Tspos binding agent

Abstract

The present invention provides a compound of formula (I) and salts, solvates and radiolabelled forms thereof, and complexes of the compound of formula (I) with TSPO, and methods for forming such complexes, and methods for detecting the compound of formula (I) in complexes with TSPO.

Description

[0001] Related applications

[0002] This application claims the rights and priority of GB 1810312.7, filed on June 22, 2018 (2018.06.22), the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention provides compounds for binding to TSPO, methods for preparing these compounds, methods for binding compounds to TSPO, and methods for detecting compounds bound to TSPO. Background Technology

[0004] The 18kDa translocator protein (TSPO), formally known as the peripheral diazepine receptor (Papadopoulos et al.), is expressed in the outer membrane of mitochondria and participates in cholesterol transport and steroid synthesis. TSPO is highly expressed in inflammatory cells such as microglia in the brain (Wilms et al.; Cosenza-Nashat et al.) and peripheral macrophages (Fujimura et al.; Bird et al.), thus TSPO has been recognized as a marker of pathological inflammation throughout the body.

[0005] Increased TSPO expression has been demonstrated in neurodegenerative diseases such as dementia and Parkinson's disease (DuPont et al.), as well as cardiovascular diseases, namely atherosclerotic plaques (Borde et al.) and post-acute myocardial infarction (DuPont et al.). Therefore, a successful imaging approach targeting TSPO with PET has significant clinical value across a wide range of pathologies. Furthermore, as a marker of inflammation, TSPO has recently been shown to play a role in neuroprotection (Thackeray et al.) and cardioprotection (Schalle et al.; and Paradis et al.), thus further expanding the application of non-invasive TSPO imaging and disease-modifying therapies in the context of disease occurrence and progression.

[0006] In the field of positron emission tomography (PET) imaging of inflammation, TSPO is one of the most widely explored targets. A typical TSPO PET radiotracer developed decades ago is... 11C-PK11195 (Charbonneau et al.). Due to the short half-life (20 min) of this radioactive isotope, requiring on-site cyclotron equipment in hospitals, the routine clinical distribution of this radioactive tracer is limited. Furthermore, 11 C-PK11195 exhibits relatively high nonspecific binding (Chauveau et al.).

[0007] Therefore, as summarized in a recent review (Alam et al.), considerable effort has been made in generating a new family of TSPO radiotracers with improved properties. Despite these developments, 11 C-PK11195 is still frequently used as a tool in clinical research. This is 11 The high inter-individual binding rates of all TSPO radiotracers synthesized and studied after the development of C-PK11195 are a result of this; as determined in the pioneering study by Owen et al., this is known to be caused by the genetic polymorphism rs6971 (Owen et al. J. Cereb. Blood Flow Metab. 2012). These common genetic polymorphisms mean that approximately 10% of the population classified as low affinity binders (LABs) is unimageable with second-generation radiotracers, while the remainder, falling between mixed affinity binders and high affinity binders (MABs and HABs), requires genetic screening and complex post-imaging correction. Significant variability exists in the sensitivity of second-generation ligands to the rs6971 genetic polymorphism. For example, 11 C-PBR28 has an in vitro LAB:HAB ratio of 55 (Owen et al. J. Nucl. Med.), however, recently designed radiotracers... 11 The in vitro ratio of analogues of C-ER176 and PK11195 is 1.3 (Zanotti-Fregonara et al.).

[0008] Therefore, PK11195 remains the only TSPO radiotracer that has consistently shown insensitivity to the rs6971 polymorphism in the human brain (Owen et al. J. Nucl. Med.; and Owen et al. J. Cereb. Blood Flow Metab. 2010). Other TSPO binders that also show insensitivity to the rs6971 polymorphism are needed. Summary of the Invention

[0009] Typically, this invention provides compounds for binding to the translocase protein (TSPO), thereby serving as a tracer of TSPO, such as a radiotracer. Therefore, the compounds of this invention can be used in methods for detecting TSPO in vivo and in vitro. Such methods can be used for the identification of inflammation or for the diagnosis of diseases associated with altered TSPO levels, such as neuroinflammatory diseases, cancer, and cardiovascular diseases.

[0010] The compounds of this invention are insensitive to the rs6971 genetic polymorphism and also exhibit an affinity for TSPO that is approximately twice that of the known TSPO binder PK11195. The compounds of this invention exhibit good kinetics and a favorable dosimetric profile in vivo, and are therefore suitable for clinical use.

[0011] In vivo characterization of the compounds of this invention also revealed specific uptake consistent with TSPO expression. Blockade studies at a single concentration of PK11195 (1 mg / kg) confirmed target engagement, with SUV values ​​measured after blockade decreasing by 64-81% compared to baseline scans.

[0012] The compounds of the present invention can be considered ideally suited as markers for TSPO, such as radiolabelers, because they have nanomolar affinity for TSPO in the human brain and heart, they can penetrate into the brain in vivo, their distribution profile in vivo is consistent with TSPO protein expression, they are slowly metabolized in plasma and have low levels of metabolites recorded in tissues.

[0013] In a first aspect of the present invention, a compound of formula (I) is provided.

[0014]

[0015] And its salt, solvate and radiolabeled forms.

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

[0017]

[0018] And its salts and solvates.

[0019] Compared to carbon radiolabeled compounds, including 11C-PK11195, and compounds known in the art, fluorine radiolabeled TSPO binders, such as those of formula (II), have a longer half-life. The dosage profile of compounds of formula (II) is such that the radiolabeled compounds are suitable for human use.

[0020] In a second aspect of the invention, a method for preparing a compound of formula (I) is provided, the method comprising the step of substituting bromine of a compound of formula (III) with fluorine:

[0021]

[0022] In a third aspect of the invention, a method for preparing a compound of formula (II) is provided, the method comprising the step of substituting chlorine of a compound of formula (IV) with 18-fluoro:

[0023]

[0024] Compounds of formula (IV) can be obtained from compounds of formula (III) by substituting bromine with chlorine.

[0025] The present invention also provides a composition comprising a compound of formula (I) and one or more pharmaceutically acceptable excipients.

[0026] In a further aspect of the invention, a method for detecting a compound of formula (I) is provided, the method comprising the steps of contacting the compound of formula (I) with TSPO to form a complex of the compound of formula (I) and TSPO, and detecting the compound of formula (I).

[0027] The TSPO can be provided in vivo or in vitro.

[0028] The compounds of this invention can be used to detect organ samples, such as heart and brain samples, taken from subjects. Accordingly, the methods of this invention can be used to detect and determine TSPO levels, for example, in neuropathology and cardiovascular pathology.

[0029] In a further aspect of the invention, a method for detecting a compound of formula (I) in a subject is provided, the method comprising administering the compound of formula (I) to the subject and subsequently detecting the compound of formula (I).

[0030] In this article, the subjects mentioned may be human subjects.

[0031] The subjects may be those with a known or suspected disease that is associated with altered TSPO levels, such as elevated TSPO levels, and diseases such as neuroinflammatory diseases, cancer, and cardiovascular diseases.

[0032] The subject may have a disease associated with altered TSPO levels, and the subject may be undergoing treatment for such disease. The method of the present invention can be used to detect changes in TSPO levels, for example, to assess the effectiveness of treatment.

[0033] These and other aspects of the invention, as well as its implementation, will now be described in further detail. Attached Figure Description

[0034] Figure 1 The structures of known TSPO binders PK11195(A), PBR28(B), and AB5186(C), and the TSPO binder LW223(D) according to embodiments of the present invention are shown. The compounds are shown in their unlabeled (non-radiolabeled) form.

[0035] Figure 2 The binding affinities of TSPO ligands from the human brain are shown for high-affinity (HAB), mixed-affinity (MAB), and low-affinity (LAB) binders, where (a) shows the average PK11195 binding affinity curve plotted using one-site fitting, HAB n=6, MBA n=8, and LAB n=4; (b) shows the average PBR28 binding affinity curve plotted using one-site fitting, excluding MAB, HAB n=4, MAB n=5, and LAB n=4; (c) shows the average AB5186 binding affinity curve plotted using one-site fitting, excluding MAB, HAB n=6, MAB n=6, and LAB n=5; and (d) shows the average LW223 binding affinity curve plotted using one-site fitting, HAB n=5, MAB n=5, and LAB n=4. n = 4; and (e) shows the PK11195 affinity values ​​(K) calculated for each human sample. i Affinity values ​​were also calculated for (f)PBR28, (g)AB5186, and (h)LW223. Unpaired t-tests were used for HAB and LAB, with ns = not significant, * = p < 0.05, *** = p ≤ 0.001. All results represent mean ± SEM.

[0036] Figure 3The binding affinities of TSPO ligands from human hearts using high-affinity (HAB), mixed-affinity (MAB), and low-affinity (LAB) ligands are shown, where (a) shows the average PK11195 binding affinity curve plotted using unit-point fitting, HAB n=4, MBA n=5, and LAB n=4; (b) shows the average PBR28 binding affinity curve plotted using unit-point fitting, HAB n=4, MAB n=5, and LAB n=4; (c) shows the average AB5186 binding affinity curve plotted using unit-point fitting, except for MAB which uses two-site fitting, HAB n=4, MAB n=5, and LAB n=4; (d) shows the average LW223 binding affinity curve plotted using unit-point fitting, HAB n=5, MAB n=5, and LAB n=4; and (e) shows the PK11195 affinity values ​​(K0) calculated for each human sample. i Affinity values ​​were also calculated for (f)PBR28, (g)AB5186, and (h)LW223. Unpaired t-tests were used for HAB and LAB, with ns = not significant and ** = p ≤ 0.01. All results represent mean ± SEM.

