The present invention relates to compounds which may be used as radioligands, in particular as GPR44 radioligands for visualizing pancreatic beta cells. The invention also relates to a process for preparing the radioligands and use of the radioligands in in vitro, ex vivo and in vivo beta cell imaging (BMI) methods.

One technique for beta cell imaging using radioligands is positron emission tomography (PET) using radioligands containing positron-emitting radioisotopes, for example ¹¹ C or 8F. Beta cell imaging may also be conducted using in vitro or ex vivo techniques, for example by autoradiography (ARG). For example, in addition to PET radioligands, tritium ( ³ H) containing radioligands, which emit beta particle radiation, can be used to image samples in vitro or ex vivo, for example by ARG analysis. Positron emission tomography (PET) is a non-invasive molecular imaging (in vivo) technique which allows the localization of a molecule labeled with positron emitting nuclides, based on the detection of positron annihilation radiation and subsequently processing of raw data into an image. PET is frequently used for early detection, characterization, and real time monitoring of diseases, as well as investigating the effectiveness of therapeutic drugs. PET can provide insights on the molecular interactions between the tracer molecule and the biological target, e.g., a protein, transporter, enzyme function and inhibition, metabolism and general biochemical function. PET has become a powerful functional imaging tool and finds particular application in oncology, neuroscience and cardiovascular diseases.

Another major utility of PET imaging is to understand and facilitate drug action and development which can be investigated in different ways. For example, existing drugs or new drug candidates can be radiolabeled and their pharmacokinetic parameters such as absorption, distribution, metabolism and excretion (ADME) can be established by PET. Based on the results of in vivo PET studies, decisions can be made in the very early stages of drug development regarding the prospects of a drug candidate. Alternatively, pharmacodynamics or dose finding studies can be evaluated through receptor occupancy using PET radioligands. Efforts have been made to develop PET radioligands for visualizing beta cells in the pancreas. Of the four majority cell types of the Islets of Langerhans of the pancreas, only beta cells secrete insulin in response to elevated blood glucose levels. Since beta cells are responsible for keeping normal glucose levels of the blood, an adequate number of functional pancreatic beta cells are required. The collective beta-cell numbers, or beta cell mass (BCM), is reduced significantly in both type 1 (T1 D) and type 2 (T2D) diabetes patients, compared to non-diabetic individuals. In T1 D, an autoimmune attack against pancreatic beta cells results in a rapid loss of endocrine BCM and in T2D, insulin resistance and beta cell dysfunction build up a progressive reduction of BCM.

Autoradiography (ARG) is a well-known in vitro or ex vivo technique by which samples taken from a subject can be imaged. ARG may be used to determine the tissue (or cell) localization of a radioligand, for example, introduced into a metabolic pathway, bound to a receptor or enzyme, or hybridized to a nucleic acid. By analysing samples in vitro or ex vivo, uptake of radioligands in the sample can be analysed and, for example, used to assess the potential of a radioligand as a PET biomarker.

G-protein coupled receptor 44 (GPR44) has been identified as one of the beta cell specific proteins by antibody-based proteomics. GPR44 is also designated as the prostaglandin D ₂ receptor 2 (DP ₂ ), chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), cluster of differentiation (CD294) is a human protein encoded by the PTGDR2 gene. GPR44 is highly expressed, exclusively in insulin producing beta cells and absent from remaining islet cells as well as exocrine cells confirmed by both immunohistochemical (human pancreatic tissues from nondiabetic, T D and T2D individuals) and immunofluorescence (human islets) staining. Therefore, GPR44 is an important target of interest for visualizing beta cells in native pancreas.

However, beta cell imaging (BCI) faces several challenges, principally relating to the anatomical location of the pancreas and dispersion of the beta cells (only 1 -2% of pancreas volume) there through. It has been estimated that the uptake of a PET radioligand should be 1 ,000 times higher in beta cells than in other cell types (exocrine pancreas, duct cells, vascular space, etc.) in order for visualization to be effective. The present invention is based on the surprising discovery of compounds that are particularly suitable for use as radioligands for in vivo, in vitro and ex vivo BCI and in diagnostic methods relating to diagnosis of beta cell-related disorders. In a first aspect, the present invention provides a compound according to formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof:

wherein x + y = 3, and x > 1 , preferably where x = 3.

In another aspect, the present invention provides a precursor to a compound according to formula (I) or formula (II), corresponding to a compound according to formula (III), or a pharmaceutically acceptable salt or solvate thereof:

In a further aspect, the present invention also provides an intermediate compound according to formula (IV), or a pharmaceutically acceptable salt or solvate thereof:

In a yet further aspect, the present invention provides an intermediate compound according to formula (V), or a pharmaceutically acceptable salt or solvate thereof:

In preferred embodiments, the compounds according to formula (I), (II), (III), (IV) and (V), or pharmaceutically acceptable salts or solvates thereof, are in the form of the (S) enantiomers at the stereogenic carbon centre, as illustrated below:

Where reference is made herein to a "pharmaceutically acceptable salt" of a compound, this may refer, for example, to a basic or acidic additional salt thereof. Examples of base addition salts include salts of alkali metals such as lithium, sodium, and potassium; salts of alkaline earth metals such as calcium and magnesium; salts of post-transition metal salts such as zinc and aluminium; and salts derived from organic bases such as benzathine, chloroprocaine, choline, tert-butylamine, diethanolamine, ethanolamine, ethyldiamine, meglumine, tromethamine and procaine. Preferred base addition salts are selected from salts of alkali metals, most preferably sodium.

Examples of specific pharmaceutically acceptable salts of compounds of formula (I) and (II) include base addition salts of formula (la) and (lla) respectively, comprising a salt of the propionic acid moiety:

wherein M ⁺ is the countercation of the base, which is for example an alkali metal cation, preferably a sodium cation.

In preferred embodiments, the pharmaceutically acceptable salt of a compound of formula (III) corresponds to a base addition salt comprising a salt of the sulfinic acid moiety and further comprising a salt of the propionic acid moiety, corresponding to a salt of formula (Ilia). In some embodiments, the pharmaceutically acceptable salt of a compound of formula (III) corresponds to a base addition salt comprising a salt of the sulfinic acid moiety, corresponding to a salt of formula (1Mb):

wherein M ⁺ is the countercation of the base, which is for example an alkali metal cation, preferably a sodium cation. Preferably, the pharmaceutically acceptable salt of a compound of formula (III) is a disodium salt corresponding to the salt of formula (Ilia). Compounds of formula (I) and (II), or pharmaceutically acceptable salts or solvates thereof, may be prepared by reaction of a compound of formula (III) with a radiolabeled methylating agent, as outlined in Scheme I below. It has been found that a soft methylating agent (i.e. where the methyl carbonium ion is bonded to a soft base) favours the formation of the desired sulfone product over an ester product which can be formed when a hard methylating agent is used. The methylating agent used in the process of the present invention is therefore ¹¹ CH ₃ R or C( ³ H) _x ( ¹ H) _y R wherein x + y = 3, x > 1 and preferably 3, and preferably selected from ¹¹ CH ₃ ! or C( ³ H) _x ( ¹ H) _y l, wherein x + y = 3, x > 1 and preferably 3.

Thus, in another aspect, the present invention also provides a process for the preparation of a compound according to formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof, as defined hereinbefore, wherein said process comprises the step of reacting a compound of formula (III), or a pharmaceutically acceptable salt or solvate thereof:

with a compound selected from ¹¹ CH ₃ R or C( ³ H) _x ( ¹ H) _y R, wherein x + y = 3, x > 1 and, R is I, preferably where x = 3 and/or R is I; and thereby forming a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof.

Whether a compound of formula (I) or (II) is prepared depends on the nature of the radiolabeled methylating agent that is employed. Typically, the methylating agent will be trapped in a solvent or reaction mixture and retained in a reaction vessel where methylation of the compound of formula (III), or a pharmaceutically acceptable salt or solvate thereof, may occur.

