Radioactive 2,7,9a-triazabenzo[cd]azurol-6(7H)one derivatives and their preparation methods and applications

Abstract

The present invention discloses a radioactive 2,7,9a-triazabenzo[cd]azurol-6(7H)-one derivative, a preparation method, and an application thereof, characterized in that the derivative has the structure of I. Compared with the prior art, the present invention provides a class of radioactive 2,7,9a-triazabenzo[cd]azurol-6(7H)-one derivatives with high affinity for PARP-1. These compounds have high affinity for PARP-1 and are novel compounds for diagnosing PARP-1-related diseases. Compared with previously reported PARP-1-targeted radiopharmaceuticals, the PARP-1-targeted imaging obtained by preparing the derivatives reported in the present invention has lower uptake in non-target organs such as the liver, muscle, and bone, and can cross the blood-brain barrier, making them applicable to whole-body imaging including the brain and abdomen.

Description

Technical Field

[0001] The present invention relates to the technical field of radiopharmaceutical chemistry and nuclear medicine, and in particular to a radioactive 2,7,9a-triazabenzo[cd]azur-6(7H)-one derivative and a preparation method and application thereof. Background Art

[0002] The earliest radioactive probe developed targeting poly (ADP-ribose) polymerase-1 (PARP-1) can be traced back to 1994 reported by Andersson et al. 11 C-labeled benzamide compounds (Appl Radiat Isot, 1994, 45: 707-714.). This type of probe has too high an uptake in the liver and kidneys, and the target to non-target ratio is low, thus failing to be clinically transformed. Subsequently, a series of new PARP-1-targeted probes were developed by modifying the structures of known high-affinity PARP-1 inhibitors such as Olaparib and Rucaparib and introducing radionuclides. Among them, the most studied ones include [ 18 F]Olaparib, [ 18 F]FTT,[ 18 F]PARPi etc. 18 F]Olaparib is achieved by replacing the 19 F is 18 F, its IC 50 It is 3.9±1.2nM. Because it is consistent with the structure of Olaparib, it has high application value in evaluating the application and efficacy of Olaparib, but it fails to penetrate the blood-brain barrier and has a low labeling yield (J.Nucl.Med.2019,60,504–510). 18 F]FTT is the most clinically studied probe targeting PARP-1. 50 =6.3nM), which has important guiding significance for the diagnosis of patients with different subtypes of breast cancer (JAMA Oncol. 2020; 6:921–923). However, the probe has a high uptake in non-target tissues such as the liver and muscle, especially the normal physiological uptake in the abdomen is high, which makes it impossible to accurately diagnose liver cancer (European Journal of Nuclear Medicine and Molecular Imaging 2022) 49:834–846, WO2015103526A1), and the probe has defluorination in vivo. Therefore, it is urgent to develop a new PARP-1-targeted probe that can penetrate the blood-brain barrier, has low uptake in non-target tissues such as the liver and muscle, does not have defluorination in vivo, and has a high target-to-non-target ratio. 18 F-labeled PET probe.

[0003] Based on overcoming the above problems, the present invention introduces diethoxy into the structure of 2,7,9a-triazabenzo[cd]azurol-6(7H)ketone. 18 F side chain, a new type of PET probe targeting PARP-1 that can penetrate the blood-brain barrier is obtained, thereby overcoming the previous unfavorable factors such as the target-to-non-target ratio and in vivo defluorination, and can be used for the diagnosis of systemic diseases. Summary of the Invention

[0004] The present invention first provides a radioactive (2,7,9a-triazabenzo[cd]azur-6(7H)ketone derivative to achieve the above purpose.

[0005] Preferably, the derivative has the structure of I,

[0006] The structural formula of I is as follows:

[0007]

[0008] Wherein, R is H or D.

[0009] The present invention provides a radioactive (2,7,9a-triazabenzo[cd]azuryl-6(7H)ketone derivative derivative,

[0010] The present invention also provides a method for preparing a compound of formula I, comprising the following steps: using a labeled precursor compound of formula II containing an activating group to obtain a compound of formula I through radioactive labeling;

[0011] The structural formula of II is as follows:

[0012]

[0013] Among them, R is H or D, and R1 is a leaving group such as OTs, OMs, etc.

[0014] The present invention also provides a method for preparing the compound of formula I, which comprises the following steps: using a labeled precursor compound of formula II containing an activated group to obtain the compound of formula I through radioactive labeling.