[0037] Figure 4 It shows in vivo 18 kinetic and metabolic curves of F-LW223, where (a) is the maximum intensity projection image of rats and (b) is the projection image of mice, showing the distribution of 18F-LW223 at basal state. B = brain, H = heart, L = lung, GB = gallbladder (mice only), A = adrenal gland, K = kidney, and G = intestine; (c) and (d) are the respective values ​​of rats and mice. 18 The time activity curve of F-LW223 is shown in (e). 18 Hemodynamics of F-LW223 rats, showing the percentage (%) of parent compounds in plasma, where results represent mean ± SEM, n = 3 at each time point; and (f) showing the effects of 2-tissue (2T) and Logan (t) * =30) and multivariate (M1, t) * =30) The distribution volume (V) in organs calculated by the model t Values, mean ± SD, n = 3.

[0038] Figure 5 This shows the binding to TSPO in vivo. 18 F-LW223, of which (a) shows the effect in the brain (B), heart (H) and lungs (L). 18(a) Image of total SUV uptake by F-LW223; (b) shows the administration of PK11195 (1 mg / kg) after blockade. 18 Total SUV images acquired by F-LW223. All images were averaged (60–120 minutes) and a Gaussian filter (1×1×1) was applied; (c) shows the main source organs at baseline. 18 F-LW223 time-activity curve; and (d) shows the major source organ at baseline after PK11195 blockade. 18 F-LW223 time-activity curves. All results represent mean ± SEM, n = 3.

[0039] Figure 6 It shows the integration into human tissues 18 F-LW223 was used to illustrate local uptake at pathological sites, where (a) shows H&E staining of brain tissue from an individual with hemorrhagic stroke, * = hemorrhagic area. Scale bar = 1000 μM; (b) shows a histological example (H&E) of a diseased coronary vessel exhibiting pathological neointimal remodeling. Dashed lines indicate the boundary between the primitive interlaminar layer and the neointimal layer. Scale bar = 1000 μM; (c) shows invasive inflammatory cells bound to the stroke tissue. 18 (d) is an autoradiography image of F-LW223; (e) shows an autoradiography image of 18F-LW223 bound to neointimal inflammatory cells in the diseased coronary vessels; (f) shows the use of PK11195 blockade in stroke. 18 (f) shows the use of PK11195 to block 18F-LW223 binding in diseased coronary tissue; (g) shows the use of LW223 to block LW223 binding in stroke. 18 F-LW223 binding; (h) shows the use of LW223 blockade in diseased coronary vessels. 18 F-LW223 binding: (i) shows binding to high and low uptake regions in stroke. 18 Quantification of F-LW223 and the targeted / untargeted ratio; and (j) showing binding to high- and low-uptake regions in diseased coronary tissue. 18 The quantification of F-LW223 and the ratio of targeted to non-targeted molecule were performed. Paired t-tests were used for targeted and non-targeted molecule analysis. Results represent mean ± SEM, n = 3, * = p < 0.05, ** = p ≤ 0.01.

[0040] Figure 7 The dissociation constant (K) is shown. d ) value and the maximum binding force of PK11195 in the human brain and heart (B max(a) shows the Kd value of PK11195 and (b) shows the B calculated from the saturated bonding test. max Unpaired t-tests were used for the brain and heart. Results are expressed as mean ± SEM. Brain n=6, heart n=5, ns= not significant, *= p<0.05.

[0041] Figure 8 The use of PK11195 in vivo was demonstrated as a replacement. 18 F-LW223, where (a) is the original substitution. 18 (a) Total SUV images captured by F-LW223; (b) Total SUV images after replacement with PK11195 (1 mg / kg), where baseline images were averaged before PK11195 administration (45–60 min), and replacement images were averaged after (95–120 min). All images have been filtered with a Gaussian filter (1×1×1); and (c) Before and after PK11195 challenge (green arrows). 18 Time-activity curve of F-LW223.

[0042] Figure 9 The mouse-derived organ was shown. 18 F-LW223 time-activity curves, where (a) represents all sites in male mice with uptake greater than background. 18 (a) F-LW223 time-activity curve; and (b) for all sites in female mice where uptake was greater than background. 18 F-LW223 time-activity curve. The raw PET data was reconstructed using a filtered back projection method without attenuation correction.

[0043] Figure 10 This shows the brain tissue of healthy humans. 18 The binding of F-LW223, where (a) is H&E staining of a healthy brain; (b) is the binding of F-LW223 in a healthy brain. 18 (c) Autoradiography of F-LW223 combined; (d) Blocking with PK11195 18 F-LW223 combination; (d) Blocking using LW223 18 F-LW223 binding; (e) is binding to the gray matter region. 18 Quantification of F-LW223 and its targeted to non-targeted ratio. Paired t-tests were used for targeted and non-targeted results. Results represent mean ± SEM, n = 3, ns = not significant. Scale bar = 1000 μm.

[0044] Figure 11 It shows 18The results of an uptake study of F-LW223 in rats with induced myocardial infarction demonstrate that myocardial infarction leads to myocardial infarction in the heart, brain, and lungs. 18 Increased F-LW223 uptake is illustrated in (A) as an example of the heart (top), brain (middle), and lung (bottom) of a healthy rat (left) and a rat with myocardial occlusion 7 days after injury (right). 18 F-LW223 SUV images; (B) shows the SUV time-activity curves in the heart, brain, and lungs of healthy rats (red circles) and rats with myocardial infarction (blue squares); (C) shows the SUVr time-activity curves in the heart, brain, and lungs relative to the blood pool of healthy rats (red circles) and rats with myocardial infarction (blue squares). Mean ± SEM, n = 5–6. Detailed Implementation

[0045] Compounds of formula (I), including compounds of formula (II), are used as TSPO binders. Accordingly, the compounds can be used to identify, for example, altered (e.g., increased) TSPO expression in subjects such as humans, and can be used to identify subjects who have or have a disease, such as neuroinflammatory diseases, cancer, or cardiovascular diseases.

[0046] The compounds of the present invention, their synthesis, and their uses are described in further detail below.

[0047] Stevenson et al. previously described compound 11, which is a compound having an iodomethyl substituent on the quinoline ring, rather than the fluoromethyl group required by the compound in this case. This compound was used in SPECT imaging at TSPO. Stevenson et al. did not describe any compound used as a radiotracer. Furthermore, this work does not suggest that such a compound or any derivative is, or may be, insensitive to the rs6971 genetic polymorphism.

[0048] Furthermore, the binding data reported by Stevenson et al. teaches the avoidance of using amide substituents present in the compounds of this invention. Therefore, a comparison between compound 11 (with methyl- and S-butyl-substituted amide nitrogen) and related compound 18 (with diethyl-substituted amide nitrogen) shows that compound 18 has the best affinity for TSPO (see Table 1 by Stevenson et al.).

[0049] Blair et al. (Chem. Sci.) previously described tracers – compounds 4 (corresponding to compound 18 of Stevenson et al.), 5, and 6 (AB5186). Blair et al.'s work showed that the binding of iodoform compounds to TSPO is improved when the halogen is positioned as a substituent on the pendant phenyl group, rather than as a halomethyl substituent on the quinoline ring. Therefore, Blair et al.'s... Figure 2 The data in the study showed that compound 5 had a better affinity than compound 4.

[0050] This case demonstrates that the compounds of the present invention have better affinity than the compounds comprising compound 6 described by Blair et al. (Chem.Sci.), which is the reference compound AB5186 in this application.

[0051] Cappelli et al. (J. Med. Lett.) describe compounds and their uses related to those described by Stevenson et al. and Blair et al. (Chem. Sci.). This document does not address the issue of polymorphic binding.

[0052] WO 02 / 26713 focuses on compounds used to treat parasitic infections. Some of the compounds disclosed therein have very minor similarities to the compounds in this case. WO 02 / 26713 does not mention TSPO or radiotracers.

[0053] compound

[0054] This invention provides a compound of formula (I).

[0055]

[0056] And its salts, solvates and radioforms.

[0057] Preferably, the compound of formula (I) is:

[0058]

[0059] And its salt, solvate and radiolabeled forms.

[0060] The inventors discovered that this particular stereoform-(R)-configuration has an affinity for TSPO that is about five times greater than that for its enantiomer.

[0061] In a preferred embodiment, the compound is a compound of formula (II).

[0062]

[0063] And its salts and solvates.

[0064] The compounds of formula (II) are radiolabeled compounds, and more specifically, are radiolabeled fluorine isotopes. 18 F mark.

[0065] Preferably, the compound of formula (II) is:

[0066]

[0067] And its salts and solvates.

[0068] Compared to unlabeled, non-radiolabeled compounds, radiolabeled compounds can be called hot compounds.

[0069] Radioactive forms, salts, solvates, and stereoforms.

[0070] Compounds of formula (I) or any other compound described herein, including compounds in which atoms are substituted with naturally occurring or non-naturally occurring isotopes. In one embodiment, the isotope is a stable isotope. Thus, compounds described herein include, for example, compounds containing deuterium. For example, H can be any isotopic form, including... 1 H, 2 H(D) and 3 H(T); C can be any isotopic form, including 11 C 12 C 13 C and 14 C and O can be in any isotopic form, including 15 O、 16 O and 18 O; F can be any isotopic form, including 18 F, etc.

[0071] Typically, the compounds of this invention contain, such as 18 F, 11 C 13 N or 15 Radioactive isotopes of O, suitable for detection by scintillation imaging methods such as positron emission tomography (PET). Therefore, in one embodiment, the compounds of the present invention comprise positron-emitting radioactive isotopes, such as... 18 F, 11 C 13 N or 15 O, and the optimal choice is 18 F.

[0072] In a preferred embodiment of the present invention, the F atom of the compound of formula (I) can be provided as 18 F. This compound is of formula (II). Due to its relationship with C 11 These compounds have a longer half-life (about 110 minutes) compared to those radiolabeled compounds (which have a half-life of about 20 minutes), making them particularly useful radioactive tracers.

[0073] Examples of salts of compounds of formula (I) or any other compounds described herein include all pharmaceutically acceptable salts, such as, but not limited to, acid addition salts of strong inorganic acids such as HCl salts and HBr salts, and addition salts of strong organic acids such as methanesulfonates. Further examples of salts include sulfates and acetates such as acetates themselves, trifluoroacetates, or trichloroacetates.

