WO2015040087A1 - Macrophage imaging - Google Patents

Abstract

The present invention concerns in vivo imaging and in particular in vivo imaging of activated macrophages. An indole-based in vivo imaging agent labelled with ¹⁸F is provided that binds with high affinity to translocator protein (TSPO). Other aspects of the invention include a method of diagnosis and a method of therapy monitoring which comprise the imaging method. In addition, the invention provides the use of the TSPO imaging agent in the methods of the invention.

Description

Macrophage Imaging.

Technical Field of the Invention.

The present invention concerns in vivo imaging and in particular in vivo imaging of activated macrophages. An indole -based in vivo imaging agent labelled with ¹⁸F is provided that binds with high affinity to translocator protein (TSPO). Other aspects of the invention include a method of diagnosis and a method of therapy monitoring which comprise the imaging method. In addition, the invention provides the use of the TSPO imaging agent in the methods of the invention.

Description of Related Art.

The mitochondrial 18 kDa translocator protein or TSPO, previously known as the peripheral benzodiazepine receptor (PBR), is known to be mainly localised in peripheral tissues and glial cells. Its' physiological function remains to be clearly elucidated.

Subcellularly, TSPO is known to localise on the outer mitochondrial membrane, indicating a potential role in the modulation of mitochondrial function and in the immune system. It has furthermore been postulated that TSPO is involved in cell proliferation, steroidogenesis, calcium flow and cellular respiration. Abnormal TSPO expression has been associated with inflammatory disease states of the central nervous system.

Rupture of atherosclerotic plaque with consequent formation of thrombi is an important risk factor for myocardial and cerebral infarction. Intra-plaque inflammation plays an important role in the progression and destabilisation of atherosclerotic plaques. Such plaques are characterised by a high degree of macrophage infiltration. The expression of TSPO is upregulated in the mitochondria of activated macrophages.

A number of TSPO ligands have been developed and PK1 1195 has been extensively studied. For in vivo imaging [ⁿC]-PKl 1195 demonstrates a low signal-to-noise ratio and high levels of non-specific binding:

Positron emission tomography (PET) imaging using the TSPO selective ligand, (R)- [ⁿC]PKl 1195 provides a generic indicator of central nervous system (CNS)

inflammation. However, (i?)-[ⁿC]PKl 1 195 is known to have high protein binding, and low specific to non-specific binding. Furthermore, the role of its radiolabelled metabolites is not known, and quantification of binding requires complex modelling.

Van der Laken et al [Arth.Rheum., 58(11), 3350-3355 (2008)] disclose that

[ⁿC]PK11 195 can be used to image macrophages in rheumatoid arthritis using PET. Fujima et al provided an autoradiography study of [ H]-PK1 1195 uptake in TSPO receptors of macrophages in patients with arterial atherosclerotic plaque [Atherosclerosis, 201 , 108-11 1 (2008)]. They concluded that macrophage and inflammatory activity in atherosclerotic plaque can be imaged via binding to TSPO.

Hatori et al [PLOS ONE, 7(9), article e45065 (2012)] disclose that the TSPO radiotracer

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[ F]FEDAC can be used to image lung inflammation using PET. They noted that, unlike prior art reports that [ⁿC]PKl 1195 images macrophage activation in COPD patients but not neutrophils, the [¹⁸F]FEDAC images had signals from both neutrophils and macrophages. Hatori et al suggested that may be due to their model of early phase lung inflammation, in which macrophage activation was slow. WO 2010/109007 discloses TSPO imaging agents of formula:

wherein:

R¹ is Ci-3 alkyl or C_1-3 fluoroalkyl;

R² is H, OH, Hal, cyano, C_1-3 alkyl, C_1-3 alkoxy, C_1-3 fluoroalkyl, or C_1-3 fluoroalkoxy;

R' and R are independently C_1-3 alkyl, C_7-10 aralkyl, or R and R , together with the nitrogen to which they are attached, form a nitrogen-containing C4-6 aliphatic ring optionally comprising 1 further heteroatom selected from nitrogen, oxygen and sulfur;

Y¹ is O, S, SO, S0₂ or CH₂; and,

Y² is CH₂, CH₂-CH₂, CH(CH₃)-CH₂ or CH₂-CH₂-CH₂;

and wherein Formula I as defined comprises an atom which is a radioisotope suitable for in vivo imaging.

WO 2010/109007 discloses that the TSPO agents are potentially useful for imaging abnormal TSPO expression, and that encompasses a wide range of disease states, including:

(i) CNS inflammatory diseases: multiple sclerosis; cerebral vasculitis; herpes encephalitis, and AIDS -associated dementia;

(ii) CNS degenerative diseases: Parkinson's disease; corticobasal degeneration;

progressive supranuclear palsy; multiple system atrophy; amyotrophic lateral sclerosis and Alzheimer's disease;

(iii) CNS ischaemic conditions: ischemic stroke; peripheral nerve injury; epilepsy; traumatic brain injury and glioma borders;

(iv) peripheral conditions: lung inflammation; chronic obstructive pulmonary disease; asthma; inflammatory bowel disease; rheumatoid arthritis; primary fibromyalgia; nerve injury; atherosclerosis; colon, prostate and breast cancer; kidney inflammation, and ischemia-reperfusion injury;

where CNS = central nervous system.

Examples 15 and 16 of WO 2010/109007 provide PBR affinity and in vivo

biodistribution data.

Wadsworth et al [Bioorg. Med. Chem., 22, 1308-1313 (2012)] discloses the synthesis and TSPO binding of the TSPO imaging agents of WO 2010/109007.

