TSPO binder

Abstract

The present invention provides compounds of formula (I), and salts, solvates and radiolabeled forms thereof, as well as complexes of compounds of formula (I) with TSPO, and methods for forming such complexes, and methods for detecting compounds of formula (I), for example, compounds of formula (I) in complex with TSPO.

Description

[Technical Field]

[0001] Related applications This case asserts the interests and priority of British Patent No. 1810312.7, filed on 22 June 2018 (22.06.2018), the contents of which are incorporated herein by reference in their entirety.

[0002] Field of Invention The present invention provides compounds for use in binding with TSPO, methods for preparing such compounds, methods for binding the compounds with TSPO, and methods for detecting compounds bound to TSPO. [Background technology]

[0003] The 18kDa transporter protein (TSPO, translocator protein), officially known as the peripheral diazepine receptor (Papadopoulos et al. Trends Pharmacol. Sci. 27, 402-409 (2006)), is expressed in the outer mitochondrial membrane and is involved in cholesterol transport and steroid synthesis (Lacapere Steroids 68, 569-585 (2003)). TSPO is highly expressed in inflammatory cells such as microglia in the brain (Wilms et al. Neurobiol. Dis. 14, 417-424 (2003) and Cosenza-Nashat et al. Neuropathol. Appl. Neurobiol. 35, 306-328 (2009)) and macrophages in the periphery (Fujimura et al. Atherosclerosis 201, 108-111 (2008) and Bird et al. Atherosclerosis 210, 388-391 (2010)), and has therefore been considered useful as a marker of inflammation in pathology throughout the body.

[0004] Increased expression of TSPO has been demonstrated in neurodegenerative diseases such as dementia and Parkinson's disease (Dupont et al. Int. J. Mol. Sci. 18, 785 (2017)), as well as in cardiovascular diseases, specifically in atherosclerotic plaques (Bird et al. Atherosclerosis 210, 388-391 (2010)) and in the heart after myocardial infarction (Thackeray et al. J. Am. Coll. Cardiol. 71, 263-275 (2018)). Therefore, a successful imaging approach targeting TSPO with PET has significant clinical value in a wide range of pathologies. In addition to its usefulness as a marker of inflammation, TSPO has recently been demonstrated to play a role in neuroprotection (Thackeray et al. J. Am. Coll. Cardiol. 71, 263-275 (2018)) and cardioprotection (Schalle et al. J. Pharmacol. Exp. Ther. 333, 696-706 (2010) and Paradis et al. Cardiovasc. Res. 98, 420-427 (2013)), thus further expanding the scope of application of non-invasive TSPO imaging in the context of disease onset and progression, as well as for targeting disease-modifying therapies.

[0005] In the field of positron emission tomography (PET) imaging of inflammation, TSPO is one of the most widely explored targets. A prototype TSPO PET radiotracer developed several decades ago... 11 This is C-PK11195 (Charbonneau et al. Circulation 73, 476-483 (1986)). The short half-life (20 minutes) of this radioactive tracer has limited its adoption in clinical routines, requiring hospitals to have on-site cyclotron facilities. In addition, 11C-PK11195 exhibits relatively high nonspecific binding (Chauveau et al. Eur. J. Nucl. Med. Mol. Imaging 35, 2304-2319 (2008)).

[0006] Therefore, as summarized in a recently published review (Alam et al. Med. Mol. Imaging (2010). 51, 283-296 (2017)), considerable effort has been made to create a novel family of TSPO radioactive tracers with improved characteristics. Despite these developments, 11 C-PK11195 is still regularly used as a clinical research tool. 11 The high inter-individual binding of all TSPO radioactive tracers synthesized and investigated after the development of C-PK11195 is now known to be caused by the genetic polymorphism rs6971, as identified in the original study by Owen et al. (Owen et al. J. Cereb. Blood Flow Metab. 32, 1-5 (2012)). This common genetic polymorphism means that approximately 10% of the human population classified as low-affinity binders (LABs) are unimagingable with second-generation radioactive tracers, and the remainder of the human population division between mixed-affinity binders and high-affinity binders (MABs and HABs) requires genetic screening and complex post-imaging correction. There is a large degree of diversity in the sensitivity of second-generation ligands to the rs6971 genetic polymorphism. For example, 11 C-PBR28 has a LAB:HAB ratio of 55 in vitro (Owen et al. J. Nucl. Med. 52, 24-32 (2011)), while a more recently designed radiotrace 11Analogues of C-ER176 and PK11195 have a ratio of 1.3 in vitro (Zanotti-Fregonara et al. ACS Chem. Neurosci. (2014), doi:10.1021 / cn500138n).

[0007] Therefore, to date, PK11195 remains the only TSPO radioactive tracer whose insensitivity to the rs6971 polymorphism in the human brain has been reliably demonstrated (Owen et al. J. Nucl. Med. 52, 24-32 (2011) and Owen et al. J. Cereb. Blood Flow Metab. 30, 1608-18 (2010)). Further binders for TSPO that also demonstrate insensitivity to the rs6971 polymorphism are needed. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Papadopoulos et al. Trends Pharmacol. Sci. 27, 402-409 (2006) [Non-Patent Document 2] Lacapere Steroids 68, 569-585 (2003) [Non-Patent Document 3] Wilms et al. Neurobiol. Dis. 14, 417-424 (2003) [Non-Patent Document 4] Cosenza-Nashat et al. Neuropathol. Appl. Neurobiol. 35, 306-328 (2009) [Non-Patent Document 5] Fujimura et al. Atherosclerosis 201, 108-111 (2008) [Non-Patent Document 6] Bird et al. Atherosclerosis 210, 388-391 (2010)

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

[0009] The present invention generally provides compounds used in the binding of transporter proteins (TSPOs) that, upon binding, act as tracers for TSPOs, such as radioactive tracers. Therefore, the compounds of the present invention may find use in methods for detecting TSPOs in vitro and in vivo. Such methods may be used for the identification of inflammation or for the diagnosis of diseases associated with changes in TSPO levels, such as neurological inflammation, cancer, and cardiovascular disease.

[0010] The compounds of the present invention are insensitive to the rs6971 genetic polymorphism and exhibit TSPO affinity approximately twice that of known TSPO binders, such as PK11195. The compounds of the present invention have favorable in vivo kinetics and a favorable dosimetry profile, and are therefore suitable for use in clinical settings.

[0011] The in vivo characterization of the compounds of the present invention also reveals specific uptake consistent with TSPO expression. Blocking studies with a single concentration of PK11195 (1 mg / kg) confirmed target engagement, along with a 64–81% reduction in SUV values ​​measured after blocking compared to baseline scans.

[0012] The compounds of the present invention may be considered ideally suited for use as labels for TSPO, such as radiolabeling, because they have nanomolar affinity for TSPO in both the human brain and heart, can penetrate the brain in vivo, have an in vivo distribution profile consistent with TSPO protein expression, their metabolism in plasma is slow, and the levels of metabolites recorded in tissues are low. [Means for solving the problem]

[0013] In a first embodiment of the present invention, formula (I):

[0014] [ka]

[0015] Compounds thereof, as well as their salts, solvates, and radiolabeled products are provided.

[0016] In a preferred embodiment, the compound of formula (I) is formula (II):

[0017] [ka]

[0018] These are compounds, as well as their salts and solvates.

[0019] A TSPO binder supporting a fluorine radiolabel such as the compound of formula (II) is: 11 It has a longer half-life than compounds carrying carbon radiolabels, such as compounds known in the art that include C-PK11195. The dosimeter profile of the compound of formula (II) is such that the radiolabeled compound is suitable for human use.

[0020] In a second embodiment of the present invention, a method for preparing a compound of formula (I), wherein formula (III):

[0021] [ka]

[0022] A method is provided which includes the step of substituting the bromine in the compound with fluorine.

[0023] In a third embodiment of the present invention, a method for preparing a compound of formula (II), wherein formula (IV):

[0024] [ka]

[0025] A method is provided which includes the step of substituting the chlorine in the compound with 18-fluorine.

[0026] The compound of formula (IV) can be obtained from the compound of formula (III) by substituting bromine with chlorine.

[0027] The present invention also provides compositions comprising a compound of formula (I) together with one or more pharmaceutically acceptable excipients.

[0028] In a further embodiment of the present invention, a method for detecting a compound of formula (I) is provided, comprising the steps of contacting the compound of formula (I) with TSPO to form a complex of the compound of formula (I) with TSPO, and detecting the compound of formula (I).

[0029] TSPO can be administered in vitro or in vivo.

[0030] The compounds of the present invention may be used to detect organ samples extracted from subjects, such as heart and brain samples. Therefore, the method of the present invention may be used, for example, in neurological and cardiovascular pathology to detect and determine the level of TSPO.

[0031] In a further embodiment of the present invention, a method is provided for detecting a compound of formula (I) in a subject, comprising the steps of administering a compound of formula (I) to the subject, and subsequently detecting a compound of formula (I).

[0032] In this case, the subject may be a human being.

[0033] The subjects may be those who are known or suspected to have diseases related to changes in TSPO levels, such as neurological inflammation, cancer, and cardiovascular disease, including elevated TSPO levels.

[0034] The subjects may have a disease associated with changes in TSPO levels, and may be receiving treatment for such a disease. The method of the present invention may be used to detect changes in TSPO levels, for example, to measure the effectiveness of treatment.

[0035] These and other aspects and embodiments of the present invention will be described in further detail below. [Brief explanation of the drawing]