A preferred methylating agent for use in the preparation of a compound of formula (I) is [ ¹¹ C]CH ₃ I. This methylating agent is particular well known and has been used for methylation on sulfur. [ ¹¹ C]CH ₃ I may be produced by "wet" or "dry" methods as described, for instance, in Link JM, Krohn KA, Clark JC. Production of [ ¹¹ C]CH ₃ I by single pass reaction of [ ¹¹ C]CH ₄ with l ₂ . Nucl Med Biol. Jan 1997; 24(1 ):93-97. In a preferred method, [ ¹¹ C]methane ([ ¹¹ C]CH ₄ ) is produced in-target via a ⁴ N(p,a) ¹¹ C reaction on nitrogen with 10% hydrogen, using, for example, 16.4 MeV protons using a GEMS PET trace cyclotron (GE, Uppsala, Sweden). Typically, the target gas is irradiated for 20-30 minutes with a beam intensity of, for instance, 35 μΑ. [ ¹¹ C]CH is then mixed with vapors from iodine crystals followed by a radical iodination reaction in a closed recirculation system to produce [ ¹¹ C]CH ₃ I.

A preferred methylating agent for use in the preparation of a compound of formula (II) is [ ³ H]CH ₃ I, which is widely available (including, for example, from American Radiolabeled Chemicals, St. Louis, MO, USA). Methods for preparing [ ³ H]CH ₃ I and similar tritium labelled methylating agents, are also known based on the use of a tritium gas reagent.

The process for preparing a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof, from reaction of a compound of formula (III), or a pharmaceutically acceptable salt or solvate thereof, may be conducted at any suitable temperature at which an acceptable rate of reaction is achieved and which avoids thermal decomposition and significant unwanted side reactions. Typically, the reaction of a compound of formula (III) is conducted at above room temperature and below 100 °C. Preferably, the reaction is conducted at a temperature of from 50 °C to 90 °C, more preferably from 60 °C to 80 °C, most preferably from 65 °C to 75 °C. The reaction of a compound of formula (III) may be conducted for any suitable period of time which provides an acceptable yield and maximises radiochemical stability of the radiolabeled product and may for instance be 15 minutes or less, preferably 10 minutes or less, more preferably 5 minutes or less. For example, the reaction of a compound of formula (III) may be conducted for a time period of from 1 minute to 15 minutes, preferably from 1 to 10 minutes, more preferably from 1 to 5 minutes, for example from 3 to 5 minutes. The reaction of a compound of formula (III), or a pharmaceutically acceptable salt or solvate thereof, with a compound selected from ¹¹ CH ₃ R or C( ³ H) _x ( ¹ H) _y R, may be conducted in the presence of a solvent. In preferred embodiments, where a solvent is used, it is an aprotic solvent, such as dimethylformamide, dimethylsulfoxide, hexamethylphosphoramide, acetonitrile, acetone, tetrahydrofuran or combinations thereof.

It is possible to convert a compound of formula (III) into a compound of formula (I) or (II) in almost quantitative yield using the process of the invention. It is also possible to prepare compounds of formula (I) and (II) with high radiochemical purity and high radiochemical stability (for example, >99% up to 2 hours from end of synthesis for a compound of formula (I) and >99% in a formulation solution after 1 week of end of synthesis for a compound of formula (II)). The crude product formed following reaction of a compound of formula (III) with a methylating agent may suitably be purified by HPLC before re-formulating.

In preferred embodiments, the time for the complete radiosynthesis, e.g. production of 1C-Mel from ¹¹ CH ₄ , ¹ C-methylation, purification, formulation and quality control is less than about three times the half-life of the relevant radioisotope. In some embodiments, the time for the complete radiosynthesis is about 1 hour or less, preferably about 30 minutes or less.

Compounds of formula (III), or pharmaceutically acceptable salts or solvates thereof, may be prepared by oxidation of a compound according to formula (V) to form a compound of formula (IV), followed by hydrolysis with a base, as outlined in Scheme II below.

Thus, in embodiments, the process for preparing a compound formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof, further comprises a preceding step of preparing the compound of formula (III), or a pharmaceutically acceptable salt or solvate thereof, by hydrolysing a compound of formula (IV), or a pharmaceutically acceptable salt or solvate thereof:

by contacting with a base.

Conversion of the compound of formula (III) to a compound formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof, may be conducted using any suitable base, including a base which may provide a base addition salt as described hereinbefore. For example, the base may be an alkali metal hydroxide, preferably sodium hydroxide, so as to generate the alkali metal salt of the compound of formula (III). The reaction may be carried out in a suitable solvent, preferably one which has a volatility to allow removal of the solvent after the reaction by evaporation under reduced pressure. Examples of preferred solvents include methanol, dichloromethane, dichloroethane and combinations thereof.

In other embodiments, the process for preparing a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof, further comprises a preceding step of preparing the compound of formula (IV) by oxidation of a compound of formula (V), or a pharmaceutically acceptable salt or solvate thereof:

Oxidation of a compound of formula (V) may be conducted using any oxidising agent suitable for converting aromatic-substituted sulphides to the corresponding sulphones, including, for example, magnesium 2-carboperoxybenzoate. The reaction may be carried out in a suitable solvent, preferably one which has a volatility to allow removal of the solvent after the reaction by evaporation under reduced pressure. Examples of preferred solvents include dichloromethane and dichloroethane.

Compounds of formula (V) may be prepared from the reaction of a sulfoxide compound of formula (VI) by means of the Pummerer rearrangement though which the sulfur is reduced and the adjacent alkyl carbon atom is oxidised. In turn, compounds of formula (VI) may be prepared from oxidation of a sulphide of formula (VII), as shown in Scheme III below.

Conversion of the compound of (VI) may be conducted in the presence of acetic anhydride and optionally also in the presence of an alkali metal acetate, such as sodium acetate, and preferably under reflux conditions. Oxidation of the compound according to formula (VII) may be conducted using any oxidising agent suitable for converting aromatic-substituted sulphides to the corresponding sulphoxide, including, for example, meta-chloroperoxybenzoic acid (mCPBA). The reaction may be carried out in a suitable solvent, preferably one which has a volatility to allow removal of the solvent after the reaction by evaporation under reduced pressure. Examples of preferred solvents include dichloromethane and dichloroethane.

Compounds of formula (VII) may be prepared from a compound of formula (VIII) by means of a Mitsunobu reaction. In turn, compounds of formula (VII) may be obtained from demethylation at the aryl methyl ether group of a compound according to formula (IX), as illustrated in Scheme IV below.

Conversion of compound of formula (VIII) may be achieved by a Mitsunobu reaction with methyl 2-hydroxypropanoate, typically in the presence of diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD), as well as triphenyl phosphine. The reaction may be carried out in a suitable solvent, preferably an aprotic solvent, such as dimethylformamide, dimethylsulfoxide, hexamethylphosphoramide, acetonitrile tetrahydrofuran, or combinations thereof. Typically, the components of the reaction are mixed at a temperature of less than 5 C, for example 0 C, before the reaction is allowed to warm to room temperature.

The Mitsunobu reaction in this case proceeds with inversion of molecular symmetry. Therefore, stereogenic control in the resulting compound of formula (VII) may be achieved by selecting the appropriate enantiomer of methyl 2-hydroxypropanoate. In preferred embodiments, the compounds of formula (I) to (VII) are in the form of the (S) enantiomers at the stereogenic carbon centre. Therefore, in order to provide compounds having this preferred stereochemistry, (R)-methyl 2-hydroxypropanoate is used in the above Mitsunobu reaction. As will be appreciated, when compounds of formula (I) to (VII) are desired in the form of the (R) enantiomers at the stereogenic carbon centre, (S)- methyl 2-hydroxypropanoate is used in the above Mitsunobu reaction. Demethylation of the compound of formula (IX) in order to provide a compound of formula (VIII) may be conducted using a suitable demethylating agent such as boron tribromide, which forms a dibromo(organo)borane group before being hydrolysed to provide the hydroxy I group. The reaction typically takes place at a temperature of less than 5 C, for example 0 C. The reaction may be carried out in a suitable solvent, preferred examples of which include dichloromethane, dichloroethane and combinations thereof.