Bird et al [Circulation, 124, (21 SuppL), Abstract 12854 (2011)] disclose that the TSPO ligand DPA-713 co-localised with macrophages in an animal model of atherosclerosis. They also report that DPA-713 exhibits much reduced non-specific binding (3% of total binding) compared to prior art TSPO ligands (typically greater than 50%).

WO 2013/138612 discloses optical dye conjugates of the TSPO ligand DPA-713 for imaging inflammation.

There is still a need for alternative imaging agents of macrophage accumulation in vivo.

Summary of the Invention.

The present invention provides TSPO imaging agents for in vivo imaging of

macrophages, especially macrophage accumulation. That is particularly useful for imaging macrophage accumulation in atherosclerosis, but also rheumatoid arthritis; osteo arthritis; pulmonary inflammation; liver inflammation; kidney inflammation; bowel inflammation, COPD (Chronic Obstructive Pulmonary Disease) and cancer.

The agents of the present invention exhibit selective TSPO binding, with higher affinity for TSPO and lower non-specific binding compared to PK1 1195. The agents of the present invention are labelled with ¹⁸F, which is more suitable for routine clinical use due to the longer half-life of ¹⁸F (110 min) compared to ⁿC (20 min) - meaning that ⁿC radiotracers require an onsite cyclotron at the clinical facility. The radiotracers of the present invention are also amenable to automated radio synthesis.

Since intra-plaque inflammation is characterised by macrophage infiltration, the methods of the present invention are expected to give information about the plaque vulnerability to rupture. Such functional information is not available from structural imaging techniques, such as CT, MRI and ultrasound. Detailed Description of the Invention.

In a first aspect, the present invention provides a method of in vivo imaging to determine the distribution of activated macrophages in a subject, said method comprising:

(i) provision of a subject to which a translocator protein (TSPO) ¹⁸F imaging agent had been previously administered;

(ii) detecting by an in vivo imaging procedure the radioactive emissions from the ¹⁸F radioisotope of the administered imaging agent of step (i);

(iii) generating an image representative of the location and/or amount of said radioactive emissions;

where said TSPO ¹⁸F imaging agent is of Formula la:

wherein:

R^2a is H, Hal or C_1-3 alkoxy;

R^Ja and R^4a are independently methyl, ethyl or benzyl, or together with the nitrogen to which they are attached form a pyrrolidinyl, piperidinyl, azepanyl, or morpholinyl ring;

Y^2a is -CH₂-, -CH₂-CH₂-, -CH(CH₃)-CH₂-, or -CH₂-CH₂-CH₂-; and;

n is 1 , 2 or 3.

An "in vivo imaging agent" in the context of the present invention is a radiolabelled compound suitable for in vivo imaging. The term "in vivo imaging" as used herein refers to those techniques that non-invasively produce images of all or part of the internal aspect of a subject.

"Administering" the in vivo imaging agent is carried out prior to imaging, and is preferably carried out parenterally, and most preferably intravenously. The intravenous route represents the most efficient way to deliver the in vivo imaging agent throughout the body of the subject. Furthermore, intravenous administration does not represent a substantial physical intervention or a substantial health risk. The in vivo imaging agent of the invention is preferably administered as a pharmaceutical composition.

The "detecting" step of the method of the invention involves detection of signals emitted by the ¹⁸F radioisotope by means of a detector sensitive to said signals. This detection step can also be understood as the acquisition of signal data. Positron-emission tomography (PET) is the most suitable in vivo imaging procedure for use in the method of the invention.

The "generating" step of the method of the invention is carried out by a computer which applies a reconstruction algorithm to the acquired signal data to yield a dataset. This dataset is then manipulated to generate images showing the location and/or amount of signals emitted by said radioisotope.

The "subject" of the invention can be any human or animal subject. Preferably the subject of the invention is a mammal. Most preferably, said subject is an intact mammalian body in vivo. In an especially preferred embodiment, the subject of the invention is a human.

If a chiral centre or another form of an isomeric centre is present in an in vivo imaging agent according to the present invention, all forms of such isomer, including enantiomers and diastereoisomers, are encompassed by the present invention. In vivo imaging agents of the invention containing a chiral centre may be used as racemic mixture or as an enantiomerically-enriched mixture, or the racemic mixture may be separated using well- known techniques and an individual enantiomer may be used alone.

Preferred Embodiments.

A preferred in vivo imaging procedure of the invention is positron emission tomography (PET). The preference for PET in the method of the invention is due to its excellent sensitivity and resolution, so that even relatively small changes in a lesion can be observed over time. PET scanners routinely measure radioactivity concentrations in the picomolar range. Micro-PET scanners now approach a spatial resolution of about 1 mm, and clinical scanners about 4-5 mm. A more preferred imaging technique is the combination of the (functional) PET image with that from an anatomical imaging modality such as CT or MRI, i.e. PET-CT or PET-MRI.

Non-limiting examples of in vivo imaging agents of Formula la are as follows:

Of the z^'« vz^'vo imaging agents 1-11 above, in vivo imaging agents 5, 6, 7, 9, 10 and 1 1 are preferred, in vivo imaging agents 5 and 10 are most preferred, and in vivo imaging agent 5 is especially preferred. For any in vivo imaging agent of the present invention, the enantiomerically pure form is particularly preferred. For imaging agent 5, the preferred enantiomer is imaging agent 5A as follows:

This is obtained by radiofiuorination of the appropriate chiral precursor.

Imaging agents of the present invention can be synthesised and radiolabelled as described by Wadsworth et al [Bioorg.Med.Chem.Let , 22(3), 1308-1313 (2012); including supplementary data thereto] and WO 2010/109007. Starting compounds and

intermediates are available commercially or are known from published scientific papers, e.g. Napper et al [J.Med.Chem., 48: 8045-8054 (2005)] and Davies et al [J.Med.Chem., 4J_: 451-467 (1998)].