[0036] [Figure 1] This figure shows the structures of known TSPO binders, PK11195(A), PBR28(B), and AB5186(C), and a TSPO binder according to an embodiment of the present invention, LW223(D). The compounds are shown in their cold (non-radioactively labeled) forms. [Figure 2]This figure shows the binding affinity of TSPO ligands in the human brain derived from high (HAB), mixed (MAB), and low (LAB) affinity binders. (a) shows the mean PK11195 binding affinity curves plotted using one-site fitting, HAB n=6, MAB n=8, and LAB n=4; (b) shows the mean PBR28 binding affinity curves plotted using one-site fitting, HAB n=4, MAB n=5, and LAB n=4, excluding MABs where two-site fitting was used; (c) shows the mean AB5186 binding affinity curves plotted using one-site fitting, HAB n=6, MAB n=6, and LAB n=5, excluding MABs where two-site fitting was used; (d) shows the mean PK11195 binding affinity curves plotted using one-site fitting, HAB n=5, MAB n=5, and LAB The mean LW223 binding affinity curves plotted using n=4 are shown, with (e) showing the affinity values ​​(Ki) individually calculated for PK11195 for each human sample. These values ​​were also calculated for (f) PBR28, (g) AB5186, and (h) LW223. Unpaired t-tests were used for HAB vs. LAB, with ns = not significant, * = p < 0.05, and *** = p ≤ 0.001. All results represent mean ± SEM. [Figure 3]This figure shows the binding affinity of TSPO ligands in the human heart derived from high (HAB), mixed (MAB), and low (LAB) affinity binders. (a) shows the mean PK11195 binding affinity curve plotted using single-site fitting, HAB n=4, MAB n=5, and LAB n=4; (b) shows the mean PBR28 binding affinity curve plotted using single-site fitting, HAB n=4, MAB n=5, and LAB n=4; (c) shows the mean AB5186 binding affinity curve plotted using single-site fitting, HAB n=4, MAB n=5, and LAB n=4, with the exception of MAB where two-site fitting was used; (d) shows the mean LW223 binding affinity curve plotted using single-site fitting, HAB n=5, MAB n=5, and LAB n=4; (e) shows the affinity values ​​(Ki) for PK11195 calculated individually for each human sample. These values ​​were also calculated for (f) PBR28, (g) AB5186, and (h) LW223. An independent t-test was used for HAB vs. LAB, with ns = not significant and ** = p ≤ 0.01. All results represent mean ± SEM. [Figure 4] This figure shows the in vivo dynamics and metabolic profiles of 18F-LW223. (a) is a maximum intensity projection image of rats, and (B) is a projection image of mice, showing the distribution of 18F-LW223 under baseline conditions. B=brain, H=heart, L=lungs, GB=gallbladder (mouse only), A=adrenal gland, K=kidney, and G=intestine. (c) and (d) are the 18F-LW223 chronological activity curves for rats and mice, respectively. (e) shows the blood kinetics of 18F-LW223 in rats, indicating the percentage of the parent compound in plasma. Results are expressed as mean ± SEM, n=3 per time point. (f) shows the volume of distribution (Vt) values ​​in organs, calculated by 2-tissue (2T), Logan (t*=30), and multivariate (M1, t*=30) modeling, mean ± SD, n=3. [Figure 5]This figure shows 18F-LW223 conjugated to TSPO in vivo. (a) shows SUV summation images of 18F-LW223 uptake in the brain (B), heart (H), and lungs (L), and (b) shows SUV summation images of 18F-LW223 uptake after blockade with PK11195 (1 mg / kg). All images are averaged (60-120 min) and Gaussian filtered (1 × 1 × 1), (c) shows the 18F-LW223 time-activity curve for the primary source organ at baseline, and (d) after PK11195 blockade. Results represent mean ± SEM, n=3. [Figure 6] This figure shows the binding of 18F-LW223 to human tissue and clarifies local uptake in pathological sites. (a) shows H&E staining of brain tissue from an individual that suffered hemorrhagic stroke, * = hemorrhagic area. Scale bar = 1,000 μM. (b) shows a histological example (H&E) of an affected coronary vessel exhibiting pathological neointima remodeling. The dotted line represents the boundary between the original medial layer and the neointima. Scale bar = 1,000 μM. (c) is an autoradiography of 18F-LW223 binding with invading inflammatory cells in stroke tissue; (d) is an autoradiography of 18F-LW223 binding with inflammatory cells in the neointimal space of affected coronary vessels; (e) shows the blockage of 18F-LW223 binding using PK11195 in stroke; (f) is from affected coronary artery tissue; (g) shows the blockage of 18F-LW223 binding using LW223 in stroke; (h) is from affected coronary vessels; (i) shows the quantification of 18F-LW223 binding in high and low uptake regions in stroke along with target vs. non-target ratios; and (j) is from affected coronary artery tissue. The results are expressed as mean ± SEM, n=3, *=p<0.05, **=p≦0.01, using a paired t-test for target vs. non-target. [Figure 7]This figure shows the dissociation constant (Kd) and maximum binding amount (Bmax) of PK11195 in the human brain and heart. (a) shows the Kd value of PK11195, and (b) shows the Bmax calculated from the saturated binding assay. Results are expressed using an unpaired t-test for brain vs. heart, meaning ± SEM, brain n=6, heart n=5, ns=not significant, *=p<0.05. [Figure 8] This figure shows in vivo replacement of 18F-LW223 using PK11195. (a) is the SUV summation image of 18F-LW223 uptake before replacement, and (b) is the SUV summation image after replacement with PK11195 (1 mg / kg). Baseline images are averaged before PK11195 administration (45-60 minutes), and replacement images are averaged after (95-120 minutes). All images are Gaussian filtered (1×1×1), and (c) is the time-activity curve of 18F-LW223 before and after PK11195 sensitization (green arrows). [Figure 9] This figure shows the 18F-LW223 time-activity curves of source organs in mice. (a) is the 18F-LW223 time-activity curve across all sites with higher uptake than the background in male mice, and (b) is the 18F-LW223 time-activity curve across all sites with higher uptake than the background in female mice. Raw PET data were reconstructed using filtered back projection without attenuation correction. [Figure 10] This figure shows the binding of 18F-LW223 in healthy human brain tissue. (a) is H&E staining of healthy brain, (b) is autoradiography of 18F-LW223 binding in healthy brain, (c) is the blocking of 18F-LW223 binding using PK11195, (d) is the blocking of 18F-LW223 binding using LW223, and (e) is the quantification of 18F-LW223 binding in gray matter regions, along with their target-to-non-target ratios. Results are shown using paired t-tests for target-to-non-target, mean ± SEM, n=3, ns=not significant. Scale bar = 1,000 μM. [Figure 11] This figure shows the results of a study on 18F-LW223 uptake in rats with induced myocardial infarction, demonstrating that myocardial infarction leads to increased 18F-LW223 uptake in the heart, brain, and lungs. (A) are examples of 18F-LW223 SUV images of the heart (upper), brain (central), and lungs (lower) in healthy (left) and myocardial infarction rats (right) 7 days after injury. (B) shows the SUV time-activity curves in the heart, brain, and lungs for healthy (red circles) and myocardial infarction rats (blue squares). (C) shows the time-activity curve of SUVr related to blood pooling in the heart, brain, and lungs for healthy (red circles) and myocardial infarction rats (blue squares). Mean ± SEM, n=5~6. [Modes for carrying out the invention]

[0037] Compounds of formula (I), which include compounds of formula (II), have been found to be useful as binders for TSPO. Therefore, the compounds may be used to identify changes such as increased TSPO expression in subjects such as humans, and may be used to identify subjects who are at risk of or have a risk of diseases such as neurological inflammation, cancer, or cardiovascular disease.

[0038] The compounds of the present invention, as well as their synthesis and use, are described in further detail below.

[0039] Stevenson et al. Bioorg. Med. Chem. Lett. 20, 954-957 (2010) previously described compound 11, which has an iodomethyl substituent on the quinoline ring rather than the fluoromethyl group required by the compound in question. This compound is intended for use in SPECT imaging of TSPO. Stevenson et al. Bioorg. Med. Chem. Lett. 20, 954-957 (2010) does not describe any compounds for use as radiotracers. Furthermore, this work does not suggest that such compounds or any derivatives are or may be insensitive to the rs6971 genetic polymorphism.

[0040] Furthermore, the bonding data reported in Stevenson et al. Bioorg. Med. Chem. Lett. 20, 954-957 (2010) teaches us independently of the use of amide substituents present in the compounds of the present invention. Therefore, a comparison between compound 11 (amide nitrogen substituted with methyl and s-butyl) and related compound 18 (amide nitrogen substituted with diethyl) shows that compound 18 has the best affinity for TSPO (see Table 1 in Stevenson et al. Bioorg. Med. Chem. Lett. 20, 954-957 (2010)).

[0041] Blair et al. Chem. Sci. 6, 4772-4777 (2015) previously described tracer compounds 4 (corresponding to compound 18 in Stevenson et al. Bioorg. Med. Chem. Lett. 20, 954-957 (2010)), 5, and 6 (AB5186). Blair et al.'s work shows that the binding of iodo compounds to TSPO is improved when the halogen is placed as a substituent on the pendant phenyl group rather than as a halomethyl substituent on the quinoline ring. Therefore, the data in Figure 2 of Blair et al. Chem. Sci. 6, 4772-4777 (2015) shows that compound 5 has better affinity than compound 4.

[0042] This case demonstrates that the compounds of the present invention have better affinity than the compounds described by Blair et al. Chem. Sci. 6, 4772-4777 (2015), which include compound 6, the reference compound AB5186 in this application.

[0043] Cappelli et al. J. Med. Lett. 14, 4055-4066 (2006) describes compounds and their uses related to those described by Stevenson et al. Bioorg. Med. Chem. Lett. 20, 954-957 (2010) and Blair et al. Chem. Sci. 6, 4772-4777 (2015). This literature says nothing about the challenges of polymorphic bonding.

[0044] International Publication No. 02 / 26713 focuses on compounds for use in treating parasitic infections. Some of the disclosed compounds have a very slight similarity to the compounds in this case. International Publication No. 02 / 26713 does not refer to TSPO or radiotracing.

[0045] compound The present invention relates to formula (I):

[0046] [ka]

[0047] The present invention provides compounds thereof, as well as their salts, solvates, and radiolabeled products.

[0048] The compound of formula (I) is

[0049] [ka]

[0050] Preferably, the salts, solvates, and radiolabeled forms thereof are also included.

[0051] The inventors have found that this particular stereoform (R) configuration has approximately five times greater affinity for TSPO compared to its enantiomer.

[0052] In a preferred embodiment, the compound is of formula (II):

[0053] [ka]

[0054] These are compounds, as well as their salts and solvates.

[0055] The compound of formula (II) is a radiolabeled compound, more specifically, a fluorine radioisotope, 18 It is labeled with F.

[0056] The compound of formula (II) is

[0057] [ka]

[0058] and salts and solvates thereof are preferred.

[0059] Radiolabeled compounds may sometimes be referred to as hot compounds, in contrast to cold non-radiolabeled compounds.

[0060] Radiation forms, salts, solvates and stereoisomers The compound of Formula (I) or any other compound described herein includes compounds in which at least one atom is replaced by a naturally occurring or non-naturally occurring isotope. In one embodiment, the isotope is a stable isotope. Therefore, the compounds described herein include, for example, deuterium-containing compounds and the like. For example, H is 1 H, 2 H(D) and 3 may be any isotope form including H(T), C is 11 C, 12 C, 13 C and 14 may be any isotope form including C, O is 15 O, 16 O and 18 may be any isotope form including O, F is 18 may be any isotope form including F and the like.

[0061] Typically, the compounds of the present invention contain 18 F, 11 C, 13 N or 15 O and the like, a radioisotope suitable for detection (or imaging) by scintigraphic imaging methods such as by positron emission tomography (PET). Therefore, in one embodiment, the compounds of the present invention contain 18 F, 11 C, 13 N or 15 O and other positron-emitting radioisotopes, most preferably contain 18 F.

[0062] In a preferred embodiment of the present invention, in the compound of Formula (I), the F atom is18 F may be provided. Such compounds are compounds of formula (II). These compounds are C 11 Compared to compounds with radioactive labels (half-live of around 20 minutes), their longer half-lives (around 110 minutes) make them particularly useful radioactive tracers.

[0063] Examples of salts of the compound of formula (I) or any other compound described herein include, but are not limited to, all pharmaceutically acceptable salts, such as acid addition salts of strong mineral acids, such as HCl and HBr salts, and addition salts of strong organic acids, such as methanesulfonates. Further examples of salts include sulfates and acetates such as the acetate itself, trifluoroacetate, or trichloroacetate.

[0064] Any reference to the compound of formula (I) or any other compound described herein also refers to the solvate of that compound. Examples of solvates include hydrates.

[0065] Unless otherwise specified, references to specific compounds include isomers, which encompass mixtures thereof (e.g., racemic mixtures). Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic means) of such isomers are either known in the art or readily obtainable by adapting methods taught herein or known methods in known ways.

[0066] One aspect of the present invention relates to a substantially purified form and / or a substantially contaminant-free form of a compound.

[0067] In one embodiment, the substantially purified form is at least 50% by weight, for example, at least 60% by weight, for example, at least 70% by weight, for example, at least 80% by weight, for example, at least 90% by weight, for example, at least 95% by weight, for example, at least 97% by weight, for example, at least 98% by weight, for example, at least 99% by weight.

[0068] Unless otherwise specified, a substantially purified form refers to a compound in any stereoisomer or enantiomer form. For example, in one embodiment, a substantially purified form refers to a mixture of stereoisomers, i.e., purified compared to other compounds. In one embodiment, a substantially purified form refers to one stereoisomer, e.g., an optically pure stereoisomer. In one embodiment, a substantially purified form refers to a mixture of enantiomers. In one embodiment, a substantially purified form refers to an equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, a substantially purified form refers to one enantiomer, e.g., an optically pure enantiomer.