Compounds of formula (IX) may be prepared from conversion of the corresponding aniline of formula (X), which in turn may be prepared from the reduction of the corresponding nitro compound of formula (XI), as illustrated in Scheme V below.

Conversion of the aniline of formula (X) to the corresponding methyl sulphide of formula (IX) may be achieved by reaction with dimethyldisulfide following initial diazotization of the aniline with isoamyl nitrite. The reaction may be carried out in a suitable nonaqueous solvent, preferred examples of which include dichloromethane, dichloroethane and combinations thereof, and preferably at elevated temperature, for example 60 C.

Reduction of the nitro compound of formula (XI) to the aniline of formula (X) may be achieved using any suitable reducing agent, including tin chloride and zinc as well through the use of catalytic hydrogenation using nickel, palladium or platinum catalysts. Preferably the conversion is achieved using zinc in acetic acid, preferably at elevated temperature, for example 50 C.

Compounds of formula (XI) may be prepared from the reaction of a compound of formula (XII) with 2-chloro-1 -fluoro-4-nitrobenzene. In turn, a compound of formula (XII) may be prepared from the corresponding boronic acid of formula (XIII), as illustrated in Scheme VI below.

The compound of formula (XII) may be reacted with 2-chloro-1 -fluoro-4-nitrobenzene in the presence of a non-nucleophilic, inorganic base, preferably potassium carbonate, at elevated temperature, for example 120 C. The reaction may be carried out in an aprotic solvent such as dimethylformamide, dimethylsulfoxide, hexamethylphosphoramide, acetonitrile, tetrahydrofuran or combinations thereof.

Conversion of the boronic acid of formula (XIII) to the compound of formula (XII) may be achieved using any suitable method for conversion of aryl boronic acids to phenols. Examples include the use of tert-butyl hydroperoxide, and promoted with potassium hydroxide, or preferably using hydrogen peroxide in ethanol under reflux conditions.

In another aspect, the present invention provides a method, or compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, for use in a method, of in vivo diagnosing a pancreatic beta cell-related disorder comprising the steps of:

i) introducing into a subject a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as defined hereinbefore;

ii) visualizing the compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, at the beta cell population in the pancreas using PET; quantifying the beta cell mass in the subject;

comparing the beta cell mass data obtained with known values representative of a healthy subject;

diagnosing the subject as having, or being at risk of having, a beta cell- related disorder based on the comparison in step iv).

In another aspect, the present invention provides a method, or compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, for use in a method, of in vivo monitoring a change in pancreatic beta cell mass comprising the steps of:

i) introducing into a subject a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as defined hereinbefore; ii) visualizing the compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, at the beta cell population in the pancreas using PET;

iii) quantifying the beta cell mass in the subject;

iv) re-examining the subject after a period of time according to steps (i) to (iii); v) comparing the beta cell mass obtained in step (iii) to the beta cell mass obtained in step (iv);

vi) monitoring a change in beta cell mass to monitor the impact of a therapeutic intervention or to monitor a beta cell-related disorder, based on the comparison in step (v).

It will be understood that methods described herein may be used for following changes in beta cell mass over time in a healthy subject or in patients suffering from a beta cell- related disorder, for example monitoring of the progression of a beta cell-related disorder. In addition, methods described here can, for example, be used for monitoring the impact of pharmaceutical interventions that directly aim to increase beta cell mass, or for monitoring pharmaceutical interventions as safety analysis to exclude negative effects on beta cell mass.

In another aspect, the present invention provides a method, or compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, for use in a method, of in vivo monitoring a change in pancreatic beta cell mass comprising the steps of:

i) introducing into a subject a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as defined hereinbefore; ii) visualizing the compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, at the beta cell population in the pancreas using

PET;

iii) quantifying the beta cell mass in the subject;

iv) re-examining the subject after a period of time according to steps (i) to (iii); v) comparing the beta cell mass obtained in step (iii) to the beta cell mass obtained in step (iv).

The radioligand of formula (I) may be introduced into a subject as part of a pharmaceutical preparation comprising the radioligand and a pharmaceutically acceptable adjuvant, diluent or carrier. Therefore, in yet a further aspect, the invention also provides pharmaceutical preparation comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as defined hereinbefore and a pharmaceutically acceptable adjuvant, diluent or carrier. Depending on the mode of administration, the pharmaceutical preparation will typically comprise from 0.01 to 100 pg/ml of the radioligand, preferably from 0.01 to 20 pg/ml, more preferably from 0.01 to 10 pg/ml, most preferably from I 0.01 to 5 pg/ml, for example from 0.02 to 2 pg/ml. The pharmaceutically acceptable adjuvant, diluent or carrier may in preferred embodiments be liquid-based for providing a pharmaceutical preparation which may be administered intravenously.

Visualization of the compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, at the beta cell population in the pancreas may be achieved by means of pure PET imaging. However a positron emission tomography-computed tomography (PET-CT) system is preferably used. The benefit of the integration of PET with CT scanning is that the distribution of the radioligand in the body can be more precisely aligned or correlated with anatomic imaging obtained by CT scanning. Distribution of the radioligand may for instance be measured in a Siemens PET/CT Biograph system. In other embodiments, a positron emission tomography-magnetic resonance imaging (PET-MRI) system is used. Similar benefits as described in relation to PET-CT may be obtained by the use of PET-MRI.

Quantification of beta cell mass may be achieved though analysis of PET data using models familiar to the skilled person. For instance, two-tissue compartment modelling, Logan's linear graphical analysis and wavelet-aided parametric imaging using plasma input (PWAPI) can be used to determine distribution volume (V _T ). Other models including one-tissue compartment models, multilinear analysis models and simplified reference tissue models may also be used. The results of beta cell mass quantification may in turn be used for comparing against known or standard reference beta cell mass data representative of a healthy subject in order to assist in diagnosing a patient with a beta cell-related disorder. In embodiments, the beta cell-related disorder is selected from type 1 diabetes mellitus, type 2 diabetes mellitus, hyperinsulinemia or pancreatic cancer such as insulinomas. An increase of the beta cell mass in the subject under investigation relative to the reference value may indicate hyperinsulinemia, while a reduction of the beta cell mass in the subject under investigation relative to the reference value may indicate diabetes mellitus of type 1 or 2.

In another aspect, the present invention also provides an in vitro or ex vivo method of visualizing pancreatic beta cells in a sample, said method comprising the steps of: a) introducing a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof, to a sample comprising pancreatic cells; and

b) visualizing the beta cells in the sample.

Beta cells in a sample may be incubated with compound of formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof, before being visualized. Visualization may be, for instance, by autoradiography techniques familiar to the skilled person. For example, a sample may be exposed to phosphor-imager screens and scanned using a phosphor imager (for example, a Cyclone Plus Phosphor imager - Perkin Elmer, or a Fujifilm BAS-5000 phosphor imager - Fujifilm, Tokyo, Japan) before being analyzed using, for instance, ImageJ (NIH). Similar benefits as described in relation to visualization by autoradiography may be obtained by the use of nonradioactive tracers such as fluorescent, luminescent etc tags and applicable visualization instruments. In ex vivo methods, it will be appreciated that a compound of formula (II) may be introduced into a subject or animal, following which a sample comprising beta cells is removed from the subject and the beta cells in the sample can be visualized ex vivo. One such clinical utility might be visualization of complete resection of an insulinoma being either the primary tumor or its metastasis. In yet another aspect, the invention also provides a kit for selectively imaging pancreatic beta cells, or for use in diagnosing a beta cell related disorder, said kit comprising a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof, as defined hereinbefore. In addition to packaging, the kit may include instructions for use of the compound of formula (I) or formula (II) in an in vitro, ex vivo or in vivo visualization method or method of diagnosis. As will be appreciated, given the radioactive stability of the compounds, the kit may be adapted for immediate use rather than storage for any length of time. In a further aspect, the invention also provides a kit for preparing a compound for use in selectively imaging pancreatic beta cells comprising: a compound of formula (III), or a pharmaceutically acceptable salt or solvate thereof, as defined hereinbefore. In addition to packaging, the kit may include instructions for use of the compound of formula (III), in the preparation of a compound of formula (I) or (II) for use in an in vitro or in vivo or ex vivo beta cell visualization method or method of diagnosis of a beta cell related disorder. In other embodiments, the kit may also comprise a compound selected from ¹¹ CH ₃ R or C( ³ H) _x ( ¹ H)yR, wherein R is Br, CI or I; and wherein i) and ii) are not in admixture in the kit. In another aspect, the present invention also provides a use of a compound according to formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof, as defined herein as a radioligand, preferably wherein the radioligand is a GPR44 radioligand. In preferred embodiments the invention provides use of a compound according to formula (I), or a pharmaceutically acceptable salt or solvate thereof, as defined herein as a PET radioligand. In preferred embodiments, the PET radioligand is used to monitor a change in beta cell mass, for example to monitor the impact of a therapeutic intervention or to monitor a beta cell-related disorder

