of endogenous H.sub.2S in the mouse brain by .sup.64Cu-ATSM-FITC(6).

[0090] FIG. 17A Results of confirming distribution by tissue at the indicated time points after administration of .sup.64Cu-ATSM-FITC(6) in normal BALB/c mice.

[0091] FIGS. 17B to 17E PET/CT images 1 hour after administration of .sup.64Cu-ATSM-FITC(6).

[0092] FIG. 17F PET/CT images 1 hour after administering .sup.64Cu-ATSM-FITC(6) to mice treated with AOAA, an H.sub.2S inhibitor.

[0093] FIG. 17G Results of quantifying the amount absorbed 1 hour after administration of .sup.64Cu-ATSM-FITC(6) in the brains of normal mice and AOAA-treated mice.

[0094] FIG. 17H A diagram showing the results of quantifying the concentration of H.sub.2S in the brains of normal mice and AOAA-treated mice by using the methylene blue method and the results of relatively quantifying same based on biodistribution according to the present invention.

[0095] FIGS. 18A to 18C are diagrams showing the PET/CT images of the coronal section (FIG. 18A), sagittal section (FIG. 18B), and transverse (FIG. 18C) section of the brain 1 hour after administration of .sup.64Cu-ATSM-FITC(6) to SD-rats.

[0096] FIGS. 19A to 19D are diagrams showing the results of detecting H.sub.2S by nuclear medicine imaging in a neuroinflammation animal model using .sup.64Cu-ATSM-FITC (6).

[0097] FIG. 19A A schematic diagram showing a method of inducing a neuroinflammatory animal model by administering LPS to the right hemisphere of the mouse brain.

[0098] FIG. 19B Results of brain PET imaging 10 minutes after administration of .sup.64Cu-ATSM-FITC(6) to control and LPS-treated animal models (white arrow: LPS administration site).

[0099] FIG. 19C Results of quantifying .sup.64Cu-ATSM-FITC(6) in the brains of the control and LPS-treated animal models.

[0100] FIG. 19D Results of quantifying H.sub.2S in the brains of control and LPS-treated animal models according to the methylene blue method immediately after PET imaging.

[0101] FIG. 20 is a schematic diagram showing the process of synthesizing ATSM-aniline (9) and radiolabeling same with Cu-64 to produce .sup.64Cu-ATSM-aniline(10).

[0102] FIG. 21 shows the results of confirming the biodistribution 4 hours after administration of .sup.64Cu-ATSM-aniline(10) to normal BALB/c mice.

[0103] FIG. 22 shows radio-TLC profiles of .sup.64Cu-ATSM-FITC(6), .sup.64CuS, and a reaction mixture of .sup.64Cu-ATSM-FITC and H.sub.2S that the decomplexation percentage was calculated using the peak integration ratios of .sup.64Cu-ATSM-FITC and .sup.64CuS. Radio-TLC conditions: silica, MeOH:EtOAc=5:95.

[0104] FIG. 23 shows coronal PET/CT images acquired 1 hour after administration of .sup.64Cu-ATSM-FITC(6) to a control (Normal rat) and a Parkinson's disease rat model.

MODE FOR INVENTION

[0105] Hereinafter, the present invention will be described in detail by the following embodiments. However, the following embodiments are only for illustrating the present invention, and the present invention is not limited thereto.

Experimental Method

1. Synthesis of 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-((E)-2-((E)-3-(2-(methylcarbamothioyl)hydrazono)butan-2-ylidene)hydrazinecarbothioamido)benzoic Acid (ATSM-FITC, 4)

[0106] Compound 3 (191 mg, 1.02 mmol) in methanol (15 mL) was added to a methanol solution of FITC-NCS (380 mg, 1.02 mmol, 15 mL), and the mixture was stirred in the dark room at room temperature. The reaction was monitored by TLC (silica, MeOH:CH.sub.2Cl.sub.2=5:95) and confirmed to be completed within 30 minutes. It was subjected to concentration under reduced pressure to obtain a light yellow solid and purified by column chromatography to obtain ATSM-FITC(4) as a yellow solid (303 mg, 52.7%).