[0069] In one embodiment, the impurities represent 50% by weight or less, for example, 40% by weight or less, for example, 30% by weight or less, for example, 20% by weight or less, for example, 10% by weight or less, for example, 5% by weight or less, for example, 3% by weight or less, for example, 2% by weight or less, for example, 1% by weight or less.

[0070] Unless otherwise specified, impurities refer to other compounds other than stereoisomers or enantiomers. In one embodiment, impurities refer to other compounds and other stereoisomers. In one embodiment, impurities refer to other compounds and other enantiomers.

[0071] In one embodiment, the substantially purified form is at least 60% optically pure (i.e., 60% of the compound is the desired stereoisomer or enantiomer on a molar basis, and 40% is the undesirable stereoisomer or enantiomer), for example, at least 70% optically pure, for example, at least 80% optically pure, for example, at least 90% optically pure, for example, at least 95% optically pure, for example, at least 97% optically pure, for example, at least 98% optically pure, for example, at least 99% optically pure.

[0072] composition The compounds of formulas (I) and (II) are suitable for binding with TSPO and can be bound in vivo. Therefore, the compounds of formula (I) or (II) may be prepared in compositions for administration to humans or animals.

[0073] If the compound of formula (I) is radiolabeled, the compound may be provided in a fluid composition at a concentration of at least 0.1, at least 0.5, at least 1, at least 2, or at least 5 MBq / mL.

[0074] If the compound of formula (I) is radiolabeled, the compound may be provided in a fluid composition at concentrations of up to 10, up to 20, up to 50, up to 100, up to 200, up to 300, or up to 200 MBq / mL.

[0075] The compound may be used at concentrations within a range having lower and upper limits selected from the values ​​described above. For example, the compound may be used at concentrations within the range of 1 to 10 MBq / mL.

[0076] In some of the cases in which this matter was implemented, 18 F-LW223 is used at a concentration of 2 MBq / mL.

[0077] Typically, the compounds of formulas (I) and (II) are used, for example, in saline solutions for intravenous injection.

[0078] While it is possible to administer the compound of formula (I) alone, it is preferable to present it as a pharmaceutical preparation (e.g., composition, preparation, pharmaceutical) containing at least one compound of formula (I) as described herein, together with one or more other pharmaceutically acceptable components known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners.

[0079] Therefore, the present invention further provides a composition as defined above, and a method for preparing the composition, comprising the step of mixing at least one compound of formula (I) as described herein with one or more other pharmaceutically acceptable components known to those skilled in the art, such as a carrier, diluent, excipient, etc. When formulated as discontinuous units (e.g., tablets), each unit contains a predetermined amount (dose) of the compound.

[0080] When used herein, the term “pharmaceutically acceptable” refers to a compound, component, material, composition, dosage form, etc., that is appropriate, within the bounds of reasonable medical judgment, for use in contact with the tissue of the subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problems or complications, and that has a reasonable benefit-to-risk ratio. Each carrier, diluent, excipient, etc., must also be “acceptable” in the sense that it is compatible with the other components of the formulation.

[0081] Appropriate carriers, diluents, excipients, etc., can be found in standard pharmaceutical textbooks, such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 2005.

[0082] The formulations may be prepared by any method well known in the field of pharmaceutical preparation. Such methods include the step of associating the compound of formula (I) with a carrier comprising one or more minor components. The formulations are generally prepared by uniformly and closely associating the compound with a carrier (e.g., a liquid carrier, a pulverized solid carrier, etc.), and then, if necessary, shaping the product.

[0083] The formulation may be prepared to provide rapid release, immediate release, delayed release, timed release, or sustained release, or a combination thereof.

[0084] The formulation may appropriately be in the form of a liquid, a solution (e.g., aqueous, non-aqueous), a suspension (e.g., aqueous, non-aqueous), an emulsion (e.g., oil-in-water, water-in-oil), a spray, a mist, or an aerosol.

[0085] The compound may be dissolved in, suspended in, or miscible with one or more other pharmaceutically acceptable components. The compound may be presented in liposomes or other microparticles designed to target, for example, blood components or one or more organs.

[0086] When the carrier is a liquid, formulations suitable for intranasal administration include, for example, nasal sprays, nasal drops, or, in the case of nebulizer aerosol administration, aqueous or oily solutions of the compound. As an alternative method of administration, dry powder delivery may be used as an alternative to spray aerosols.

[0087] When the carrier is solid, formulations suitable for intranasal administration include, for example, those presented as coarse powders having particle sizes in the range of approximately 20 to 500 microns, which are administered by inhalation through the nose, i.e., by rapid inhalation through the nasal cavity from a container of powder held near the nose.

[0088] Formulations suitable for pulmonary administration (e.g., by inhalation or inhalation therapy) include those presented as aerosol sprays from a pressurized pack with the use of a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichloro(dichoro)-tetrafluoroethane, carbon dioxide, or other suitable gas. Additionally or alternatively, formulations for pulmonary administration may be formulated for administration from a nebulizer or dry powder inhaler. For example, the formulation may be provided with a carrier or liposomes to provide an appropriate particle size that reaches a reasonable portion of the lung to aid in the delivery of a reasonable dose and enhance retention in lung tissue.

[0089] Formulations suitable for parenteral administration (e.g., intravenously or subcutaneously by injection or infusion) include aqueous or nonaqueous, isotonic, pyrogen-free sterile solutions (e.g., solutions, suspensions) in which the compound is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohol, sugar, polyol, glycerol, and vegetable oil. Examples of suitable isotonic carriers for use in such formulations include sodium chloride injection, Ringer's solution, or Ringer's lactate injection. Typically, the concentration of the compound in liquid is about 1 ng / mL to about 500 μg / mL, for example, about 1 ng / mL to about 100 μg / mL, for example, about 10 ng / mL to about 10 μg / mL, for example, about 10 ng / mL to about 1 μg / mL. The formulation may be presented in unit dose or multi-dose sealed containers, such as ampoules and vials, and may be stored under freeze-drying conditions requiring only the addition of a sterile liquid carrier, such as sterile water for injection, immediately before use. Immediate injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0090] Preparation method The present invention also provides methods for preparing compounds of formula (I) and compounds of formula (II).

[0091] In the preparation method, fluorine in the compounds of formula (I) and (II) may be introduced in the final step of synthesis. This is particularly important when the compound of formula (I) contains fluorine as a radioactive label, such as in the compound of formula (II). The method for preparing the compounds of the present invention does not require the handling of intermediate radioactive labels, which is generally avoided, thanks to the loss of radioactivity over time. This would occur if the preparation and purification of radioactive labels were required.

[0092] The compound of formula (I) may be prepared from the compound of formula (III) by substitution of bromine in the compound of formula (III) with fluorine. The compound of formula (III) is

[0093] [ka]

[0094] Also, salts, solvates, and radiolabeled forms thereof.

[0095] Halogen substitution reactions and fluorine substitution reactions are well known in the art.

[0096] The compound of formula (III) can be reacted with a fluorine anion to obtain the compound of formula (I). The fluorine anion can be provided from an alkali metal salt, such as a fluorine salt with potassium fluoride. The substitution reaction may be carried out in the presence of a catalyst such as 18-crown-6.

[0097] The compound of formula (II) is obtained from the compound of formula (IV), where the chlorine in the compound of formula (IV) is... 18 - May be prepared by substitution with fluorine. Compounds of formula (IV) are

[0098] [ka]

[0099] Also, salts, solvates, and radiolabeled forms thereof.

[0100] For example, compounds of formula (IV) are well known for use in the art [ 18 F]KF etc. 18 Treat with F anion. 18 It is permissible to introduce F radioactive labeling.

[0101] The compound of formula (IV) is derived from the compound of formula (III) IIIIt may be prepared by substituting bromine in the compound with chlorine.

[0102] For example, the formula ( III The compound may be treated with a chloride anion such as LiCl.

[0103] The present invention is based on the formula ( R- The compounds of formula (III) and (IV), as well as their salts, solvates, and radiolabeled forms, are also provided.

[0104] complex In a further embodiment of the present invention, a complex is provided comprising a compound of formula (I) together with TSPO, where the compound of formula (I) is non-covalently bonded to TSPO.

[0105] The complex of the compound of formula (I) with TSPO may be provided in vitro or in vivo. TSPO may be located on the outer mitochondrial membrane.

[0106] Typically, the compound of formula (I) is provided with a bond pair with TSPO. Therefore, the stoichiometry of the compound and TSPO in the complex is 1:1.

[0107] The compound of formula (I) may be detectable if it is radiolabeled, or if it is bound to TSPO in the complex.

[0108] If the compound of formula (I) is radioactively labeled, as in the compound of formula (II), the compound can be detected by a method appropriate for radioactive labeling, such as positron emission tomography.

[0109] If the compound of formula (I) is not radiolabeled, the compound can be detected by other methods, including, for example, NMR.

[0110] TSPO is a transporter protein. It can be referred to as a peripheral benzodiazepine receptor.

[0111] TPSO may be human or mammalian TPSO such as a rodent.

[0112] TSPO may be a protein containing the NCBI reference sequence: NP_000705.2.

[0113] Method and Use The compounds of this invention are intended for use in binding with TSPO. The compounds are detectable, and are detectable when present as a complex with TSPO.

[0114] The compound of the present invention may be brought into contact with TSPO, which may be present in vivo or in vitro, to form a complex such as those described. The complex may then be detected by detection of the compound of the present invention.

[0115] If the compound of formula (I) is radiolabeled, for example, if the compound is the compound of formula (II), the compound may be detected by positron emission tomography (PET).

[0116] Therefore, the method in question generally includes the step of imaging compounds of formula (I), such as those of formula (II), using scintigraphy imaging methods, including PET (positron emission tomography). The scintigraphy imaging method may include the use of a camera or scanner to detect radioactivity in a single plane. The PET imaging system may include a circular array of detectors that can detect radioactivity in multiple dimensions.

[0117] Advantageously, the compounds of the present invention are insensitive to mutations within TSPO, such as the rs6971 genetic polymorphism.

[0118] The method may include a step of administering the compound of formula (I) to a human or animal subject.

[0119] The method of the present invention may include the step of detecting a compound of formula (I), such as one present in complex with TSPO, at a site in an object selected from the group consisting of the brain, heart, lungs, gallbladder, adrenal gland, kidney, and intestine.

[0120] The present invention also provides a method for imaging TSPO expression in a subject, comprising the steps of administering a compound of formula (I) or a composition containing said compound to the subject, and producing one or more images of the distribution of the compound within the subject.

[0121] In some embodiments, for example with respect to neurological inflammation, the disease rate and the degree of TSPO expression may be determined using imaging. This may be useful, for example, in predicting the outcomes of methods for treating neurological inflammation.

[0122] Here, the binding of the compound of formula (I) to a certain site in the target indicates the amount and degree of TSPO expression at that site.

[0123] The administration of the compound of formula (I) to the target subject is described in more detail below.

[0124] One or more images of the distribution of imaging agents within the subject may be produced using molecular imaging techniques, such as those that may use radiolabeling.

[0125] Using appropriate molecular imaging techniques, one or more images can be produced showing the distribution of the compound of formula (I) within all or part of a subject over a period of time after administration of the active substance. The amount or concentration of a detectable label in a tissue or region of the body indicates the amount of TSPO expression in that tissue or region. An increase in the concentration of the compound of formula (I) in a tissue or region of the body indicates that the cells in that tissue or region are experiencing increased TSPO expression relative to other tissues or regions in the body. Therefore, the imaging agent of the present invention may be useful in TSPO expression and may be suitable for use in detecting diseases associated with changes in TSPO expression levels.