[0074] Compounds of formula (I) or any other compound described herein, as well as solvates of said compounds, are also involved. Examples of solvates include hydrates.

[0075] Unless otherwise stated, the specific compounds involved include isomers, including mixtures thereof (e.g., racemic mixtures). The methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic methods) of such isomers are either known in the art or readily available by means of methods taught herein or known methods.

[0076] One aspect of the invention relates to compounds in substantially purified and / or substantially contaminant-free forms.

[0077] In one implementation, the basic 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.

[0078] Unless otherwise stated, the basic purified form refers to any stereoisomer or enantiomeric form of the compound. For example, in one embodiment, the basic purified form refers to a mixture of stereoisomers, i.e., purified relative to other compounds. In one embodiment, the basic purified form refers to a single stereoisomer, such as an optically pure stereoisomer. In one embodiment, the basic purified form refers to a mixture of enantiomers. In one embodiment, the basic purified form refers to an equimolar mixture of enantiomers (i.e., a racemic mixture, a racemic compound). In one embodiment, the basic purified form refers to a single enantiomer, such as an optically pure enantiomer.

[0079] In one embodiment, the contaminant percentage refers to no more than 50% by weight, for example, no more than 40% by weight, for example, no more than 30% by weight, for example, no more than 20% by weight, for example, no more than 10% by weight, for example, no more than 5% by weight, for example, no more than 3% by weight, for example, no more than 2% by weight, for example, no more than 1% by weight.

[0080] Unless otherwise stated, a contaminant refers to other compounds, that is, compounds other than stereoisomers and enantiomers. In one embodiment, a contaminant refers to other compounds and other stereoisomers. In another embodiment, a contaminant refers to other compounds and other enantiomers.

[0081] In one embodiment, the basic purified form is at least 60% optically pure (i.e., on a molar basis, 60% of the compound is the desired stereoisomer or enantiomer, while 40% is the undesired 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.

[0082] Composition

[0083] The compounds of formulas (I) and (II) are suitable for binding to TSPO and can be bound in vivo. Accordingly, the compounds of formulas (I) and (II) can be prepared into compositions for administration to humans or animals.

[0084] When the compounds of formula (I) are radiolabeled, they may be provided to the fluid composition at a concentration of at least 0.1 Mbq / mL, at least 0.5 Mbq / mL, at least 1 Mbq / mL, at least 2 MBq / mL, or at least 5 MBq / mL.

[0085] When the compounds of formula (I) are radiolabeled, they may be provided to the fluid composition at concentrations of up to 10 Mbq / mL, up to 20 Mbq / mL, up to 50 Mbq / mL, up to 100 Mbq / mL, up to 200 Mbq / mL, up to 300 Mbq / mL or up to 200 Mbq / mL.

[0086] The compound can be used at concentrations selected from the lower and upper limits given above. For example, the compound can be used at concentrations in the range of 1-10 MBq / mL.

[0087] In some examples of this case, it was used at a concentration of 2 MBq / mL. 18 F-LW223.

[0088] Compounds of formulas (I) and (II) are typically used in saline solutions, for example, for intravenous injection.

[0089] Although the compound of formula (I) can be administered alone, it is desirable to have it as a pharmaceutical dosage form (e.g., composition, formulation, pharmaceutical agent) comprising at least one compound of formula (I) described herein, and one or more other pharmaceutically acceptable ingredients known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, diluents, excipients, auxiliaries, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners.

[0090] Therefore, the present invention further provides compositions as defined above, and methods for preparing the compositions, the methods comprising mixing at least one compound of formula (I) as described herein with one or more other pharmaceutically acceptable ingredients known to those skilled in the art, such as carriers, diluents, excipients, etc. If formulated into discrete units (e.g., tablets), each unit contains a predetermined amount (dosage) of the compound.

[0091] As used in this article, "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc., that, within reasonable medical judgment, are suitable for contact with the tissues of subjects in question (e.g., humans), without excessive toxicity, irritation, allergic reactions, or other problems or complications, and are proportionate to a reasonable benefit / risk ratio. In the sense of compatibility with other components in the dosage form, carriers, diluents, excipients, etc., also need to be "acceptable."

[0092] Suitable carriers, diluents, excipients, etc., can be found in standard pharmaceutical texts, such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pennsylvania, 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 2005.

[0093] The dosage form can be prepared by any method known in the pharmaceutical field. These methods include the step of combining a compound of formula (I) with a carrier constituting one or more auxiliary components. Typically, the dosage form is prepared by uniformly and tightly combining the compound with a carrier (e.g., a liquid carrier, a dispersed solid carrier, etc.) and, if necessary, subsequently shaping the product.

[0094] The dosage forms can be prepared to provide rapid release; immediate, delayed, timed, or sustained release; or combinations thereof.

[0095] The dosage form may be, appropriately, a liquid, a solution (e.g., aqueous or non-aqueous), a suspension (e.g., aqueous or non-aqueous), an emulsion (e.g., oil-in-water or water-in-oil), a spray, a mist, or an aerosol.

[0096] The compound may be dissolved in, suspended in, or incorporated into one or more other pharmaceutically acceptable ingredients. The compound may be present in liposomes or other microparticles designed to target the compound to, for example, blood components or one or more organs.

[0097] When the carrier is a liquid, dosage forms suitable for intranasal administration include, for example, nasal sprays, nasal drops, or aerosol administration using a spray, including aqueous or oil solutions of the compound. As an alternative administration method, dry powder delivery can be used as a substitute for atomized aerosols.

[0098] When the carrier is solid, dosage forms suitable for intranasal application include, for example, formulations present as coarse powder having a particle size, for example, in the range of about 20 to about 500 micrometers, which are applied in such a way that nasal smoke is rapidly inhaled from a powder container near the nose through the nasal passage.

[0099] Dosage forms suitable for pulmonary administration (e.g., via inhalation or blowing therapy) include those formulations that use suitable propellants, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases, in the form of aerosol sprays from pressurized packaging. Additionally or alternatively, dosage forms for pulmonary administration can be formulated for administration from a nebulizer or dry powder inhaler. For example, the dosage forms may be provided with carriers or liposomes, providing suitable particle size to reach the appropriate portions of the lung, aiding in the delivery of an appropriate dose to enhance retention in lung tissue.

[0100] Dosage forms suitable for parenteral administration (e.g., via intravenous or subcutaneous injection or infusion) include aqueous or anhydrous liquids, isobaric liquids, pyrogen-free liquids, and sterile liquids (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, antibacterial agents, suspending agents, thickeners, and solutes that bring the formulation to isobaric levels with the blood (or other relevant bodily fluids) of the designated recipient. Examples of excipients include, for example, water, alcohols, sugars, polyols, glycerol, vegetable oils, etc. Examples of suitable isobaric carriers for such dosage forms include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Typically, the concentration of the compound in the liquid is from about 1 ng / mL to about 500 μg / mL, for example from about 1 ng / mL to about 100 μg / mL, for example from about 10 ng / mL to about 10 μg / mL, for example from about 10 ng / mL to about 1 μg / mL. The dosage form can be present in single-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use. Temporary injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0101] Preparation method

[0102] The present invention also provides methods for preparing compounds of formula (I) and formula (II).

[0103] In the preparation method, fluorine can be introduced into compounds of formula (I) and formula (II) in the final step of the synthesis. This is particularly important when the compound of formula (I) contains fluorine as a radiolabel, such as in the compound of formula (II). The method for preparing the compounds of the present invention does not require treatment of intermediate radiolabeled compounds, which should generally be avoided due to the loss of radioactivity over time. This situation arises when it is necessary to prepare and purify the radiolabeled intermediate compound.

[0104] The compound of formula (I) can be prepared from the compound of formula (III) by replacing the bromine in the compound of formula (III) with fluorine. The compound of formula (III) is:

[0105]

[0106] And its salt, solvate and radiolabeled forms.

[0107] Halogen substitution reactions, as well as fluorine substitution reactions, are well known in the art.

[0108] The compound of formula (III) can react with a fluoride anion to give the compound of formula (I). The fluoride anion can be provided by a fluoride salt, such as with an alkali metal salt, for example, potassium fluoride. The substitution reaction can be carried out in the presence of a catalyst such as 18-crown ether-6.

[0109] The compound of formula (II) can be prepared from the compound of formula (IV) by substituting chlorine in the compound of formula (IV) with 19-fluorine. The compound of formula (IV) is:

[0110]

[0111] And its salt, solvate and radiolabeled forms.

[0112] For example, compounds of formula (IV) can be used in ways such as [ 18 F]KF 19 F anion treatment, the 19 F anions are known in the art for their use in introducing... 18 F radioactive labeling.

[0113] Compounds of formula (IV) can be prepared from compounds of formula (III) by substituting bromine in compounds of formula (III) with chlorine.

[0114] For example, compounds of formula (IV) can be treated with chloride anions such as LiCl.

[0115] The present invention also provides compounds of formula (III) and formula (IV), and their salts, solvates and radiolabeled forms.

[0116] complex

[0117] In a further aspect of the invention, a complex of a compound of formula (I) and TSPO is provided. In this document, the compound of formula (I) is non-covalently bonded to TSPO.

[0118] The complex of the compound of formula (I) with TSPO can be provided in vitro or in vivo. The TSPO may be present on the outer layer of the mitochondrial membrane.

[0119] Typically, the compound of formula (I) is provided as a binding pair with TSPO. Therefore, the stoichiometry of the compound and TSPO in the complex is 1:1.

[0120] When a compound of formula (I) is bound to TSPO in a complex, the compound of formula (I) can be detectable, such as when the compound is radiolabeled.

[0121] In the case where the compound of formula (I) is radiolabeled, such as in the compound of formula (II), the compound can be detected by a suitable radiolabeling method, such as positron emission tomography.

[0122] When the compound of formula (I) is not radiolabeled, it can be detected by other methods, including NMR, for example.

[0123] TSPO is a transport protein. It can be referred to as a peripheral benzodiazepine receptor.

[0124] The TSPO can be a mammalian TSPO, such as a human or rodent TSPO.