[0107] .sup.1H NMR (500 MHz; DMSO-d.sub.6): 2.28 (s, 3H), 2.32 (s, 3H), 3.05 (d, 3H, J=4.5 Hz), 6.57-6.62 (m, 4H), 6.69 (s, 1H), 6.70 (s, 1H), 7.27 (d, 1H, J=8.5 Hz), 8.02 (dd, 1H, J=2 Hz, J=8.5 Hz), 8.35 (d, 1H, J=2 Hz), 8.44-8.43 (m, 1H), 10.14 (s, 2H), 10.23 (s, 1H), 10.34 (s, 1H), 10.87 (s, 1H) ppm; .sup.13C NMR (125 MHz; DMSO-d.sub.6): 12.26, 12.68, 31.71, 83.54, 102.76, 110.00, 113.10, 120.23, 124.12, 126.60, 129.42, 133.02, 141.10, 148.10, 149.39, 150.55, 152.30, 159.96, 168.87, 177.15, 178.99 ppm; HRMS (FAB) m/z calcd. 577.1332 for C.sub.27H.sub.24N.sub.6O.sub.5S.sub.2 [M+H].sup.+, found 577.1332.

2. Synthesis of Copper(II) Complex of 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-((E)-2-((E)-3-(2-(methylcarbamothioyl)hydrazono)butan-2-ylidene)hydrazinecarbothioamido)benzoic Acid (Cu-ATSM-FITC (5))

[0108] CuCl.sub.2.Math.2H.sub.2O (46 mg, 0.27 mmol) in methanol (1 mL) was added to a solution of ATSM-FITC(4) (103 mg, 0.18 mmol). A dark brown precipitate was formed immediately. The mixture was then heated at reflux for 2 hours. It was cooled to room temperature, and the precipitate was collected, washed with methanol (31 mL), and dried under vacuum to obtain Cu-ATSM-FITC(5) as a brown solid (38 mg, 33%). The purity of the compound was confirmed by an HPLC profile (Waters, Grace smart RP C18 column [4.6 mm250 mm, 5 m], isocratic mobile phase MeOH:H.sub.2O=60:40, flow rate of 1 mL/min), and identified by HRMS. HRMS (FAB) m/z calcd. 638.0462 for C.sub.27H.sub.22CuN.sub.6O.sub.5S.sub.2 [M+H].sup.+, found 638.0470.

3. Radiolabeling of ATSM-FITC(4) Using .SUP.64.Cu

[0109] Stock solutions of chelators (1 g/L) were prepared in anhydrous DMSO. Complexation of .sup.64Cu with ATSM-FITC(4) was performed by mixing .sup.64CuCl.sub.2 (18.5 to 111 MBq) to which a carrier was not added in 0.01 M HCl (1 to 5 L) with a chelator solution (10 to 20 g) in 0.1 M ammonium acetate (pH 6.8, 100 L). It was subjected to incubation at 60 C. for 20 min in a thermomixer (800 rpm, Eppendorf). Completion of the reaction was confirmed by radio-TLC [silica, MeOH:EtOAc=5:95]. The formation of .sup.64Cu-ATSM-FITC(6) was confirmed by comparing the HPLC profiles of .sup.64Cu-ATSM-FITC(6) and Cu-ATSM-FITC(5) (Waters, Grace Smart RP C18 column [4.6 mm250 mm, 5 m], isocratic mobile phase consisting of MeOH:H.sub.2O=60:40, flow rate of 1 mL/min).

4. In Vitro Serum Stability

[0110] .sup.64Cu-ATSM-FITC(6) (10 L, 37 MBq) was first mixed with FBS (500 L) or PBS (pH 7.4) at the same volume, and the mixture was incubated at 37 C. Demetalization was monitored using radio-TLC (silica, MeOH:EtOAc=5:95) for up to 12 hours.

5. Animal Testing

[0111] All animal experiments were conducted in accordance with the approved animal protocols and guidelines established by the Kyungpook National University Animal Care Committee (Nos. KNU 2017-0096, 2019-0009, 2019-0101, and 2020-0079).

