18f-CXCR2 pet tracer

Abstract

The present invention relates to a PET tracer of formula (I).

Description

[0001] 18F-CXCR2 PET Tracer

[0002] BACKGROUND

[0003] The present invention relates to novel18F-CXCR2 PET tracers for functional imaging.

[0004] The innate immune system represents the first set of biological response against pathogenic and non-pathogenic microbes and can be divided into four distinct defence-mechanisms, anatomic, physiologic, phagocytic / endocytic and inflammatory barriers.1One key player of innate immunity are neutrophils, representing phagocytic response.2While neutrophils primarily eliminate bacteria and fungi to protect the host organism at an early stage of infiltration, recent reports highlight the ambivalent role of neutrophils in inflammation-induced disease.3-7Hence, several studies investigated the therapeutical potential of neutrophil manipulation, aiming the treatment of pulmonary, cardiovascular or autoimmune diseases, as well as sepsis, cancer or neutropenia.3’5’8'11Pharmaceutical companies transferred numerous neutrophil-affecting small molecules to clinical trials, predominantly targeting chemokine receptors.7’12’13Neutrophils have fast turnover, unexpected plasticity or reduced expression and / or recruitment due to endogenous modulators.14'17

[0005] Therefore, to further understand the role of neutrophils, to quantify and correlate neutrophil population to disease and disease progression, and to diagnose diseases, functional imaging by positron emission tomography (PET) offers a dynamic technique. PET offers high detection sensitivity, spatial and temporal resolution and can be combined with computed tomography (CT) and magnetic resonance imaging (MRI) making it a superior platform for neutrophil imaging. But, as PET imaging is dependent on the utilized tracer, carrying a positron-emitting nuclide, a target structure that is primarily expressed on neutrophils has to be selected prior to tracer development. Interleukin 8 receptor, beta (CXCR2), first described in 1991 , is expressed predominantly on neutrophils, making CXCR2 a promising target structure for PET imaging.18’19Regarding this approach, Rennen et al. developed a [99mTc]Tc-CXCL8 radiotracer to target the CXC chemokine receptors CXCR1 and 2 from their previous candidate based on123l for single-photon emission computed tomography (SPECT).20'23Although the targeting of CXCR2 shows high potential due to its almost exclusive expression of neutrophils, reducing the risk of off-target binding and therefore enhancing image resolution, this approach suffers from significant drawbacks. While non-modified polypeptides, such as CXCL8, containing 72 amino acids resulting in a molecular weight of 8.4 kDa, are prone to a rapid degradation in vivo due to cleavage by endogenous proteases, the approach is also restricted by the use of99mTc, limited to SPECT. An established technique for the imaging of leukocytes routinely used, is the autologous labelling by11ln introduced in 1976.24This ex vivo technique, relying on the labelling of all circulating white blood cells and planar scintigraphy, represents a time-consuming, complex and often painful procedure for the patient, as well as the handling with possibly contaminated blood.21 23

[0006] Consequently, it was an object of the present invention to provide a PET tracer, that enables in vivo diagnosis of various diseases, and in particular enables the in vivo molecular imaging of neutrophil population.

[0007] However, a key factor for PET imaging is the choice of a suitable radionuclide for tracer development. Fluorine-18 (18F) exhibits a suitable half-life for in vivo imaging of 110 min, enabling transport from cyclotron facilities to satellite imaging centres, thus increasing cost effectiveness and the implementation of delayed imaging protocols.25Furthermore,18F outperforms most PET radionuclides with its low maximum positron energy of 0.634 MeV, resulting in reduced tissue penetration, only being matched by52Mn, a barely utilized nuclide with a half-life of 5.6 days.26-28

[0008] Since the visualization of neutrophils by CXCR2 targeting is aimed for, a suitable ligand that allows fluoride incorporation, while keeping CXCR2 affinity and having high CXCR2 selectivity, is pivotal for the development of a novel radiotracer. In the last two decades, pharmaceutical companies have developed several ligands targeting CXCR2, that have been tested in clinical studies, investigating their effect on various diseases, e.g. COPD, neutrophilic asthma or breast cancer.29-32The investigated compounds can be divided into three main classes, phenyl acetamides (A), pyrimidine-2-thiols (B) and urea- and bioisosteric squaramides (C).12

[0009] Thus, it was an object of the present invention to provide a PET tracer that has a high CXCR2 affinity. In addition, it was an object of the present invention to provide a PET tracer that has a high CXCR2 selectivity. Furthermore, it was an object to provide a PET tracer that has a sufficient stability for in vivo diagnosis, i.e. has high metabolic and chemical stability. It was a further object to provide a PET tracer that can easily be manufactured and wherein the radionuclide can easily be introduced.

[0010] SUMMARY OF THE INVENTION The present invention specifically relates to a PET tracer of formula (I): wherein

[0011] R1and R2are independently selected from alkyl and Hydrogen,

[0012] A is -A1-A2,

[0013] A1is selected from O, NH, or a direct bond,

[0014] A2is selected from alkyl, aryl, heteroaryl, alkylaryl, and alkylheteroaryl,

[0015] B is -B1-B2,

[0016] B1is selected from sulfonyl, C(O), or a direct bond,

[0017] B2is selected from heterocyclyl, NH2, or -N(alkyl)2 n is 0 or 1 ,

[0018] R3is selected from18F, I, Br, Cl, and F, with the proviso that when n is 0 or R3is not18F, then A2or B2is substituted with a18F group.