[0125] The TSPO can be a protein that includes the NCBI reference sequence: NP_000705.2.

[0126] Methods and uses

[0127] The compounds of the present invention are used to bind to TSPO. The compounds are detectable and are detectable when present in the complex with TSPO.

[0128] The compounds of the present invention can be contacted with TSPO present in vivo or in vitro to form complexes such as those described. These complexes can then be detected by detecting the compounds of the present invention.

[0129] In the case where the compound of formula (I) is radiolabeled, for example, if the compound is a compound of formula (II), the compound can be detected by positron emission tomography (PET).

[0130] Therefore, the method in this case typically involves imaging compounds of formula (I) (such as compounds of formula (II)) using scintillation imaging, including PET (positron emission tomography). Scintillation imaging may include using a camera or scanner to detect radioactivity in a single plane. PET imaging systems may include a ring-shaped detector array that can also detect radioactivity in multiple dimensions.

[0131] Advantageously, the compounds of the present invention are insensitive to mutations within TSPO, such as the rs6971 genetic polymorphism.

[0132] The method may include the step of administering a compound of formula (I) to a subject, such as a human or animal.

[0133] The method of the present invention may include the step of detecting a compound of formula (I) (such as one present in a complex with TSPO) at a site selected from the group consisting of the brain, heart, lungs, gallbladder, adrenal glands, kidneys and intestines of a subject.

[0134] The present invention also provides a method for imaging TSPO expression in a subject, the method comprising the steps of applying a compound of formula (I) or a composition containing said compound to the subject and generating an image of the distribution of one or more said compounds within the subject.

[0135] In some implementations, the onset rate and extent of TSPO expression, such as that associated with neuroinflammatory conditions, can be determined by imaging. This can, for example, be used to predict the outcome of methods for treating neuroinflammatory conditions.

[0136] Here, the binding of the compound of formula (I) to the subject site indicates the amount or extent of TSPO expression at that site.

[0137] The administration of compound (I) to a subject is described in more detail below.

[0138] Molecular imaging techniques, such as those utilizing radiolabeling, can be used to generate one or more images of the distribution of the imaging agent within the subject.

[0139] Using appropriate molecular imaging techniques, one or more images can be generated showing the distribution of compound (I) of formula (I) in all or part of a subject over a period of time following administration of an imaging agent. The amount or concentration of a detectable marker in a body tissue or region indicates the amount of TSPO expressed in that tissue or region. An increase in the concentration of compound (I) of formula (I) in a body tissue or region relative to other tissues or regions in the body indicates that the cells in that tissue or region are undergoing increased TSPO expression. Therefore, the imaging agent of the present invention can be used for TSPO expression and can be applied to the detection of diseases associated with altered TSPO expression levels.

[0140] The subjects may have a disease characterized by the presence of sites with elevated or decreased TSPO expression (elevated TSPO expression), and one or more images show the distribution of the compound of the present invention at one or more sites.

[0141] The present invention also provides a method for determining the effectiveness of a therapy for a disease symptom associated with elevated or decreased TSPO expression (e.g., elevated TSPO expression), the method comprising the following steps:

[0142] Before, during, or after treatment, administer a compound of formula (I) or a composition containing said compound to a subject; and,

[0143] One or more images of the compound distribution are generated at one or more sites where TSPO expression is elevated or decreased (e.g., elevated TSPO expression) in the subject.

[0144] During or after treatment, one or more images can be generated showing the distribution of the compound of formula (I) at TSPO expression sites in the subject.

[0145] Changes in the binding of the compounds of the present invention (such as decreases or increases) at one or more sites of elevation or decrease after the treatment, relative to before the treatment, indicate that the treatment is effective in alerting the subject to TSPO expression.

[0146] In cases where disease symptoms are associated with elevated TSPO expression, treatment may be considered effective if the binding of the compound is reduced at one or more sites.

[0147] In early clinical trials and subsequent clinical settings, compounds of formula (I) can be used to evaluate drug efficacy, which can then guide treatment. Ineffective treatments can be abandoned at an early stage, allowing for the selection of more effective drugs.

[0148] A method for determining the efficacy of a treatment regimen in a subject is also provided, the method comprising:

[0149] The initial treatment protocol that subject the subject to the treatment; and

[0150] To determine the amount or extent to which compound (I) binds to TSPO in the subject.

[0151] The change in the amount or degree of binding to the regimen indicates that the regimen is effective in the subjects.

[0152] This method may include additional steps:

[0153] A treatment regimen that alters the protocol and subjectes the patient to the alteration.

[0154] Determine the amount or extent to which the compound of formula (I) in the subject binds to TSPO;

[0155] The process involves repeatedly altering the regimen and determining the binding until a change in the amount or extent of compound binding is observed.

[0156] The effectiveness of the regimen in subjects is indicated by changes in the amount or extent to which the compound is bound in response to the regimen.

[0157] The subjects described in the above method may have neuroinflammatory conditions, and a decrease in the amount or extent of binding of the compound of formula (I) in response to the protocol indicates that the protocol is effective in the subjects.

[0158] The subjects may have disease symptoms characterized by increased TSPO expression levels, and a decrease in the amount or extent of binding of one or more disease sites to the imaging agent in response to the protocol indicates that the protocol is effective in the subjects.

[0159] dose

[0160] Typically, the method of the present invention may include administering an effective amount of the compound of formula (I) to a subject, thereby effectively labeling TSPO in the target region.

[0161] Those skilled in the art will understand that the appropriate dosage of compounds of formula (I) and compositions containing compounds of formula (I) can vary from patient to patient. Determining the optimal dosage typically involves balancing TSPO marker levels with any risk or harmful side effects.

[0162] The selected dose level will depend on a variety of factors, including but not limited to the route of administration, timing of administration, rate of excretion of the compound, duration of treatment, drug, compound used, and / or materials used by the subject, as well as species, sex, age, weight, condition, general health status, and patient history. Although the dose is usually selected to achieve a local concentration at the site of action, the amount and route of administration of the compound of formula (I) are ultimately determined by the physician, veterinarian, or clinician to achieve the desired effect without causing substantial harmful or toxic side effects.

[0163] During the entire time required for labeling and detection, administration may be continuous or intermittent (e.g., divided doses at appropriate intervals) in the form of a single dose. Methods for determining the most effective administration method and dosage are well known to those skilled in the art and will vary depending on the dosage form, target cells and / or organs, and the subject being treated. Single or multiple administrations may be performed at dose levels and in a manner chosen by the attending physician, veterinarian, or clinician.

[0164] Typically, the appropriate dose of a compound of formula (I) is in the range of about 10 μg to about 250 mg per kilogram of subject body weight per day (more typically about 100 μg to about 25 mg). In the case where the compound of formula (I) is a salt or solvate, the dosage is calculated based on the parent compound, and therefore the actual weight used is increased proportionally.

[0165] When the compounds of formula (I) are radiolabeled, they may be used in the amounts listed below, for example, in dosage forms.

[0166] The compound may be used in amounts of at least 0.1 MBq, at least 0.5 MBq, at least 1 MBq, at least 5 MBq, at least 10 MBq, or at least 20 MBq.

[0167] The compound may be used in amounts of up to 50 MBq, up to 100 MBq, up to 200 MBq, up to 200 MBq, or up to 500 MBq.

[0168] The compound can be used in amounts selected from the lower and upper limits of the above values. For example, the compound can be used in amounts from 20 to 100 MBq.

[0169] The biological dose experienced by the subject may be low, such as 20 mSv per dose, such as per scan. Preferably, the biological dose is 15 mSv or less per dose, 10 mSv or less per dose, such as 7 mSv or less per dose.

[0170] Reagent test kit

[0171] The present invention also provides a kit comprising (a) a compound of formula (I) typically provided in a suitable container and / or suitable packaging, or a composition comprising a compound defined by any of formula (I); and (b) instructions for use, such as written instructions on how to administer the compound or composition.

[0172] Application route

[0173] Compounds of formula (I) or compositions comprising compounds of formula (I) may be administered to subjects via any convenient route of administration, whether systemic / peripheral or local (i.e. at the site of desired action).

[0174] Routes of administration include, but are not limited to, pulmonary (e.g., via mouth or nose, using, for example, a spray, or by inhalation or blowing); parenteral, such as by injection or infusion, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrasheath, intraspinal, intracapsular, intraorbital, intraperitoneal, intratracheal, subepidermal, intra-articular, subarachnoid, and intrasternal; and implantation of a depot or reservoir, for example, by subcutaneous or intramuscular injection.

[0175] Subjects

[0176] The compound of formula (I) can be administered to the subject for the detection of TSPO, including TSPO expression level and TSPO expression distribution.

[0177] The subject can be a chordate, vertebrate, mammal, placental mammal, marsupial (e.g., kangaroo, wombat), rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), lagomorph (e.g., rabbit), avian (e.g., bird), canid (e.g., dog), feline (e.g., cat), equidistant (e.g., horse), swine (e.g., pig), sheep (e.g., sheep), bovine (e.g., cow), primate, ape (e.g., monkey or ape), monkey (e.g., lesser ape, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human. Furthermore, the subject can be in any stage of development, such as a fetus. The subject can be a vertebrate, such as an aquatic vertebrate, like a fish (e.g., zebrafish).

[0178] It is also envisioned that the invention can be implemented on non-human animals. Non-human mammals can be rodents. Rodents include rats, mice, guinea pigs, chinchillas, and other small rodents of similar size used for laboratory research.

[0179] In one implementation, the subject is a human, such as an adult human.

[0180] In one implementation, the subject is a rodent, such as a mouse.

[0181] Other presets

[0182] In this document, each suitable combination of the above embodiments is explicitly disclosed as if each combination were listed separately and explicitly.

[0183] In view of what has been disclosed, various further aspects and embodiments of the present invention will be apparent to those skilled in the art.

[0184] As used herein, “and / or” is considered to be a specific disclosure of each of the two specified features or components, or one of the two specified features or components. For example, “A and / or B” will be considered to be a specific disclosure of (i) A, (ii) B, and (iii) A and B, as each is listed separately herein.