[0112] Male BALB/c mice (6 to 9 weeks old) were purchased from Hyochang Bioscience (Daegu, Korea). All mice were housed at 20 to 24 C. together with sufficient water and commercial food under a 12-h day/night cycle. For studies on H.sub.2S inhibition, male BALB/c mice were injected intraperitoneally with AOAA ((O-(carboxymethyl) hydroxylamine hemihydrochloride, 10 mg/kg, Sigma-Aldrich (C13408-1G)), 1 hour before administered with .sup.64Cu-ATSM-FITC(6) into the tail vein.

6. Preparation of Neuroinflammation Animal Models

[0113] Mice were mounted in a stereotaxic frame under isoflurane anesthesia. Sterile saline (control) or LPS (5 mg/mL, Sigma-Aldrich, Saint Louis, MO, USA, catalog number: L2630) was injected unilaterally into the right striatum (anterior-posterior [AP], 0.5 mm, medial) of each mouse by using a 28-gauge Hamilton syringe attached to an automatic microinjector. Injection was carried out at a rate of 0.2 L/min for 10 minutes. After each injection, the needle was left in place for 5 minutes before being slowly retracted.

7. Study on Biodistribution of .SUP.64.Cu-ATSM-FITC(6) in Normal Mice

[0114] BALB/c mice (male, 9 weeks old) were used to investigate the systemic distribution and clearance rate of .sup.64Cu-ATSM-FITC (6). .sup.64Cu-ATSM-FITC (6) with a radioactivity of 0.56 to 0.74 MBq in a saline solution (5% DMSO, 200 L) was injected into mice through the tail vein under anesthesia. Mice were sacrificed 5, 30, 1, and 2 hours after the injection (n=4). Blood was drawn, and organs comprising heart, lung, muscle, fat, bone, spleen, kidney, liver, intestine, and brain were harvested, weighed, and analyzed by using a -counter (Wallac Wizard 1480, PerkinElmer, France). Radioactivity was calculated as percentage of injected dose per gram (% ID/g).

8. Quantification of H.SUB.2.S in Brain by Methylene Blue Method

[0115] The methylene blue method was used to directly measure H.sub.2S concentration in brain tissues. Briefly, PET imaging was performed, brains were harvested, weighed, and homogenized in liquid N.sub.2. An ice-cold PBS solution in a volume equivalent to the weight of the homogenate (w/v, approximately 260 to 300 mg) was added, followed by vortex for 3 min and centrifugation (14,000 rpm, 5 min, 4 C.). Then, 100 L of the supernatant was incubated together with 100 L of 1% zinc acetate dihydrate at 37 C. for 10 minutes to immobilize H.sub.2S. 100 L of 0.1 M sodium tetraborate buffer (pH 9) and 200 L of 20 mM N, N-dimethyl-p-phenylenediamine sulfate were added to 7.2 M HCl, and 200 L of 30 mM iron (III) chloride was added to 1.2 M HCl, and the mixture was incubated at 37 C. for 15 min to form methylene blue dyes. The mixture was centrifuged again (14,000 rpm, 5 minutes, 4 C.). 100 L of supernatant was transferred in triplicate to 96-well plates, and absorbance was recorded spectrophotometrically at 670 nm. The H.sub.2S concentration was calculated by using a standard curve obtained by plotting absorbance (A670) versus NaHS concentration (0 to 30 M). For quantification of H.sub.2S in the left and right hemispheres of the brains, PET imaging was performed, and only the cerebral hemispheres were harvested and processed separately. The protocols were as described above, except that cold PBS weighed 3 times than each hemisphere (w/v, approximately 50 to 90 mg) was added to the homogenate.

9. Animal PET/CT Imaging

[0116] Animal PET/CT images were obtained by using a nanoScan PET/CT scanner (PET 82S, Mediso, Budapest, Hungary). Radiolabeled .sup.64Cu-ATSM-FITC(6) (11 MBq, 200 L of 5% DMSO/PBS) was injected via tail vein into normal and AOAA-treated BALB/c mice (n=4), and the mice were scanned for 20 minutes at 1 hour after the injection. For PET image-based quantification, regions of interest (ROIs) were manually drawn on the whole brain.