[0019] It has surprisingly been found that squaramide derivative of formula (I) solves the above objects. The PET tracer of formula (I) has a high CXCR2 affinity. Furthermore, the18F can efficiently be introduced. In addition, it has been found that the PET tracers of formula (I) have a high metabolic stability, and excellent renal clearance.

[0020] In an embodiment, the present invention relates to a composition comprising the PET tracer.

[0021] In an embodiment, the present invention relates to a process comprising the steps of:

[0022] (i) providing an intermediate compound of formula (III) (III), wherein R4is a leaving group selected from aryloxy, halo, and heterocyclyloxy.

[0023] (ii) coupling said intermediate compound of formula (III) to a compound of formula (IV)

[0024] In an embodiment, the present invention relates to the PET tracer or the PET tracer composition use in the in vivo diagnosis of a disease.

[0025] Furthermore, the invention relates to the use of the PET tracer or PET tracer composition for in vitro diagnosis.

[0026] DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Synthesis and radioligand binding data of pyrrolidine derivative 3a, b.. Reactions and conditions: i) 3-amino-2-hydroxybenzoic acid, zinc triflate, DI PEA, DMF, rt, overnight; ii) 3-aminopentane, ethanol, rt, overnight; iii) pyrrolidines, HATLI, TEA, DMF, rt, overnight.

[0028] Figure 2: Indirect labelling using PFP ester 5 and [18F]-(R)-3-fluoropyrrolidine to yield [18F]3b. Reactions and conditions: i) pentafluorphenol, EDC HCI, CH2CI2, 0°C to rt, 16 h, 39%; ii) 1) [18F]TEAF, MeCN, 100°C, 10 min; 2) TFA, 110°C, 10 min; ii) DIPEA, MeCN, 80°C, 10 min, RCY: 16.9 ± 8.9%. An indirect approach was developed, utilizing tert-butyl (S)-3- (tosyloxy)pyrrolidine-carboxylate 6 as a prosthetic group, that, after deprotection, was coupled using pentafluorophenyl ester 5, as it had a lower susceptibility to degradation when compared to 4-nitrophenol and / V-hydroxysuccinimid (NHS) esters. Compound 4 was treated with / \ / -(3- dimethylaminopropyl)- / \ / '-ethylcarbodiimid (EDC)-hydrochloride and pentafluorophenol in CH2CI2, taking advantage of the low pKavalue of pentafluorophenol when compared to the salicylic acid component of 4. The prosthetic group [18F]-(R)-3-fluoropyrrolidine was obtained by generating [18F]TEAF by elution of [18F] F’ with tetraethylammonium bicarbonate from using an anion exchange cartridge (Sep-Pak QMA Cartridge). [18F]TEAF was treated with compound 5 in MeCN at 110°C for 10 min, followed by deprotection using trifluoroacetic acid (TFA). The corresponding [18F]-(R)-3-fluoropyrrolidine was carefully concentrated at 90°C under nitrogen flow to remove the solvent and TFA, taken up in MeCN and treated with PFP ester 5 and DI PEA, leading to the corresponding amide. After purification by semipreparative high- performance liquid chromatography (HPLC), the labelled target compound [18F]3b (compound Ila) was obtained in a RCY of 16.9 ± 8.9%, radiochemical purity >99% and specific activity of 2.37 GBq / pmol (n = 4)

[0029] Figure 3: Uptake of [18F]3b in different cell types. (A) Time dependent uptake of [18F]3b in HEK-CXCR2 cell. All quantitative data are presented as means ± SEM with n = 6. Statistical significance between different treatments was calculated by using two-way analysis of variance (ANOVA); ** p < 0.01 , *** p < 0.001 , **** p < 0.0001 HEK CXCR2 compared to CXCR2-negative HEK293 cells; (B) Time dependent uptake of [18F]3b in human peripheral blood neutrophils. All quantitative data are presented as means ± SEM with n = 2.

[0030] Figure 4: In vivo studies on [18F]3b. (A) Dynamic PET / CT fusion imaging in healthy Wistar rat; (B) Time-activity curves of [18F]3b in major organs; (C) Biodistribution studies 60 min after tracer injection.

[0031] DETAILED DESCRIPTION

[0032] Definitions

[0033] The following definitions are relevant in connection with the embodiments of the present invention.

[0034] The prefix "Cy-Cx" as used herein refers to the number of carbon atoms of the respective group. For heteroaryl and heterocyclyl groups, the prefix "Cy-Cx" refers to the number of atoms (including heteroatoms) in the aromatic or cyclic system.

[0035] As used herein, singular forms, such as "a", "an", and "the", include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a pharmaceutically acceptable excipient" means one or more than one pharmaceutically acceptable excipient.

