WO2017111402A1 - Radioactive compound containing fluorine-18 radioisotope as tracer for pet for diagnosing brain neural inflammation, and method for producing said radioactive compound - Google Patents

Abstract

The present invention relates to a radioactive compound to which [¹⁸F]alkyl is introduced as a tracer for PET for diagnosing brain neural inflammation, and to a method for producing said radioactive compound.

Description

Radioactive compounds containing fluorine-18 isotopes as tracers for PET for diagnosing neuroinflammation and methods for their preparation

The present invention relates to a radioactive compound incorporating [ ¹⁸ F] alkyl as a tracer for PET for diagnosing neuroinflammation, and a method for preparing the same.

Recently, there has been increasing interest in neuroinflammation imaging in degenerative brain diseases. Microglia, which make up about 10% of brain cells, are known to function as phhargocytosis and to remove fragments of cells and external antigens, such as macropharges that exist outside the brain. .

Microglia originate from monocytes during the birth and move from the bone marrow to the brain, reacting to stimuli in the brain and changing from a basal state to an activated state. Macrophage macrophages function receptor-dependent and play an important role in overall immune function at the central nervous system. Microglial activation is observed in various degenerative brain diseases. Microglial cells are collected around amyloid plaque, which is the main pathology of Alzheimer's disease, and microglial cells activated in Parkinson's disease have been reported. Microglia secrete neurons by releasing substances such as free radicals, nitric oxide, neurotoxics, cytokines, interleukin-1 and MCP-1, which can exacerbate neuronal destruction or pathology. It is known that it can exacerbate degenerative brain disease because it affects function and viability, and recent studies have reported that activated microglia remove antibodies from immunotherapy of Alzheimer's disease. Until now, the role of microglia in degenerative brain disease has been mainly studied through pathological tissues, but the development of molecular imaging has led to the activation of microglia using radiopharmaceuticals that bind to the peripheral benzodiazepine receptor (PBR). The cells can be measured by PET imaging. PBR was named after diazepam's binding to the central benzodiazepine receptor located in the GABA receptor of the central nervous system after discovering the strong affinity of diazepam to the rat kidney. It is located in the outer membrane of mitochondria of astrocytes and is considered part of the mitochondrial permeability transition pore. Recently called translocator protein (TSPO, 18 kDa), the function of PBR in the central nervous system is not yet known, but it is used to modulate neurosteroid synthesis, mitochondrial function, and neuroinflammatory in microglia. Is being presented. Activation of microglia in the central nervous system occurs due to increased expression of TSPO of 18 kDa in the mitochondrial membrane, and has been reported to start within several hours after the disease and continue for several days. Therefore, the measurement of TSPO expression level of microglia in various central nervous system diseases can be used as a biomarker in vivo to evaluate cell activation during neuroinflammatory process.

A representative substance known as a molecular imaging tracer for imaging peripheral benzodiazepine receptors in activated microglia is [ ¹¹ C] PK11195, which is known to bind to isoquinoline binding protein (IBP).

These compounds are classified into two types according to their stereochemical properties, and among them, (R)-[ ¹¹ C] PK11195 has a high affinity for posterior spheres. In addition to the PK11195 series, [ ¹⁸ F] FEDAA1106 and [ ¹¹ C] DPA-713, which are [ ¹⁸ F] fluorine-labeled derivatives of [ ¹¹ C] DAA1106 and DAA1106, have been developed as candidates. Branches have not yet been found to replace the (R)-[ ¹¹ C] PK11195 derivatives with the highest affinity (Ki 28.8 nM) among radiopharmaceuticals.

Therefore, animal models and clinical studies using [ ¹¹ C] PK11195 PET have been reported in various neurological diseases. In particular, one of the characteristic pathological findings of degenerative brain disease is increased PBR, and degenerative diseases such as Alzheimer's disease and Parkinson's disease are observed. A number of studies on the activation and mechanism of microglia cells using [ ¹¹ C] PK11195 PET in brain disease have been published.

However, [ ¹¹ C]-(R) -PK11195 is limited to widespread use due to the short half-life of radioisotope carbon-11 contained therein and the problems of nonspecific binding of ligand PK11195 and low signal to noise ratio.