[0117] Brain inflammation and control models were prepared 2 days prior to the study on PET imaging (n=5 each). .sup.64Cu-ATSM-FITC(6) (18 MBq, 200 L of 5% DMSO/PBS) was injected into mice, and the mice were scanned for 10 minutes at 10 minutes after the injection. In addition, Sprague Dawley rats were injected with radiolabeled .sup.64Cu-ATSM-FITC(6) (18 MBq, 200 L of 5% DMSO/PBS) through the tail vein, and the mice were scanned for 20 minutes at 1 hour after the injection. CT images were generated immediately without the use of additional contrast agents. All PET images were reconstructed by using the Mediso Tera-Tomo 3D iterative algorithm, together with corrections for interaction depth, radionuclide decay, detector normalization, decision dead time, and attenuation.

[0118] Detection was performed in 1:3 matching smode with four iterations with six subsets. Analysis of the acquired PET/CT images was performed by using the Mediso InterView Fusion software package. The uptake of .sup.64Cu-ATSM-FITC(6) in each tissue was normalized to the administered radioactive dose and animal body weight and expressed as the average percent injected dose per gram (% ID/g).

10. Histological Analysis of Brain Cytotoxicity Using Cu-ATSM-FITC

[0119] Animals were anesthetized by using isoflurane, and injected with 100 g of Cu-ATSM-FITC(5) in 100 L of 10% DMSO/PBS, and 500 g of Cu-ATSM-FITC(5) in 100 L of 40% DMSO/PBS, respectively, through tail vein injection. The animals were perfused transcardially with saline 1, 3, and 7 days after the injection. The brains were dissected, fixed in 4% paraformaldehyde, and embedded in paraffin to make blocks. Serial coronal sections in paraffin with the thickness of 7 m were obtained. The sections were stained with H&E by using a staining kit (BBC ClearView, BBC Eosin Y Alcoholic). The stained brain tissues were photographed under a Carl Ziess microscope at 400 magnification.

11. Cytotoxicity Analysis of Cu-ATSM-FITC(5) and CuS

[0120] The cytotoxicity of Cu-ATSM-FITC(5) was evaluated through 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT; sigma-aldrich) analysis. Human cervical cancer cells (HeLa), human hepatoma cells (HepG2), and human embryonic kidney cells (HEK293) were prepared in 96-well plates at a density of 10,000 cells per well. As compared to the control group treated with PBS alone, they were treated with Cu-ATSM-FITC (5) at increasing concentrations of 1, 10, and 25 M. Afterwards, they were incubated for 1, 4, and 24 hours (37 C., 5% CO.sub.2), respectively. For each treatment time, each well was added with 10 L of MTT (5 mg/mL in PBS) solution and incubated for 3 hours. The supernatant was carefully aspirated, and the purple insoluble formazan crystals in the wells were dissolved using DMSO for 30 minutes. Absorbance was measured at 550 nm using a spectrophotometer (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany). CuS was also performed four times in a similar manner by using HeLa, HEK293, HepG2, and human brain glioblastoma (U87MG) cells.

12. In Vitro Serum Stability

[0121] Radioactively labeled .sup.64Cu-ATSM-FITC(6) (10 L, 37 MBq) was mixed with 100 L of FBS or PBS at pH 7.4 in the same amount and incubated at 37 C. using a Thermomixer (500 rpm). In addition, demetalization was measured by radio-TLC (silica, MeOH:EtOAC=5:95) every 4 hours for up to 24 hours.

13. Measurement of Quantum Yield

[0122] The background levels of methanol used in the experiment were measured by using a SpectraMaxi-3 spectraphotometer, and Cu-ATSM-FITC(5) was measured at concentrations of 0.02, 0.04, 0.06, 0.08, and 0.1 M. A fluorescein standard line graph was obtained according to the concentration and intensity of Cu-ATSM-FITC(5). The quantum yield was calculated according to the following equation: =ST (Grad x/Grad ST) (2/ST 2). Here, the subscripts ST and X represent the measured values of the standard and test solutions, respectively. In addition, represents the quantum yield and represents the refractive index of the solution.

14. Preparation of a Rat Model of Parkinson's Disease

[0123] The Parkinson's disease-induced rat model was prepared by taking 7-week-old SD rats and injecting 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle using a Hamilton syringe after drilling a small 1.5 mm hole in the rat's skull near the parietal region at 8 weeks of age.