[0036] The term "aryl" as used herein by itself or as part of another group refers to a monocyclic, bicyclic or tricyclic aromatic ring system having from six to fourteen carbon atoms (i.e., C6-C14 aryl)- Non-limiting exemplary aryl groups include phenyl (abbreviated as "Ph"), naphthyl, phenanthryl, anthracenyl, indenyl, biphenylyl including fluorenyl. The term “aryl” as used herein encompasses phenyls, naphthyls, phenanthryls, anthracenyls, and biphenylyls including fluorenyls that are substituted by one or more substituents, e.g. halo, nitro, cyano, hydroxy, alkoxy, and alkyl. It is to be understood that the term “aryl” thus also encompasses “optionally substituted aryl”.

[0037] The term "alkyl" as used herein by itself or as part of another group refers to a straight- or branched-chain aliphatic hydrocarbon containing one to twelve carbon atoms (i.e., C1-C12 alkyl). The alkyl may be substituted by one or more substituents selected from halo, nitro, cyano, hydroxy, and alkoxy. It is to be understood that the term “alkyl” thus also encompasses “optionally substituted alkyl”.

[0038] In one embodiment, the alkyl group is chosen from a straight chain C1-C10 alkyl group. In another embodiment, the alkyl group is chosen from a branched chain C3-C10 alkyl group. In another embodiment, the alkyl group is chosen from a straight chain Ci-Ce alkyl group. In another embodiment, the alkyl group is chosen from a branched chain C3-C6 alkyl group. In another embodiment, the alkyl group is chosen from a straight chain C1-C4 alkyl group. In another embodiment, the alkyl group is chosen from a branched chain C3-C4 alkyl group. In another embodiment, the alkyl group is chosen from a straight or branched chain C3-C4 alkyl group. Non-limiting exemplary C1-C10 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, iso-butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like. Non-limiting exemplary C1-C4 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, secbutyl, tert-butyl, and iso-butyl.

[0039] The skilled person will understand that the term "alkyl" as used herein also encompasses "alkylene", including as non-limiting examples e.g. methylene (-CH2-), ethylene (-CH2CH2-), or propylene (-CH2CH2CH2-), when the alkyl group is not a terminal group, but is part of a chain, such as e.g. A1in -A1-A2. The same considerations also apply to other groups or substituents as defined in the present application when being part of a chain, and not a terminal group. For example, the term (hetero)aryl also encompasses “(hetero)arylene”.

[0040] The term "heteroaryl" as used herein by itself or as part of another group refers to a monocyclic or bicyclic heteroaromatic ring system having from five to ten atoms (including 1 , 2, 3, or 4 heteroatoms independently chosen from oxygen, nitrogen and sulfur, i.e. C5-C10 heteroaryl). In one embodiment, the heteroaryl is a C5 heteroaryl. In another embodiment, the heteroaryl is a Ce heteroaryl. Non-limiting exemplary heteroaryl groups include but are not limited to furyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thienyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1 ,2,3-triazinyl, 1 ,2,4,5-tetrazinyl, benzofuryl, indolyl, benzothienyl, quinolinyl, isoquinolinyl, cinnolinyl, quinaziolinyl, quinoxalinyl, and phthalazinyl. The term “heteroaryl” as used herein encompasses heteroaryls that are substituted by one or more substituents, e.g. by halo, nitro, cyano, hydroxy, and alkyl. It is to be understood that the term “heteroaryl” thus also encompasses “optionally substituted heteroaryl”.

[0041] The term “heterocyclyl" as used by itself or as part of another group refers to saturated and partially unsaturated (e.g., containing one or two double bonds) cyclic groups containing one, two, or three rings having from three to fourteen ring members (i.e., a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13- or 14-membered heterocyclyl) and at least one heteroatom. Each heteroatom is independently selected from the group consisting of oxygen, sulfur, including sulfoxide and sulfone, and / or nitrogen atoms. The term "heterocyclyl" encompasses heteroaryl groups. The heterocycle may be unsubstituted or optionally substituted with one to four substituents independently selected from halo, nitro, cyano, hydroxy, and alkyl. It is to be understood that the term “heterocyclyl” thus also encompasses “optionally substituted heterocyclyl”. Nonlimiting examples of heterocycles include 2-oxopyrrolidin-3-yl, 2-imidazo-lidinone, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and indolinyl.

[0042] Composite terms as used herein, such as "alkylheteroaryl", are to be understood in their broadest technically sensible way and may refer to a connectivity of -alkyl heteroaryl or alkylheteroaryl-. However, it is preferred that composite terms are interpreted in that the last- mentioned group is connected "first", e.g. -heteroaryl-alkyl for “alkylheteroaryl".

[0043] Preferred embodiments according to the invention are defined hereinafter. The preferred embodiments are preferred alone or in combination. Further, it is to be understood that the following preferred embodiments refer to all aspects of the present invention, i.e. the PET tracer, the PET tracer composition, the process for obtaining the PET tracer as well as the use for in vivo diagnosis or for in vitro diagnosis.