In a second aspect, the present invention provides a method of diagnosis of a site of activated macrophage accumulation in vivo which comprises the method of imaging of the first aspect.

Preferred embodiments of the imaging agent and method of imaging in the second aspect are as described in the first aspect (above).

For this aspect, the in vivo imaging procedure is preferably PET. PET has excellent sensitivity and resolution, so that even relatively small changes in a lesion can be observed over time, which is particularly advantageous for treatment monitoring. Quantification is also more easily achieved with PET. In a third aspect, the present invention provides a method of therapy monitoring, which comprises carrying out the method of the first aspect on a subject undergoing therapy to treat a condition associated with macrophage accumulation in vivo.

Preferred embodiments of the imaging agent and method of imaging in the third aspect are as described in the first aspect (above).

Conditions associated with macrophage accumulation in vivo include inflammation and atherosclerosis; rheumatoid arthritis; osteo arthritis; pulmonary inflammation; liver inflammation; kidney inflammation; bowel inflammation, COPD (Chronic Obstructive Pulmonary Disease) and cancer. Treatments for inflammation are known in the art, and include anti-inflammatory drugs [Shahbaz-Samavi and McKenna, Nonsteroidal Antiinflammatory Drugs pages 1307-1316 in Clinical Immunology Rich et al (Eds), Elsevier (2008)]. Treatments for atherosclerosis are known in the art, and are described in Atherosclerosis: Treatment and Prevention, Weber & Soehnlein (Eds), Pan Stanford Publishing (2013) and Coronary Atherosclerosis: Current Management and Treatment, Arampatzis et al (Eds), Informa Healthcare (2012).

In a fourth aspect, the present invention provides the TSPO ¹⁸F imaging agent of Formula la as defined in the first aspect for use in either:

(i) the method of in vivo imaging of the first aspect;

(ii) the method of diagnosis of the second aspect; or

(iii) the method of therapy monitoring of the third aspect.

Preferred embodiments of the TSPO ¹⁸F imaging agent in the fourth aspect are as described in the first aspect (above). In a fifth aspect, the present invention provides a radiopharmaceutical composition comprising the TSPO 18 F i·magi·ng agent of Formula la as defined in the first aspect, together with a biocompatible carrier medium, for use in either:

(i) the method of in vivo imaging of the first aspect;

(ii) the method of diagnosis of the second aspect; or

(iii) the method of therapy monitoring of the third aspect.

Preferred embodiments of the TSPO ¹⁸F imaging agent in the fifth aspect are as described in the first aspect (above).

The "biocompatible carrier medium" is a fluid, especially a liquid, in which the in vivo imaging agent is suspended or dissolved, such that the composition is physiologically tolerable, i.e. can be administered to the mammalian body without toxicity or undue discomfort. The biocompatible carrier is suitably an injectable carrier liquid such as sterile, pyrogen-free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is either isotonic or not hypotonic); an aqueous solution of one or more tonicity-adjusting substances (e.g. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or other non-ionic polyol materials (e.g. polyethyleneglycols, propylene glycols and the like). The biocompatible carrier may also comprise biocompatible organic solvents such as ethanol. Such organic solvents are useful to solubilise more lipophilic compounds or formulations. Preferably the biocompatible carrier is pyrogen-free water for injection, isotonic saline or an aqueous ethanol solution. The pH of the biocompatible carrier for intravenous injection is suitably in the range 4.0 to 10.5.

The "radiopharmaceutical composition" is a composition comprising the in vivo imaging agent of the invention, together with a biocompatible carrier in a form suitable for mammalian administration. The biocompatible carrier is as defined above. The radiopharmaceutical composition may be administered parenterally, i.e. by injection, and is most preferably an aqueous solution. Such a composition may optionally contain further ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g.

cyclodextrins or surfactants such as Pluronic, Tween or phospholipids); pharmaceutically acceptable stabilisers or antioxidants (such as ascorbic acid, gentisic acid or para- aminobenzoic acid). For parenteral administration, steps to ensure that the

radiopharmaceutical composition is sterile and apyrogenic also need to be taken.

The invention is illustrated by a series of non-limiting examples:

Example 1 describes the synthesis of imaging agent 5 and its' precursor. Example 2 describes enantiomeric separation of precursor compound 5. Example 3 provides the

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TSPO affinity of the compounds. Example 4 shows that [ F]-Compound 5A is taken up in atherosclerotic plaque in a mouse model of atherosclerosis. Example 5 provides autoradiography data, showing that [¹⁸F]-Compound 5A accumulates in macrophage-rich areas of atherosclerotic plaques. Example 6 shows that the uptake of [¹⁸F]-Compound 5A is blocked by non-radioactive Compound 5A - showing that the uptake is specific.

Brief Description of the Figures.

Figure 1 shows that the uptake of Compound 5A (expressed as count density) correlates with the amount of macrophages (expressed as mac-3 staining).

Figure 2 shows the uptake of Compound 5A in macrophage-positive and macrophage- negative areas of the atherosclerotic plaque.

Abbreviations.

aq aqueous

DCM dichloro methane

DMAP 4-Dimethylaminopyridine

DMF dimethylformamide EDC 1 -Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride

EOS end of synthesis

EtOAc ethyl acetate

IPA isopropyl alcohol

LC-MS liquid chromatography-mass spectrometry

NMR nuclear magnetic resonance

OBn benzyloxy

OMs mesylate

OTs to sy late

RT room temperature

TLC thin layer chromatography

Tol toluene.