Experiment Results

[0124] The present inventors have designed a novel chelator in which radioactive copper(II)-diacetyl-bis(N4-methylthiosemicarbazone) (ATSM) backbone is directly conjugated to fluorescein isothiocyanate (FITC-NCS). By combining the strengths of two imaging modalities, the high spatial resolution of fluorescence imaging and the unlimited tissue penetration capability of nuclear imaging, we intended to detect H.sub.2S from the cellular level to the whole-body scale using a single imaging probe. Such attempt has never been successful before.

[0125] A new chelator, ATSM-FITC (4), was synthesized through four sequential steps (FIG. 1).

[0126] Briefly, Intermediate 3 was prepared according to a previously reported procedure and then the terminal amine of Intermediate 3 was conjugated with the NCS group of FITC to obtain the ATSM-FITC conjugate (4). The overall yield of ATSM-FITC conjugate (4) from N-methyl hydrazinecarbothioamide (1) was 25.9%.

[0127] The chemical structure and high purity were confirmed through .sup.1H and .sup.13C NMR spectroscopy and HRMS (FIGS. 2 to 4). ATSM-FITC (4) was refluxed with CuCl.sub.2 in methanol to obtain the Cu(II) complex Cu-ATSM-FITC (5) as a dark brown solid in 33% yield. Purity was determined by HRMS (FIG. 5) and RP-HPLC analysis.

[0128] The optical properties of the free ligand ATSM-FITC (4) were evaluated. UV-Vis spectroscopy showed an absorbance band at 494 nm and a maximum fluorescence emission at 524 nm upon excitation at 480 nm (FIG. 6A). The Cu-ATSM-FITC(5) complex did not show a detectable fluorescence signal Since the paramagnetic Cu.sup.2+ ions coordinated to the ATSM-FITC chelator quenched the fluorescence signal of fluorine-rescein. When H.sub.2S sequestered Cu(II) ions from the copper complex (5) and precipitated CuS, the ATSM-FITC ligand was released and fluorescence was emitted (FIG. 7).

[0129] To confirm the sensitivity, Cu-ATSM-FITC(5) was incubated with a wide range of NaHS concentrations (0-100 M) at 37 C. and the fluorescence intensity was measured (FIG. 6B). The probe showed a gradual increase in fluorescence signals at 524 nm, together with up to 3500-fold fluorescence enhancement at 100 M of NaHS, as compared to that at the concentration of 0, which is much higher than that reported for most fluorescent H.sub.2S probes. Cu-ATSM-FITC(5) showed rapid reactivity toward H.sub.2S. When reacted with 50 M NaHS solution, the fluorescence intensity of Cu-ATSM-FITC(5) reached a plateau within 30 seconds (FIG. 6C). In contrast, the reactivity of Cu-ATSM-FITC(5) toward L-cysteine (1 mM) and glutathione (GSH) (10 mM) was minimal, as compared to H.sub.2S, even though their concentrations were at least 20 times higher than H.sub.2S. The detection limit of Cu-ATSM-FITC(5) was calculated to be 0.20 M using the regression equation (FIG. 8). The quantum yield was confirmed to be 0.33 according to the equation, as compared to the fluorescein standard value (FIG. 9).

[0130] The selectivity of Cu-ATSM-FITC(5) was investigated against a variety of potentially competing biological species in the presence and absence of H.sub.2S (FIG. 6D). Cu-ATSM-FITC(5) did not show reactivity (fluorescence signals) with other species such as biothiols-L-cysteine (Cys), L-homocysteine (Hcys), glutathione (GSH), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME), inorganic sulfur species (S.sub.2O.sub.3.sup.2 and S.sub.2O.sub.5.sup.2), reducing agents such as ascorbic acid (AA), inorganic and organic nucleophiles (Cl, I, OAc, ClO4-, HCO.sub.3.sup.2, N.sup.3 or NO.sup.2), and reactive oxygen and nitrogen species, including hydrogen peroxide (H.sub.2O.sub.2), peroxynitrite (ONOO.sup.), and nitric oxide (NO). However, when NaHS (50 M) was added to the mixture along with other biologically abundant species, full fluorescence intensity was observed without interference from other biological entities (FIG. 6D). Cu-ATSM-FITC(5) also showed high stability and reactivity over a wide physiological pH range (pH 5 to 10) (FIG. 10). All these results show that Cu-ATSM-FITC (5) has excellent sensitivity, reactivity, and selectivity to be used as a biosensor for H.sub.2S.