[0044] PET tracer

[0045] In an embodiment, the PET tracer is of formula (I): wherein

[0046] R1and R2are independently selected from alkyl and Hydrogen,

[0047] A is -A1-A2,

[0048] A1is selected from O, NH, or a direct bond,

[0049] A2is selected from alkyl, aryl, heteroaryl, alkylaryl, and alkylheteroaryl,

[0050] B is -B1-B2,

[0051] B1is selected from sulfonyl, C(O), or a direct bond,

[0052] B2is selected from heterocyclyl, NH2, or -N(alkyl)2 n is 0 or 1 ,

[0053] R3is selected from18F, I, Br, Cl, and F, with the proviso that when n is 0 or R3is not18F, then A2or B2is substituted with a18F group.

[0054] In an embodiment, R1is H and R2is ethyl, n is 0 and B1is C(O). As n is 0, A2or B2is substituted with a18F group.

[0055] In an embodiment, A1is a direct bond, and A2is selected from alkyl, preferably methyl, and A2is a alkylheteroaryl group, wherein the alkylheteroarylgroup is prefereably (C1-C3 alkyl) heteroaryl, more preferably (C1-C2 alkyl)furyl, (C1-C2 alkyl)pyrrolyl, (C1-C2 alkyl)pyrazolyl, (Ci- 02 alkyl)imidazolyl, (C1-C2 alkyl)triazolyl, (C1-C2 alkyl)oxazolyl, (C1-C2 alkyl)thienyl, and (C1-C2 alkyl)pyridyl and wherein the alkyl group is substituted with18F. Late-stage18F-labeling may be achieved from the corresponding tosylate using tetraethylammonium [18F]fluoride ([18F]TEAF] for nucleophilic fluorination.

[0056] In preferred embodiment, A1is a direct bond, A2is selected from alkyl or alkylheteroaryl, and B2is heterocyclyl, preferably pyrrolidinyl. It is furthermore preferred that n is 0 and the heterocyclyl moiety of the B2substituent is substituted with a18F group. It has been found that the incorporation of the18F group on a heterocyclic group B2increases metabolic stability compared to terminal fluoroalkyls.

[0057] In a particularly preferred embodiment, A1is a direct bond,

[0058] A2is alkyl,

[0059] B1is C(O),

[0060] B2is selected from heterocyclyl, n is 0, wherein B2is substituted with a18F group.

[0061] In an embodiment, R1is H and R2is ethyl, n is 0 and B1is C(O). As n is 0, A2or B2is substituted with a18F group.

[0062] In an even more preferred embodiment, PET tracer of the present invention is of formula (II)

[0063] Even more preferred, the PET tracer is the enantiomer of formula (Ila)

[0064] The above compound of formula (Ila) is the R-enantiomer PET tracer also referred to as compound [18F]3b. In an alternative embodiment, the PET tracer is the S-enantiomer [18F]3a.

[0065] PET tracer composition

[0066] The PET tracer composition comprises the PET tracer of the present invention and further comprises a pharmaceutically acceptable excipient.

[0067] The PET tracer composition is preferably a liquid formulation, i.e. a solution, particularly a formulation for systemic administration. Although the route of administration is not particularly limited, intravenous (i.v.) injection or infusion is preferred. Suitable infusion solutions are for example electrolyte solutions selected from the group consisting of 0.9 wt.-% aqueous NaCI solution, Ringer’s solution, Ringer’s lactate solution and Ringer’s acetate solution. The PET tracer composition of the present invention does not need to be manually prepared in situ and can be used as is without an additional step. However, in an alternative embodiment, the PET tracer composition may be a concentrate that is reconstituted or diluted before administration.

[0068] In addition, the PET tracer composition may comprise a solubilizing agent selected from alcohols, polyalcohols, and nonionic surfactants. In an embodiment, the composition is sterile in accordance with the European Pharmacopoeia 5.1.1 , edition 10.0, 2019. In an embodiment the composition is sterile, i.e. it contains less than 10'4wt.-% of nonsterile material, such as microbial contamination.

[0069] Process for preparing the PET tracer8F]3a,b

[0070] In an embodiment, the invention relates to a process for preparing the PET tracer [18F]3a,b, comprising the steps of:

[0071] (iii) providing an intermediate compound of formula (III) wherein R4is a leaving group selected from aryloxy, halo, and heterocyclyloxy; and

[0072] (iv) coupling said intermediate compound of formula (lll)to a compound of formula (IV) R4is not particularly limited. However, it is preferred that R4is pentafluorophenoxy.

[0073] PET Imaging

[0074] In an embodiment, the present invention relates to the use of the PET tracer of the present invention or the PET tracer composition of the present invention for use in the in vivo diagnosis of a disease. The PET tracer can be administered to a subject as an i.v. infusion or injection. The distribution of the PET tracer can be imaged using PET imaging alone or combined with computed tomography (CT) or magnetic resonance imaging (MRI). A "subject" or "patient" to whom the PET tracer is administered may be a mammal, such as a non-primate (e.g. cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g. monkey or human). It is preferred that the subject is a human. In certain aspects, the human is a pediatric patient. In other aspects, the human is an adult patient.