Example 1: Synthesis of Methanesulphonic acid 2-(4-diethylcarbamyl-5-methoxy- l,2.,3.,4-tetrahvdro-carbazol-9-yl) ethyl ester (precursor compound 5) and 9-(2- [¹⁸FlFluoro-ethyl)-5-methoxy-2.,3.,4.,9-tetrahvdro-lH-carbazole^t-carboxylic acid diethylamide (imaging agent 5).

Example 1(a): Benzyloxy acetyl chloride (1)

To benzyloxyacetic acid (10.0 g, 60.0 mmol, 8.6 mL) in dichloromethane (50 mL) was added oxalyl chloride (9.1 g, 72.0 mmol, 6.0 mL) and DMF (30.0 mg, 0.4 mmol, 32.0 μί) and stirred at RT for 3 h. There was initially a rapid evolution of gas as the reaction proceeded but evolution ceased as the reaction was complete. The dichloromethane solution was concentrated in vacuo to give a gum. This gum was treated with more oxalyl chloride (4.5 g, 35.7 mmol, 3.0 mL), dichloromethane (50 mL), and one drop of DMF. There was a rapid evolution of gas and the reaction was stirred for a further 2 h. The reaction was then concentrated in vacuo to afford 1 1.0 g (quantitative) of Benzyloxy acetyl chloride (1) as a gum. The structure was confirmed by ^1JC NMR (75 MHz, CDC1₃) 5c 73.6, 74.8, 128.1, 128.4, 128.6, 130.0, and 171.9.

Example 1(b): 2-Benzyloxy-N-(2-chloro-5-metnhoxy-phenyl) acetamide (2).

Benzyloxy acetyl chloride (1) (1 1.0 g, 60.0 mmol) and 2-chloro-5-methoxyaniline hydrochloride (11.7 g, 60.2 mmol) in dichloromethane (100 mL) at 0°C, was stirred and triethylamine (13.0 g 126.0 mmol, 18.0 mL) added slowly over 15 min. The stirred reaction was allowed to warm to RT over 18 h. There was a heavy precipitation of triethylamine hydrochloride. The dichloromethane solution was washed with 10% aqueous potassium carbonate (50 mL), dried over magnesium sulfate and concentrated in vacuo to afford 18.9 g (quantitative) of 2-Benzyloxy-N-(2-chloro-5-methoxy-phenyl)

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acetamide (2) as a gum. The structure was confirmed by C NMR (75 MHz, CDCI₃): 5c 55.6, 69.6, 73.6, 106.2, 1 11.1 , 1 14.1 , 127.7, 128.3, 128.6, 129.2, 134.6, 136.5, 158.9, and 167.7.

Example 1(c): (2-Benzyloxy-ethyl)-(2-chloro-5-methoxyphenyl) amine (3).

2-Benzyloxy-N-(2-chloro-5-methoxy-phenyl) acetamide (2) (18.9 g, 62.0 mmol) in THF (100 mL) was stirred and lithium aluminium hydride (4.9 g, 130.0 mmol) was added slowly over 15 min. There was a rapid evolution of hydrogen gas as the first of the lithium aluminium hydride was added. The reaction was then heated to reflux for 4 h and allowed to stand at RT over the weekend. The reaction was then quenched by the dropwise addition of water (50 mL) to the stirred solution. There was a violent evolution of hydrogen causing the reaction mixture to reflux. The reaction was then concentrated in vacuum to a slurry. Water (200 mL) and ethyl acetate (200 mL) were added and the mixture vigorously shaken. The reaction was then filtered through celite to remove the precipitated aluminium hydroxide and the ethyl acetate solution was separated, dried over magnesium sulfate and concentrated in vacuo to afford 18.4 g (quantitative) of (2- Benzyloxy-ethyl)-(2-chloro-5-methoxyphenyl) amine (3) as a gum. The structure was confirmed by ¹³C MR (75 MHz, CDC1₃) 5_C 43.3, 55.3, 68.2, 73.0, 98.1, 101.8, 1 11.6, 127.6, 127.7, 128.4, 129.3, 137.9, 144.8, and 159.5. Example 1(d): 3-Bromo-2-hydroxy-cyclohex-l-enecarboxylic acid ethyl ester (4)

Ethyl 2-oxocyclohexanecarboxylate (30 g, 176 mmol, 28 mL) was dissolved in diethyl ether (30 mL) and cooled to 0°C under nitrogen. Bromine (28 g, 176 mmol, 9.0 mL) was added dropwise over 15 min and the reaction mixture was allowed to warm to RT over 90 min. The mixture was slowly poured into ice-cold saturated aqueous potassium carbonate (250 mL) and extracted with ethyl acetate (3 x 200 mL). The combined organic layers were dried over magnesium sulfate, filtered, concentrated in vacuo and dried on the vacuum line for 18 h to afford 41.4 g (94%) of 3-Bromo-2 -hydro xy-l-enecarboxylic acid

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ethyl ester (4) as a yellow oil. The structure was confirmed by ^1JC NMR (75 MHz, CDCls): 5c 14.1, 17.7, 21.8, 32.0, 60.0, 60.8, 99.7, 166.3, and 172.8.

Example 1 (e) : 3 [ (2 -B enzyloxy-ethyl)- (2 -chlo ro -5 -methoxy-phenyD-amino ] -2 -hy droxy- cyclohex- 1 -ene carboxylic acid ethyl ester (5).