[0015] The structural formula of II is as follows:

[0016]

[0017] Among them, R is H or D, and R1 is a leaving group such as OTs, OMs, etc.

[0018] The present invention also provides a product comprising derivatives I and II.

[0019] Preferably, the product is a medicament, a drug, a kit or a mixture.

[0020] Preferably, the agent is an imaging agent for diseases such as liver cancer, brain glioma, stroke, prostate cancer, and PARP-1-related diseases; the kit comprises one or more bottles; and the mixture comprises one or more compounds of Formula I and Formula II according to any one of claims 1 to 3.

[0021] The present invention also provides a compound of derivatives I and II, or a method for preparing the same, or an application of a product containing the same;

[0022] Preferably, the application is (1) preparing a medicament, a drug, a kit or a mixture; or (2) for imaging of a disease, and the application is for non-diagnostic purposes.

[0023] Preferably, when R is 19 F. The application is to prepare a control or comparison substance of a radioactive 2,7,9a-triazabenzo[cd]azur-6(7H)-one derivative.

[0024] The beneficial effects of the above technical solution of the present invention are as follows:

[0025] The present invention provides a class of high-affinity 2,7,9a-triazabenzo[cd]azur-6(7H)-one derivatives. These compounds possess high affinity and are novel compounds for diagnosing PARP-1-related diseases. Compared to previously reported PARP-1-targeted radiopharmaceuticals, the PARP-1-targeted imaging obtained from these derivatives exhibits lower uptake in non-target organs such as the liver, muscle, and bone, and can cross the blood-brain barrier, making them suitable for whole-body imaging, including the brain and abdomen. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the MicroPET imaging of 22Rv1 tumor-bearing nude mice in Example 3; the white circle area is the tumor. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0028] Example 11-(4-(2-fluoroethoxy)ethoxyphenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azur-6(7H)-one

[0029] The structural formula is as follows:

[0030]

[0031] The synthetic route is as follows:

[0032]

[0033] 1. (2-(2-(4-(6-oxo-6,7,8,9-tetrahydro-2,7,9a-triazabenzo[cd]azur-1-yl)phenoxy)ethoxy)ethyl-4-methylbenzenesulfonate

[0034] The compound 1-(4-hydroxyphenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azurol-6(7H)-one (279 mg, 1 mmol) was dissolved in 10 mL of anhydrous DMF. Potassium carbonate (276 mg, 2 mmol) and oxybis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (828 mg, 2 mmol) were added sequentially. The mixture was allowed to react overnight at room temperature. The reaction solution was filtered through celite, and the filtrate was extracted with a large amount of ethyl acetate and washed three times with saturated brine. The organic phase was collected. The organic phase was dried, spin-dried, mixed, and purified by column chromatography (dichloromethane / methanol = 20 / 1) to give (2-(2-(4-(6-oxo-6,7,8,9-tetrahydro-2,7,9a-triazabenzo[cd]azur-1-yl)phenoxy)ethoxy)ethyl-4-methylbenzenesulfonate (210 mg, 40.29% yield). 1 H NMR (300MHz, DMSO-d6) δ8.49(t,J=5.7Hz,1H),7.91-7.87(m,2H),7.85-7.73(m,4H),7.46(d,J=8.0Hz,2H),7.36(t,J=7 .8Hz,1H),7.13(d,J=8.8Hz,2H),4.47(d,J=4.6Hz,2H),4.18-4.13(m,4H),3.80-3.64(m,4H),3.56(s,2H),2.39(s,3H). 13 C NMR(75MHz,DMSO-d6)δ163.21,150.34,142.01,139.24,134.40,131.89,130 .16,130.02,129.88,114.04,85.96,85.22,84.71,70.12,68.10,65.60.HRMS calcd for:C27H28N3O6S+,522.1693[M+H] + ;found,522.1691.