[0075] The effective dosage is determined by factors, such as age and weight of the subject, and is linked to the radiation that the total dosage should have. For adult, human patients, a typical radiation is typically from 100 to 500 MBq. The determination of the dosage is determined by known methods, no undue experimentation is required.

[0076] The diagnosis with the PET tracer of the present invention allows the imaging of neutrophils.

[0077] The disease is a CXCR2 mediated disease, preferably the disease is selected from inflammatory diseases, cancer, autoimmune diseases, cardiovascular diseases, and neutropenia.

[0078] The potential applications of CXCR2-targeted PET imaging encompass a wide range of diseases linked to inflammatory processes. This includes cancer, ischemic diseases, trauma, bowel disease, respiratory infections, atherosclerosis, and various autoimmune and degenerative diseases. It is more preferred that the is an acute or chronic inflammatory disease, even more preferably selected from psoriasis, rheumatoid arthritis, radiation-induced fibrotic pulmonary disease, autoimmune bullous skin disease (AIBD), chronic obstructive pulmonary disease, and ozone-induced airway inflammation.

[0079] It is preferred that the disease is a cancer, even more preferably selected from rhabdomyosarcoma, Lewis Lung Cancer (LLC), non-small cell lung cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma (RCC), colorectal cancer (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, prostate small cell neuroendocrine carcinoma (SCNC), liver cancer, glioblastoma, liver tumor, oral squamous cell carcinoma, squamous cell carcinoma, pancreatic cancer, papillary thyroid cancer, intrahepatic cholangiocellular carcinoma, hepatocellular carcinoma, bone cancer, and nasopharyngeal carcinoma.

[0080] It is preferred that the disease is an autoimmune disease, more preferably selected from neutrophilic asthma.

[0081] EXAMPLES

[0082] EXAMPLE 1 - Chemistry

[0083] All reagents were purchased from SigmaAldrich (St. Louis, Missouri), ABCR (Karlsruhe, Germany) and Fluorochem (Hadfield, United Kingdom) and used without further purification. THF was dried by refluxing over sodium under an argon atmosphere and DCM was dried over magnesium sulfate. Thin-layer chromatography was performed on silica gel 60 (alumina foils with fluorescent indicator 254 nm). For detection, staining by potassium permanganate, Ninhydrin or UV light (254 and 366 nm) were used. For column chromatography, silica gel 60 (particle size 0.040-0.063 mm) was used. Nuclear magnetic resonance spectra were recorded with a Bruker AV-400 NMR instrument (Bruker, Karlsruhe, Germany) in deuterated solvents, and chemical shifts are expressed in ppm relative to the solvent residues used for NMR. Purity was determined by HPLC (Shimadzu Products), containing a DGU-20A3R degassing unit / controller, a LC20AB solvent delivery unit, and a SPD-20A UVA / IS detector. UV detection was measured at 254 nm. Mass spectra were obtained by a LCMS 2020 (Shimadzu Products). As a stationary phase, a Synergi 4U fusion-RP (150 mm x 4.6 mm) column was used, and as a mobile phase, a gradient of MeOH / water with 0.1 % formic acid was used. Parameters: A= water, B = MeOH, V(B) / (V(A) + V(B)) = from 5% to 90% over 10 min, V(B) / (V(A) + V(B)) = 90% for 5 min, V(B) / (V(A) + V(B)) = from 90% to 5% over 3 min. The method was performed with a flow rate of 1 .0 mL / min. Compounds were only used for biological evaluation if the purity was > 95% and were dried under high vacuum (< 0.1 mbar) beforehand.

[0084] EXAMPLE 1.1 - Synthesis of Compound 3a, b

[0085] 3-((2-Ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-2-hydroxybenzoic acid (1): 3-Amino-2- hydroxybenzoic acid (160 mg, 1.05 mmol, 1.0 eq.) was dissolved in DMF (7 mL) before diethyl squarate (234 pL, 1.50 g, 8.82 mmol, 1.5 eq.) and zinc triflate (38.2 mg, 105 pmol, 0.1 eq.) were added slowly. After one hour DIPEA (1.05 mmol, 1.0 eq.) The solution was stirred overnight at rt. The solvent was evaporated under reduced pressure. Subsequent column chromatography (CF^Ch / methanol, 19:1 to 8:2) yielded the product 3-((2-ethoxy-3,4- dioxocyclobut-1-en-1-yl)amino)-2-hydroxybenzoic acid (1) (286 mg, 1.03 mmol, 98%) as a brown oil.1H-NMR (400 MHz, methanol-d4): 5 = 7.76 (dd, J = 7.9, 1.6 Hz, 1 H), 7.51 (d, J = 7.5 Hz, 1 H), 6.82 (t, J = 7.8 Hz, 1 H), 4.82 (q, J = 7.1 Hz, 2H), 1.48 (t, J = 7.1 Hz, 3H);13C-NMR (101 MHz, methanol-d4): 5 = 188.20, 183.67, 177.80, 171.56, 170.48, 157.23, 127.12, 125.40, 125.29, 119.18, 114.47, 69.07, 15.64 ppm; ESI-MS: m / z = 278.05 [M+H]+, 316.00 [M+K]+, calc. 278.06.