[0131] Next, the present inventor tested the toxicity and biocompatibility of Cu-ATSM-FITC(5) before fluorescence imaging of H.sub.2S in HeLa cells. According to MTT analysis, the survival rate of HeLa cells exceeded 85% at concentrations of 1, 10, and 25 M even after 24 hours of incubation, indicating its excellent biocompatibility (FIG. 6E). No cytotoxicity was observed in other cell lines comprising human hepatoma cell line (HepG2) and human embryonic kidney 293 (HEK293) cells (FIGS. 11A to 11C). In addition, CuS was subjected to MTT analysis in the same manner by adding human brain glioblastoma (U87MG), and, as a result, no cytotoxicity was found in all cell lines (FIGS. 12A to 12D).

[0132] H&E staining data of mouse brain tissues confirmed the low toxicity of CuS precipitated for up to 7 days even after injection of a high-dose, 500 g, of Cu-ATSM-FITC(5) (FIG. 13).

[0133] Next, we investigated the usefulness of Cu-ATSM-FITC(5) for monitoring H.sub.2S levels in living cells (FIG. 14). HeLa cells incubated with Cu-ATSM-FITC(5) at a low toxic (25 M) concentration showed a faint fluorescence signal due to the tightly controlled low levels of H.sub.2S in healthy cells (a). In contrast, a linear rise in green fluorescence intensity was detected upon addition of NaHS at two different concentrations (50 and 100 M) to probed HeLa cells (b and c). Cu-ATSM-FITC(5) also successfully detected the increase in endogenous H.sub.2S concentration induced by L-cysteine (100 M) (d). When H.sub.2S-generating cystathionine -lyase (CSE) enzyme activity was blocked by DL-propargylglycine (PPG, 450 M), green fluorescence due to endogenous H.sub.2S was completely reduced (e). In cells co-stained with Hoechst (f-j) and LysoTracker (k-o), an overlap was observed between Cu-ATSM-FITC(5) and LysoTracker (p-t), indicating that Cu-ATSM-FITC(5) predominantly co-stained with lysosomes. Taken together, the fluorescence imaging results indicate that the fluorescent probe Cu-ATSM-FITC (5) can be used to noninvasively monitor small fluctuations in H.sub.2S concentration at the cellular level.

[0134] Next, the potential of Cu-ATSM-FITC(5) as a nuclear imaging agent for in vivo imaging was confirmed.

[0135] The ligand ATSM-FITC(4) was radiolabeled with .sup.64CuCl.sub.2 ions in 0.1M ammonium acetate buffer (pH 6.8) at 60 C., with a radiochemical yield of >97% (FIG. 3). Among the various positron-emitting radioisotopes of copper (.sup.60Cu, .sup.61Cu, .sup.62Cu, or .sup.64Cu), Cu-64 has a moderately long half-life (12.7 hours) and a decay mode favorable for PET imaging (17.8% + decay). The identity of radioactively labeled .sup.64Cu-ATSM-FITC(6) was confirmed by comparing the retention time with that of Cu-ATSM-FITC(5), a non-radioactive standard compound, by HPLC (FIG. 15B). .sup.64Cu-ATSM-FITC(6) reacted immediately with H.sub.2S, fixing gaseous H.sub.2S into an insoluble copper sulfide (.sup.64CuS) precipitate (FIG. 15A). The proportion of demetalized .sup.64CuS in the intact .sup.64Cu-ATSM-FITC(6) complex may be easily quantified by radio-TLC analysis. The Rf value of .sup.64Cu-ATSM-FITC(6) was 0.72 on a silica plate with mobile phase (ethyl acetate methanol (95:5)), and .sup.64CuS remained at the origin of the TLC plate (FIG. 22). As the concentration of H.sub.2S increased, the decomplexation percentage of .sup.64Cu-ATSM-FITC(6) increased proportionally and reached a plateau at 20 M of H.sub.2S (FIG. 15C). Radiolabeled .sup.64Cu-ATSM-FITC (6) showed high stability in both fetal bovine serum (FBS) and phosphate-buffered saline (PBS) solutions at 37 C. Less than 10% degradation of .sup.64Cu-ATSM-FITC(6) was observed for up to 24 hours after incubation (FIG. 16). The lipophilicity of the radiotracer was measured using the general octanol-PBS partitioning method.