[0185] Unless the context otherwise indicates, the description and definition of the above features are not limited to any particular aspect or implementation of the invention, and are equally applicable to all aspects and implementations described.

[0186] Some aspects and embodiments of the invention will now be described by way of implementation and with reference to the accompanying drawings described above.

[0187] Experiments and Results

[0188] General Experiment

[0189] All reagents and starting materials were obtained from commercial sources and used directly. All dry solvents were purified using a PureSolv 500MD solvent purification system. Unless otherwise specified, all reactions were carried out under argon atmosphere. Brine was defined as a saturated solution of sodium chloride.

[0190] Flash column chromatography was performed using Fisher Matrix silica 60. A Macherrey-Nagel aluminum backing plate, pre-coated with silica 60 (UV254), was used for thin-layer chromatography and made visible using UV light.

[0191] Record using a Bruker DPX 400 spectrometer or a Bruker 500 spectrometer. 1 H NMR spectra and 13 C10 NMR spectra, chemical shift ppm values ​​relative to tetramethylsilane (δ¹⁺ ...), H 0.00 and δ C 0.0) or residual chloroform (δ H 7.26 and δ C 77.2) as the standard. 1 H and 13 The C assignments are based on two-dimensional COZY and DEPT experiments, respectively.

[0192] Infrared spectra were recorded using a JASCO FTIR 410 spectrometer.

[0193] Mass spectrometry is recorded using electron collision, chemical ionization, or rapid atomic bombardment techniques. HRMS spectra are recorded using a dual-focusing magnetic analyzer mass spectrometer.

[0194] The melting point was determined using a Gallenkamp melting point apparatus.

[0195] The chiral HPLC method was calibrated using the corresponding racemic mixture.

[0196] 3-Methyl-4-phenylquinoline-2-carboxylic acid was prepared using a previously reported method (Stevenson et al.).

[0197] Preparation of compounds

[0198] The compound of formula (I) was prepared according to the scheme shown below. The compound is referred to as LW233.

[0199] Preparation of Scheme 1 - LW233

[0200]

[0201] (R)-(N-sec-butyl)-3-methyl-4-phenylquinoline-2-carboxamide

[0202]

[0203] O-(benzotriazol-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 to anhydrous N,N-dimethylformamide (250 mL) of 3-methyl-4-phenylquinoline-2-carboxylic acid (2.54 g, 9.65 mmol). The reaction mixture was stirred at room temperature for 0.5 h before the addition of (R)-(-)-sec-butylamine (1.10 mL, 10.6 mmol), followed by heating to 40 °C and holding for 4 h. 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 (MgSO4), filtered, and concentrated under vacuum to obtain a brown oil. Purification was performed by rapid column chromatography (petroleum ether / ethyl acetate, 4:1) to give (R)-(N-sec-butyl)-3-methyl-4-phenylquinoline-2-carboxamide (2.81 g, 91%) as a white solid.

[0204] Mp 152-154℃ (lit.mp 157-158℃ - see Cappelli et al. (J. Med. Chem.)); IR (KBr) 3287 (NH), 2968 (CH), 1641 (CO), 1539, 1448, 1157, 761 cm -1 ;[α] D 25 -26.7 (c 1.0, CHCl3); 1H 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 CNMR (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 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 / z319(M+H) + , 100%), 220(19), 202(5), 148(6), 113(16), 85(77); C calculated by HRMS(CI) 21 H 23 N2O(M+H + The value is 319.1810, and the value found is 319.1809.

[0205] (R)-(N-sec-butyl)-N-methyl-3-methyl-4-phenylquinoline-2-carboxamide

[0206]

[0207] Sodium hydride (60% dispersed in mineral oil, 0.710 g, 17.6 mmol) was added to a tetrahydrofuran (176 mL) solution of (R)-(N-sec-butyl)-3-methyl-4-phenylquinoline-2-carboxamide (2.81 g, 8.82 mmol). The mixture was stirred at room temperature for 0.5 h before the addition of iodomethane (2.75 mL, 44.1 mmol). The resulting solution was stirred at room temperature for 3 h 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 10% sodium thiosulfate aqueous solution (10 mL), brine (10 mL), dried (Na₂SO₄), filtered, and concentrated under vacuum. Purification by rapid column chromatography (petroleum ether / ethyl acetate, 3:1) gave (R)-(N-sec-butyl)-N-methyl-3-methyl-4-phenylquinoline-2-carboxamide as a white solid (2.75 g, 94%).

[0208] NMR spectra showed a 1:1 mixture of rotational isomers. Signals for both rotational isomers were recorded. Mp 114–117 °C (lit.mp 117–118 °C – see Capelli et al. (J. Med. Chem.); IR (KBr) 2969 (CH), 1637 (CO), 1466, 1072, 731 cm⁻¹ -1 ;[α] D 23 -6.3 (c 1.0, CHCl3); 1 H 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, CH2CH3), 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); 13C 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), 1 26.8(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 / z333(M+H) + , 100%), 291(48), 250(41), 220(14), 86(23); C calculated by HRMS(CI) 22 H 25 N2O(M+H + The value is 333.1967, and the value found is 333.1972.

[0209] (R)-3-bromomethyl-(N-sec-butyl)-N-methyl-4-phenylquinoline-2-carboxamide

[0210]

[0211] N-bromosuccinimide (2.17 g, 12.2 mmol) and dibenzoyl peroxide (0.20 g, 0.812 mmol) were added to a stirred, 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), and the solution was heated to reflux for 6 h. Then, another portion of N-bromosuccinimide (1.00 g, 5.61 mmol) was added, and the solution was further heated to reflux for 16 h. 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 (MgSO4), filtered, and concentrated under vacuum. Purification was performed by rapid column chromatography using a fractionated eluent of dichloromethane > dichloromethane / ethyl acetate (95:5) to obtain (R)-3-bromomethyl-(N-sec-butyl)-N-methyl-4-phenylquinoline-2-carboxamide as an orange solid (2.76 g, 83%).

[0212] The NMR spectrum showed a 2:1 mixture of rotational isomers. Only the signal of the major rotational isomer was recorded. Mp 160-164℃; IR (KBr) 2970 (CH), 1631 (CO), 1484, 1397, 1046, 766 cm⁻¹ -1 ;[α] D 28 -9.0 (c 1.0, CHCl3); 1 H NMR (400MHz, CDCl3) δ1.09 (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 4.87 (1H, sextet, J = 6.8 Hz, CHCH3), 7.37-7.48 (4H, m, ArH), 7.51-7.59 (3H, m, ArH), 7.70 (1H, ddd, J = 8.3, 6.7, 1.5 Hz, ArH), 8.10 (1H, dd, J = 8.8, 8.3 Hz, ArH); 13CNMR (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 / z410(M) + ,5%),298(15),296(14),217(57),189(28),151(10),86(100);C calculated by HRMS(EI) 22 H 23 79 BrN2O(M + The value was 410.0994, and the value found was 410.0992.

[0213] (R)-(N-sec-butyl)-3-chloromethyl-N-methyl-4-phenylquinoline-2-carboxamide

[0214]

[0215] Lithium chloride (0.160 g, 3.66 mmol) was added to an anhydrous tetrahydrofuran (10 mL) solution of (R)-3-bromomethyl-(N-sec-butyl)-N-methyl-4-phenylquinoline-2-carboxamide (0.500 g, 1.22 mmol), and the reaction mixture was stirred at room temperature for 16 h. The reaction was quenched with water (30 mL) and extracted to ethyl acetate (3 × 30 mL). The organic layer was washed with brine (90 mL), dried (MgSO4), filtered, and concentrated under vacuum. The product was purified by rapid column chromatography (dichloromethane / ethyl acetate, 95:5) to give (R)-3-chloromethyl-(N-sec-butyl)-N-methyl-4-phenylquinoline-2-carboxamide as a white solid (0.327 g, 73%).

[0216] The NMR spectrum showed a 1.5:1 mixture of rotational isomers. Only the signals of the major rotational isomers were recorded. Mp 140-142℃; IR (KBr) 2970 (CH), 1620 (CO), 1481, 1404, 1219, 748 cm⁻¹ -1 ;[α] D 24 -11.6 (c 1.0, CHCl3); 1H 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), 4.6 7(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); 13 C NMR (101MHz, CDCl3) δ11.1(CH3), 17.1(CH3), 26.6(CH2), 30.4(CH3), 40.4(CH2), 50.1(CH), 125.8(C), 126.8(CH), 127.2(CH), 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(EI)mz 389(M+Na + (100%); C calculated by HRMS (ESI) 22 H 23 35 ClN2NaO(M+Na + The value is 389.1391, and the value found is 389.1381.

[0217] LW223-(R)-(N-sec-butyl)-3-fluoromethyl-N-methyl-4-phenylquinoline-2-carboxamide

[0218]

[0219] Potassium fluoride (0.036 g, 0.61 mmol) was added to a solution of 18-crown-6 (0.032 g, 0.12 mmol) in acetonitrile (2.5 mL), and the resulting suspension was stirred at room temperature for 0.5 h. Subsequently, an acetonitrile:dichloromethane (2:1, 9.0 mL) solution of (R)-3-bromomethyl-(N-sec-butyl)-N-methyl-4-phenylquinoline-2-carboxamide (0.050 g, 0.12 mmol) was added dropwise, and the reaction mixture was heated under reflux for 72 h. After the reaction was complete, 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 under vacuum. The (R)-N-(sec-butyl)-3-(fluoromethyl)-N-methyl-4-phenylquinoline-2-carboxamide was purified by rapid column chromatography (petroleum ether / ethyl acetate, 7:3) to obtain a white solid (0.023 g, 53%).