[0126] The subjects may have a medical condition characterized by the presence of sites with increased or decreased TSPO expression, such as elevated TSPO expression, and one or more images show the distribution of the compound of the present invention in one or more sites.

[0127] The present invention relates to a method for determining the effectiveness of a therapy for a medical condition associated with an increase or decrease in TSPO expression, such as an increase in TSPO expression. A step of administering a compound of formula (I) or a composition containing said compound to a subject before, during, or after therapy, A step of producing one or more images of the distribution of a compound in one or more sites where TSPO expression is increased or decreased, such as an increase in TSPO expression in the target, and We also provide methods that include this.

[0128] During or after therapy, one or more images of the distribution of the compound of formula (I) at the target TSPO expression site may be produced.

[0129] A change such as a decrease or increase in the binding of the compound of the present invention at one or more sites of increase or decrease after the therapy, relative to before the therapy, indicates that the therapy is effective in altering TSPO expression in the subject.

[0130] If the disease is associated with elevated TSPO expression, therapy may be considered effective if there is a reduction in compound binding at one or more sites.

[0131] The compound of formula (I) may be used to evaluate its efficacy in early-stage clinical trials and, where applicable, in a clinical setting. Ineffective treatments may be discontinued early to allow for the selection of more effective drugs.

[0132] A method for determining the effectiveness of a treatment regimen for a subject, The step of providing the subject to the initial treatment regimen, A step to determine the amount and degree of binding of the compound of formula (I) to TSPO in the subject, Includes, A change in the amount or degree of binding in response to a regimen indicates that the regimen is effective in the subject. Methods will also be provided.

[0133] This delicious, The steps include changing the treatment regimen and subjecting the patient to the regimen change, A step of determining the amount or degree of binding of the compound of formula (I) to TSPO in the subject, The steps of changing the regimen and determining the bond are repeated until a change in the amount or degree of bonding of the compound is observed. It may also include, A change in the amount or degree of compound binding in response to a regimen indicates that the regimen is effective in the target.

[0134] In the above method, the subject may have neurological inflammation, and a decrease in the amount or degree of binding of the compound of formula (I) in response to the regimen indicates that the regimen is effective in the subject.

[0135] Subjects may have a condition characterized by increased TSPO expression levels, and a decrease in the amount or degree of binding of imaging agents to one or more disease sites in subjects in response to the regimen indicates that the regimen is effective in the subjects.

[0136] Dosage The method of the present invention may generally include the step of administering an effective amount of the compound of formula (I) to a subject so as to enable effective labeling of TSPO in a target area.

[0137] Those skilled in the art will understand that the appropriate dosage of the compound of formula (I) and the composition containing the compound of formula (I) may vary from patient to patient. Determining the optimal dosage will generally involve balancing the level of TSPO labeling against any risk or adverse side effects.

[0138] The selected dosage level will depend on a variety of factors, including but not limited to the route of administration, time of administration, compound excretion rate, duration of treatment, use of drugs, compounds and / or materials depending on the subject, and the patient's species, sex, age, weight, condition, overall health, and medical history. The amount and route of administration of the compound in formula (I) will ultimately be at the discretion of the physician, veterinarian, or clinician, but the dosage will generally be selected to achieve a local concentration that produces the desired effect at the site of action without causing substantially harmful or adverse side effects.

[0139] Throughout the entire time required for labeling and detection, administration can be achieved in a single dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Methods for determining the most effective means and dose of administration are well known to those skilled in the art and will vary depending on the formulation, target cells and / or organs, and the subject being treated. Single or multiple doses may be administered at dose levels and patterns selected by the treating physician, veterinarian, or clinician.

[0140] The appropriate dose of the compound of formula (I) is generally in the range of approximately 10 μg to 250 mg (more typically approximately 100 μg to 25 mg) per kilogram of body weight per day. If the compound of formula (I) is a salt or solvate, the amount administered is calculated based on the parent compound, and therefore the actual weight used increases proportionally.

[0141] If the compound of formula (I) is radioactively labeled, the compound may be used in the dosage form in the amounts specified below.

[0142] The compound may be used in an amount of at least 0.1, at least 0.5, at least 1, at least 5, at least 10, or at least 20 MBq.

[0143] The compound may be used in amounts of up to 50, up to 100, up to 200, up to 200, or up to 500 MBq.

[0144] The compound may be used in amounts within a range having lower and upper limits selected from the values ​​described above. For example, the compound may be used in amounts of 20 to 100 MBq.

[0145] The biological dose experienced by the subject may be as low as 20 mSv per dose, for example, per scan. Preferably, the biological dose is 15 mSv or less per dose, 10 mSv or less per dose, for example, 7 mSv or less per dose.

[0146] kit The present invention also provides (a) a composition comprising a compound of formula (I) or a compound as defined in any one of formulas (I), typically provided, for example, in a suitable container and / or suitable packaging, and (b) a kit comprising instructions for use, for example, written instructions on how to administer the compound or composition.

[0147] Route of administration Compounds of formula (I), or compositions containing compounds of formula (I), may be administered to subjects by any convenient route of administration, whether systemically / peripherally or topically (i.e., to the desired site of action).

[0148] The routes of administration include, but are not limited to, transpulmonary (e.g., via aerosol, or by inhalation or inhalation, for use via mouth or nose); parenteral administration by injection or infusion, for example, subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, subarachnoid, intrathecal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subepidermal, intraarticular, subarachnoid, and intrasternal; and subcutaneous or intramuscular administration by implantation of a depot or reservoir, for example.

[0149] subject The compound of formula (I) may be administered to a subject for the detection of TSPO, including the level and distribution of TSPO expression.

[0150] The subjects may be chordates, vertebrates, mammals, placental mammals, marsupials (e.g., kangaroos, wombats), rodents (e.g., guinea pigs, hamsters, rats, mice), murids (e.g., mice), lagomorphs (e.g., rabbits), birds (e.g., birds), canids (e.g., dogs), felines (e.g., cats), equids (e.g., horses), pigs (e.g., pigs), sheep (e.g., sheep), cats (e.g., cows), primates, true monkeys (e.g., monkeys or apes), monkeys (e.g., marmosets, baboons), apes (e.g., gorillas, chimpanzees, orangutans, gibbons), or humans. Furthermore, the subjects may be any of their developmental stages, for example, a fetus. The subjects may be cephalothodes, for example, aquatic cephalothodes such as fish (e.g., zebrafish).

[0151] It is also conceivable that the present invention may be applied to non-human animals. Non-human mammals may be rodents. Rodents include rats, mice, guinea pigs, chinchillas, and other small rodents of similar size used in laboratory research.

[0152] In one embodiment, the subject is a human being, such as an adult.

[0153] In one embodiment, the subject is a rodent such as a mouse.

[0154] Other priorities Any suitable combination of the embodiments described above is expressly disclosed herein, as if every possible combination were individually and explicitly enumerated.

[0155] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in consideration of this disclosure.

[0156] When used herein, “and / or” should be interpreted as a specific disclosure of each of the two designated features or components, with or without the other. For example, “A and / or B” should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) A and B, as if each were individually specified herein.

[0157] Unless the context indicates otherwise, the descriptions and definitions of features set forth above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0158] Herein, certain aspects and embodiments of the present invention are illustrated by example and with reference to the above-described figures.

[0159] Experiment and Results General experiment All reagents and starting materials were obtained from commercial sources and used as received. All dry solvents were purified using a PureSolv 500 MD solvent purification system. All reactions were carried out under argon unless otherwise noted. Brine is defined as a saturated solution of aqueous sodium chloride.

[0160] Flash column chromatography was performed using Fisher Matrix silica 60. Macherey-Nagel aluminum-backed plates pre-coated with silica gel 60 (UV254) were used for thin-layer chromatography and visualized using UV light.

[0161] 1 1H NMR and 13 The 13C NMR spectrum was obtained using a Bruker DPX 400 spectrometer or a Bruker 500 spectrometer, with tetramethylsilane (δ) used as a standard substance. H 0.00 and δ C 0.0) or residual chloroform (δ H 7.26 and δ C The chemical shift values ​​were recorded in ppm relative to 77.2). 1 H and 13 The C assignments are based on two-dimensional COSY and DEPT experiments, respectively.

[0162] The infrared spectrum was recorded using a JASCO FTIR 410 spectrometer.

[0163] Mass spectra were recorded using electron collision, chemical ionization, or fast atomic impact techniques. HRMS spectra were recorded using a dual-focusing magnetic analysis mass spectrometer.

[0164] The melting point was determined using a melting point analyzer manufactured by Gallenkamp.

[0165] The chiral HPLC method was calibrated with the corresponding racemic mixture.

[0166] 3-methyl-4-phenylquinoline-2-carboxylic acid was prepared as previously reported (Stevenson et al. Bioorg. Med. Chem. Lett. 20, 954-957 (2010)).

[0167] compound preparation The compound of formula (I) was prepared as shown in the following scheme. The compound is known as LW223.

[0168] Preparation of Scheme 1-LW223

[0169] [ka]

[0170] (R)-(N-sec-butyl)-3-methyl-4-phenylquinoline-2-carboxamide

[0171] [ka]

[0172] To a solution of 3-methyl-4-phenylquinoline-2-carboxylic acid (2.54 g, 9.65 mmol) in anhydrous N,N-dimethylformamide (250 mL), O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.49 g, 14.5 mmol) and N,N'-diisopropylethylamine (3.40 mL, 19.3 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 hours, then (R)-(-)-sec-butylamine (1.10 mL, 10.6 mmol) was added, and the mixture was then heated at 40°C for 4 hours. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (300 mL), and washed with water (3 × 200 mL) and brine (200 mL). The organic layer was dried over (MgSO4), filtered, and concentrated under vacuum to obtain a brown oil. Purification by flash column chromatography (petroleum ether / ethyl acetate, 4:1) yielded (R)-(N-sec-butyl)-3-methyl-4-phenylquinoline-2-carboxamide as a white solid (2.81 g, 91%). Melting point 152~154℃ (melting point literature value 157~158℃-(Cappelli et al. J. Med. Chem. 40, 2910-2921 (1997)) for reference); IR(KBr) 3287(NH), 2968(CH), 1641(CO), 1539, 1448, 1157, 761cm -1 [α] D 25 -26.7 (c 1.0, CHCl3); 1 H NMR(400MHz,CDCl3)δ1.03(3H,t,J=7.4Hz,CHCH2CH3), 1.33(3H,d,J=6.6Hz,CHCH 3), 1.61~1.75(2H,m,CH2CH3), 2.56(3H,s,3-CH3), 4.08~4.20(1H,m,CHCH3), 7.21 ~7.25(2H,m,ArH), 7.35(1H,d,J=8.3Hz,ArH), 7.40~7.55(4H,m,ArH), 7.65(1H,d dd,J=8.3,6.8,1.4Hz,ArH), 7.90(1H,d,J=8.3Hz,NH), 8.09(1H,d,J=8.3Hz,ArH); 13 C NMR (101MHz, CDCl3) δ10.6(CH3), 17.6(CH3), 20.5(CH3), 29.9(CH2), 46.8(CH), 126.1(CH), 127.5(CH), 127.9(CH), 128.6( C), 128.6(2×CH), 128.7(CH and びC), 129.3(2×CH), 129.5(CH), 137.3(C), 144.7(C), 149.5(C), 150.1(C), 166.2(C); MS(CI)m / z 319(M+H + ,100%), 220(19), 202(5), 148(6), 113(16), 85(77); HRMS(CI)C 21 H 23 N2O(M+H + ), the calculated value is 319.1810, and the measured value is 319.1809.