(2-Benzyloxy-ethyl)-(2-chloro-5-methoxyphenyl) amine (3) (10.0 g, 34.2 mmol) was stirred in dry THF (100 mL) at -40°C under nitrogen and potassium bis(trimethylsilyl) amide (143.0 mL of a 0.5 M solution in toluene, 72.0 mmol ) was added over 30 min. 3- bromo-2-hydroxycyclohex-l -enecarboxylic acid ethyl ester (4) (8.5 g, 34.2 mmol) in dry THF (10 mL) was then added and allowed to warm to RT over a period of 1.5 h. Acetic acid (10.0 g, 166 mmol, 10.0 mL) was added and concentrated in vacuo to remove the THF. Ethyl acetate (200 mL) and 10% aqueous potassium carbonate (100 mL) was added and the mixture vigorously shaken. The ethyl acetate solution was separated, dried over magnesium sulfate and concentrated in vacuo to afford 16.5 g (quantitative) of 3[(2- Benzyloxy-ethyl)-(2-chloro-5-methoxy-phenyl)-amino]-2-hydroxy-cyclohex-l-ene carboxylic acid ethyl ester (5) as a gum which was used crude in the next step. HPLC (Gemini 150 x 4.6 mm, 50-95% methanol/water over 20 min) of crude reaction mixture, 18.9 min (38%), 19.2 min (25%), 23.1 min (28%).

One component of the reaction was isolated ¹³C NMR (75 MHz, CDC1₃) 5_C 14.3, 20.6, 21.8, 26.4, 38.6, 43.0, 55.8, 60.5, 68.7, 73.3, 93,4, 106.3, 108.2, 119.3, 121.5, 127.5, 127.6, 128.3, 135.7, 137.0, 137.9, 155.7, and 175.0. Example 1 (f): 9-(2-Benzyloxy-ethyl)-8-chloro-5-methoxy-2,3,4,9,-tetrahydro-l H- carbazole-4-carboxylic acid ethyl ester (6).

Zinc chloride (7.1 g, 52.0 mmol) was added to 3[(2-Benzyloxy-ethyl)-(2-chloro-5- methoxy-phenyl)-amino]-2-hydroxy-cyclohex-l-ene carboxylic acid ethyl ester (5) (8.0 g, 17.0 mmol) in dry diethyl ether (150 mL) under nitrogen and heated at reflux for 5.5 h. As the reaction was refluxed a thick brown dense oil formed in the reaction. The reaction was then cooled and the supernatant diethyl ether decanted off, ethyl acetate (100 mL) was added, washed with 2 N HC1 (50 mL) and with 10% aqueous potassium carbonate (50 mL). The diethyl ether layer was separated, dried over magnesium sulfate and concentrated in vacuo to afford an oil (2.0 g). The crude material was purified by silica gel chromatography eluting with petrol (A): ethyl acetate (B) (10-40% (B), 340 g, 22 CV, 150 mL/min) to afford 1.8 g of 9-(2-Benzyloxy-ethyl)-8-chloro-5-methoxy-2,3,4,9,- tetrahydro-lH-carbazole-4-carboxylic acid ethyl ester (6). The thick dense brown layer was treated with ethyl acetate (100 mL) and 2 N HC1 (50 mL). The ethyl acetate solution was separated, washed with 10% aqueous potassium carbonate (50 mL), dried over magnesium sulfate and concentrated in vacuo to give an oil (5.2 g). Diethyl ether (100 mL) and anhydrous zinc chloride (7.0 g) were added. The mixture was heated at refiux for a further 5 days. The ether layer was decanted off from the dark gum, was washed with 2 N HC1 (50 mL), dried over magnesium sulfate and concentrated in vacuo to give a gum (2.8 g). This gum was purified by silica gel chromatography eluting with petrol (A): ethyl acetate (B) (5-35% (B), 340 g, 150 mL/min) to afford 2.1 g of 9-(2-Benzyloxy- ethyl)-8-chloro-5-methoxy-2,3,4,9,-tetrahydro-lH-carbazole-4-carboxylic acid ethyl ester (6). Total material obtained was 4.1 g (50%) of 9-(2-Benzyloxy-ethyl)-8-chloro-5- methoxy-2,3,4,9,-tetrahydro-lH-carbazole-4-carboxylic acid ethyl ester (6). The structure was confirmed by ¹³C NMR (75 MHz, CDC1₃): 5_C 14.4, 20.5, 22.3, 27.5, 40.2, 43.9, 55.0, 60.2, 70.7, 73.3, 100.2, 107.5, 108.4, 120.1 , 122.8, 127.4, 127.5, 128.2, 132.0, 137.4, 138.1 , 152.6, and 175.8. Example Kg): 9-(2-Benzyloxy-ethyl)-8-chloro-5-methoxy-2 ,3,4,9 ,-tetrahydro-lH- carbazole-4-carboxylic acid (7).

To 9-(2-Benzyloxy-ethyl)-8-chloro-5-methoxy-2,3,4,9,-tetrahydro-lH-carbazole-4- carboxylic acid ethyl ester (6) (2.0 g, 4.1 mmol) in ethanol (50 mL) was added sodium hydroxide (1.1 g, 27.1 mmol) and water (5 mL) and heated at 80 C for 18 h. The ethanol was then removed by evaporation in vacuo and the residue partitioned between diethyl ether (50 mL) and water (50 mL). The diethyl ether layer was separated, dried over magnesium sulfate and concentrated in vacuo to give a gum (71.0 mg). The aqueous layer was acidified to pH 1 with 2N HC1 (20 mL) and extracted with dichloromethane (2 x 100 mL). The dichloromethane layer was dried over magnesium sulfate and concentrated in vacuo to afford 1.6 g (87%) of 9-(2-Benzyloxy-ethyl)-8-chloro-5-methoxy-2,3,4,9,- tetrahydro-lH-carbazole-4-carboxylic acid (7) as a foam. The structure was confirmed by ¹³C NMR (75 MHz; CDC1₃): 5_C 20.2, 22.2, 27.1 , 39.7, 44.0, 55.1 , 70.7, 73.3, 100.6, 106.3, 108.9, 123.0, 127.4, 127.5, 128.3, 132.0, 138.0, and 152.0.