[Prior literature]

James, ML et al. , Synthesis and in vivo evaluation of a novel peripheral benzodiazepine receptor PET radioligand, Bioorg. Med. Chem., 2005, 13: 6188-6194.

2. Roeda, D. et al. , Synthesis of fluorine-18-labelled TSPO ligands for imaging neuroinflammation with Positron Emission Tomography, J. Fluorine Chem., 2012, 134: 107-114.

3. Damont, A. et al. , Synthesis of 6- [ ¹⁸ F] fluoro-PBR28, a novel radiotracer for imaging the TSPO 18 kDa with PET, Bioorg. Med. Chem. Lett., 2011, 21 (8): 4819-4822.

4. Zhang, M.-R. et al. ¹¹ C-AC-5216: A Novel PET Ligand for Peripheral Benzodiazepine Receptors in the Primate Brain, J. Nucl. Med., 2007, 48 (11): 1853-1861.

5. Kita, A. et al. , Antianxiety and antidepressant-like effects of AC-5216, a novel mitochondrial benzodiazepine receptor ligand, Br. J. Pharmacol., 2004, 142 (7): 1059-1072.

6. Tiwari, AK et al. , [ ¹⁸ F] FEBMP: Positron Emission Tomography Imaging of TSPO in a Model of Neuroinflammation in Rats, and in vitro Autoradiograms of the Human Brain, Theranostics, 2015, 5 (9): 961-969.

The present inventors have diligently researched to find a substance that can replace a short-lived [ ¹¹ C] -containing radioactive compound as a tracer for PET for diagnosing neuroinflammation, and then introduced [ ¹⁸ F] alkyl instead of [ ¹¹ C] methyl. It was found that one radioactive compound could be produced and the present invention was completed.

One object of the present invention is to provide a radioactive compound for PET for diagnosing cerebral neuritis, comprising a [ ¹⁸ F] alkyl group.

The radioactive compound incorporating [ ¹⁸ F] alkyl of the present invention, through a longer half-life of [ ¹⁸ F], exhibits a similar effect as the radioactive compound comprising [ ¹¹ C] as a tracer for PET for diagnosing cerebral neuroinflammatory inflammation. The short half-life can compensate for the shortcomings of radioactive compounds including [ ¹¹ C], which cannot be used in many patients, and thus can be usefully used as a PET tracer for diagnosing neuroinflammatory diseases.

1 is a PET tracer for the diagnosis of neuroinflammatory diseases, in accordance with the present invention, a diagram showing a novel radioactive compound.

2 is a diagram showing a [ ¹⁸ F] labeled radioactive compound derived from a PBR28 compound, according to the present invention.

3 is a diagram showing a [ ¹⁸ F] labeled radioactive compound derived from an AC-5216 compound, according to the present invention.

As one embodiment for achieving the above object, the present invention provides a radioactive compound for PET for diagnosing cerebral neuritis, comprising a [ ¹⁸ F] alkyl group.

Preferably, the compound may be a compound represented by Formula 1:

[Formula 1]

Where

n is 1 or 2;

R ₁ is hydrogen or fluoro; And

R ₂ is hydrogen or hydroxy.

More preferably, the compound

,

And

It may be selected from the group consisting of.

Also, preferably, the compound may be a compound represented by Formula 2:

[Formula 2]

Where

R ₃ to R ₆ are each independently hydrogen or ¹⁸ F,

Except where both R ₃ to R ₆ are hydrogen.

More preferably, the compound

,

,

And

It may be selected from the group consisting of.

The compounds according to the present invention can specifically bind to 18 kDa translocator protein (TSPO), the marker of cerebral neuroinflammatory disease, and has a longer half-life than compounds containing [ ¹¹ C] It can be used as a tracer for positron emission tomography (PET) for inflammation diagnosis.

To facilitate administration in the body of an individual including a human, excipients and / or diluents and the like may be further provided as an injection formulation.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example 1: [ ¹⁸ F] Preparation of labeled PBR28 derivatives

PBR28 derivatives were synthesized from 4-chloro-3-nitropyridine according to Scheme 1 below.