[0173] (R)-(N-sec-ブチル)-N-メチル-3-メチル-4-フェニルキノリン-2-カルボキサミド

[0174]

change

[0175] To a solution of (R)-(N-sec-butyl)-3-methyl-4-phenylquinoline-2-carboxamide (2.81 g, 8.82 mmol) in tetrahydrofuran (176 mL), sodium hydride (60% dispersed in mineral oil, 0.710 g, 17.6 mmol) was added. The mixture was stirred at room temperature for 0.5 hours, after which iodomethane (2.75 mL, 44.1 mmol) was added. The resulting solution was stirred at room temperature for 3 hours, and then quenched by the addition of water. The aqueous phase was extracted with diethyl ether (3 × 10 mL). The combined organic phases were washed with a 10% aqueous solution of sodium thiosulfate (10 mL) and brine (10 mL), dried to (Na₂SO₄), filtered, and concentrated under vacuum. Purification by flash column chromatography (petroleum ether / ethyl acetate, 3:1) yielded (R)-(N-sec-butyl)-N-methyl-3-methyl-4-phenylquinoline-2-carboxamide as a white solid (2.75 g, 94%).

[0176] The NMR spectrum showed a 1:1 mixture of rotational isomers. Signals were recorded for both rotational isomers. Melting point 114–117°C (see literature value 117–118°C - (see Cappelli et al. J. Med. Chem. 40, 2910–2921 (1997)); IR(KBr) 2969(CH), 1637(CO), 1466, 1072, 731 cm⁻¹ -1 ;[α] D 23 -6.3 (c 1.0, CHCl3); 1H NMR(400MHz,CDCl3)δ0.86(3H,t,J=7.3Hz,CH2CH3), 1.03(3H,t,J=7.3Hz,CH2CH3), 1.24(3H,d,J=6.6Hz,CHCH3), 1.28(3H,d,J=6.6Hz,CHCH3), 1.36~1.71(4H,m,2×CH2CH3), 2.21(3H,s,3-CH3), 2.23(3H,s,3-CH3), 2.73(3H,s,N CH3), 3.04(3H,s,NCH3), 3.42~3.53(1H,m,CHCH3), 4.84~4.94(1H,m,CHCH3), 7.25~7.31(4H,m,ArH), 7.38~7.44( 4H,m,ArH), 7.45~7.57(6H,m,ArH), 7.60~7.67(2H,m,ArH), 8.09(1H,d,J=8.3Hz,ArH), 8.11(1H,d,J=8.3Hz,ArH); 13 C NMR (101MHz, CDCl3) δ11.2(CH3), 11.3(CH3), 16.0(CH3), 16.4(CH3), 17.3(CH3), 18.6(CH3), 25.5(CH3), 26.5 (CH3), 27.2(CH2), 29.3(CH2), 49.6(CH), 55.8(CH), 124.6(C), 125.3(C), 125.9(CH), 126.0(CH), 126.7(2×CH ), 126.8(2×CH), 127.4(2×C), 128.0(2×CH), 128.6(2×CH), 128.7(2×CH), 129.2(4×CH), 129.4(2×CH), 136.7(C ), 136.8(C), 145.8(C), 146.1(C), 148.0(C), 148.1(C), 156.1(C), 156.6(C), 169.4(C), 169.7(C); MS(CI)m / z 333(M+H + ,100%), 291(48), 250(41), 220(14), 86(23); HRMS(CI)C 22 H 25 N2O(M+H + ), the calculated value is 333.1967, and the measured value is 333.1972.

[0177] (R)-3-ブロモメチル-(N-sec-ブチル)-N-メチル-4-フェニルキノリン-2-カルボキサミド

[0178] [ka]

[0179] To a stirred and degassed solution of (R)-(N-sec-butyl)-N-methyl-3-methyl-4-phenylquinoline-2-carboxamide (2.70 g, 8.12 mmol) in chloroform (300 mL), N-bromosuccinimide (2.17 g, 12.2 mmol) and dibenzoyl peroxide (0.20 g, 0.812 mmol) were added, and the solution was heated under reflux for 6 hours. Then, an additional portion of N-bromosuccinimide (1.00 g, 5.61 mmol) was added, and the solution was heated under reflux for a further 16 hours. The reaction mixture was cooled to room temperature, filtered, and the solvent was removed under vacuum. The crude residue was then diluted with ethyl acetate (100 mL) and washed with water (3 × 100 mL). The organic layer was dried with (MgSO4), filtered, and concentrated under vacuum. Purification by flash column chromatography using a graduated eluent of dichloromethane > dichloromethane / ethyl acetate (95:5) yielded (R)-3-bromomethyl-(N-sec-butyl)-N-methyl-4-phenylquinoline-2-carboxamide as an orange solid (2.76 g, 83%).

[0180] The NMR spectrum showed a 2:1 mixture of rotational isomers. Only the signals for the major rotational isomer are recorded. Melting point 160–164°C; IR(KBr)2970(CH), 1631(CO), 1484, 1397, 1046, 766 cm⁻¹ -1 ;[α] D 28 -9.0 (c 1.0, CHCl3); 1¹H NMR (400MHz, CDCl₃) δ 1.09 (3H,t, J=7.4Hz, CH₂CH₃), 1.32 (3H,d, J=6.8Hz, CHCH₃), 1.51~1.80 (2H,m, CH₂CH₃), 2.86 (3H,s, NCH₃), 4.60 (1H,d, J=10.2Hz, 3-CHH), 4.67 (1H,d, J=10.2Hz, 3-CHH), 4.87 (1H, hexapex, J=6.8Hz, CHCH₃), 7.37~7.48 (4H,m, ArH), 7.51~7.59 (3H,m, ArH), 7.70 (1H,ddd, J=8.3, 6.7, 1.5Hz, ArH), 8.10 (1H,dd, J 8.8, 8.3 Hz, ArH); 13 C NMR (101MHz, CDCl3) δ11.1(CH3), 17.1(CH3), 26.6(CH2), 27.7(CH2), 30.5(CH3), 50.1(CH), 126.3(C), 126.7(2×CH), 127.4(CH), 128 .6(2×CH), 128.7(CH), 129.0(CH), 129.1(CH), 129.5(CH), 130.1(C), 134.9(C), 146.4(C), 149.3(C), 156.0(C), 168.4(C); MS(EI)m / z 410(M + ,5%), 298(15), 296(14), 217(57), 189(28), 151(10), 86(100); HRMS(EI)C 22 H 23 79 BrN2O(M + ), the calculated value is 410.0994, and the measured value is 410.0992.

[0181] (R)-(N-sec-ブチル)-3-クロロメチル-N-メチル-4-フェニルキノリン-2-カルボキサミド

[0182]

change

[0183] To a solution of (R)-3-bromomethyl-(N-sec-butyl)-N-methyl-4-phenylquinoline-2-carboxamide (0.500 g, 1.22 mmol) in dry tetrahydrofuran (10 mL), lithium chloride (0.160 g, 3.66 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with water (30 mL) and extracted in ethyl acetate (3 × 30 mL). The organic layers were combined, washed with brine (90 mL), dried, filtered, and concentrated under vacuum. The product was purified by flash column chromatography (dichloromethane / ethyl acetate, 95:5) to yield (R)-3-chloromethyl-(N-sec-butyl)-N-methyl-4-phenylquinoline-2-carboxamide as a white solid (0.327 g, 73%).

[0184] The NMR spectrum showed a 1.5:1 mixture of rotational isomers. Only the signals for the major rotational isomer are recorded. Melting point 140-142°C; IR (undiluted) 2970(CH), 1620(CO), 1481, 1404, 1219, 748 cm⁻¹ -1 ;[α] D 24 -11.6 (c 1.0, CHCl3); 1 H NMR(400MHz,CDCl3)δ1.08(3H,t,J=7.4Hz,CH2CH3), 1.30(3H,d,J=6.8Hz,CHCH3), 1.49~1.79(2H,m,CH2CH3), 2.84(3H,s,NCH3), 4.67(1H,d ,J=10.6Hz,3-CHH), 4.72(1H,d,J=10.6Hz,3-CHH), 4.82~4.92(1H,m,CHCH3), 7.36~7.61(7H,m,ArH), 7.69~7.75(1H,m,ArH), 8.11(1H,dd,J 9.0,8.4Hz,ArH); 13C NMR (101 MHz, CDCl3) δ 11.1 (CH3), 17.1 (CH3), 26.6 (CH2), 30.4 (CH3), 40.4 (CH2), 50.1 (CH), 125.8 (C), 126.8 (CH), 127.2 (C), 127.4 (CH), 128.5 (2×CH), 128.7 (CH), 129.4 (CH), 129.6 (2×CH), 130.1 (CH), 134.9 (C), 146.6 (C), 149.5 (C), 156.1 (C), 168.5 (C); MS (ESI) m / z 389 (M+Na + , 100%); HRMS (ESI) C 22 H 23 35 ClN2NaO (M+Na + ) calculated value, 389.1391; found value 389.1381.

[0185] LW223-(R)-(N-sec-butyl)-3-fluoromethyl-N-methyl-4-phenylquinoline-2-carboxamide

[0186]

Chemical formula

[0187] To a solution of 18-crown-6 (0.032 g, 0.12 mmol) in acetonitrile (2.5 mL) was added potassium fluoride (0.036 g, 0.61 mmol), and the resulting suspension was stirred at room temperature for 0.5 hours. Then, a solution of (R)-3-bromomethyl-(N-sec-butyl)-N-methyl-4-phenylquinoline-2-carboxamide (0.050 g, 0.12 mmol) in acetonitrile:dichloromethane (2:1, 9.0 mL) was added dropwise, and the reaction mixture was heated under reflux for 72 hours. Upon completion, the reaction mixture was cooled to ambient temperature, and water (20 mL) was added. The solution was extracted with dichloromethane (3 × 20 mL), dried (MgSO4), filtered, and concentrated in vacuo. Purification by flash column chromatography (petroleum ether / ethyl acetate 7:3) afforded (R)-N-(sec-butyl)-3-(fluoromethyl)-N-methyl-4-phenylquinoline-2-carboxamide as a white solid (0.023 g, 53%).

[0188] The NMR spectrum showed a 3:1 mixture of rotamers. Only the signals for the major rotamer are reported. Melting point 146~148°C; IR (neat) 2972 (CH), 1628 (CO), 1559, 1485, 1398, 1049, 970 cm -1 ; [α] D 30 -12.6 (c 0.5, CHCl3); 1 1H NMR (400 MHz, CDCl3) δ 1.05 (3H, t, J=7.4 Hz, CH2CH3), 1.29 (3H, d, J=6.8 Hz, CHCH3), 1.41~1.78 (2H, m, CH2CH3), 2.77 (3H, s, NCH3), 4.84~4.95 (1H, m, NCH), 5.31 (1H, dd, J 20.8, 10.8 Hz, 3-CHH), 5.44 (1H, dd, J 20.8, 10.8 Hz, 3-CHH), 7.31~7.40 (2H, m, ArH), 7.43~7.58 (5H, m, ArH), 7.74 (1H, t, J 7.6 Hz, ArH), 8.17 (1H, d, J 8.4 Hz, ArH); 13C NMR (101 MHz, CDCl3) δ 10.9 (CH3), 17.4 (CH3), 26.5 (CH3), 29.9 (CH2), 50.0 (CH), 79.2 (CH2, 1 J C-F =162.8 Hz), 123.0 (C, 2 J C-F =15.1 Hz), 127.0 (CH), 127.1 (C, 4 J C-F =2.3 Hz), 127.4 (CH, 5 J C-F= 1.2 Hz), 128.5 (2×CH), 128.7 (CH), 129.6 (2×CH), 129.7 (CH), 130.4 (CH), 134.8 (C, 4 J C-F =1.5 Hz), 147.4 (C, 3 J C-F =2.5 Hz), 150.8 (C, 3 J C-F =4.7 Hz), 156.6 (C, 5 J C-F =2.1 Hz), 168.8 (C); MS (ESI) m / z 373 (M+Na + , 100%); HRMS (ESI) C 22 H 23 FN2NaO (M+Na + ) calculated value, 373.1687, found 373.1670.