[0035] 2.11-(4-(2-fluoroethoxy)ethoxyphenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azur-6(7H)-one

[0036] (2-(2-(4-(6-oxo-6,7,8,9-tetrahydro-2,7,9a-triazabenzo[cd]azur-1-yl)phenoxy)ethoxy)ethyl-4-methylbenzenesulfonate (260 mg, 0.5 mmol) was dissolved in 10 mL of anhydrous THF. After nitrogen protection, 2 mL of TBAF (1 mol / L, dissolved in tetrahydrofuran) was added at room temperature and reacted at 50 degrees overnight. A large amount of ethyl acetate was added to the reaction solution for extraction, and the organic phase was washed three times with saturated brine to collect. The organic phase was dried, spin-dried, mixed, and purified by column chromatography (dichloromethane / methanol = 20 / 1) to obtain the product (90 mg, yield 48.76%). 1 H NMR (300MHz, CDCl3) δ8.28(s,2H),7.68(dd,J=8.6,2.1Hz,2H),6.77(d,J=8.6Hz ,2H),4.61-4.38(m,4H),4.06-3.90(m,4H),3.20-3.13(dd,J=12.9,7.2Hz,4H). 13 C NMR (75MHz, CDCl3) δ163.25,150.39,142.09,112.48,111.12,80.31,78.99,68.74,62.05.HRMS calcd for:C20H21FN3O3+, 370.1561[M+H] + ;found,370.1563.

[0037] Example 21-(4-(2-(2-fluoroethoxy-1,1,2,2-d4)ethoxy-1,1,2,2-d)phenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azur-6(7H)-one

[0038] The structural formula is as follows:

[0039]

[0040] The synthetic route is as follows:

[0041]

[0042] Compound synthesis:

[0043] 2-(2-(4-(6-oxo-6,7,8,9-tetrahydro-2,7,9a-triazabenzo[cd]azur-1-yl)phenoxy)ethoxy-1,1,2,2-d4)ethyl-1,1,2,2-d4-methylbenzenesulfonate

[0044] The compound 1-(4-hydroxyphenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azurol-6(7H)-one (279 mg, 1 mmol) was dissolved in 10 mL of anhydrous DMF, and potassium carbonate (276 mg, 2 mmol) and oxybis(ethane-2,1-diyl-1,1,2,2-d4)bis(4-methylbenzenesulfonate) (844 mg, 2 mmol) were added sequentially. The mixture was allowed to react overnight at room temperature. The reaction solution was filtered through celite, and the filtrate was extracted with a large amount of ethyl acetate and washed three times with saturated brine. The organic phase was collected. The organic phase was dried, then spin-dried, mixed, and purified by column chromatography (dichloromethane / methanol = 20 / 1) to give 2-(2-(4-(6-oxo-6,7,8,9-tetrahydro-2,7,9a-triazabenzo[cd]azur-1-yl)phenoxy)ethoxy-1,1,2,2-d4)ethyl-1,1,2,2-d4-methylbenzenesulfonate (213 mg, 40.3% yield). HRMS calculated for: C27H20D8N3O6S+, 522.1693 [M+H] + ;found,522.1694.

[0045] 1-(4-(2-(2-fluoroethoxy-1,1,2,2-d4)ethoxy-1,1,2,2-d)phenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azur-6(7H)-one

[0046] 2-(2-(4-(6-oxo-6,7,8,9-tetrahydro-2,7,9a-triazabenzo[cd]azur-1-yl)phenoxy)ethoxy-1,1,2,2-d4)ethyl-1,1,2,2-d4-methylbenzenesulfonate (268 mg, 0.5 mmol) was dissolved in 10 mL of anhydrous THF. Under nitrogen protection, 2 mL of TBAF (1 mol / L, dissolved in tetrahydrofuran) was added at room temperature and reacted at 50°C overnight. A large amount of ethyl acetate was added to the reaction solution for extraction, and the organic phase was washed three times with saturated brine and collected. The organic phase was dried, spin-dried, mixed, and purified by column chromatography (dichloromethane / methanol = 20 / 1) to obtain the product (92 mg, 48.7% yield). HRMS calculated for: C20H12D8FN3O3, 377.1991 [M+H] + ; found,377.1994.

[0047] Example 3 1-(4-(2-(2-(fluoro-18F)ethoxy)ethoxy)phenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azur-6(7H)-one

[0048] The structural formula is as follows:

[0049]

[0050] The synthetic route is as follows:

[0051]

[0052] Experimental steps: a) 18 F - 1 mL K 222 / K2CO3 solution (160 mg K 222 in 18.6 mL acetonitrile / 29 mg K2CO3 in 3.4 mL H2O) into the reaction tube;

[0053] b) Evaporate the above K at 110°C under N2 purge 222 / K2CO3 eluent; add 1 mL of anhydrous acetonitrile to the evaporation residue and evaporate to dryness again, repeat the above process three times to obtain dry 18 F - / K 222 / K2CO3 complex;