Scheme 1

Example 2: [ ¹⁸ F] Preparation of Labeled AC5216 Derivatives

AC5216 derivatives were synthesized from 2- (5- (ethoxycarbonyl) -2-phenylpyrimidin-4-ylamino) acetic acid according to the scheme of any of Schemes 2-4 below.

Scheme 2

Scheme 3

Scheme 4

Experimental Example  1: Identification of Synthetic Radioactive Compounds

Eight compounds (including three precursor compounds) synthesized from Examples 1 and 2 were identified by ¹ H-NMR, ¹⁹ F-NMR and LC-Mass. The results for each compound are shown in Table 1 below. All compounds were obtained with a yield of at least 95% and a purity of at least 95%.

Entry	Analysis method	Measurement data
FAO	1 H-NMR	(300 MHz, DMSO + D 2 O): 8.45 (d, 1H), 8.23-8.28 (m, 2H), 7.40-7.49 (m, 4H), 6.93-7.30 (m, 3H), 4.86-4.91 (d, 4H), 3.23-3.55 (m, 5H), 0.93-1.27 (dt, 3H), 4.54-4.75 (d, 2H)
	19 F-NMR	(300MHz, DMSO + D 2 O): (-177.16 s, -118-34 s)
	LC-Mass	calculated for C 23 H 22 FN 5 O 2 , 419.18; found [M + H] 420.10
FAC	1 H-NMR	(300 MHz, CDCl 3 ): 8.34-8.38 (m, 2H), 8.23-8.26 (d, 1H), 7.45-7.50 (m, 5H), 7.36-7.41 (m, 2H), 7.26-7.27 (m, 1H ), 4.91-4.97 (d, 2H), 4.76-4.82 (d, 2H), 4.52-4.71 (dt, J = 47.5 Hz, 4.9 Hz, 2H), 3.63-3.79 (m, 2H), 3.49-3.52 ( d, 3H)
	19 F-NMR	(300 MHz, CDCl 3 ): (-221.11--220.69.m) (-222.80--2.28.m)
	LC-Mass	calculated for C 23 H 22 FN 5 O 2 , 419.18; found [M + H] 420.25
FAM	1 H-NMR	(300 MHz, CDCl 3 ): 8.33-8.37 (m, 2H), 8.23-8.27 (d, 1H), 7.41-7.47 (m, 3H), 6.71-7.24 (m, 4H), 4.92-4.82 (d, 2H ), 4.61-4.66 (d, 2H), 3.44-3.52 (m, 5H), 6.12-1.38 (dt, 3H)
	19 F-NMR	(300MHz, CDCl 3 ): (-111.36, -112.68)
	LC-Mass	calculated for C 23 H 22 FN 5 O 2 , 419.18; found [M + H] 420.10
FAP (precursor)	1 H-NMR	(300 MHz, CDCl 3 ): 8.33-8.35 (m, 2H), 8.24-8.27 (d, 1H), 7.44-7.50 (m, 3H), 7.34-7.38 (m, 1H), 7.11-7.24 (m, 2H ), 6.91-6.97 (m, 1H), 4.83-4.90 (d, 2H), 4.58-4.64 (d, 2H), 3.41-3.52 (m, 5H), 1.11-1.37 (dt, 3H)
	19 F-NMR	(300MHz, CDCl 3 ): (-114.22, -115.02)
	LC-Mass	calculated for C 23 H 22 FN 5 O 2 , 419.18; found [M + H] 420.10
BAC (precursor)	1 H-NMR	8.76 (t, J = 5.8 Hz, 1H), 8.55 (s, 1H), 8.31-8.36 (m, 2H), 7.48-7.52 (m, 3H), 7.19-7.28 (m, 5H), 4.62 (s, 2H), 4.33 (d, J = 5.9 HZ, 2H), 3.44 (s, 3H)
	LC-Mass	calculated for C 21 H 19 N 5 O 2 , 373.15; found [M + H] 374.20
EAC	1 H-NMR	(300MHz, DMSO): 8.55 (s, 1H), 8.31-8.34 (m, 2H), 8.25 (t, J = 5.3 Hz, 1H), 7.45-7.52 (m, 3H), 4.51 (s, 2H), 3.43 (s, 3H), 3.07-3.18 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H)
	LC-Mass	calculated for C 16 H 17 N 5 O 2 , 311.14; found [M + H] 312.10
HEPA (precursor)	1 H-NMR	(300MHz, CDCl 3 ): 9.12 (s, 1H), 7.93 (s, 1H), 7.38-7.44 (m, 2H), 7.23-7.32 (m, 2H), 6.92-6.96 (m, 1H), 6.67- 6.79 (m, 4H), 6.17 (d, J = 1.2 Hz, 1H), 4.79 (dd, J = 16.3 Hz, 4.7 Hz, 2H), 2.03 (s, 3H)
	19 F-NMR	(300MHz, CDCl 3 ): -62.11
	LC-Mass	calculated for C 20 H 17 FN 2 O 3 , 352.12; found [M + H] 353.10, [M + Na] 375.10
FEPA	1 H-NMR	(300 MHz, CDCl 3 ): 7.77 (s, 1 H), 7.19-7.46 (m, 5 H), 6.89-6.94 (m, 3 H), 6.73-6.76 (m, 1 H), 6.10 (d, J = 1.2 Hz, 1H), 5.23 (d, J = 14.1 Hz, 1H), 4.82 (d, J = 14.1 Hz, 1H), 4.43-4.73 (m, 2H), 3.90-4.16 (m, 2H), 1.98 (s3H)
	19 F-NMR	(300MHz, CDCl 3 ): -64.09, -222.68--223.15
	LC-Mass	calculated for C 22 H 20 F 2 N 2 O 3 , 398.14; found [M + H] 399.10, [M + Na] 421.10