[0189] The enantiomeric excess was determined by HPLC analysis using a Chiralcel AD-H column (hexane: i iPrOH 97.5:2.5, flow rate 1.0 mL / min), t major =30.68 and 32.22 min, t minor =27.15 and 38.38 min; er=99.5:0.5.

[0190] The compound of formula (II) was prepared as shown in the following scheme. The compound is 18 known as F-LW223.

[0191] Scheme 2- 18 Preparation of F-LW223

[0192] [ka]

[0193] 18 F-LW223-(R)-(N-sec-butyl)-3-[ 18 -Fluoromethyl-N-methyl-4-phenylquinoline-2-carboxamide (R)-(N-sec-butyl)-3-chloromethyl-N-methyl-4-phenylquinoline-2-carboxamide, 18 F-fluoride ([ 18 F)KF)) was reacted with potassium carbonate and Kryptofix 222 in a commercially available synthesizer, GE TRACERlab® FX-FN, at 100°C for 10 minutes. The radiolabeled product was purified by semi-preparative high-performance liquid chromatography (HPLC) under the following conditions: C18 Synergi Hydro-RP 80Å, 150×10mm, 4μm column (Phenomenex, UK), using acetonitrile / water (70:30 v / v) at a flow rate of 3 mL / min.

[0194] The final product was formulated in a physiological solution containing 10% ethanol in physiological saline. 18 F-LW223, after a total synthesis time of 55 minutes, is 35% (15±5 GBq) 18 Starting with F-fluoride, the average undamped yield was obtained (n=20). At the end of the synthesis, 18 The identification of F-LW223, its radiochemical purity (over 99%), and specific activity (410-810 GBq / μmol; 11-22 Ci / μmol) were determined by HLPC analysis.

[0195] Biological Tests In vitro competition and saturation binding assays using human tissue. All studies using human tissue were conducted according to the East of Scotland Research Ethics Service (Edinburgh Brain Bank, 11 / ES / 0022). Samples of 51 brains (78% male, age 53.9±9.5) and 29 hearts (83% female, age 48.2±13.2) were obtained and screened for rs6971 genetic polymorphisms, and grouped into high-affinity binders (HAB), mixed-affinity binders (MAB), or low-affinity binders (LAB) as previously described (Owen et al. J. Cereb. Blood Flow Metab. 32, 1-5 (2012)). Only male samples were used in this study to exclude sex-dependent differences in TSPO binding (Fairweather et al. J. Cardiovasc. Transl. Res. 7, 192-202 (2014)). The tissue samples were homogenized in 10×w / v buffer (50 mM Tris-Base, pH 7.4, 4°C) and then centrifuged (32,000 g, 10 min, 4°C). The tissue pellets were then resuspended in 10×w / v buffer, centrifuged again, and then resuspended in buffer (2 mL). The samples were then evaluated for protein concentration using a Bio-Rad protein assay (Bio-Rad, USA), parceled, and stored at -80°C until use.

[0196] The competitive binding assay was performed as previously reported (Blair et al. Med. Chem. Commun. 4, 1461-1466 (2013)). Briefly, 250 μg of protein / mL of each sample was prepared in buffer, and then 200 μL of this solution was mixed with 100 μL of 1 nM 3H-PK11195 (PerkinElmer, USA) was added to 100 μL of our test ligands PK11195 (Sigma-Aldrich, USA), PBR28 (ABX, Germany), AB5186, or LW223 at 14 different concentrations (ranging from 0.001 to 3,000 nM) over 90 minutes at 4°C. An 8 μM concentration was used to determine the nonspecific binding of each ligand. After terminating binding with the addition of 2 mL of ice-cold buffer, the samples were immediately filtered using a Brandel sampling device (Brandel, USA) through a Whatman GF / B filter (Whatman, UK) pre-treated with 0.3% polyethyleneimine (Sigma-Aldrich, USA). Next, the filter paper was removed and the sample was placed in 2.5 mL of Optiphase HiSafe 3 (Perkin Elmer, USA), and counted after 48 hours using Hidex 300 SL (Hidex, Finland). To determine nonspecific binding, six concentrations of PK11195 were used alongside 10 μM PK11195. 3 Except for using H-PK11195 (1.6~200 nM), a saturation assay was performed using a similar protocol to determine the K of PK11195. d This was determined. All binding assays were performed in triplicate.

[0197] All binding affinity curves were fitted using GraphPad Prism version 6 (GraphPad Software, USA). A least-squares algorithm was used to compare one-site and two-site fits, and an F-test was used to compare model selection. The null hypothesis (one-site fit being more appropriate) was rejected if p < 0.05. The appropriateness of one-site or two-site fitting was determined using the mean %SB normalized to minimum inhibition for each group (HAB, MAB, or LAB), and then the LAB:HAB ratio was calculated. Affinity values ​​(K) were determined by fitting individual tissue samples based on the mean group fit. i ) was calculated. PK11195 K at 13.95 nM d The values ​​used were based on the saturated coupling curve results.

[0198] findings Using competitive binding assays, the affinity (K) of established TSPO ligands PK11195 and PBR28, as well as our own ligands AB5186 and LW223 (as shown in Figure 1), was determined. i The K of PK11195 was calculated to assess their susceptibility to genetic polymorphisms. A saturation assay was performed using the inventors' experimental conditions to assess the K of PK11195. d The value was determined to be 13.95 nM (see Figure 7).

[0199] In competitive binding assays using human brains, PK11195 had a LAB:HAB affinity ratio of 1 (Figure 2(a)), unlike PBR28 (Figure 2(b)), which was unaffected by genetic polymorphism and had a ratio of 49. Ligand AB5186 was affected by genetic polymorphism, with a ratio of 9 (Figure 2(b)), unlike LW223, which was unaffected and had a ratio of 1. Individually calculated affinity values ​​for all brain samples did not demonstrate significant differences between HAB and LAB in binding studies of PK111195 and LW223 (Figures 2(e) and (h)H, respectively). The mean affinity of LW223 was 0.6 nM, which was twice as high as that of PK11195. Comparison of individually calculated affinity values ​​for HAB and LAB for PBR28 and AB5186 revealed significant differences between groups (Figures 2(f) and (g)). The MAB group in PBR28 and AB5186 fit with two-site fitting, while all other experiments fit better with one-site fitting.

[0200] Similar to the brain, PK11195 was unaffected by genetic polymorphisms in the heart, with a LAB:HAB ratio of 1 (Figure 3(a)). PBR28 and AB5186 were similarly affected in the heart versus brain, with ratios of 48 and 7, respectively (Figures 3(b) and (c)). LW223 was unaffected by polymorphisms in the heart, with a ratio of 1 (Figure 3(d)). Individually calculated affinity values ​​for all cardiac samples did not demonstrate significant differences between HAB and LAB in the binding studies of PK111195 and LW223 (Figures 3(e) and (h)). In the heart, the mean affinity of LW223 was 1.7 nM, the same as PK11195. In addition, in the AB5186 experiment, only the MAB group was a good fit for two-site fitting.

[0201] Up until now, 11 In vivo imaging evidence suggested that C-PK11195 binding is sensitive to polymorphisms in peripheral organs such as the heart and lungs, but not in the brain (Kreisl et al. Neuroimage 49, 2924-2932 (2010)). Sensitivity of PK11195 to polymorphisms in the brain is also suggested to be limited by the low signal-to-noise ratio, the inability to image due to low brain uptake compared to the heart and lungs, and the unavoidable limitation of the sample size due to the low incidence of LAB (Kobayashi et al. J. Cereb. Blood Flow Metab. 38, 393-403 (2018)). However, brain uptake levels are not a major issue in susceptibility ex vivo competitive binding assays. In this study, differences in affinity for LW223 between the brain and heart (0.6 and 1.2 nM, respectively) were demonstrated, which differed from PK11195, which had the same affinity (1.2 nM). Improved LW223 affinity in the brain would offer advantages when targeting neurological inflammation.

[0202] Animal and surgical procedures All experiments were conducted in accordance with the Animal Ethics Committee of the University of Edinburgh and authorized by the Home Office under the Animals (Scientific Procedures) Act 1986. Twenty-eight mature male Sprague-Dolly rats (357.1±8.1g and 10.2±0.4 weeks) and two C57bl / 6 rats (25.3±3.7g and 10.1±0.0 weeks) were used in this study, and two C57bl / 6 rats (26.0±5.3g and 13.5±5.7 weeks) were used in additional studies. The animals were housed under standard 12-hour light:12-hour dark conditions with free access to food and water. On the day of the experiment, anesthesia was induced and maintained with 1.5–2.5% isoflurane (50 / 50 oxygen / nitrous oxide, 1 L / min). For imaging experiments, an intravenous (IV) line for injecting the radioactive tracer was established in the femoral vein or tail vein, and as previously described (Warnock et al. EJNMMI Res. 1, 1-11 (2011)), a cannula was inserted into the femoral artery to enable automated blood sample collection.

[0203] In a separate experimental setup (radioactive metabolite study), a cannula was inserted into the femoral artery for blood collection, and a radioactive tracer was administered intravenously via the tail vein. Surgical cannulation of the femoral vein and artery was performed as follows: a polyethylene catheter (PE50) filled with heparinized saline (20 IU / mL) was inserted into the left femoral artery or vein with the help of a stereomicroscope and securely fixed with ligation (6-0 silk thread). The catheter was held in place with surgical adhesive. Body temperature was maintained by a heated scanner bed or heated mat and monitored with a rectal thermometer. Vital signs, including heart rate and respiratory rate, were continuously monitored throughout the experiment.

[0204] PET research Research design: 13 PET scans, 18 The procedure was performed using F-LW223 (average injected radioactivity of 19.55 ± 2.1 MBq). This was performed in rats via tail vein or femoral vein shunts. 18Scans were acquired after intravenous bolus injection of F-LW223 (invasive kinetic modeling experiment using a β-probe automatic blood sampler). Test-retest scans were obtained after intravenous bolus of the radiotracer, with a 2-week interval between test and retest imaging sessions in 3 rats. Blocking studies were performed in rats 18 by intravenous administration of PK11195 (1 mg / kg) 30 minutes before receiving F-LW223 intravenously. These scans were compared to baseline scans where PK11195 was not used. For displacement studies, rats were 18 administered F-LW223 intravenously and scanned continuously for 120 minutes. After 60 minutes, a single dose of PK11195 (1 mg / kg) was administered. Male and female mice were initially used to derive 18 dosimetry estimates for F-LW223 with a 4-hour dynamic scan, followed by additional mice used in further studies (in studies using an average injected radioactivity of 10.83 ± 5.6 MBq).

[0205] Arterial input function using a β-probe automatic blood sampler A commercially available β-probe system (Twilite2, Swisstrace, Switzerland) was used for the measurement of blood radioactivity, as previously described (Warnock et al. EJNMMI Res. 1, 1-11 (2011)). This system enables whole-blood arterial input function measurement with 1-second time resolution, without blood loss via a surgically induced arteriovenous shunt. Using data acquired in a separate study (radiometabolite experiment), the whole-blood arterial input function measured by the automatic blood sampler was corrected for plasma-to-whole blood ratio and for in vivo metabolism.