[0220] The NMR spectrum showed a 3:1 mixture of rotational isomers. Only the signals of the major rotational isomers were recorded. Mp 146–148 °C; IR (pure) 2972 ​​(CH), 1628 (CO), 1559, 1485, 1398, 1049, 970 cm⁻¹ -1 ;[α] D 30 -12.6 (c 0.5, CHCl3); 1 H NMR (400MHz, CDCl3) δ1.05 (3H, t, J=7.4Hz, CH2CH3), 1.29 (3H, d, J=6.8Hz, CHCH3), 1.41-1.78 (2H, m, C H2CH3), 2.77 (3H, s, NCH3), 4.84-4.95 (1H, m, NCH), 5.31 (1H, dd, J20.8, 10.8Hz, 3-CHH), 5.44 (1H, dd, J 20.8, 10.8Hz, 3-CHH), 7.31-7.40 (2H, m, ArH), 7.43-7.58 (5H, m, ArH), 7.74 (1H, t, J 7.6Hz, ArH), 8.17 (1H, d, J 8.4Hz, ArH); 13 C NMR (101MHz, CDCl3) δ10.9(CH3), 17.4(CH3), 26.5(CH3), 29.9(CH2), 50.0(CH), 79.2(CH2, 1 J C-F =162.8Hz), 123.0 (C, 2 J C-F =15.1Hz), 127.0(CH), 127.1(C,4 J C-F =2.3Hz), 127.4 (CH, 5 J C-F =1.2Hz), 128.5(2×CH), 128.7(CH), 129.6(2×CH), 129.7(CH), 130.4(CH), 134.8(C, 4 J C-F =1.5Hz), 147.4 (C, 3 J C-F =2.5Hz), 150.8 (C, 3 J C-F =4.7Hz), 156.6 (C, 5 J C-F =2.1Hz), 168.8(C); MS(ESI)m / z373(M+Na + (100%); C calculated by HRMS (ESI) 22 H 23 FN2NaO(M+Na + The value was 373.1687, and the value found was 373.1670.

[0221] Chiralcel AD-H column (hexane: i PrOH 97.5:2.5, flow rate 1.0 mL / min) was analyzed by HPLC to determine the enantiomeric excess. 主要的 = 30.68 and 32.22 minutes, t 次要的 =27.15 and 38.38 minutes; er = 99.5:0.5.

[0222] The compound of formula (II) was prepared according to the scheme shown below. The compound is referred to as... 19 F-LW233.

[0223] Option 2 - 19 Preparation of F-LW233

[0224]

[0225] 18 F-LW223-(R)-(N-sec-butyl)-3-[19-fluoro]-methyl-N-methyl-4-phenylquinoline-2-carboxamide

[0226] Using the commercial synthesizer GE in the presence of potassium carbonate and Kryptofix 222. FX-FN, (R)-(N-sec-butyl)-3-chloromethyl-N-methyl-4-phenylquinoline-2-carboxamide and 18F-fluoride ([ 18 The reaction mixture (F]KF) was reacted at 100℃ for 10 min. The following conditions were used: C18 series column, Hydro-RP. Radiolabeled products were purified by semi-preparative high-performance liquid chromatography (HPLC) using a 150×10 mm, 4 μm column (Phenomenex, UK), acetonitrile / water (70:30 v / v), and a flow rate of 3 mL / min.

[0227] The final product was prepared in physiological saline containing 10% ethanol. After a total synthesis time of 55 minutes, a yield with an average non-decay corrected yield of 35% was obtained (from...). 18 Starting with 15±5 GBq of F-fluoride (n=20), 18 F-LW223. Identified by HLPC analysis at the end of the synthesis. 18 F-LW223's identity, radiochemical purity (>99%), and specific radioactivity (410-810 GBq / μmol; 11-22 Ci / μmol).

[0228] Biological tests

[0229] In vitro competition and saturation binding assays with human tissues

[0230] All studies using human tissue were conducted in accordance with the East of Scotland Research Ethics Service (Edinburgh Brain Bank, 11 / ES / 0022). Samples were obtained from 51 brains (78% male, age 53.9 ± 9.5) and 29 hearts (83% female, age 48.2 ± 13.2), and were screened for rs6971 genetic polymorphism and categorized as high-affinity binders (HAB), mixed-affinity binders (MAB), or low-affinity binders (LAB) as previously described (Owen et al. J. Cereb. Blood FloW Metab. 2012). To exclude sex-dependent differences in TSPO binding (Fairweather et al.), only male samples were used in this study. Before centrifugation (32000g, 10 min, 4℃), tissue samples were homogenized in 10x w / v buffer (50 mM Tris-Base, pH 7.4, 4℃). Tissue pellets were then resuspended in 10x w / v buffer and centrifuged again before resuspending in buffer (2 mL). Protein concentration was then assessed using a Bio-Rad protein assay (Bio-Rad, USA), aliquoted, and stored at -80℃ until use.

[0231] A competitive binding assay, as previously reported (Blair et al. (Med. Chem. Commun.)), was performed. Briefly, each sample was prepared at 250 μg protein / mL in buffer solution, and then 200 μL of this solution along with 10 μL of our test ligands PK11195 (Sigma-Aldrich, USA), PBR28 (ABX, Germany), AB5186, or 14 different concentrations of LW223 (range 0.001–3,000 nM) were added to 100 μL of 1 nM buffer. 3H-PK11195 (PerkinElmer, USA) was incubated at 4°C for 90 min. Non-specific binding of each ligand was determined using a concentration of 8 μM. Binding was terminated by adding 2 mL of ice-cold buffer, followed by immediate filtration using a Brandel harvester (Braudel, USA) through a Waterman GF / B filter (Whatman, UK) pretreated with 0.3% polyethyleneimine (Sigma-Aldrich, USA). The filter paper was then removed and placed in 2.5 mL of Optiphase HiSafe 3 (PerkinElmer, USA) and counted on Hidex 300SL (Hidex, Finland) for 48 hours. Saturation assays were performed using a similar protocol to determine the Kelvin of PK11195. d The difference lies in using 6 concentrations. 3 H-PK11195 (1.6–200 nM) was used in conjunction with 10 μM PK11195 to determine nonspecific binding. All binding assays were performed in triplicate.

[0232] All binding affinity curves were fitted using GraphPad Prism version 6 (GraphPad Software, USA). The least squares algorithm was used to compare single-point and two-site fits, and the F-test was used to compare model selection. If p < 0.05, the null hypothesis was rejected (single-point fit was considered more appropriate). The mean SB% of minimum suppression normalized for each group (HAB, MAB, or LAB) was used to determine whether a single-point or two-site fit was appropriate, and subsequently used to calculate the LAB:HAB ratio. Individual tissue samples were fitted using mean-based group fitting to calculate affinity values ​​(K0). i Based on the saturation binding curve results, PK11195K at 13.95 nM was used. d value.

[0233] Commentary

[0234] Competition binding assays were used to calculate the established TSPO ligands PK11195 and PBR28, as well as our ligands AB5186 and LW223 (e.g. Figure 1 Affinity (K) shown i To assess their sensitivity to genetic polymorphism, a saturation test was performed using our experimental conditions to determine the K0 of PK11195. d PK11195K d It is 13.95 nM (see Figure 7 ).

[0235] In competitive binding experiments using human brains, PK11195 was unaffected by genetic polymorphism, and the LAB:HAB affinity ratio was 1 ( Figure 2 (a)), the ratio of PBR28 to PBR28 is 49 ( Figure 2 (b)). The ligand AB5186 is affected by genetic polymorphism, with a ratio of 9 ( Figure 2 (b)), unlike the unaffected LW223, its ratio was 1. In the PK111195 and LW223 combination study, the affinity values ​​of all brain samples calculated individually showed no significant difference between HAB and LAB (respectively). Figure 2 (e) and (h)H). The average affinity of LW223 was 0.6 nM, which was twice that of PK11195. Comparison of the affinity values ​​of PBR28 and AB5186 calculated separately in HAB and LAB showed significant differences between the groups. Figure 2 (f) and (g)). The MAB group in PBR28 and AB5186 is suitable for two-site fitting, while all other experiments are better suited for single-point fitting.

[0236] Similar to its effect on the brain, PK11195 is not affected by cardiac genetic polymorphisms, with a LAB:HAB ratio of 1 ( Figure 3 (a)). PBR28 and AB5186 were affected to similar degrees in the heart and brain, with ratios of 48 and 7, respectively (respectively). Figure 3 (b) and (c)). LW223 is unaffected by cardiac polymorphism, with a ratio of 1 ( Figure 3 (d)). In the combined PK111195 and LW223 study, the affinity values ​​of all cardiac samples calculated individually demonstrated no significant difference between HAB and LAB (respectively). Figure 3 (e) and (h)). In the heart, the mean affinity of LW223 was the same as that of PK11195 at 1.7 nM. Furthermore, only the MAB group in the AB5186 experiment was a good fit for a two-site fit.

[0237] Previous in vivo imaging evidence has shown that 11The binding of C-PK11195 is sensitive to polymorphisms in peripheral organs such as the heart and lungs, but not in the brain (Kreisl et al.). It has been argued that the sensitivity to PK11195 polymorphisms in the brain cannot be imaged due to the low signal-to-noise ratio, lower brain uptake compared to the heart and lungs, and the inevitably limited number of subjects due to the low prevalence of LAB (Kobayashi et al.). However, using sensitive in vitro competitive binding assays, brain uptake levels are no longer an issue. In this study, it was demonstrated that the brain and heart have different affinities for LW223 (0.6 and 1.2 nM, respectively), while the affinity for PK11195 is the same (1.2 nM). Improved affinity for LW223 in the brain would provide an advantage when targeting neuroinflammatory conditions.

[0238] Animals and Surgery

[0239] All experiments were conducted in accordance with the local University of Edinburgh Animal Ethics Committee and approved by the Home Office under the Animals (Scientific Procedures) Act 1986. Twenty-eight adult male Sprague-Dawley rats (357.1 ± 8.1 g and 10.2 ± 0.4 weeks old) and two C57bl / 6 rats (25.3 ± 3.7 g and 10.1 ± 0.0 weeks old) were used in this study, with two C57bl / 6 rats (26.0 ± 5.3 g and 13.5 ± 5.7 weeks old) used for additional research. Animals were housed under standard 12-hour light:12-hour darkness conditions, with food and water provided at all times. On the day of the experiments, anesthesia was induced and maintained using 1.5–2.5% isoflurane (50 / 50 oxygen / nitrous oxide, 1 L / min). For imaging experiments, as previously described (Wamock et al.), intravenous (iv) lines were established in the femoral or tail vein for the injection of radioactive tracers, and the femoral artery was cannulated to allow for automated blood sampling.