Example 1(h): 9-(2-Benzyloxy-ethyl)-8-chloro-5-methoxy-2 ,3,4,9 ,-tetrahydro-lH- carbazole-4-carbonyl chloride (8).

9-(2-Benzyloxy-ethyl)-8-chloro-5-methoxy-2,3,4,9,-tetrahydro-lH-carbazole-4- carboxylic acid (7) (1.5 g, 3.7 mmol) was dissolved in dichloromethane (50 mL) and oxalyl chloride (700 mg, 5.5 mmol, 470 μί) and DMF (1 drop) were added and the reaction stirred at 20°C for 2 h. There was a moderate evolution of gas for about 30 min as the reaction proceeded. The reaction was then concentrated in vacuo to give 9-(2- Benzyloxy-ethyl)-8-chloro-5-methoxy-2,3,4,9,-tetrahydro-lH-carbazole-4-carbonyl chloride (8) as a gum which was used into the next step without purification. The structure was confirmed by ¹³C NMR (75 MHz; CDC1₃): 5_C 20.8, 22.1 , 26.4, 44.2, 51.8, 55.1 , 70.7, 73.3, 100.7, 106.0, 108.6, 1 19.5, 123.4, 127.3, 127.7, 128.3, 131.9, 138.0, 138.2, 152.0. and 176.3. Example l(i): 9-(2-Benxyloxy-ethyl)-8-chloro-5-methoxy-2,3,4,9-tetrahydro-lH- carbazole-4-carboxylic acid diethylamide (9).

9-(2-Benzyloxy-ethyl)-8-chloro-5-methoxy-2,3,4,9,-tetrahydro-lH-carbazole-4-carbonyl chloride (8) (1.6 g, 3.7 mmol) was then dissolved in dichloromethane (50 mL), cooled to 0 C, stirred and diethylamine (810 mg, 1 1.0 mmol, 1.1 mL) was added dropwise. The reaction was allowed to warm to room temperature over a period of 18 h. The reaction mixture was then washed with 10% aqueous potassium carbonate (50 mL), separated, dried over magnesium sulfate and concentrated in vacuo to a gum. The crude material was crystallized from diethyl ether to afford 1.2 g (71%) of 9-(2-Benxyloxy-ethyl)-8- chloro-5-methoxy-2,3,4,9-tetrahydro-lH-carbazole-4-carboxylic acid diethylamide (9) as a white crystalline solid. The structure was confirmed by ¹³C NMR (75 MHz; CDCI3): 5c 13.0, 14.5, 19.8, 22.2, 27.9, 36.4, 40.4, 41.9, 43.8, 55.0, 70.8, 73.3, 100.2, 108.5, 108.6, 119.9, 122.5, 127.4, 127.5, 128.3, 131.5, 137.8, 138.2, 152.4, and 174.5.

Example l(j): 9-(2-Benzyloxy-ethyl)-5-methoxy-2,3,4,9-tetrahydro-lH-carbazole-4- carboxylic acid diethylamine (10).

9-(2-Benxyloxy-ethyl)-8-chloro-5-methoxy-2,3,4,9-tetrahydro-lH-carbazole-4- carboxylic acid diethylamide (9) (1.0 g, 2.1 mmol) in methanol (100 ml) was shaken with 10% palladium on charcoal (1.0 g), triethylamine (2.9 mg, 2.9 mmol, 4 μί) under an atmosphere of hydrogen gas for 18h at 55 C. The reaction was then filtered through a pad of celite and the filtrate concentrated in vacuo to give a gum (908 mg). The gum was then taken up in dichloromethane (100 ml) and washed with 5% aqueous potassium carbonate solution (50 ml). The dichloromethane solution was then separated, dried over magnesium sulfate and concentrated in vacuo to afford a gum. The gum was then crystallised from diethyl ether (50ml) and the crystals collected by filtration to afford 523 mg (57%) of 9-(2-Benzyloxy-ethyl)-5-methoxy-2,3,4,9-tetrahydro-lH-carbazole-4- carboxylic acid diethylamine (10). The structure was confirmed by ¹³C NMR (75 MHz; CDCI3): 5c 13.1, 14.6, 20.1, 22.0, 28.1 , 36.4, 40.5, 42.0, 43.0, 54.7, 68.8, 73.3, 99.4, 102.4, 107.8, 116.4, 121.2, 127.6,127.6, 128.3, 135.6, 137.8, 138.0 153.6, and 175.0. Example l(k): 9-(2-hydroxyethyl)-5-methoxy-2,3,4,9-tetrahydro-lH-carbazole-4- carboxylic acid diethylamine (11).

9-(2-Benzyloxy-ethyl)-5-methoxy-2,3,4,9-tetrahydro-lH-carbazole-4-carboxylic acid diethylamine (10) (1.0 g, 2.1 mmol) in methanol (50 ml) was shaken with 10% palladium on charcoal (300 mg), and hydrogen gas excess for 18h at 55°C. The reaction was then filtered through a pad of celite and the filtrate concentrated in vacuo to give 578 mg (100%) 9-(2-hydroxyethyl)-5-methoxy-2,3,4,9-tetrahydro-lH-carbazole-4-carboxylic acid diethylamine (11) as a foam. The structure was confirmed by ¹³C NMR (75 MHz; CDCls): 5c 13.0, 14.4, 20.0, 22.0, 28.0, 36.4, 40.6, 42.0, 54.7, 60.6, 99.2, 102.6, 107.0, 116.7, 121.1, 136.1 , 137.5, 138.0 153.5, and 175.7.