[0206] Image acquisition and reconstruction All PET data were acquired using a preclinical PET / CT small animal scanner (nanoPET / CT, Mediso, Hungary). CT scans (semicircular full trajectory, maximum field of view, 480 projection, 50kVp, 300ms, and 1:4 binning) were acquired for attenuation correction. Immediately after radiotracer administration, a 120-minute radioscan was obtained using 3D 1:5 mode and rebinned as follows: 18×10 sec, 2×30 sec, 1×60 sec, 2×2 minutes, 10×5 minutes, and 6×10 minutes. PET images were reconstructed using the Mediso iterative Tera-Tomo 3D reconstruction algorithm with the following settings: 4 iterations, 6 subsets, full detector model, low regularization, spike filter on, voxel size 0.4 mm, and 400-600 keV energy window. PET data were corrected for randomness, scattering, and attenuation.

[0207] Image processing and data analysis The reconstructed scans were imported into PMOD 3.8 software (PMOD Technologies, Switzerland). Volumes of interest (VOI) were manually plotted around the desired organs. Time-activity curves (TAC) were generated, and the standardized uptake value (SUV), calculated as the concentration at the VOI, was divided by the injection dose and then by the animal's weight. Dynamic modeling was performed using compartment analysis (1-tissue (1T) and 2-tissue (2T) models) and graphical analysis (Logan plot and Ichise multivariate analysis) to determine the volume of distribution (V) in different tissues. TThe following were estimated (Innis et al. J. Cereb. Blood Flow Metab. 27, 1533-1539 (2007), Logan Nucl. Med. Biol. 27, 661-670 (2000), and Ichise et al. J. Cereb. Blood Flow Metab. 22, 1271-1281 (2002)). The Akaike Information Criterion (AIC) and Model Selection Criterion (MSC) were used to determine the fit performance of the models, and the preferred models had the lowest AIC and the highest MSC. The selected identifiability criterion was V T The percentage standard error (%SE) was used for estimation. Dynamic modeling was also performed using graphical analysis to estimate the distribution volume ratio (DVR) in tissue regions and organs, and the reference region VOI was the blood pool VOI in the left ventricle (Logan et al. J. Cereb. Blood Flow Metab. 16, 834-840 (1996)).

[0208] DVR test-retest reproducibility was calculated as the absolute value of the mean measurement divided by the standard deviation: ABS(mean test / retest) / SD(test / retest).

[0209] Dosimetry The reconstructed whole-body PET scans were imported into PMOD 3.8 software (PMOD Technologies, Switzerland), and VOIs were plotted around organs showing higher radioactivity concentrations than the background, i.e., around the source organs. The following organs were identified as source organs: brain, heart, lungs, gallbladder, liver, intestines, adrenal glands, kidneys, and bladder. Whole-body VOIs were plotted around the animal body and used as whole-body activity minus source organ activity to quantify residual whole-body activity. At each time point, the measured activity of the source organs was expressed as a percentage of the injected dose (%ID).

[0210] The residence time τ, defined as the ratio of accumulated activity (A(bar)) to injected activity (A0) in the target organ; τ = A(bar) / A0, was calculated as the area under the tissue time-activity curve normalized to %ID from time zero to infinity. Trapezoidal rule was used to estimate τ and after the last measured time point, assuming that the radioactive tracer underwent only physical decay without biological elimination from the source organ. The calculated τ was normalized as a percentage of total body weight for the difference in organ weight between mouse and human (based on known data from Khanuja et al. Genet. Commun. 92, 7729-7733 (1995), Bielohuby et al. Am. J. Physiol. Metab. 293, E139-E146 (2007)), (Stabin et al. J. Nucl. Med. 47, 655-9 (2006), Hindorf et al. J. Nucl. Med. 45, 1960-5 (2004), and Hui et al. Cancer 73, 951-957 (1994)), and entered into the OLINDA / EXM 1.0 software, which was used to estimate organ doses and effective doses according to the male or female models implemented in OLINDA / EXM 1.0.

[0211] findings In mice and rats, after intravenous bolus injection, 18 F-LW223 was rapidly distributed to TSPO-expressing tissues, including the brain, heart, lungs, and adrenal glands (see Figures 4(a) and (b)), and eliminated via both urinary and hepatic biliary excretion pathways. After peak uptake, elimination of the radioactive tracer was faster in the brain and lungs compared to the heart (see Figures 4(c) and (d)).

[0212] In rat arterial blood 18The radiometabolism of F-LW223 was slow, with approximately 70% being parental at 120 minutes post-injection (Figure 4(e)). Less than 10% of radioactive metabolites were measured in the brain, heart, and lungs at 60 and 120 minutes post-administration. The measured parental-free fraction in plasma was 38.5 ± 7.0% (mean ± SEM, n=XX).

[0213] In vivo 18 F-LW223 dynamics are reversible, and can be analyzed using a 2-tissue compartment model, as well as graphical Logan plots (t * (30 minutes) and Ichise multivariate analysis (t * V was better fitted / described by (=30 minutes) (see Figure 4(f)). The graphical method was preferred over the compartment model (lower % standard error, lower Akaike selection criterion, and higher model selection criterion). V measured in the brain, heart, and lungs. T The values ​​were 1.46±0.16, 9.48±0.03, and 5.13±0.29, respectively (Logan modeling, mean ± SEM, n=3). The volume-of-distribution ratio (DVR) related to blood pooling in the brain, heart, and lungs was 0.53±0.06, 2.96±0.14, and 1.84±0.13, respectively (mean ± SEM, n=3). Test-retest analysis showed good agreement of measurements, as well as between-subject variability of 16% in the brain, 2% in the heart, and 15% in the lungs, when DVR was used as the outcome measure.

[0214] 18 Administration of PK11195 prior to F-LW223 injection demonstrated in vivo target binding (see Figure 5). SUV values ​​measured after blocking showed a 64–81% reduction compared to baseline scans. V measured after blocking in the brain, heart, and lungs. TThe values ​​were 1.29±0.12, 6.19±0.38, and 2.27±0.07, respectively (mean ± SEM, n=3). This corresponds to a 12%, 35%, and 56% reduction in radiotracer binding for the brain, heart, and lungs, respectively. The same dose of PK11195 resulted in only 29%, 52%, and 40% reductions in the brain, heart, and lungs, respectively, from the target site at equilibrium. 18 F-LW223 could be replaced (see Figure 8). Pharmacokinetic / pharmacodynamic studies using PK11195 in rats at 1 hour showed that the exposure level in the brain was 11.01 ± 1.99 ng / mL, compared to 29.31 ± 3.93 ng / mL and 38.86 ± 2.24 ng / mL (mean ± SEM, n=3) in the heart and lungs, respectively. This is consistent with blocking and replacement measured in PET experiments, and the difference in target binding % in the brain and peripheral organs was consistent with the K values ​​measured in vitro for LW223 and PK11195. i This matches.

[0215] The whole-body effective time activity curve showed that the highest peak injectable dose % occurred in the colon, followed by the liver, lungs, kidneys, heart, brain, gallbladder, bladder, and adrenal glands (see Figure 9). Dosemetric estimations using normalized tau values ​​and adult human phantoms for males and females indicated that the determinant organ was the adrenal gland. The whole-body effective dose was estimated to be 15.3 μSv / MBq and 18.4 μSv / MBq for male and female phantoms, respectively (Table 1).

[0216] Further work with mice revealed that the determinant organ was the lower intestinal wall. The whole-body effective dose was estimated to be 20.5 μSv / MBq and 23.7 μSv / MBq for male and female phantoms, respectively (Table 2).

[0217] [Table 1]

[0218] [Table 2]

[0219] Processing and analysis of radioactive metabolites and arterial blood Arterial blood samples were collected at 2, 5, 10, 20, 30, 60, and 120 minutes after administration of the radioactive tracer (69.8 ± 9.2 MBq, n=17 rats). All blood samples were 1 mL each, and population curves were generated by manually collecting samples from different animals to adhere to the total blood volume limit for terminal arterial blood collection in rats. After blood collection, all samples were kept on ice until analysis. Radioactivity in whole blood and plasma was evaluated using a well-type gamma counter with a 400–1400 keV window (Perkin Elmer Wizzard 2, USA). Plasma samples (400 μL) were treated with acetonitrile denaturation and analyzed by HPLC (Ultimate2000, ThermoFisher, UK) on a Luna C18(2) column (Luna C18(2), 10 × 250 mm, 10 μm, Phenomenex, UK) using an acetonitrile / water 70 / 30 mobile phase at a flow rate of 4 mL / min to estimate the parent fraction. Plasma protein-bound free fraction (f p The results were determined using an ultrafiltration unit (Centrifree® 30K, Millipore, UK).

[0220] 18 F-LW223 Human Autoradiography After dewaxing and rehydrating 1-2 levels of paraffin-embedded human deep brain stroke tissue and affected coronary artery tissue sections, and incubating in buffer (50 mM Tris-Base, pH 7.4) for 30 minutes, a 2 MBq / mL solution was administered in the presence or absence of either PK11195 (30 μM in stroke tissue and 10 μM in coronary artery tissue) or LW223 (10 μM, nonspecific binding group). 18The slides were incubated with F-LW223 (fully bound group). After washing the slides twice with buffer, they were air-dried and exposed to a Fujifilm BAS-IP MS 2040 fluorescence screen (Fujifilm, Japan). The fluorescence screen was imaged using a Fujifilm FLA5100 imaging plate reader (Fujifilm, Japan). The desired global region was plotted around the entire tissue section, and %SB was calculated. Localized sampling analysis was used within the same tissue at the highest and lowest uptake areas to calculate the target:non-target ratio.

[0221] findings Using autoradiography, 18 The binding of F-LW223 to ex vivo human affected tissue was imaged. Histological staining of the tissue revealed areas of hemorrhage containing migratory inflammatory cells in deep brain stroke specimens (see Figure 6(a)). In affected coronary vessels from patients with sudden cardiac death, there were distinct areas of pathological remodeling (e.g., neointimal hyperplasia) containing infiltrating inflammatory cells (Figure 6(b)). 18 F-LW223 binding was highest in the hemorrhagic region, and some uptake was highest in the distant region (Figure 6(c)). In affected coronary vessels, 18 F-LW223 binding was the best in area pathological remodeling (Figure 6(d)). 18 F-LW223 binding could not be adequately blocked using PK11195 in deep brain tissue samples (Figure 6(e)), while specific binding % (%SB) of 75.3±3.2% was achieved in coronary arteries (Figure 6(f)). Blocking with LW223 in stroke samples achieved 42.5±7.0%SB (Figure 6(g)) and 88.1±3.4%SB in coronary arteries (Figure 6(h)). Importantly, a high target:non-target ratio was achieved in both stroke (7.5) and affected coronary vein (4.5) tissue (Figures 6(i) and (j)). Compared to the brain of a healthy human, there were no regions of high uptake except for some nonspecific binding in the white matter (see Figure 10).

[0222] General statistical analysis Graphpad Prism version 6 (GraphPad Software Inc., USA) was used for all fitting, statistical analysis, and graph generation. Outliers within the experimental triplicate in competitive binding and saturation assays were removed using Grubbs tests with an alpha of 0.2. Unpaired and paired t-tests were used in this study for comparisons between two groups, as indicated in the figure captions; p<0.05 was considered statistically significant. All error bars represent the standard error mean (SEM) of the mean unless otherwise indicated in the figure and table captions.

[0223] findings Recently, Thr 148 A study detailing the crystalline structures of TSPO with and without polymorphism has been published for the first time (Li et al.). In this study, the authors elegantly demonstrated that PK11195 binding stabilizes the binding pocket, while polymorphism has a significant impact on the cholesterol binding site. This evidence suggests that ligands involved with the cholesterol site may exhibit greater sensitivity to polymorphism. Through various binding studies, it is known that the TSPO binding site has three main binding pockets: a benzodiazepine site, an isoquinoline site, and a cholesterol site (Li et al., Lin et al. Genomics 18, 643-50 (1993), and Luus et al. J. Label. Compd. Radiopharm. 53, 501-510 (2010)). Since LW223 is insensitive to polymorphisms in the brain and heart and is structurally similar to PK11195, we hypothesize that LW223 targets the isoquinoline site rather than cholesterol. However, the differences in overall affinity values ​​for LW223 in the brain and heart suggest organ-specific conformational changes in the TSPO isoquinoline binding pocket across different sites.