[0240] In another set of experiments (radiometabolite studies), femoral artery cannulation was used for blood sampling, and a radioactive tracer was administered via intravenous injection into 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 using a stereomicroscope and securely fixed with a bandage (6-0 suture). The catheter was then secured in place with surgical adhesive. Body temperature was maintained using a heated scanning bed or heating pad and monitored via rectal thermometer. Vital signs, including heart rate and respiratory rate, were continuously monitored throughout the experiment.

[0241] PET study

[0242] Research Design:

[0243] use 18 F-LW223 underwent 13 PET scans (mean injected radioactivity was 19.55 ± 2.1 MBq). The drug was administered via intravenous bolus injection into rats through a tail vein or femoral vein shunt. 18 Scans were performed following F-LW223 (an invasive kinetic model experiment using a β-probe automated blood sampler). Test-retest scans were performed in three rats following intravenous bolus injection of the radiotracer, with a two-week interval between the test and retest imaging periods. Rats received intravenous injections... 18 For F-LW223, blockade studies were conducted 30 minutes prior to administration of PK11195 (1 mg / kg) intravenously. These scans were compared to baseline scans without PK11195. For replacement studies, rats received intravenous administration of PK11195. 18 F-LW223 was administered, followed by continuous scanning for 120 minutes. Sixty minutes later, a single dose of PK11195 (1 mg / kg) was administered. Initially, one male and one female mouse were used, and dynamic scanning was performed over 4 hours to obtain... 18 The dose estimate for F-LW223 was subsequently used in additional studies using mice (in a study with a mean injection of radioactivity of 10.83 ± 5.6 MBq).

[0244] Artery input function using a β-probe automated blood sampler

[0245] As previously described (Warnock et al.), a commercially available β-probe system (Twilite2, Swisstrace, Switzerland) was used to measure blood radioactivity. This system allows for whole blood arterial input function measurements with a time resolution of 1 second and avoids blood loss due to surgically induced arteriovenous shunts. Using data obtained in a separate study (radioactive metabolism experiments), the whole blood arterial input function, measured by an automated blood sampler, was corrected for the plasma-to-whole blood ratio and in vivo metabolism.

[0246] Image acquisition and reconstruction

[0247] All PET data were acquired using a preclinical PET / CT small animal scanner (nanoPET / CT, Mediso, Hungary). CT scans for attenuation correction were obtained (semi-circular full trajectory, maximum field of view, 480 projections, 50 kVp, 300 ms, and 1:4 binning). Immediately after radiotracer administration, 120 minutes of radiation scans were acquired in 3D 1:5 mode and re-binded as follows: 18 × 10 s; 2 × 30 s; 1 × 60 s; 2 × 2 min; 10 × 5 min; 6 × 10 min. PET images were reconstructed using Mediso's iterative Tera-Tomo 3D reconstruction algorithm with the following settings: 4 iterations, 6 subsets, full detector model, low regularization, narrowband filter enabled, stereo pixel size of 0.4 mm, and 400–600 keV energy window. PET data were then subjected to randomization, scattering, and attenuation correction.

[0248] Image processing and data analysis

[0249] Reconstructed scans were imported into PMOD 3.8 software (PMOD Technologies, Switzerland). Volumes of interest (VOI) were manually plotted around the organ of interest. Time-activity curves (TACs) were generated, and standardized uptake values ​​(SUVs) were calculated by dividing the VOI concentration by the injection dose and then by the animal's body weight. Kinetic modeling was performed using compartmental analysis (1-tissue (1T) and 2-tissue (2T) models) and graphical analysis (Logan plots and Ichise multivariate analysis) to estimate the volume of distribution (VT) in different tissues (Innis et al.; Logan; and Ichise et al.). Model fit performance was evaluated using the Akaike information criterion (AIC) and model selection criterion (MSC), with the preferred model having the lowest AIC and the highest MSC. The selected identifiability criterion was V. T The percentage standard error (%SE) of the estimates was calculated. Dynamic modeling was also performed using graphical analysis to estimate the distribution volume ratio (DVR) in tissue regions and organs, with the reference region VOI being the blood pool VOI of the left ventricle (Logan et al.).

[0250] The test-retest reproducibility of a DVR is calculated by dividing the absolute value of the average measurement by the standard deviation: ABS(average test-retest) / SD(test / retest).

[0251] Dosimetry

[0252] The reconstructed whole-body PET scans were imported into PMOD 3.8 software (PMOD Technologies, Switzerland), and VOIs were plotted around organs exhibiting higher radiation concentrations than the background (i.e., source organs). The following organs were defined as source organs: brain, heart, lungs, gallbladder, liver, intestines, adrenal glands, kidneys, and bladder. Whole-body VOIs were plotted around the animal and used to quantify systemic residual activity, i.e., systemic activity minus source organ activity. At each time point, the measured activity of the source organ was expressed as a percentage of the injected dose (%ID).

[0253] Residence time τ is defined as the cumulative activity in the target organ. The ratio to injectable activity (A0); The area under the tissue time-activity curve, normalized to %ID from time zero to infinity, was used for calculation. A trapezoidal method was employed to estimate τ, and it was assumed that the radioactive tracer underwent only physical decay and was not biologically expelled from the source organ after the last measurement time point. τ calculated for the organ weight difference between mice and humans was normalized to a percentage of total weight (based on known data from Khanuja et al.; Bielohuby et al.; Stabin et al.; Hindorf et al.; and Hui et al.) and then entered into OLIDA / EXM 1.0 software, which is used to estimate organ dose and effective dose according to the male or female model applied in OLIDA / EXM 1.0.

[0254] Commentary

[0255] In mice and rats, after intravenous bolus injection 18 F-LW223 rapidly distributes to TSPO-expressing tissues, including the brain, heart, lungs, and adrenal glands (see [link to relevant documentation]). Figure 4 (a) and (b)); and is excreted via urine and hepatobiliary excretion. Post-peak up, the brain and lungs excrete radiotracers faster than the heart (see […]). Figure 4 (c) and (d)).

[0256] 120 minutes after injection, the rat arterial blood... 18 F-LW223 has a slow radioactive metabolism, with approximately 70% of the parent compound being metabolized. Figure 4(e)). At 60 and 120 minutes after administration, the levels of radioactive metabolites in the brain, heart, and lungs were less than 10%. The parent free fraction in plasma was 38.5 ± 7.0% (mean ± SEM, n = XX).

[0257] 18 The in vivo kinetics of F-LW223 are reversible and better fit / describe the 2-tissue compartment model and graphical Logan plots (t). * =30 minutes) and Ichise multivariate analysis (t * =30 minutes) (see) Figure 4 (f)). The graphical method outperforms the atrioventricular 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 distribution volume ratios (DVRs) in the brain, heart, and lungs relative to the blood pool were 0.53±0.06, 2.96±0.14, and 1.84±0.13, respectively (mean ± SEM, n=3). When DVRs were used as the outcome measure, test-retest analysis showed good agreement between measurements and inter-subject variability of 16% in the brain, 2% in the heart, and 15% in the lungs.

[0258] During injection 1 Prior to the administration of PK11195 to wF-LW223, in vivo targeted binding was demonstrated (see...). Figure 5 Compared to baseline scans, the SUV values ​​measured after blockade were reduced by 64-81%. The measured VTs after blockade in the brain, heart, and lungs 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 in the brain, heart, and lungs, respectively. The same dose of PK11195 was able to reduce SUV values ​​in the brain, heart, and lungs at equilibrium time points. 18 F-LW223 replaced 29%, 52%, and 40% of the target site (see [link to relevant documentation]). Figure 8 Pharmacokinetic / pharmacodynamic studies of PK11195 in rats over 1 hour showed that the exposure level in the brain was 11.01 ± 1.99 ng / mL, while in the heart and lungs it was 29.31 ± 3.93 ng / mL and 38.86 ± 2.24 ng / mL, respectively (mean ± SEM, n = 3). This is consistent with the blockade and substitution measured in PET experiments, and the difference in % target binding between the brain and peripheral organs is consistent with the K+ levels of LW223 and PK11195 measured in vitro. i Consistent.

[0259] The systemic effective time-activity curves show that the highest peak % of the injected dose occurs in the large intestine, followed by the liver, lungs, kidneys, heart, brain, gallbladder, bladder, and adrenal glands (see [link to relevant data]). Figure 9 Using normalized tau values ​​and dosimetric estimates from male and female adult human phantoms, the adrenal gland was identified as the critical organ. The estimated whole-body effective dose for the male and female phantoms was 15.3 μSv / MBq and 18.4 μSv / MBq, respectively (Table 1).

[0260] Further studies on mice revealed that the key organ was the lower intestinal wall. The estimated systemic effective dose for male and female phantoms was 20.5 μSv / MBq and 23.7 μSv / MBq, respectively (Table 2).

[0261] Table 1 - Estimated absorbed dose in selected target organs

[0262]

[0263]

[0264] Table 2 - Estimated absorbed dose in selected target organs

[0265]

[0266]

[0267] Radioactive metabolites and arterial blood processing and analysis

[0268] Arterial blood samples (69.8 ± 9.2 MBq, n = 17 rats) were collected at 2, 5, 10, 20, 30, 60, and 120 minutes after administration of the radioactive tracer. All blood samples were 1 mL in volume and were collected manually from different animals to generate population curves, thus adhering to the total blood volume limit of terminal arterial blood in rats. After collection, all samples were stored on ice until analysis. Radioactivity in whole blood and plasma was assessed using a well-type gamma counter with a 400–1400 keV window (PerkinElmer Wizzard2, USA). Plasma samples (400 μL) were denatured with acetonitrile and analyzed by HPLC (Ultimate2000, Thermo Fisher Scientific, UK) on a Luna C18(2) column (Luna C18(2), 10 × 250 mm, 10 μm, Pheromon, UK) at a flow rate of 4 mL / min to estimate the parent fraction. Ultrafiltration units were used. Millipore (UK) measures the fraction of free plasma proteins bound (f) p ).