The invention will now be illustrated by the following non-limiting examples and Figures wherein:

Figures 1 A to 1 E: show the results of in vitro autoradiography (ARG) of binding of a compound of formula (I) in pancreas from human non-diabetic subjects (Figure 1 A), human subjects with T2D (Figure 1 B) and rat pancreas (Figure 1 C), as well as the uptake in the whole pancreatic sections (Figure 1 D) and enrichment in islet hotspots (Figure 1 E);

Figure 2: shows the results of islet-to-exocrine specific binding ratio for a nanomolar concentration of radioligand of formula (I);

Figure 3a: shows the results of a competition binding assay to determine the potency of antagonists at human GPR44 in vitro by quantifying the ability of unlabeled ligand, (2S)- 2-(4-chloro-2-(2-chloro-4-(methylsulfonyl)phenoxy)phenoxy)pr opanoic acid, to displace binding of [ ³ H]ProstaglandinD ₂ (PGD ₂ ) from membranes of HEK293 cells transfected with human recombinant GPR44 and Ga16;

Figure 3b: shows the results of a competition binding assay to quantify the ability of the unlabelled ligand, (2S)-2-(4-chloro-2-(2-chloro-4-(methylsulfonyl)phenoxy)pheno xy)- propanoic acid, to displace a radioligand of formula (II);

Figure 4: shows the results of a dynamic mass redistribution (DMR) assay with unlabelled ligand, (2S)-2-(4-chloro-2-(2-chloro-4-(methylsulfonyl)phenoxy)pheno xy)- propanoic acid in displacing the effect of PGD ₂ on human clonal beta cells (EndoC cells);

Figure 5a: shows the results of a fluorescence-activated cell sorting (FACS) analysis of human islet cells demonstrating GPR44 positive cells to be insulin positive cells. An isotype control showed the GPR44 antibody to be specific. Figure 5b: shows the results of experiments to determine the levels of insulin (INS) mRNA expression in FACS sorted GPR44 positive cells and GPR44 negative cells;

Figures 6a and 6b: show the results of investigations into quantification of binding in pancreas for a radioligand of formula (I) using <20 min PET data and the 2-tissue compartment model for baseline (Figure 6a) and following pretreatment with a GPR44 specific compound (Figure 6b); and

Figure 7: shows the results of PET data analysis using the two-tissue compartment model, Logan's linear graphical analysis and wavelet-aided parametric imaging using plasma input (PWAPI) for deriving the distribution volume (V _T ), for base line experiments with a radioligand of formula (I) and experiments where there has also been a pretreatment with a GPR44 specific compound.

In the below examples, the following abbreviations are used:

EtOAc - ethylacetate

DCM - dichloromethane

DCE - dichloroethane

EtOH - ethanol

THF - tetrahydrofuran

AcOH - acetic acid

Ac ₂ 0 - acetic anhydride

DMF - N, N-dimethylformamide

ACN - acetonitrile

AF - ammonium formate

PVT - polyvinyltoluene

DMSO - dimethyl sulphoxide

mCPBA - meta-chloroperoxybenzoic acid Example 1

(S)-methyl 2-(2-(4-((acetoxymethyl)thio)-2-chlorophenoxy)-4-chloropheno xy)- propanoate, compound according to formula (V) i) 5-chloro-2-methoxyphenol, compound according to formula (XII)

Hydrogen peroxide (0.55 ml_, 5.38 mmol) was added to (5-chloro-2- methoxyphenyl)boronic acid (5 g, 26.82 mmol) in EtOH (100 ml_). The resulting solution was stirred at 80 C for 30 minutes, then cooled to room temperature naturally. The solvent was removed under reduced pressure. The residue was dissolved in DCM (200 mL), then washed with saturated brine (100 mL x2), The organic layer was dried over Na ₂ S0 ₄ , filtered and evaporated to afford 5-chloro-2-methoxyphenol (4.19 g, 99 %) ii) 2-chloro- 1 -(5-chloro-2-methoxyphenoxy)-4-nitrobenzene, compound according to formula (XI)

K ₂ C0 ₃ (5.48 g, 39.63 mmol) was added to 5-chloro-2-methoxyphenol (4.19 g, 26.42 mmol) from step ii) and 2-chloro-1 -fluoro-4-nitrobenzene (4.64 g, 26.42 mmol) in DMF (3 mL). The resulting solution was stirred at 120 C for 6 hours and then cooled to room temperature naturally. The reaction mixture was diluted with water. The precipitate was collected by filtration, washed with MeOH (20mL) and dried under vacuum to afford 2- chloro-1 -(5-chloro-2-methoxyphenoxy)-4-nitrobenzene (6.80 g, 82 %) as a yellow solid, which was used in step iii) below without further purification. iii) -chloro-4-(5-chloro-2-methoxyphenoxy)aniline, compound according to formula

Zinc (7.08 g, 108.24 mmol) was added to 2-chloro-1-(5-chloro-2-methoxyphenoxy)-4- nitrobenzene (6.8 g, 21.65 mmol) from step iii) in AcOH (100 mL). The resulting solution was stirred at 50 C for 1 hour and then cooled to room temperature naturally. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford 3-chloro-4-(5-chloro-2- methoxyphenoxy)aniline (5.50 g, 89 %) as a yellow solid.

(3-chloro-4-(5-chloro-2-methoxyphenoxy)phenyl)(methyl)sul fane, compound according to formula (IX)

Isoamyl nitrite (2.72 g, 23.23 mmol) was added to dimethyldisulfide (2.61 ml_, 29.04 mmol) and 3-chloro-4-(5-chloro-2-methoxyphenoxy)aniline (5.5 g, 19.36 mmol) from step iii) in DCE (100 mL). The resulting solution was stirred at 60 °C for 1 hour and then stirred for an hour at room temperature. The reaction mixture was diluted with DCM (100 mL) and washed sequentially with water (100 mL x1 ) and saturated brine (100 mL x2). The organic layer was dried over Na ₂ S0 ₄ , filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford (3-chloro-4-(5-chloro-2-methoxyphenoxy)phenyl)(methyl)sulfan e (3.15 g, 51.6 %) as a yellow oil.