Experimental Example 2: Binding Affinity to 18 kDa Translocation Proteins (TSPO) in Vitro

Leukocytes were isolated by Pack (Ficoll-Hypaque) gradient centrifugation. Isolated white blood cells were frozen and preserved. The cells were thawed the day before analysis, diluted with an equal amount of buffer (50 mM HEPES, pH 7.4) and homogenized, and centrifuged at 20,000 g for 15 minutes at 4 ° C. The obtained leukocytes were resuspended in 2.4 ml buffer and stored at -70 ° C, and protein concentration was confirmed by the Bradford assay.

In vitro binding was determined from leukocytes (100 nL resuspended membrane) from 100 nL of radioligand (1 × PBS solution of [ ³ H] PK11195 (non-radioactivity: 83.4 Ci / mmol) as control) and Examples 1 and 2 above. 1 ml of the reaction mixture containing the synthesized compound and 50 ml of 0.07 nM radioligand ([ ³ H] PK11195) was reacted at room temperature for 30 minutes. After washing twice using a cell harvester, the amount of bound radioactivity was measured by a beta counter. The proportion of specific binding fractions under the assay conditions was less than 20% of the total ³ H radioactivity. Results for the in vitro binding calculated were subjected to nonlinear regression analysis with PRISM software to calculate IC ₅₀ values. As a result, HEPA and FEPA showed good binding affinity at 20 nM level (24.85 and 29.14 nM, respectively) and FAC at 40 nM level (47.72 nM).

Claims (5)

1. A radioactive compound for PET for diagnosing cerebral neuritis, comprising a [ ¹⁸ F] alkyl group.
2. The method of claim 1,

   Compound represented by the following formula (1):

   [Formula 1]

   

   Where

   n is 1 or 2;

   R ₁ is hydrogen or fluoro; And

   R ₂ is hydrogen or hydroxy.
3. The method of claim 2,

   The compound is

   

   ,

   

   And

   

   Compound selected from the group consisting of.
4. The method of claim 1,

   Compound represented by the following formula (2):

   [Formula 2]

   

   Where

   R ₃ to R ₆ are each independently hydrogen or ¹⁸ F,

   Except where both R ₃ to R ₆ are hydrogen.
5. The method of claim 4, wherein

   The compound is

   

   ,

   

   ,

   

   And

   

   Compound selected from the group consisting of.

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