[0086] 3-((3,4-Dioxo-2-(pentan-3-ylamino)cyclobut-1-en-1-yl)amino)-2-hydroxybenzoic acid (2): 3- Aminopentane (241 pL, 180 mg, 2.06 mmol, 2.0 eq.) was dissolved in ethanol (5 mL) before 3-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-2-hydroxybenzoic acid (1) (286 mg, 1.03 mmol was added in one portion. The solution was stirred overnight at rt. The solvent was evaporated and the residue was taken up in ethyl acetate (50 mL) and washed with hydrochloric acid (3.0 M in water, 2x40 mL) and brine. Subsequent column chromatography (C^Ch / methanol / acetic acid, 6:1 :0.1) yielded the product 3-((3,4-dioxo-2-(pentan-3- ylamino)cyclobut-1-en-1-yl)amino)-2-hydroxybenzoic acid (2) (decomposition 222.9°C) (198 mg, 632 pmol, 60%) as a off-white solid.1H-NMR (400 MHz, methanol-d4): 5 = 8.19 (d, J = 8.0 Hz, 1H), 7.63 (dd, J = 7.9, 1.5 Hz, 1 H), 6.93 (td, J = 8.0, 1.5 Hz, 1 H), 4.05 (dt, J = 8.8, 4.0 Hz, 1 H), 1.82 - 1.68 (m, 2H), 1.66 - 1.50 (m, 2H), 1.03 (t, J = 7.4 Hz, 6H);13C-NMR (101 MHz, methanol-d4): 5 = 185.34, 181.71 , 173.94, 171.16, 164.43, 152.83, 128.73, 125.95, 125.87, 119.78, 101.39, 59.52, 29.63, 10.54 ppm; ESI-MS: m / z = 319.05 [M+H]+, calc. 319.12.

[0087] (R)-3-((3-(3-Fluoropyrrolidine-1-carbonyl)-2-hydroxyphenyl)amino)-4-(pentan-3- ylamino)cyclobut-3-ene-1 , 2-dione (3b): 3-((3,4-Dioxo-2-(pentan-3-ylamino)cyclobut-1-en-1- yl)amino)-2-hydroxybenzoic acid (2) (105 mg, 330 pmol, 1.0 eq.), TEA (137 pL, 100 mg, 3.0 eq.) and HATU (150 mg, 396 pmol, 1.2 eq.) were dissolved in DMF (4.5 mL) and stirred for 10 min at rt, before (R)-3-fluoropyrrolidine (49.9 mg, 396 pmol, 1.2 eq.) was added. The solution was stirred overnight at rt. The solvent was evaporated under reduced pressure. Subsequent column chromatography (ethyl acetate) yielded the product (R)-3-((3-(3- fluoropyrrolidine-1-carbonyl)-2-hydroxyphenyl)amino)-4-(pentan-3-ylamino)cyclobut-3-ene- 1 ,2-dione (3b) (32 mg, 82.3 pmol, 25%) as an off-white oil.1H-NMR (400 MHz, methanol-d4): 5 = 8.08 (d, J = 8.0 Hz, 1H), 7.25 (d, J = 7.9 Hz, 1 H), 6.98 (t, J = 8.0 Hz, 1H), 5.37 (d, J = 52.7 Hz, 1 H), 4.06 (td, J = 8.8, 4.7 Hz, 1 H), 4.02 - 3.80 (m, 4H), 2.40 - 2.08 (m, 2H), 1.66 - 1.53 (m, 2H), 1.66 - 1.54 (m, 2H), 1.04 (t, J = 7.4 Hz, 6H);13C-NMR (101 MHz, methanol-d4): 6 = 185.27, 181.68, 171.10, 170.93, 164.48, 129.58, 124.06, 123.68, 121.18, 120.30, 59.52, 29.58, 10.54 ppm; ESI-MS: m / z = 319.10 [M+H]+, 428.05 [M+K]+, calc. 390.18.

[0088] (S)-3-((3-(3-Fluoropyrrolidine-1-carbonyl)-2-hydroxyphenyl)amino)-4-(pentan-3- ylamino)cyclobut-3-ene-1, 2-dione (3a): 3-((3,4-Dioxo-2-(pentan-3-ylamino)cyclobut-1-en-1- yl)amino)-2-hydroxybenzoic acid (2) (75.0 mg, 236 pmol, 1.0 eq.), TEA (97.9 pL, 71.5 mg, 3.0 eq.) and HATLI (98.8 mg, 260 pmol, 1.1 eq.) were dissolved in DMF (4 mL) and stirred for 10 min at rt, before (S)-3-fluoropyrrolidine (32.8 mg, 260 pmol, 1.1 eq.) was added. The solution was stirred overnight at rt. The solvent was evaporated under reduced pressure. Subsequent column chromatography (ethyl acetate / petroleum ether, 9:1) yielded the product (S)-3-((3-(3-fluoropyrrolidine-1-carbonyl)-2-hydroxyphenyl)amino)-4-(pentan-3- ylamino)cyclobut-3-ene-1 ,2-dione (3a) (34.2 mg, 87.9 pmol, 37%) as an off-white oil.1H-NMR (400 MHz, methanol-d4): 5 = 8.07 (dd, J = 8.1 , 1.4 Hz, 1 H), 7.33 - 7.18 (m, 1 H), 6.96 (t, J = 8.0 Hz, 1 H), 5.35 (d, J = 52.6 Hz, 1 H), 4.05 (td, J = 8.5, 4.4 Hz, 1 H), 4.00 - 3.77 (m, 4H), 2.44 - 2.09 (m, 2H), 1.80 - 1.69 (m, 2H), 1.64 - 1.53 (m, 2H), 1.03 (t, J = 7.4 Hz, 6H);13C-NMR (101 MHz, CDCI3): 5 = 185.29, 181.72, 171.15, 170.97, 164.50, 129.63, 124.02, 123.70, 121.15, 120.32, 59.51 , 29.62, 10.55 ppm; ESI-MS: m / z = 390.10 [M+H]+, 412.10 [M+Na]+, 428.05 [M+K]+, 779.20 [2M+H]+, 801.25 [2M+Na]+, 817.25 [2M+K]+, calc. 390.18.