4-chloro-2-(2-chloro-4-(methylthio)phenoxy)phenol, compound accordi formula (VIII)

A solution of BBr ₃ (4.72 mL, 49.97 mmol) in DCM (20 mL) was added dropwise to a stirred solution of (3-chloro-4-(5-chloro-2-methoxyphenoxy)phenyl)(methyl)sulfan e (3.15g, 9.99 mmol) from step iv) in DCM (80 mL) at 0 °C. The resulting solution was stirred at 0 °C for 2 hours. Water (20 mL) was added dropwise at 0 C to quench the reaction. The reaction mixture was diluted with DCM (50 mL), and washed sequentially with water (75 mL x1 ) and saturated brine (75 mL x1 ). The organic layer was dried over Na ₂ S0 ₄ , filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 15% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford 4-chloro-2-(2-chloro-4- (methylthio)phenoxy)phenol (2.300 g, 76 %) as a white solid. vi) (S)-methyl 2-(4-chloro-2-(2-chloro-4-methylthio)phenoxy)phenoxy)propano ate, compound according to formula (VII)

A solution of diethyl azodicarboxylate (DEAD) (1 .262 mL, 7.97 mmol) in THF (8mL) was added to a stirred solution of 4-chloro-2-(2-chloro-4-(methylthio)phenoxy)phenol (2.0 g, 6.64 mmol) from step v), (R)-methyl 2-hydroxypropanoate (0.726 g, 6.97 mmol) and triphenyl phosphine (1 .742 g, 6.64 mmol) in THF (60 mL) at 0 °C. The resulting solution was stirred at 25 °C for 16 hours. The solvent was removed under reduced pressure. The residue was dissolved in petroleum ether-ethyl ether (2:1 ,150 mL). The solids were filtered off and the filtrate was concentrated under vacuum. The crude product was purified by flash silica chromatography, elution gradient 0 to 15% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford (S)-methyl 2-(4-chloro-2-(2- chloro-4-(methylthio)phenoxy)phenoxy)propanoate (2.50 g, 97 %) as a yellow oil, m/z (ES+), [M+Hf = 387. vii) (2S) methyl-2-(4-chloro-2-(2-chloro-4-(methylsulfinyl)phenoxy)phe noxy)- propanoate, compound according to formula (VI)

Meta-chloroperoxybenzoic acid (mCPBA) (1 .176 g, 6.82 mmol) was added to (S)-methyl 2-(4-chloro-2-(2-chloro-4-(methylthio)phenoxy)phenoxy)propan oate (2.4 g, 6.20 mmol) from step vi) in DCM (30 mL). The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 50 to 90% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford (2S)-methyl 2-(4-chloro-2-(2- chloro-4-(methylsulfinyl)phenoxy)phenoxy)propanoate (1 .90 g, 76 %) as a pale yellow oil, m/z (ES+), [M+H] ⁺ = 403. viii) (S)-methyl 2-(2-(4-((acetoxymethyl)thio)-2-chlorophenoxy)-4-chloropheno xy)- propanoate, compound according to formula (V)

Sodium acetate (1 .159 g, 14.13 mmol) was added to (2S)-methyl 2-(4-chloro-2-(2- chloro-4-(methylsulfinyl)phenoxy)phenoxy)propanoate (1 .9 g, 4.71 mmol) from step vii) in AC2O (25 mL). The resulting mixture was stirred at 140 °C for 4 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 10 to 40% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford (S)-methyl 2-(2-(4-((acetoxymethyl)thio)-2- chlorophenoxy)-4-chlorophenoxy)propanoate (1.400 g, 66.7 %) as a colourless gum, m/z (ES+), [M+Naf = 476.

Example 2

(S)-methyl 2-(2-(4-((acetoxymethyl)sulfonyl)-2-chlorophenoxy)-4-chlorop henoxy)- propanoate, compound of formula (IV)

Magnesium 2-carboperoxybenzoate (1.823 g, 4.72 mmol) was added to (S)-methyl 2-(2- (4-((acetoxymethyl)thio)-2-chlorophenoxy)-4-chlorophenoxy)pr opanoate (1.4 g, 3.14 mmol) from Example 1 in MeOH (10 mL) and DCM (20 mL). The resulting mixture was stirred at room temperature for 12 hours. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC using decreasingly polar mixtures of water (containing 0.1 % Formic acid) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford (S)-methyl 2-(2- (4-((acetoxymethyl)sulfonyl)-2-chlorophenoxy)-4-chlorophenox y)propanoate (1.000 g, 66.6 %) as a colourless gum, m/z (ES+), [M+Naf = 499.

Example 3

(2S)-2-(4-chloro-2-(2-chloro-4-sulfinophenoxy)phenoxy)propan oic acid, compound of formula (Ml)

Sodium hydroxide (0.168 g, 4.19 mmol) was added to (S)-methyl 2-(2-(4- ((acetoxymethyl)sulfonyl)-2-chlorophenoxy)-4-chlorophenoxy)p ropanoate (0.8 g, 1.68 mmol) from Example 2 in THF (10 ml_) and water (3 ml_). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC, using decreasingly polar mixtures of water (containing 0.1 % NH ₄ HC0 ₃ ) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford, as a white solid, the disodium salt of (2S)-2-(4-chloro-2-(2-chloro-4-sulfinophenoxy)phenoxy)propan oic acid (0.530 g, 72.3 %).

Example 4 - production of [ ¹¹ C]methyl iodide ([ ¹¹ C]CH ₃ I)

[ ¹¹ C]Methane ([ ¹¹ C]CH ₄ ) was produced in -target via the ⁴ N(p,c/.) ¹¹ C reaction on nitrogen with 10% hydrogen, with 16.4 MeV protons using a GEMS PET trace cyclotron (GE, Uppsala, Sweden). Typically the target gas was irradiated for 20-30 minutes with a beam intensity of 35 μΑ. [ ¹¹ C]CH ₄ was released from the target and isolated on a Porapak Q trap cooled with liquid nitrogen. Following collection [ ¹¹ C]CH ₄ was released by warming the trap with pressurized air. [ ¹¹ C]CH ₄ was then mixed with vapors from iodine crystals followed by a radical iodination reaction in a closed recirculation system to produce [ ¹¹ C]CH ₃ I. The formed [ ¹¹ C]CH ₃ I was collected on a Porapak Q trap. [ ¹¹ C]CH ₃ I was released from the Porapak Q trap by heating the trap using a custom-made oven.

Preparation of PET radioligands according to formula (I) and (ID The HPLC system used in the below examples for the preparation of radioligands according to formula (I) and (II) included a semi-preparative reverse phase (RP) ACE column (C18, 10 ^x 250 mm, 5 μιτι particle size) and a Merck Hitachi UV detector (λ= 254 nm) (VWR, International, Stockholm, Sweden) in series with a GM-tube (Carroll- Ramsey, Berkley, CA, USA) used for radioactivity detection. Radiomethylation, purification, and formulation were performed using a computer controlled automated system (Scansys, Denmark).

Example 5

(2S)-2-(4-chloro-2-(2-chloro-4-([ ¹¹ C]methylsulfonyl)phenoxy)phenoxy)-propanoic acid, compound of formula (I) Carbon-1 1 labelled [ ¹¹ C]CH ₃ I was obtained by the method of Example 4 above and the released [ ¹¹ C]CH ₃ I was trapped at room temperature in a reaction vessel containing the disodium salt of the compound of formula (III), (2S)-2-(4-chloro-2-(2-chloro-4- sulfinophenoxy)phenoxy)-propanoic acid, from Example 3 (1.0 mg-2.0 mg, 2.48 μιηοΙ- 4.95 μιηοΙ) in DMF (300 μΙ_). The reaction mixture was then heated at 70°C for 5 minutes. The reaction mixture was diluted with sterile water (500 μΙ_) before injecting the mixture into a built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled title compound. The product was eluted with a mobile phase of 35% acetonitrile (ACN) in ammonium formate (AF, 0.1 M) containing sodium-L- ascorbate (500 mg/L) with a flow rate of 4 mL/min which gave a radioactive fraction corresponding to pure (2S)-2-(4-chloro-2-(2-chloro-4-([ ¹¹ C]methylsulfonyl)phenoxy)- phenoxy)propanoic acid. The collected fraction from HPLC was evaporated to dryness and re-formulated in 6-8 mL phosphate buffered saline (PBS) (pH 7.4).