Example 1(1): Methanesulphonic acid 2-(4-diethylcarbamyl-5-methoxy-1.2.3.4- tetrahydro-carbazol-9-yl) ethyl ester (precursor compound 5).

9-(2-Hydroxyethyl)-5-methoxy-2,3,4,9-tetrahydro-lH-carbazole-4-carboxylic acid diethylamine (11) (478 mg, 1.4 mmol) in dichloromethane (30 ml) was cooled to 0°C and methanesulfonyl chloride (477 mg, 4.2 mmol, 324 μί) and triethylamine (420 mg, 4.2 mmol, 578 μί) were added and allowed to warm to RT overnight. The reaction was washed with 5% aqueous potassium carbonate solution. The layers were separated. The combined organics were dried over magnesium sulfate and concentrated in vacuo to give a gum (696 mg). The crude material was purified by silica gel chromatography eluting with petrol (A): ethyl acetate (B) (75-100% B, 22 CV, 120 g, 85 mL/min) to afford Methanesulphonic acid 2-(4-diethylcarbamyl-5-methoxy-l ,2,3,4-tetrahydro-carbazol-9- yl) ethyl ester (precursor compound 5) as a gum that crystallised from diethyl ether to give 346 mg (59%) of a colourless solid. The structure was confirmed by ¹³C NMR (75 MHz; CDC1₃): 5_C 13.1, 14.5, 20.0, 21.9, 28.0, 36.3, 36.7, 40.3, 41.8, 41.9, 54.7, 68.1, 100.0, 102.0, 109.0, 1 16.4, 122.0 135.1, 137.3, 153.8, and 174.6. 18

Example Urn): 9-(2-[ F]Fluoro-ethyl)-5-methoxy-2,3,4,9-tetrahydro-lH-carbazole-4- carboxylic acid diethylamide (imaging agent 5).

18

[ FJFluoride was supplied from GE Healthcare on a GE PETrace cylcotron. Kryptofix 2.2.2 (2 mg, 5 μηιοΐ), potassium bicarbonate (0.1 mol dm^"3, 0.1ml, 5 mg, 5 μηιοΐ) and acetonitrile (0.5 ml) was added to [¹⁸F]F7H₂0 (ca. 400 MBq, 0.1 -0.3 ml) in a COC reaction vessel. The mixture was dried by heating at 100°C under a stream of nitrogen for 20-25mins. After drying and without cooling, precursor compound 5 (0.5-1 mg, 1.2-2.4 μηιοι) in acetonitrile (1 ml) was added to the COC reaction vessel and heated at 100°C for 10 mins. After cooling, the reaction mixture was removed and the COC reaction vessel rinsed with water (1.5 ml) and added to the main crude reaction.

Following this, the crude product was applied to semi-preparative HPLC: HICHROM ACE 5 CI 8 column (100 x 10 mm i.d.), particle size 5 μηι; mobile phase A: Water, mobile phase B: Methanol; flow gradient: 3ml/min; 0-1 min 40 %B; 1 -20 mins 40-95 %B; Wavelength 254 nm; t_R imaging agent 5 16 mins. The imaging agent 5 HPLC purified-peak was diluted to a volume of 10 ml with water and adsorbed on a tC18 Sep- Pak (lite) cartridge. The cartridge was washed with water (2 ml), and eluted with anhydrous ethanol (0.5 ml) followed with Dulbecco's phosphate buffered saline(4.5 ml). Radiochemical yield 30±7% (n=4) non-decay corrected, time 90-120 mins, radiochemical purity >99%.

Analytical-HPLC: Phenomenex Luna C18 column (150 x 4.6 mm i.d.), particle size 5μηι; mobile phase A: Water, mobile phase B: Methanol; flow gradient: lml/min; 0-1 min 40 %B; 1 -20 mins 40-95 %B; Wavelength 230 nm; t_R imaging agent 5 16 mins. Figure 1 shows co-elution of imaging agent 5 and non-radioactive imaging agent 5. Example 2: Enantiomeric Separation of Precursor Compound 5.

Precursor compound 5 Enantiomer 1 Enantiomer 2

Precursor compound 5 (obtained as described in Example 1) was separated into its enantiomers using chiral supercritical fluid (C0₂) chromatography on a Kromasil Amycoat, 250x10 mm, 5 μηι, 100 A column using 30 % IPA at 40°C at 13ml a min with a run time of 6 min. Precursor compound 5 (60 mg) was dissolved in 1.4-Dioxane (2ml) and up to 200 μΐ at a time was as injected for each run. Baseline separation between the two enantiomers was achieved. Analytical HPLC determination of the enantiomeric purity of the two separated enantiomers on an IC from Chiral Technologies, 250x4.6 mm, 5 μηι, run isocratic, 80:20 - MeOH: IPA at 0.5 ml / min and room temperature indicated an enantiomeric purity of 99.5% of each of the enantiomers.

Example 3: In Vitro Potency Assay.

Affinity for TSPO was screened using a method adapted from Le Fur et al (Life Sci. 1983; USA 33: 449-57). Non-radioactive analogues of in vivo imaging agents of the invention were tested. Each test compound (dissolved in 50mM Tris-HCl, pH 7.4, lOmM MgCl₂ containing 1 %DMSO) competed for binding to Wistar rat heart PBR against 0.3 nM [³H] PK-11 195. The reaction was carried out in 50mM Tris-HCl, pH 7.4 lOmM MgCl₂ for 15 minutes at 25°C. Each test compound was screened at 6 different concentrations over a 300-fold range of concentrations around the estimated IQ. The following data were observed: Imaging Agent Ki (nM)

Imaging agent 5 2.35

Imaging agent 6 18.30

Imaging agent 7 1.25

Imaging agent 9 3.79

Imaging agent 10 7.62

Imaging agent 11 2.12

Example 4: Uptake of [ Fl-Compound 5A in Atherosclerotic Plague Inflammation in Mice.