[0269] 18 F-LW223 Human Autoradiography

[0270] Human deep stroke tissue and diseased coronary artery tissue sections embedded in grade 1-2 paraffin were dewaxed, rehydrated, and cultured in buffer solution (50 mM Tris-Base, pH 7.4) for 30 minutes. Then, in the presence or absence of PK11195 (30 μM in stroke tissue, 10 μM in coronary tissue) or LW223 (10 μM, nonspecific binding group), 2 MBq / mL of [a specific antibiotic / conjugate] was used. 18 F-LW223 (total binding group) was cultured. Slides were washed twice with buffer solution and exposed to a Fujifilm BAS-IP MS 2040 fluorescent screen (Fujifilm, Japan) before air drying. The fluorescent screen was imaged on a Fujifilm FLA5100 imaging plate reader (Fujifilm, Japan). The total region of interest was plotted around the entire tissue section to calculate %SB. To calculate the targeted vs. untargeted ratio, regional sampling analysis was used within the same tissue at the highest and lowest uptake areas.

[0271] Commentary

[0272] Autoradiography for imaging 18 F-LW223 binds to isolated human diseased tissue. Histological staining of the tissue showed that the hemorrhagic areas in deep stroke samples contained migratory inflammatory cells (see...). Figure 6 (a) In the diseased coronary vessels of patients with sudden cardiac death, there are obvious areas of pathological remodeling (e.g., neointimal hyperplasia) containing infiltrative inflammatory cells. Figure 6 (b)). Stroke 18 F-LW223 binding was highest in the hemorrhage area, with some uptake in more distant areas. Figure 6 (c)). In diseased coronary vessels, 18 F-LW223 binding was highest in areas of pathological remodeling. Figure 6 (d) PK11195 was not sufficiently blocking in deep brain tissue samples. 18 The combination of F-LW223 ( Figure 6 (e)), however, it had a specific binding rate of 75.3 ± 3.2% in the coronary arteries (%SB). Figure 6 (f)). The LW223 segment in the stroke sample reached 42.5 ± 7.0% SB ( Figure 6 (g)), reaching 88.1±3.4% SB in the coronary arteries ( Figure 6(h)). Importantly, high targeting rates were achieved in both stroke (7.5) and diseased coronary artery (4.5) tissues: the non-targeting rate ( Figure 6 (i) and (j)). Compared to the healthy human brain, there are no other high-uptake areas except for some non-specific binding in white matter (see [i]). Figure 10 ).

[0273] General statistical analysis

[0274] Graphpad Prism version 6 (GrafPad Software, USA) was used for all fitting, statistical analysis, and graph generation. In the competitive binding and saturation experiments, outliers in the three experimental groups were removed using the Grubbs test with alpha of 0.2. As shown in the relevant legends, unpaired and paired t-tests were used to compare the two groups, and p < 0.05 was considered statistically significant. Unless otherwise stated in the legends of the figures or tables, all error bars represent the standard error mean (SEM).

[0275] Commentary

[0276] Recently, for the first time, a detailed description of having and not having Thr was published. 148 A study of the crystal structure of polymorphic TSPO (Li et al.). In this study, the authors demonstrated that binding to PK11195 stabilizes the binding pocket, however, polymorphism has a significant impact on the cholesterol binding site. This evidence suggests that ligands binding to cholesterol sites may exhibit greater sensitivity to polymorphism. Despite various binding studies, three main binding pockets are known for TSPO binding sites: benzodiazepine sites, isoquinoline sites, and cholesterol sites (Li et al.; Lin et al.; and Luus et al.). Since LW223 is insensitive to polymorphisms in the brain and heart and is structurally similar to PK11195, we hypothesized that LW223 targets the isoquinoline site rather than cholesterol. However, the difference in overall affinity values ​​of LW223 in the brain and heart suggests that the TSPO isoquinoline binding pocket undergoes organ-specific conformational changes across different sites.

[0277] As the first fluorinated TSPO ligand insensitive to the rs6971 polymorphism, 18 The unique feature of F-LW223 is that it exists as a rotational isomer. This is evident from the two peaks present on HPLC and confirmed by chiral HPLC. This unusual characteristic may be... 18The reason behind F-LW223's excellent kinetic and metabolic properties is that, despite being administered via intravenous bolus, it exhibits characteristics similar to those of infusion administration. 18 Another unique aspect of F-LW223 is its high plasma free fraction of 38.5%, while 11 The free fraction of C-PK11195 in humans is 1-6% (Owen et al.; Endres et al.). Clearly, further research is needed to determine this unique aspect and its contribution to favorable kinetics and metabolic profiles. Although these characteristics do not preclude… 18 F-LW223 has entered clinical trials, but other criteria must still be met. First, toxicological testing is required to determine the safety of this radiotracer. It should be noted that administration into the body... 18 No negative effects were observed after F-LW223. Secondly, the dosimetric estimates presented in this study did not imply any radiation safety issues.

[0278] The limitations of this study are similar to those faced by most research developing novel TSPO radiotracers. First, the prevalence of LAB in this study was approximately 14%, which, while higher than reported in other studies (Fujita et al.), limited the samples available for testing. However, this study found that the LAB:HAB ratio of PK11195 and PBR28 in the brain was similar to previously reported values ​​(Owen et al. J. Cereb. Blood Flow Metab. 2012; Owen et al.; Owen et al. J. Cereb. Blood Flow Metab.), and that PBR28 and AB5186 showed statistically significant differences between LAB and HAB. Some controversy has also arisen in the area where in vitro binding studies do not reflect in vivo binding studies. TSPO radiotracers 11 C-ER176 exhibited a LAB:HAB ratio of 1.3 in vitro (Zanotti-Fregonara et al.); however, subsequent in vivo studies found the ratio to be approximately 3 (Ikawa et al.). Nevertheless, even with this slight polymorphism, it has been reported that... 11 C-ER176 still possesses the ability to perform imaging lab (LAB), which is not possible with other ligands. If the same phenomenon also applies... 18 F-LW223, this radiotracer, still benefits from fluorination, thus enabling its broader clinical applications. One of the ultimate limitations of this study is that it was conducted in rodents, which typically exhibit faster kinetics and metabolism than non-human primates and humans. However, even with this caveat, this study...18 The kinetics and metabolic profile of F-LW223 also suggest that these aspects will not be a problem when it enters clinical trials.

[0279] Given the roles of inflammation, phagocytes such as macrophages, and microglia in a range of pathologies, the development of successful TSPO imaging methods holds great promise. In this study, we demonstrate... 18 In vitro uptake of F-LW223 is evident in areas of pathology and inflammation-driven events. Previously, despite limitations, 11 C-PK11195 has been successfully used to demonstrate increased inflammation in neurological diseases. For example, it has been shown to increase inflammation in patients with Alzheimer's disease and progressive supranuclear palsy compared to controls. 11 Increased uptake of C-PK11195 (Calsolaro et al.). Areas of increased uptake correlated 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 study atherosclerotic plaque inflammation. In in vitro studies, it was found... 11 C-PK11195 binding is associated with macrophage-rich areas (Berd et al.). In in vivo clinical studies, plaque-bound... 11 C-PK11195 uptake can distinguish between recent symptomatic and asymptomatic lesions (Gaemperli et al.). These positive clinical studies, in addition to the novel role of TSPO as a therapeutic target (Schalle et al.; and Paradis et al.), highlight the potential of successful TSPO radiotracers as research tools, diagnostic agents, and therapeutic companion imaging agents.

[0280] all in all, 18 F-LW223 is insensitive to the rs6971 genetic polymorphism. Its long radiolabeled half-life and favorable kinetics make this novel TSPO radiotracer a promising breakthrough in the field of TSPO imaging, and it deserves further clinical application.

[0281] Additional experiments and results

[0282] In a rat model of myocardial infarction 18 F-LW223 combined

[0283] Rats underwent permanent ligation of the left anterior descending coronary artery to induce myocardial occlusion, and were then treated with [treatment] 7 days later. 18 Imaging was performed using F-LW223 PET / CT (see...) Figure 11 Young rats were used as healthy controls for comparison. Quantification of the standard uptake value (SUV) relative to the blood pool (measured in the left ventricle) confirmed its presence in the heart, brain, and lungs. 18 Increased binding to F-LW223. This suggests that myocardial infarction mediates increased TSPO expression in these organs, which could also be explained by increased systemic inflammation.

[0284] References

[0285] All references cited in this specification are incorporated herein by reference in their entirety.

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Claims

1. A compound of formula (I): And its salt and radioactive labeling forms.

2. The compound according to claim 1, wherein it is a compound of formula (II): And its salt.

3. A composition comprising a compound of formula (I) according to claim 1 or 2 and one or more pharmaceutically acceptable excipients.

4. A complex comprising a compound according to claim 1 or 2 non-covalently bonded to TSPO.

5. A method for preparing the complex according to claim 4, the method comprising the step of contacting the compound according to claim 1 or 2 with TSPO.

6. Use of the compound of claim 1 or 2 in the preparation of a complex for the detection of TSPO by a method comprising the step of forming a complex comprising the compound of claim 1 or 2 non-covalently bound to TSPO, and the method comprising the step of detecting the compound.

7. A method for preparing a compound of formula (I) according to claim 1, the method comprising the step of substituting bromine of a compound of formula (III) with fluorine:

8. A method for preparing a compound of formula (II) according to claim 2, the method comprising the step of substituting the chlorine of the compound of formula (IV) with 18-fluoro:

9. The method according to claim 8, wherein the compound of formula (IV) is prepared from the compound of formula (III) by substituting bromine in the compound of formula (III) with chlorine:

10. A compound of formula (IV): And its salt and radioactive labeling forms.

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