Example 6

(2S)-2-(4-chloro-2-(2-chloro-4-([ ³ H]rnethylsulfonyl)phenoxy)phenoxy)propanoic acid, compound of formula (II)

[ ³ H] Methyl Iodide ([ ³ H]CH ₃ I) (available from American Radiolabeled Chemicals, St. Louis, MO, USA) was added to a reaction vessel containing the disodium salt of the compound of formula (III), (2S)-2-(4-chloro-2-(2-chloro-4-sulfinophenoxy)phenoxy)- propanoic acid, from Example 3 (1.0 mg-2.0 mg, 2.48 μιηοΙ-4.95 μιηοΙ) in DMF (300 μί) at room temperature. The reaction mixture was then heated at 70°C for 5 minutes. The reaction mixture was then purified using high performance liquid chromatography (HPLC). The title compound was eluted using 30:70 [acetonitrile (ACN):0.1 M ammonium formate (AF)] mobile phase composition at a flow rate of 5 mL/min. The solvent from the collected HPLC fraction was removed by evaporation under vacuum below 40°C which afforded pure title compound which was subsequently formulated with 70% EtOH in water. Example 7 - radiochemical purity, radiochemical identity, specific radioactivity and stability investigations

The radiochemical purity, radiochemical identity and stability of the title compound of Example 5 were determined using an analytical HPLC system which included an Eclips XDB RP column (Agilent, C18, 4.6 * 150 mm, 5 pm particle size), Merck-Hitatchi L-7100 Pump, L-7400 UV detector and GM-tube for radioactivity detection (VWR International). A wavelength of 254 nm and a mobile phase system of 25% acetonitrile (ACN) in ammonium formate (AF) (0.1 M) at a flow rate of 3 mUmin was used for the analysis. The radiochemical purity, radiochemical identity and the stability of the title compound of Example 6 were determined using analytical HPLC system which included an ACE RP column (C18, 4.6 * 150 mm, 5 μιη particle size), Merck-Hitatchi L-7100 Pump, L-7400 UV detector and GM-tube for radioactivity detection (VWR International). A wavelength of 254 nm and a mobile phase system of 30% ACN in AF (0.1 M) at flow rate of 2 mL/min was used for the analysis. The specific radioactivity (SA) was calibrated for UV absorbance (λ = 254 nm) response per mass of ligand and calculated as the radioactivity of the radioligand (GBq) divided by the amount of associated carrier substance (μιτιοΙ). Each sample was analyzed two times and compared to a reference standard analyzed two times. The identity and the purity of the title compound of Example 5 was confirmed by co- injection with unlabeled reference standard, (2S)-2-(4-chloro-2-(2-chloro-4- (methylsulfonyl)phenoxy)phenoxy)-propanoic acid. The unlabeled reference standard, (2S)-2-(4-chloro-2-(2-chloro-4-(methylsulfonyl)phenoxy)pheno xy)-propanoic acid may be produced by any suitable method, for example by the method described in WO 2005/018529, or by the method of Example 6 using unlabeled methyl iodide. At the end of the synthesis, the radiochemical purity was determined to be 98±0.9% (n=20) and the specific radioactivity obtained was 1351 ±575 GBq/pmol (n=18). The stability of the title compound of Example 5 in the formulated solution of that example was tested at different time intervals; 30, 60, 90, 120 minutes after the synthesis using radio-HPLC. The formulated title compound of Example 5 was found to be radiochemically stable for up to 2 h.

The identity and the purity of title compound of Example 6 was confirmed by co-injection with unlabeled reference standard, (2S)-2-(4-chloro-2-(2-chloro-4- (methylsulfonyl)phenoxy)phenoxy)-propanoic acid. The formulation from Example 6 was stored at -18°C with specific radioactivity of 2 GBq/pmol and the purity of the radioligand was >99.9% up to one week after radiosynthesis. Example 8 - Liquid chromatography-mass spectrometry (LC-MS/MS) analysis

LC-MS/MS analysis of the radiolabeled compound of Example 5, and of the reference standard, (2S)-2-(4-chloro-2-(2-chloro-4-(methylsulfonyl)phenoxy)pheno xy)-propanoic acid, was performed using a Waters Acquity™ ultra performance LC system connected with a Micromass premier™ Quadrupole time of flight (TOF) mass spectrometer (Waters, Milford, MA, USA). LC was performed using a Waters Acquity UPLC™ BEH column (C18, 2.1 * 50 mm, 1.7 μιη particle size) kept at 50°C. The mobile phase consisted of 0.1 % formic acid in water (A) and 0.1 % formic acid in ACN (B). Samples were analyzed using a linear gradient (0 to 100% B in 5 min) at a flow rate of 0.5 mL/min. The MS was operated in electrospray ionization (+ESI) mode, with the following settings: capillary voltage 3.0 kV; cone voltage 35 V; source temperature 100°C; dissolvation temperature 400°C and collision energy 20 eV. The retention time of the formulated product of Example 5 and fragmentation pattern of the parent peak were identical to those of the reference standard, unlabeled (2S)-2-(4-chloro-2-(2-chloro-4- (methylsulfonyl)phenoxy)phenoxy)propanoic acid.

Example 9 - Frozen tissue autoradiography (ARG) experiments

Pancreatic biopsies were collected from deceased human donors of healthy subjects, subjects with type 2 diabetes (T2D), as well as from Sprague Dawley rats. The biopsies were frozen to -80°C and processed into 20 pm slices. The rat pancreas was used as a negative control, since rat pancreatic islets does not express GPR44. The use of human tissue was approved by the Uppsala Ethical Review Board (Dnr 2015-401 ; #201 1/473, #Ups 02-577) and tissues obtained from Uppsala Biobank. For ARG experiments using the radioligand of formula (I), corresponding to the title compound of Example 5, all sections (healthy subjects, n=6 and subjects with T2D, n=6), as well as from Sprague Dawley rats (n=4) were pre-incubated in 100 ml_ 50 mM PBS (pH 7.4) for 10 minutes. Then, radioactivity corresponding to 1 nM of the radioligand (n=3) was added, and the sections were incubated with the radiotracer for 30 minutes at room temperature. Non-displaceable binding was assessed in a separate assay, by co-incubation with 20 μΜ of a GPR44 antagonist compound of formula (XIV). The compound of formula (XIV) may be synthesized by any suitable method, for example the method described in WO 2005/018529.

For ARG experiments using the radioligand of formula (II), corresponding to the title compound of Example 6, all sections (healthy subjects, n=3 and Sprague Dawley rats n=3) were pre-incubated in 100 ml_ 50 mM PBS (pH 7.4) for 10 minutes. Then, 1 nM of the radioligand (n=3) was added, and the sections were incubated with the radiotracer for 3 hours at room temperature. Non-displaceable binding was assessed in a separate assay, by co-incubation with 20 μΜ of a GPR44 antagonist compound of formula (XIV). Following incubation, tissue sections were washed 3 times for 2 minutes in 50 mM PBS at 4°C. The sections were dried and exposed to phosphor-imager screen for 40 minutes in case of the radioligand of formula (I), and 90 h in case of the radioligand of formula (II). The screens were scanned using a Cyclone Plus Phosphor imager (Perkin Elmer) at 600 dpi in case of the radioligand of formula (I), or a Fujifilm BAS-5000 phosphor imager (Fujifilm, Tokyo, Japan) in case of the radioligand of formula (II), and analyzed using ImageJ (NIH). Regions of interest (ROIs) were drawn over the entire pancreatic sections as well as over regions corresponding to islets of Langerhans and exocrine tissue. Radioligand binding in Bq/g tissue was normalized against aliquots of the incubation solution as Bq/cc and expressed as a unit less measurement of enrichment (similar to Standardized Uptake Value (SUV) for in vivo PET examinations). Specific binding was defined by subtracting non-displaceable binding from total binding. In vitro ARG for binding of a compound of formula (I) showed that pancreatic uptake in humans is heterogeneous and localized to hotspots likely corresponding to islets of Langerhans in both non-diabetic subjects (Figure 1 A) and subjects with T2D (Figure 1 B), as opposed to rat pancreas which has no GPR44 expression (Figure 1 C). The uptake in the whole pancreatic sections (which is analogous with what is measured in a PET scanner) was almost completely displaceable in the human sections, but not the negative control rat pancreas (Figure 1 D). The enrichment in islet hotspots was greater than 7 times higher than the exocrine background in both healthy subjects and subjects with T2D (Figure 1 E). The hotspots obtained in non-diabetic subject were co-localized with insulin staining and therefore representing Islets of Langerhans.