[0089] EXAMPLE 1.2 - Synthesis of [18F]3b

[0090] [18F] F’ was obtained by proton bombardment of H218O and isolated by trapping on a Sep-Pak Accell Plus QMA Plus Light cartridge (Waters GmbH, Eschborn, Germany, preconditioned by 0.5 M NaHCOs 5 mL and water 10 mL). The cartridge was washed with 2 mL of ddH2O and [18F]F_was eluted with a solution of tetraethylammoniumbicarbonat (1 mg, 5.0 eq.) in methanol (600 pL). After the solvent was removed at 110°C under nitrogen flow, MeCN (600 pL) was added to the V-vial and was dried again. Labelling was carried out by the addition of a solution of 6 (300 pg, 1.0 eq.) in dry MeCN (200 pL) and heating to 110°C for 10 minutes, followed by removal of the Boc group by addition of TFA (200 pL) and heating to 110°C for 10 minutes. Then, the solvent was removed at 90°C by nitrogen flow. Amide coupling was then carried out by addition of PFP ester 5 (500 pg, 1.0eq.) and DI PEA (10 pL) in dry MeCN (300 pL). The solution was heated to 80°C for 10 min, diluted with 30% MeCN / water (1.5 mL) and purified via semipreparative HPLC (column: COSMOSIL C18 ARII 6.0 x 150 mm; mobile phase: Phase A: Water with 0.1% TFA, Phase B: MeCN with 0.1 % TFA; 0-12 min, 30% to 55% B, 12-14 min, 55% to 95% B, 14-17 min 95% B, 17-18 min 95% to 30%, 18-20 min 30%, Flow rate: 1.5 ml / min). The collected fraction was diluted with 10 mL and trapped on a Sep-Pak C18 Plus light cartridge (Waters GmbH, Eschborn, Germany, preconditioned by 5 mL ethanol and 10 mL water). The cartridge was washed by water (5 mL) followed by the elution with ethanol (600 pL). After purification by semipreparative high-performance liquid chromatography (HPLC), the labelled target compound [18F]3b was obtained in a RCY of 16.9 ± 8.9%, radiochemical purity >99% and specific activity of 2.37 GBq / pmol (n = 4).18F3b was diluted with saline or PBS to proper concentration for further application.

[0091] REFERENCE EXAMPLE 1 - Synthesis of compound 8

[0092] 3-((2-Hydroxy-3-(pyrrolidine-1-carbonyl)phenyl)amino)-4-(pentan-3-ylamino)cyclobut-3-ene- 1,2-dione (8): 3-((3,4-Dioxo-2-(pentan-3-ylamino)cyclobut-1-en-1-yl)amino)-2-hydroxybenzoic acid (2) (50.0 mg, 127 pmol, 1.0 eq.), TEA (51.9 pL, 38.5 mg, 3.0 eq.) and HATU (72.6 mg, 191 pmol, 1.1 eq.) were dissolved in DMF (3 mL) and stirred for 10 min at rt, before pyrrolidine (20.7 pL, 17.6 mg, 140 pmol, 1.1 eq.) was added. The solution was stirred overnight at rt. The solvent was evaporated under reduced pressure. Subsequent column chromatography (ethyl acetate / petroleum ether, 6:1) yielded the product 3-((2-hydroxy-3-(pyrrolidine-1- carbonyl)phenyl)amino)-4-(pentan-3-ylamino)cyclobut-3-ene-1 ,2-dione (8) (16.3 mg, 35 %) as a beige oil.1H-NMR (400 MHz, acetone-d6): 5 = 12.81 (s, 1 H), 8.32 (s, 1 H), 8.17 (td, J = 8.2, 1.9 Hz, 1 H), 7.42 (d, J = 9.2 Hz, 1 H), 7.36 (dd, J = 8.0, 1.4 Hz, 1 H), 6.87 (t, J = 8.1 Hz, 1 H), 4.28 - 4.01 (m, 1 H), 3.88 - 3.59 (m, 4H), 1 .99 - 1.94 (m, 4H), 1.76 - 1.65 (m, 2H), 1.61 - 1 .48 (m, 2H), 0.97 (t, J = 7.4 Hz, 6H);13C-NMR (101 MHz, methanol-d4): 5= 185.27, 181.69, 171.09, 170.52, 164.51 , 148.47, 129.45, 124.10, 123.48, 121.48, 120.13, 61.53, 59.53, 29.60, 14.44, 10.54 ppm; ESI-MS: m / z = 372.15 [M+H]+, 410.00 [M+K]+, 743.25 [2M+H]+, calc. 372.19.