[0054] c) cooling the reaction solution and adding the dried 18 F - / K 222 1 mg of labeled precursor solution (dissolved in 1 mL of DMSO) was added to the / K2CO3 complex and reacted at 110 °C for 10 min;

[0055] d) Add 9 mL of water to the reaction tube, and then pass the diluted reaction solution through an Oasis HLB solid phase extraction column;

[0056] e) Rinse the Oasis HLB solid phase extraction cartridge with 10 mL of water;

[0057] f) The solid phase extraction column was eluted with 1 mL of methanol solution and then purified by semi-preparative HPLC (Phenomenex Gemini-Nx C18 110A (250×4.6 mm×5 μm, acetonitrile / 0.1% formic acid water = 25 / 75). The peak elution time was 25.6 min and the purity was greater than 95%.

[0058] Example 4 1-(4-(2-(2-(fluoro- 18 F) Ethoxy-1,1,2,2-d4)Ethoxy-1,1,2,2-d4)phenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azur-6(7H)-one

[0059] The structural formula is as follows:

[0060]

[0061] The synthetic route is as follows:

[0062]

[0063] Experimental steps: a) 18 F - 1 mL K 222 / K2CO3 solution (160 mg K 222 in 18.6 mL acetonitrile / 29 mg K2CO3 in 3.4 mL H2O) into the reaction tube;

[0064] b) Evaporate the above K at 110°C under N2 purge 222 / K2CO3 eluent; add 1 mL of anhydrous acetonitrile to the evaporation residue and evaporate to dryness again, repeat the above process three times to obtain dry 18 F - / K 222 / K2CO3 complex;

[0065] c) cooling the reaction solution and adding the dried 18 F - / K 222 1 mg of labeled precursor solution (dissolved in 1 mL of DMSO) was added to the / K2CO3 complex and reacted at 110 °C for 10 min;

[0066] d) Add 9 mL of water to the reaction tube, and then pass the diluted reaction solution through an Oasis HLB solid phase extraction column;

[0067] e) Rinse the Oasis HLB solid phase extraction cartridge with 10 mL of water;

[0068] f) The solid phase extraction column was eluted with 1 mL of methanol solution and then purified by semi-preparative HPLC (Phenomenex Gemini-Nx C18 110A (250×4.6 mm×5 μm, acetonitrile / 0.1% formic acid water = 25 / 75). The peak elution time was 25.6 min and the purity was greater than 95%.

[0069] Example 5 Affinity Experiment (PARP-1)

[0070] In the presence of DNA damage, PARR can catalyze the binding of poly(ADP-ribose) to histones coated on the bottom of a 96-well plate in an NAD+-dependent manner. Poly(ADP-ribose) is biotin-labeled, and HRP is linked to the histones through the Biotin-Strep hinge. After the reaction is washed with PBS, the HRP fixed on the bottom of the 96-well plate can catalyze the conversion of the substrate TACS-Sapphire into a blue substance that absorbs light at 450nm. When an inhibitor is present, PARP enzyme activity is inhibited. By configuring different concentrations of inhibitors and co-incubating with the PARP enzyme, an inhibition curve can be obtained, and then the IC value of the inhibitor on the PARP enzyme can be calculated. 50 value.

[0071] Experimental steps: IC 50 For the specific determination method, refer to the protocol provided in the instructions of the Universal PARP Colorimetric Assay Kit (Catalog Number: 4677-096-K). As can be seen from Table 1, the compounds of the present invention all have a high affinity for PARP-1.

[0072] Table 1: Affinity of compounds for PARP-1

[0073]