Example 10 - Binding of a compound of formula (!) to pancreatic tissue homogenates

Isolated pancreatic islets and exocrine tissue were obtained within the Nordic network for Clinical Islet Transplantation Laboratory in Uppsala, Sweden. Pancreatic islets (93% islet purity) and exocrine tissues were homogenized in ice-cold 0.32 M sucrose by hand using a Dounce glass homogenizer using a polytron tissue homogenizer (Polytron® PT 3000, Kinematica AG, Littau, Switzerland) in ice-cold 0.32 M sucrose at a concentration of 6 mg/ml and then by hand using a Dounce glass homogenizer. Aliquots of the homogenates were stored at -80°C until used.

2 mg of homogenized Islets of Langerhans and exocrine tissue were separately incubated for 30 minutes at room temperature with radioactivity corresponding to 2.3, 1 1 or 23 nM of a compound of formula (I), corresponding to the title compound of Example 5, in 50 mM TRIS (pH 7.4) in a final incubation volume of 1 mL. 20 μΜ of a GPR44 antagonist compound of formula (XIV) was added for determination of non-specific binding. The samples were filtered using a Brandel M-48 cell harvester with Whatman GF/C filter (presoaked with 50 mM TRIS) and washed four times with 3 mL 50 mM TRIS (RT). All samples were performed in triplicates. Filters were measured in a well counter (Uppsala Imanet AB, Uppsala, Sweden). The specific binding was calculated by subtracting the non-specific binding from the total binding, and the islet-to-exocrine binding ratio was calculated.

Binding of the radioligand to the preparations of Islets of Langerhans was displaceable (60-100%) by 20 μΜ GPR44 antagonist compound for all tracer concentrations. The binding to exocrine preparations was magnitudes lower, but also displaceable. The specific binding was 8-20 times higher in islets of Langerhans than in exocrine preparations (Figure 2). Example 11 - Ligand Binding Assay

The potency of antagonists at human GPR44 was determined in vitro by quantifying the ability of unlabeled (2S)-2-(4-chloro-2-(2-chloro-4-(methylsulfonyl)phenoxy)pheno xy)- propanoic acid to displace binding of [ ³ H]ProstaglandinD ₂ (PGD ₂ ) from membranes of HEK293 cells transfected with human recombinant GPR44 and Ga16. [ ³ H]PGD ₂ was purchased from Perkin Elmer Life Sciences with a specific activity of 100-210 Ci/mmol. All other chemicals were of analytical grade.

HEK cells expressing recombinant human GPR44/Ga16 were routinely maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Foetal Bovine Serum (FBS, HyClone), 1 mg/mL G418 (geneticin), 2 mM L-glutamine and 1 % non-essential amino acids. For the preparation of membranes, adherent transfected HEK cells were grown to confluence in two layer tissue culture factories (Fisher, catalogue number TKT- 170-070E). Maximal levels of receptor expression were induced by addition of 500 mM sodium butyrate for the last 18 hours of culture. The adherent cells were washed once with phosphate buffered saline (PBS, 50 mL per cell factory) and detached by the addition of 50 mL per cell factory of ice-cold membrane homogenisation buffer [20 mM HEPES (pH 7.4), 0.1 mM dithiothreitol, 1 mM EDTA, 0.1 mM phenyl methyl sulphonyl fluoride and 100 pg/mL bacitracin]. Cells were pelleted by centrifugation at 220x g for 10 minutes at 4°C, re-suspended in half the original volume of fresh membrane homogenisation buffer and disrupted using a Polytron homogeniser for 2 x 20 second bursts keeping the tube in ice at all times. Unbroken cells were removed by centrifugation at 220x g for 10 minutes at 4°C and the membrane fraction pelleted by centrifugation at 90000x g for 30 minutes at 4°C. The final pellet was re-suspended in 4 mL of membrane homogenisation buffer per cell factory used and the protein content determined. Membranes were stored at -80°C in suitable aliquots.

The assay was performed in white 384-well (Non-Binding surface (NBS) plates with clear bottom (Corning)). Prior to assay, the HEK cells membranes containing CRTh2 were coated onto wheat germ agglutinin coated polyvinyltoluene (PVT) scintillation proximity assay (SPA) beads (Perkin Elmer). For coating, membranes were incubated with beads at typically 200 ^ig membrane protein per mg beads at 4°C with constant agitation 3-4 hours. The optimum coating concentrations were determined for each batch of membranes. The beads were pelleted by centrifugation (800x g for 10 minutes at 4°C), washed once with assay buffer (50 mM HEPES pH 7.4 containing 5 mM magnesium chloride) and finally re-suspended in assay buffer at a bead concentration of 10 mg/mL. Compounds, dissolved in dimethyl sulphoxide (DMSO), were added as 3x dilution series, starting at 3 μΜ. The volumes were normalised to 1 % DMSO in the final assay volume (50 μ!_). 1 μΜ of unlabelled (i.e. radioactively "cold") (2S)-2-(4-chloro-2-(2- chloro-4-(methylsulfonyl)phenoxy)phenoxy)-propanoic acid was used as max control and 1 % DMSO as min control. 5 μΙ_ assay buffer was dispensed into the plate, followed by centrifugation at 200 xg, 1 min. 20 μΙ_ of 6.25 nM [ ³ H]PGD ₂ (final assay concentration 2.5 nM) and 25 μΙ_ membrane saturated SPA beads, both in assay buffer, were added. The assay plate was incubated at room temperature for 2-4 hours and counted on a Wallac Microbeta liquid scintillation counter (30 seconds per well). The result of the assay confirmed that unlabeled compound of formula (II), (2S)-2-(4- chloro-2-(2-chloro-4-(methylsulfonyl)phenoxy)phenoxy)propano ic acid, has a pSC ₅ o of 8.6 in the binding assay, as illustrated in Figure 3a.

Example 12 - Competition assay

To quantify the ability of unlabeled (2S)-2-(4-chloro-2-(2-chloro-4- (methylsulfonyl)phenoxy)phenoxy)-propanoic acid, to displace a radioligand of formula (II), corresponding to the title compound of Example 6, a radio filtration assay was set up. A 96-well flat bottom non-bind PS plate from Corning was used as assay plate and a 96-well Multiscreen HTS+HiFlow FB plate from Millipore was used as filter plate. The compound was tested on HEK membranes (similar to what was used in Example 12) at 7 g/well, and 3nM of radioligand of formula (II). The compound (in DMSO) was dispensed into the assay plate in a 3x dilution series, starting at 1 μΜ. The volumes were normalised to 1 % DMSO of the final assay volume (200 μΙ_). 1 μΜ of unlabeled compound of formula (II) (2S)-2-(4-chloro-2-(2-chloro-4-

(methylsulfonyl)phenoxy)phenoxy)-propanoic acid was used as max control and 1 % DMSO as min control. 20 μΙ_ of buffer (50 mM HEPES pH 7.4 containing 5 mM magnesium chloride, 10% BSA, w/v) was added, followed by 20μΙ_ of 30 nM compound of formula (II) and 160 μΙ_ of membrane. The plate was incubated for 3 hours, shaking. 170 μΙ_ of the reaction was transferred to the filter plate followed by 4 times washing with ice cold PBS. The filter plate was dried prior to addition of scintillation solution. After 30 min of incubation the plate was counted on a Wallac Microbeta liquid scintillation counter (60 seconds per well). The unlabeled (2S)-2-(4-chloro-2-(2-chloro-4- (methylsulfonyl)phenoxy)phenoxy)-propanoic acid competed out the compound of formula (II) in a dose response manner with full inhibition, as illustrated in Figure 3b. The unlabeled compound of formula (II), (2S)-2-(4-chloro-2-(2-chloro-4- (methylsulfonyl)phenoxy)phenoxy)propanoic acid has a plC50 of 8.6 in the competition assay (Figure 3b). Example 13 - Potency measure using dynamic mass redistribution (DMR) assay