Six atherosclerotic mice deficient in low density lipoprotein receptor and expressing only apolipoprotein B100 (LDLR^~'^~ApoB^luu,luu) and six healthy C57BL/6N mice were injected with 10 MBq of [¹⁸F]-Compound 5A. Dynamic 30-minute PET scan was performed followed by contrast-enhanced CT. Mice were sacrificed at 60 minutes post-injection. Tissue samples were obtained for ex vivo biodistribution measurements, and aortas were frozen and cut into serial cryosections for digital autoradiography. Tracer uptake in areas of plaque, media and adventitia were analyzed based on haematoxylin-eosin stain.

Macrophages in atherosclerotic aortas were detected by Mac-3 immuno staining. Tracer uptake in Mac3- positive and negative areas was analyzed. Three LDLR^' ApoB ^100/100 mice and three C57BL/6N mice received unlabelled Compound 5 A five minutes before

18

[ F] -Compound 5 A injection, and similar study protocol was performed.

Results.

18

[ F] -Compound 5 A was rapidly cleared from the blood circulation. Both in vivo and ex vivo biodistribution measurements showed the highest radioactivity uptake in adrenal glands, kidneys and lungs, respectively, which is in accord with the high TSPO expression in these tissues. 18

Example 5: Localisation of [ Fl -Compound 5 A in the Aorta of Atherosclerotic Plague Inflammation in Mice.

18

Autoradiography revealed uptake of [ F] -Compound 5 A in the aortic atherosclerotic plaques as well as in the non-atherosclerotic media (Table 1):

Table 1 : Results of [¹⁸F]-Compound 5A aortic autoradiography. Results expressed as count densities (photo-stimulated luminescence/mm²)

The tracer uptake was significantly higher in macrophage-rich plaque areas than in non- inflamed areas - using hematoxylin-eosin staining to detect macrophage accumulation. There was also a significant correlation between percentage of Mac-3-positive area and

18

[ F] -Compound 5A-derived count density - see Figures 1 and 2.

Example 6: Effect of Blocking of [ Fl-Compound 5A with Non-radioactive

Compound 5A.

Blocking with unlabelled Compound 5A reduced the uptake of [¹⁸F] -Compound 5A in all tissues with high TSPO expression, whereas significantly more tracer remained in the circulation. Blocking also lowered the difference between uptake in positive and negative plaque areas and the correlation between percentage of Mac-3-positive area and [¹⁸F] -Compound 5 A -derived count density was diminished.

Claims

Claims.

1. A method of in vivo imaging to determine the distribution of activated macrophages in a subject, said method comprising:

(i) provision of a subject to which a translocator protein (TSPO) F imaging agent had been previously administered;

(ii) detecting by an in vivo imaging procedure the radioactive emissions from the 1⁸F radioisotope of the administered imaging agent of step (i);

(iii) generating an image representative of the location and/or amount of said radioactive emissions;

where said TSPO 18 F i·maging agent is of Formula la:

wherein:

R^2a is H, Hal or C_1-3 alkoxy;

R^Ja and R^4a are independently methyl, ethyl or benzyl, or together with the nitrogen to which they are attached form a pyrrolidinyl, piperidinyl, azepanyl, or morpholinyl ring;

Y^2a is -CH₂-, -CH₂-CH₂-, -CH(CH₃)-CH₂-, or -CH₂-CH₂-CH₂-; and;

n is 1 , 2 or 3. The method of claim 1, wherein R ^a and R ^a are both ethyl, or R ^a is methyl and R ^a is benzyl, or together with the nitrogen to which they are attached form an azepanyl ring.

The method of claim 1 or claim 2, wherein R^2a is H, methoxy or fiuoro.

The method of any one of claims 1 to 3, wherein Y^2a is -CH₂-CH₂- or -CH(CH₃)-CH₂-.

The method of claim 1 or claim 2, wherein:

R^3a and R^4a are both ethyl, or R^3a is methyl and R^4a is benzyl, or together with the nitrogen to which they are attached form an azepanyl ring;

R^2a is H, methoxy or fiuoro;

Y^2a is -CH2-CH2- or -CH(CH₃)-CH₂-; and,

n is 2.

6. The method of any one of claims 1 to 6, where the TSPO ¹⁸F imaging agent is:

7. The method of any one of claims 1 to 6, where the activated macrophages are located at sites of inflammation and/or atherosclerosis in vivo.

8. A method of diagnosis of a site of activated macrophage accumulation in vivo which comprises the method of imaging of any one of claims 1 to 7.

9. The method of therapy monitoring, which comprises carrying out the method of any one of claims 1 to 7 on a subject undergoing therapy to treat a condition associated with macrophage accumulation in vivo.

10. The TSPO ¹⁸F imaging agent of Formula la as defined in any one of claims 1 to 6 for use in either:

(i) the method of in vivo imaging of any one of claims 1 to 7;

(ii) the method of diagnosis of claim 8; or

(iii) the method of therapy monitoring of claim 9.

1 1. A radiopharmaceutical composition comprising the TSPO ¹⁸F imaging agent of Formula la as defined in any one of claims 1 to 6, together with a biocompatible carrier medium, for use in either:

(i) the method of in vivo imaging of any one of claims 1 to 7;

(ii) the method of diagnosis of claim 8; or

(iii) the method of therapy monitoring of claim 9.