[0074] Example 6 Biodistribution

[0075] To further explore the brain uptake, tumor uptake and pharmacokinetics of radioactive compounds, we conducted biodistribution experiments. 18 F]FTT brain, tumor uptake and pharmacokinetics. 18 F]FTT was diluted with physiological saline to 20 μCi / 0.1 mL. MCF-7 subcutaneous tumors weighing about 20 g were randomly divided into groups of 5. The drug from Example 3, Example 4, or [ 18 F]FTT, the animals were decapitated at 5, 30, 60, and 120 minutes after injection. Blood, tumor, heart, liver, spleen, lung, kidney, brain, muscle, bone, fur, and other tissues and organs were collected, cleaned, weighed, and radioactivity counted. The uptake of each organ and tissue (%ID / g) and the tumor / muscle ratio were calculated. The results are shown in Table 2 below. As can be seen from Table 2, at 60 minutes, the uptake of samples 3, 4, or [ 18 The tumor uptake of F]FTT was similar, but the muscle uptake of cases 3 and 4 was lower. Therefore, the tumor uptake of cases 3 and 4 was higher than the muscle uptake, indicating that the imaging contrast of cases 3 and 4 was better than that of [ 18 F]FTT. In addition [ 18The liver uptake of F]FTT was higher than that of Examples 3 and 4. The tumor-to-liver ratio of Examples 3 and 4 can be applied to the imaging of abdominal tumors, while [ 18 Examples 3 and 4 showed high uptake in the mouse brain at 2 minutes and were rapidly cleared at 60 minutes. Therefore, Examples 3 and 4 can be applied to the diagnosis of PARP-1-related brain diseases.

[0076] Table 2: Biodistribution results of the compounds 60 min after administration (% ID / g)

[0077]

[0078] Example 7 Imaging Experiment

[0079] MCF-7 subcutaneous tumors and 22Rv1 subcutaneous tumors were anesthetized and fixed on mouse plates. After anesthesia, the cells were injected via the tail vein with the following examples: 3, 4 or [ 18 F]FTT (~1.5mCi / 0.1mL), and perform micro PET / CT dynamic scanning for 2 hours after administration, and transmit the acquired and reconstructed PET / CT images to the workstation for image fusion and display, and judge whether the results of Example 3, 4 or [ 18 F]FTT radioactivity distribution and binding to PARP. 60 minutes after administration, the 18 In the MCF-7 model, the tumor uptake of F]FTT was 2.01±0.21%ID / g, 1.99±0.32%ID / g, and 3.11±0.82%ID / g, respectively; the muscle uptake was 0.24±0.32%ID / g, 0.25±0.11%ID / g, and 1.43±0.32%ID / g, respectively; the corresponding tumor-to-muscle ratios were 8.37, 7.96, and 2.17, respectively; in the 22Rv1 model, the tumor uptake of F]FTT was 2.01±0.21%ID / g, 1.99±0.32%ID / g, and 3.11±0.82%ID / g, respectively. The tumor uptake was 8.68±1.86%ID / g, 8.37±0.65%ID / g, 7.04±0.11%ID / g, respectively; the muscle uptake was 0.82±0.22%ID / g, 0.79±0.29%ID / g, 1.03±0.36%ID / g, respectively; the corresponding tumor-to-muscle ratio was 10.57, 10.59, 6.83, respectively; As can be seen from the above, the tumor-to-muscle ratio of Examples 3 and 4 is higher than [ 18 F]FTT, and thus the imaging contrast is better than [ 18 F]FTT. Consistent with the biodistribution results, samples 3 and 4 were metabolized primarily by the kidneys, whereas [ 18 F]FTT is primarily metabolized by the liver (see Figure 1 ), therefore, Examples 3 and 4 have advantages in the imaging of abdominal tumors. In the PET imaging of the two models, [ 18F]FTT has defluorination phenomenon, while Examples 3 and 4 do not have defluorination phenomenon. In summary, Examples 3 and 4 overcome the [ 18 F]FTT has low uptake in non-target organs such as liver, muscle, and bone and can cross the blood-brain barrier, so it can be used for whole-body imaging including the brain and abdomen.

[0080] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A radioactive 2,7,9a-triazabenzo[cd]azurol-6(7H)-one derivative provided, characterized in that: The derivative has the structure of I, The structural formula of I is as follows: Wherein, R is H or D.

2. A method for preparing the compound of formula I according to claim 1, characterized in that: The method comprises the following steps: using a labeled precursor compound of formula II containing an activated group to obtain a compound of formula I through radiolabeling; The structural formula of II is as follows: Wherein, R is H or D; R1 is OTs or OMs leaving group.

3. A product, characterized in that The product comprises the derivative according to claim 1.

4. The product according to claim 3, characterized in that The product is a medicament, a drug, or a kit.

5. The product according to claim 4, characterized in that The agent is an imaging agent for a disease; the disease is one or more of liver cancer, brain glioma, stroke, and prostate cancer; and the kit comprises one bottle or multiple bottles.

6. Use of the compound according to any one of claims 1-2 or the product according to claim 3, characterized in that: The application is the preparation of a medicament, a drug or a kit.

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