DMR is measured by resonant waveguide grating (RWG) using an Epic biosensor (Corning). This is a label-free technology capturing the integrated cell response as a consequence of morphological changes and changes in distribution of cellular components after receptor stimulation (Schroder R, Janssen N, Schmidt J, Kebig A, Merten N, Hennen S, et al. Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat Biotechnol. 2010 Sep; 28(9):943-9). Human EndoC-βΗΙ cells (clonal beta cell line) endogenously expressing hGPR44 were used to determine antagonist activity at the human GPR44 receptor. The cells were plated at a density of 2x10 ⁴ cells/well in 384-well fibronectin-coated Epic biosensor plates (Corning) and cultured at 37°C, 5% CO ₂ for 24 h. On the day of experiment, the cells were washed with assay buffer (IxHBSS, 20 mmol/L HEPES (pH 7.4) and 0.2 % BSA) and allowed to equilibrate for 1 h inside the Corning Epic Biosensor at 26°C. Following equilibration, a 5 min scan was performed to create a baseline read before applying unlabeled compound of formula (II), (2S)-2-(4-chloro-2-(2-chloro-4- (methylsulfonyl)phenoxy)phenoxy)-propanoic acid, at a concentration range of 10 pmol/L to 38 pmol/L diluted in assay buffer containing 150 pmol/L 15f?-15-methyl-PGD ₂ using a CyBi-Well vario. The real-time measurement of the dynamic mass response (DMR) was detected during a 60 min scan. Data were fitted according to a 4 parameter logistic fit using the equation y = A + ((B-A)/1 + ((C/x) ^A D))) where A is no activation, B is full activation, C is the IC50 and D is the Hill slope. The unlabeled compound of formula (II), (2S)-2-(4-chloro-2-(2-chloro-4- (methylsulfonyl)phenoxy)phenoxy)-propanoic acid, has a plC ₅ o of 8.1 in the DMR potency assay (Figure 4).

Example 14 - Identification of GPR44 expressing cell in primary human islet

Human primary islet cells were purchased from Prodo Laboratories, USA. Dissociated human islets cells were fixed with Fixation buffer I (BD Biosciences) at 37°C for 10 minutes. Cells were washed in DPBS without calcium and magnesium and permeabilized with Perm/Wash buffer (BD Biosciences). Cells were incubated over night at 4°C with primary antibodies, mouse anti-human GPR44 AlexaFluor 647 (BD Biosciences) or isotype AF647 control, rabbit anti-insulin-PE (Cell Signaling technology, Beverly, MA). Cells were washed twice in BD Perm/wash buffer and resuspended in DPBS without calcium and magnesium containing 2% FBS and 5mM EDTA. Analysis was performed on a BD LSR Fortessa (BD Biosciences).

Fluorescence-activated cell sorting (FACS) analysis revealed that the GPR44 positive cells were also insulin positive. The isotype control showed that the GPR44 antibody was specific (Figure 5a). For FACS sorting of viable dissociated human islets cells were stained only with mouse anti-human GPR44 AlexaFluor 647 and the viability marker 7AAD (BD Biosciences) in DPBS without calcium and magnesium containing 2% FBS and 5mM EDTA. Sorting was performed using a BD FACS Arialll (BD Biosciences).

To further strengthen GPR44 to be specifically expressed in human beta cells, the expression of insulin was measured in the FACS sorted human islet cells stained for GPR44. RNA was isolated from the sorted cells (GPR44 positive and GPR44 negative, respectively) using Qiagen RNeasy microkit with on-column DNAse digestion (Qiagen, Hombrechtikon, Switzerland). Complementary DNA (cDNA) was generated using the High-Capacity cDNA reverse transcription kit (Applied Biosystems) and RT-qPCR analysis was performed with an ABI Prism 7900 (Applied Biosystems) using Taqman Gene expression assays for insulin following manufacturer's instructions. The data was normalized against acidic ribosomal phosphoprotein P0 (m36B4) expression for each sample. GPR44 positive cells were found to have high insulin mRNA expression while the GPR44 negative cells had low levels of insulin mRNA expression (Figure 5b). Example 15 - In vivo testing

PET measurements were performed in anesthetized cynomolgus monkeys. Anesthesia was induced with an intramuscular (im) injection of Ketalar (Ketamine) and maintained by a continuous intravenous infusion (1 ml/kg/hr) of ketamine (4 mg/ml) and xylazine (0.4 mg/ml) using a syringe pump. Body temperature was maintained by a Bair Hugger model 505 (Arizant Healthcare, MN) and monitored by an oesophageal thermometer. Monkeys were fitted with indwelling catheters to allow for intravenous injection of radioligand and pretreatment drug, and blood sampling for determination of radioactivity in plasma, metabolism and plasma protein binding of radioligand. The monkeys were observed continuously during the PET-CT experimental session. Continuous physiological monitoring was performed including non-invasive blood pressure, oxygen saturation (Sp0 ₂ ), ECG and pulse. The fluid balance was controlled during the whole experimental session by a continuous intravenous infusion of saline 9 mg/mL (2 mL/kg monkey/h) using a syringe pump. The monkeys were also monitored for any adverse effects related to the infusion of the radioligand of formula (I), corresponding to the title compound of Example 5, and GPR44 drug pretreatment with a compound of formula (XIV). After the experiment the monkeys were returned to the stables and observed for changes in appearance and behaviour, signs of ill health, and mortality.

The monkeys were positioned in the PET-CT system to allow PET imaging over the lung to kidney area. Distribution of the radioligand was measured in a Siemens PET/CT Biograph system. One low-dose CT was performed before intravenous administration of the radioligand, and this CT data was used for attenuation correction. The radioligand was injected as a bolus into a sural vein during 5 seconds with simultaneous start of PET-data acquisition. In a subsequent experiment monkeys were pretreated with a 10- min infusion of the GPR44 specific compound of formula (XIV), (1 mg/kg), starting 20 min before administration of the radioligand. Injected radioactivity was in the range of 130 to 260 MBq.

Distribution of radioactivity was measured for 93 minutes according to a preprogrammed series of time frames starting immediately after injection of the radioligand. The initial frames were 4 ^χ 30 sec; followed by 4 * 60 sec; 1 1 * 180 sec and finally 9 * 360 sec. PET images were reconstructed with the OSEM algorithm, with four iterations and eight subsets, using a 5-mm Gaussian filter. Regions of interest (ROIs) were delineated for pancreas and spleen on the CT scan which was in register with the PET images. Radioactivity concentration (nCi/cm ³ ) in the ROIs for each PET measurement was decay-corrected to the time of injection and plotted versus time. PET data were analysed using the two-tissue compartment model. The outcome parameter was the distribution volume (V _T ). Alternative approaches for V _T estimation were also evaluated including Logan's linear graphical analysis and wavelet-aided parametric imaging using plasma input (PWAPI) (calculation of parametric image being restricted to voxels in the area of pancreas and spleen). The latter approach provided quantified images of voxel-wise estimates of V _T .

The results from the PET experiments demonstrate the following: a PET signal was detected in the pancreas;

following pretreatment of a GPR44 specific drug of formula (XIV), a clear effect on the uptake in pancreas was observed supporting radioligand GPR44 target engagement; and

the radioligand showed favourable kinetics for quantification of binding in the pancreas using <20 min PET data and the 2-tissue compartment model (Figures 6a and 6b).

In the pretreatment experiments, quantification using three approaches were evaluated. Results from linear graphical analysis (Logan) and wavelet-aided parametric imaging using plasma input (PWAPI) overall agree well with two-tissue compartment modelling, as illustrated in Figure 7. Total binding in the pancreas and spleen is reduced following pretreatment. The maximum reduction of total binding after pretreatment is substantial (>70%) using any of the models. Thus, assuming that essentially all specific binding could be blocked, specific binding should be a large part of total binding so that BP _ND at baseline may be 2 or higher.

The above in vivo experiments demonstrate that the radioligand of formula (I), corresponding to the title compound of Example 5, is suitable for quantification of GPR44 receptors in pancreas for either drug occupancy measurements or as an imaging biomarker for beta cell mass.