[0136] The log D.sub.7.4 value of .sup.64Cu-ATSM-FITC(6) was 1.700.05, and the range of 1.5 to 2.7 is desirable for optimal BBB penetration.

[0137] The body distribution and elimination pattern of .sup.64Cu-ATSM-FITC(6) was accurately measured in a biodistribution study in normal BALB/c mice (FIG. 17A). High brain uptake was observed as early as 5 minutes after injection. Brain uptake of greater than 9% ID/g at 5 minutes was maintained for up to 2 hours after injection, and more than 3.3% of the total injected activity was found in the whole brain, confirming that it is significantly higher, as compared to existing brain-targeted radio-tracers including .sup.64Cu-ATSM (2 to 5% ID/g). The initially high uptake of .sup.64Cu-ATSM-FITC(6) in the heart, lungs, and kidneys gradually decreased over time.

[0138] Next, a whole-body imaging study was performed using an animal PET/computed tomography (CT) scanner. The radiotracer .sup.64Cu-ATSM-FITC(6) was injected into normal BALB/c mice via the tail vein, and PET/CT images were acquired 1 hour after injection (FIGS. 17B to 17E). As expected from the biodistribution data, significant brain uptake was clearly observed in PET/CT images using Maximum Intensity Projection (MIP) (FIG. 17B). Evenly distributed brain uptake of radioactivity was observed in coronal, sagittal, and cross-sectional images (FIGS. 17C to 17E). In addition, images not only in mice but also in SD-rats were confirmed with the radioactive tracer .sup.64Cu-ATSM-FITC(6), and, as a result, similar brain absorption was observed (FIGS. 18A to 18C). However, when the concentration of H.sub.2S in the brain was lowered by intraperitoneal injection of cystathionine -synthase (CBS), which is an H.sub.2S generating enzyme, and aminooxyacetic acid (AOAA, 10 mg/kg), which is an inhibitor of CSE, the brain uptake of .sup.64Cu-ATSM-FITC(6) was significantly reduced (FIG. 17F). In PET imaging, the brain uptake of .sup.64Cu-ATSM-FITC(6) was 15.01.8 vs. 11.42.0% ID/g in control and AOAA-treated mice, respectively (FIG. 17G). The H.sub.2S concentration in the brains of AOAA-treated mice was measured directly by the methylene blue method and was reduced by 32% compared to controls (3.10.5 vs. 2.10.5 g/g brain), which is consistent with brain biodistribution data in control and AOAA-treated mice (FIG. 17H).

[0139] These data clearly indicate that brain uptake of .sup.64Cu-ATSM-FITC(6) is closely correlated with brain H.sub.2S levels.

[0140] Finally, its potential as an imaging agent for detection of increased H.sub.2S concentrations in disease models was evaluated. Neuroinflammation has been linked to traumatic brain injury, stroke, Alzheimer's disease, Parkinson's disease, and many other brain disorders. In this study, the neuroinflammation model was induced by intracerebroventricular injection of LPS (FIG. 19A).

[0141] As a control group, a saline solution was injected instead of LPS. Coronal and transverse PET images showed higher activity in the LPS-treated model, as compared to the control group, especially near the injection site (FIG. 19B). As a result of quantifying the amount of radioactivity in the right hemisphere (injection site) and left hemisphere of the brain by using -counter, the absorption amount was similar in both sides in the control group, but a 31% increase was observed at the injection site in LPS-treated animals (FIG. 19C). When H.sub.2S concentrations in the brains were measured ex vivo by the methylene blue method immediately after PET scanning, a 39% increase in H.sub.2S concentrations was detected only in the LPS-treated cerebral hemisphere (4.90.5 vs. 3.20.7 g/g brain), which is consistent with PET imaging and biodistribution results.