[0224] Not only is the first fluorinated TSPO ligand insensitive to the rs6971 polymorphism, 18 F-LW223 is also unique in that it exists as a rotational isomer. This is evident from the presence of two peaks in HPLC, which was confirmed by chiral HPLC. This rare characteristic is 18 This may be behind the excellent kinetic and metabolic properties of F-LW223, which exhibits a similar profile to infusion administration despite being administered by intravenous bolus. 18 Another unique aspect of F-LW223 is that it has a free fraction of 1-6% in humans. 11 Compared to C-PK11195, it had a higher plasma free fraction of 38.5% (Owen et al., Endres et al. J. Nucl. Chem. 50, 1276-82 (2009)). Clearly, further investigation is needed to determine this unique characteristic and its contribution to the favorable kinetic and metabolic profile. These features are 18 This does not preclude the transfer of F-LW223 to the clinic, but other criteria must still be met. Firstly, a toxicology package has not yet been prepared to determine the safety of this radioactive tracer. 18 It should be noted that no adverse effects were observed after in vivo administration of F-LW223. Furthermore, secondly, the dose estimations presented in this study do not suggest any radiological safety issues.

[0225] The limitations of this study are similar to those faced by most studies developing novel TSPO radioactive tracers. Firstly, the incidence of LAB in this study is around 14%, which is higher than the incidence reported in other studies (Fujita et al. EJNMMI Res. 7 (2017), doi:10.1186 / s13550-017-0334-8), but this limits the samples available for testing. However, this study found that the LAB:HAB ratios for PK11195 and PBR28 in the brain were similar to those previously reported (Owen et al. J. Cereb. Blood Flow Metab. 32, 1-5 (2012), Owen et al. J. Nucl. Med. 52, 24-32 (2011), Owen et al. J. Cereb. Blood Flow Metab. 30, 1608-18 (2010)), and that PBR28 and AB5186 reached statistically significant differences between LAB and HAB. Some controversy has arisen in areas where in vitro binding results have not been reflected in in vivo studies, such as TSPO radioactive tracers. 11 C-ER176 showed a LAB:HAB ratio of 1.3 in vitro (Zanotti-Fregonara et al. ACS Chem. Neurosci. (2014), doi:10.1021 / cn500138n), but later in vivo studies showed that this ratio is around 3 (Ikawa et al. J. Nucl. Med. (2016), doi:10.2967 / jnumed.116.178996). However, even with this slight influence of polymorphism, 11 It has been reported that C-ER176 still possesses the ability to image LABs, achieving results not possible with other ligands. The same phenomenon 18If this also applies to F-LW223, this radiotrace still benefits from fluorination, thereby leading to broader clinical use. One of the final limitations of this study was that it was conducted in non-human primates and rodents, which typically exhibit faster kinetics and metabolism than humans. However, even with this caveat, in this study... 18 The kinetic and metabolic profiles of F-LW223 suggest that these aspects will not be problematic during transitions.

[0226] Given the roles of inflammatory phagocytic cells such as macrophages and microglia in a wide range of pathologies, the development of a successful TSPO imaging approach holds great potential. In this study, the inventors of this invention... 18 Ex vivo uptake of F-LW223 is evident in the area of ​​pathological inflammation-driven events. To date, 11 Despite its limitations, C-PK11195 has been successfully used and demonstrated its ability to increase inflammation in neurological disorders. For example, 11 Increased C-PK11195 uptake has been demonstrated in patients with Alzheimer's disease and progressive supranuclear palsy compared to controls (Calsolaro et al. Alzheimer's Dement. 11, P792 (2015)). The areas of increased uptake were associated with established neuropathological distribution patterns in both diseases and were negatively correlated with episodic memory in Alzheimer's disease and positively correlated with disease severity in progressive supranuclear palsy. 11 C-PK11195 has also been successfully used to investigate plaque inflammation in atherosclerosis. In ex vivo studies, 11 C-PK11195 binding was found to correlate with macrophage-rich regions (Bird et al. Atherosclerosis 210, 388-391 (2010)). In in vivo clinical studies, within plaques... 11C-PK11195 uptake has recently been shown to differentiate between symptomatic and asymptomatic lesions (Gaemperli et al. Eur. Heart J. 33, 1902-1910 (2012)). These proactive clinical studies, in addition to the new role of TSPO as a therapeutic target (Schalle et al. J. Pharmacol. Exp. Ther. 333, 696-706 (2010) and Paradis et al. Cardiovasc. Res. 98, 420-427 (2013)), highlight the potential that can be derived from successful TSPO radiotracers as research tools, diagnostic agents, and therapeutic companion imaging agents.

[0227] In conclusion, regarding the rs6971 genetic polymorphism 18 The insensitivity of F-LW223, the longer half-life of the radiolabel, and favorable dynamics make this novel TSPO radiotrace a promising breakthrough in the field of TSPO imaging, worthy of further deployment in clinical settings.

[0228] Additional experiments and results In a rat model of myocardial infarction 18 F-LW223 combination Rats were subjected to permanent ligation of the left anterior descending coronary artery to induce post-myocardial infarction, and 7 days later, 18 Imaging was performed using F-LW223 PET / CT (see Figure 11). Naive blood was used as a healthy control for comparison. Quantification of standard uptake value (SUV) related to the blood pool (measured in the left ventricle) was performed across the heart, brain, and lungs. 18 We demonstrated increased F-LW223 binding. This indicates that myocardial infarction mediated increased TSPO expression in these organs, which can also be interpreted as increased systemic inflammation.

[0229] (References) All references made herein are incorporated herein by reference in their entirety. Alam et al. Med. Mol. Imaging (2010). 51, 283-296 (2017) Bielohuby et al. Am. J. Physiol. Metab. 293, E139-E146 (2007) Bird et al. Atherosclerosis 210, 388-391 (2010) Blair et al. Med. Chem. Commun. 4, 1461-1466 (2013) Blair et al. Chem. Sci. 6, 4772-4777 (2015) Calsolaro et al. Alzheimer’s Dement. 11, P792 (2015) Cappelli et al. J. Med. Chem. 40, 2910-2921 (1997) Cappelli et al. J.Med. Lett. 14, 4055-4066 (2006) Charbonneau et al. Circulation 73, 476-483 (1986) Chauveau et al. Eur. J. Nucl. Med. Mol. Imaging 35, 2304-2319 (2008) Cosenza-Nashat et al. Neuropathol. Appl. Neurobiol. 35, 306-328 (2009) Dupont et al. Int. J. Mol. Sci. 18, 785 (2017) Endres et al. J. Nucl. Chem. 50, 1276-82 (2009) Fairweather et al. J. Cardiovasc. Transl. Res. 7, 192-202 (2014) Fujimura et al. Atherosclerosis 201, 108-111 (2008) Fujita et al. EJNMMI Res. 7 (2017), doi:10.1186 / s13550-017-0334-8 Gaemperli et al. Eur. Heart J. 33, 1902-1910 (2012) Hindorf et al. J. Nucl. Med. 45, 1960-5 (2004) Hui et al. Cancer 73, 951-957 (1994) Ichise et al. J. Cereb. Blood Flow Metab. 22, 1271-1281 (2002) Ikawa et al. J. Nucl. Med. (2016), doi:10.2967 / jnumed.116.178996 Innis et al. J. Cereb. Blood Flow Metab. 27, 1533-1539 (2007) Khanuja et al. Broom. Common. 92, 7729-7733 (1995) Kobayashi et al. J. Cereb. Blood Flow Metab. 38, 393-403 (2018) Kreisl et al. Neuroimage 49, 2924-2932 (2010) Lacapere Steroids 68, 569-585 (2003) Li et al. Clin. Exp. Pharmacol. Physiol. 42, 1068-1074 (2015) Li et al. Science 347, 555-558 (2015) Lin et al. Genomics 18, 643-50 (1993) Logan et al. J. Cereb. Blood Flow Metab. 16, 834-840 (1996) Logan Nucl. Med. Biol. 27, 661-670 (2000) Luus et al. J. Label. Compd. Radiopharm. 53, 501-510 (2010) Owen et al. J. Cereb. Blood Flow Metab. 30, 1608-18 (2010) Owen et al. J. Cereb. Blood Flow Metab. 32, 1-5 (2012) Owen et al. J. Nucl. Med. 52, 24-32 (2011) Papadopoulos et al. Trends Pharmacol. Sci. 27, 402-409 (2006) Paradis et al. Cardiovasc. Res. 98, 420-427 (2013) Schalle et al. J. Pharmacol. Exp. Ther. 333, 696-706 (2010) Stabin et al. J. Nucl. Med. 47, 655-9 (2006) Stevenson et al. Bioorg. Med. Chem. Lett. 20, 954-957 (2010) Thackeray et al. J. Am. Coll. Cardiol. 71, 263-275 (2018) Warnock et al. EJNMMI Res. 1, 1-11 (2011) Wilms et al. Neurobiol. Dis. 14, 417-424 (2003) WO 02 / 26713 Zanotti-Fregonara et al. ACS Chem. Neurosci. (2014), doi:10.1021 / cn500138n

Claims

1. Equation (I): 【Chemistry 1】 Compounds thereof, as well as their salts, solvates, and radiolabeled forms.

2. structure: 【Chemistry 2】 The compound according to claim 1, having the following characteristics.

3. Formula (II): 【Transformation 3】 The compound according to claim 1, which is a compound of the same, as well as a salt and solvate thereof.

4. structure: 【Chemistry 4】 The compound according to claim 3, having the following characteristics.

5. A composition comprising a compound of formula (I) according to any one of claims 1 to 4, together with one or more pharmaceutically acceptable excipients.

6. A complex comprising the compound according to any one of claims 1 to 4, which is non-covalently bonded to TSPO.

7. A method for preparing the complex described in claim 6 in vitro, comprising the step of contacting a compound described in any one of claims 1 to 4 with TSPO.

8. A method for preparing the complex according to claim 6, comprising the step of contacting a compound according to any one of claims 1 to 4 with TSPO, wherein the subject is non-human.

9. A method for detecting TSPO, comprising the steps of forming a complex in vitro containing a compound according to any one of claims 1 to 4 that is non-covalently bound to TSPO, and detecting the compound.

10. A method for detecting TSPO, comprising the steps of forming a complex containing a compound according to any one of claims 1 to 4 that is non-covalently bound to TSPO, and detecting the compound, wherein the subject is non-human.

11. The method according to claim 9, wherein the compound of formula (I) is administered to a subject.

12. The method according to claim 10, wherein the compound of formula (I) is administered to a subject.

13. The method according to claim 11, wherein the subject is a human subject.

14. The method according to any one of claims 8 to 13, wherein the compound of formula (I) is radiolabeled, and the compound is detected by positron emission tomography.

15. A method for preparing a compound of formula (I) as described in claim 1, wherein formula (III): 【Transformation 5】 The method comprising the step of substituting the compound bromine with fluorine.

16. A method for preparing the compound of formula (II) described in claim 3, wherein formula (IV): 【Transformation 6】 The method comprising the step of substituting the chlorine in the compound with 18-fluorine.

17. The compound of formula (IV) is of formula (III) 【Transformation 7】 The method according to claim 16, wherein the compound is prepared by chlorine substitution of the bromine in the compound of formula (III) from the compound of (III).

Images