[0142] These PET imaging results clearly show that the radiotracer according to the present invention can detect a sufficiently wide range of H.sub.2S fluctuations (decrease and increase) in the brain.

[0143] In addition, we tested whether .sup.64Cu-ATSM-FITC(6) can be used to diagnose Parkinson's disease. Several studies have already reported that the concentration and distribution of hydrogen sulfide in the brain changes with the onset and progression of Parkinson's disease. However, due to the lack of a radiolabeled probe that can actually detect hydrogen sulfide in the brain, there have been no reports of hydrogen sulfide-based diagnostic studies for Parkinson's disease. In this study, we used a .sup.64Cu-ATSM-FITC radioprobe developed by our group to observe brain uptake changes in a Parkinson's disease model and a normal rat model through PET/CT nuclear medicine imaging studies.

[0144] Coronal PET/CT images of .sup.64Cu-ATSM-FITC were acquired 1 hour after injection of about 1 mCi of .sup.64Cu-ATSM-FITC into the tail vein of normal rats and a Parkinson's disease rat model, and it was found that the uptake in brain regions was significantly lower in the Parkinson's disease rat model. In addition, the uptake pattern was also different between normal rats and the Parkinson's disease rat model (FIG. 23).

Comparative Data: Body Distribution of .SUP.64.Cu-ATSM-aniline

(1) Synthesis of (E)-N-(4-(dimethylamino)phenyl)-2-((E)-3-(2-(methylcarbamothioyl)hydrazineylidene)butan-2-ylidene)hydrazine-1-carbothioamide, ATSM-aniline (9)

[0145] Compound 8 (420 mg, 2 mmol) in methanol (20 mL) was added dropwise to a cold methanolic solution of Compound 2 (350 mg, 2 mmol, dissolved in 20 mL methanol) in the presence of catalyst HCl. When TLC (silica, MeOH:CH2Cl2=5:95) showed complete consumption of starting material, the mixture was stirred at the same temperature for 1 hour. It was then concentrated under reduced pressure and purified by column chromatography to obtain ATSM-aniline (9) as a yellow solid (260 mg, 36%) (FIG. 20).

(2) Cu-64 Labeling of ATSM-aniline (9)

[0146] A stock solution (1 g/L) of the chelator was prepared in anhydrous DMSO. Complexation of .sup.64Cu and ATSM-aniline(9) was carried out by reacting in 0.1 M ammonium acetate (pH 6.8, 100 L) at 60 C. for 20 minutes in a heat mixer (800 rpm). The completion of the reaction was monitored by radio-TLC [silica, MeOH:EtOAc=5:95] (FIG. 20).

(3) Confirmation of Body Distribution of .SUP.64.Cu-ATSM-aniline

[0147] The distribution of .sup.64Cu-ATSM-aniline in the body was confirmed by using the same method as described in Experimental Methods 5 and 7 above.

[0148] As a result, as shown in FIG. 21, it was confirmed that .sup.64Cu-ATSM-aniline was most widely distributed in the lungs and liver, while its distribution in the brain was very low.

[0149] For reference, it has been reported that the degree to which Copper Bis(thiosemicarbazonato)-stilbenyl Complexes were absorbed into the brain was very low at 1 to 2% ID/g 2 minutes after injection, and 0.2 to 0.3% ID/g 1 hour after injection (Inorg. Chem. 2020, 59, 16, 11658-11669).

INDUSTRIAL APPLICABILITY

[0150] The complex compound provided by the present invention selectively binds to hydrogen sulfide and can selectively image areas where hydrogen sulfide is abnormally increased within cells or tissues. In particular, it has a very high blood-brain barrier permeability to prevent various neuroinflammatory diseases. It can very effectively detect brain hydrogen sulfide, which is detected at high levels in the brain. In addition, it can be used as a dual-modality contrast agent capable of simultaneous nuclear and fluorescence imaging and, thus, can be very useful for diagnosis and research of various diseases mediated by hydrogen sulfide. Therefore, the industrial applicability of the present invention is very high.