[0093] EXAMPLE 2 - In vitro (or ex vivo) Biology

[0094] EXAMPLE 2.1 - IC50 measurement of compounds 3a, b

[0095] Activity of compounds 3a, b and 8 was evaluated revealing an interesting profile. While simple pyrrolidine derivative 8 showed an IC50 value of 992 nM, fluorination at the 3-position, for both enantiomers, improved CXCR2 binding. (S)-enantiomer 3a already has an IC50 value of 428 nM, but is exceeded by (R)-enantiomer 3b.

[0096] The results of the activity measurement are depicted in Table 1 below. Table 1

[0097] In the assay conditions used, compound 3b shows an IC50 value of 273 nM, only a factor of 1.5 higher compared to reference compound 10, reported in the literature with an IC5O( / 7CXCR2) of 5 nM.39

[0098] EXAMPLE 2.2 - Time-dependent uptake of [18F]3b in HEK cells

[0099] To evaluate CXCR2-specificity, the time-dependent uptake of [18F]3b in recombinant human embryonic kidney (HEK293) cells stably overexpressing / ?CXCR2 (HEK-CXCR2) was measured with and without the reference ligand 10 and commonly used CXCR2 specific inhibitor AZD5069. CXCR2-negative HEK293 cells were used as control. Therefore, the respective cells were incubated with vehicle, AZD5069 or 10 for 30 min, followed by addition of [18F]3b. In more detail:

[0100] Time-dependent uptake assay was performed in adherent HEK-CXCR2 and HEK293 (CXCR2 negative) cells on 24 well cell culture plates (Greiner bio-one, Frickenhausen, Germany). Cells were plated at a density of 1.5 x 105cells per well 48-hours prior to the experiment to achieve 90% growth confluence. Each well was washed once with 500 pl pre-warmed (37°C) high glucose (4.5 g / L) DMEM / 0.1% BSA uptake buffer (uptake medium). Then, 400 pl (if using inhibitor) or 450 pl Uptake Medium (vehicle) were added to each well (3 replicates / time point; total volume in the end: 500 pl / well). AZD5069 (MedChemExpress, Monmouth Junction, NJ, USA) or compound 10 3-((2-Hydroxy-3-(dimethylcarbamoyl-1 -carbonyl)phenyl)amino)- 4-(pentan-3-ylamino)cyclobut-3-ene-1 ,2-dione (final concentration of 1 pM) were then incubated at rt for 30 min with adherent HEK-CXCR2 and HEK293 (CXCR2 negative) cells prior to addition of the [18F]3b tracer (50 pL). For each time point, respective wells were washed twice with ice-cold PBS and lysed by addition of aqueous sodium hydroxide (0.1 M, 200 pL). After transfer to Eppendorf tubes, radioactivity was measured using a gamma counter (2470 Wizard2, PerkinElmer, Rodgau, Germany). After the measurement, the cell lysates were used for protein determination by BCA Protein Assay with bovine serum albumin standards (Pierce™ Bovine Serum Albumin, Biotylated, ThermoFisher Scientific GmbH, Dreieich, Germany).

[0101] For HEK-CXCR2, the vehicle-treated cells showed a significant uptake of radiotracer [18F]3b, while both the AZD5069- and 10-treated cells blocked uptake of the developed radiotracer. The same behaviour is apparent in CXCR2-negative HEK cells. Compound [18F]3b can therefore be considered as a specific ligand for CXCR2.

[0102] Example 2.3 - Time-dependent uptake of [18F]3b in human peripheral blood neutrophils

[0103] T o further verify the application of radiotracer [18F]3b as an inflammation marker of neutrophils, human peripheral blood cells were incubated with the radiotracer. In a similar fashion, when compared to HEK-CXCR2 experiments, blocking was examined using AZD5069. Compound [18F]3b was taken up quickly by human neutrophils, while blocking with AZD5069 prohibited binding promptly, therefore proving the specific and time-dependent uptake of radiotracer [18F]3b in human neutrophils and its potential as neutrophil-specific tracer. In more detail:

[0104] Human peripheral blood neutrophils (Asian male, non-smoker, blood type B+ provided by STEMCELL Technologies Inc., Vancouver, Canada) were distributed to Eppendorf tubes in 500 pl prewarmed RPMI1640 Medium and centrifuged. The supernatant was taken up and added to prewarmed RPMI1640 Medium. Neutrophils were either incubated with AZD5069 (1 pM, 50 pl) for 30 min at rt, before [18F]3b (50 pl) was added, followed by vortexing and incubation at 37°C for one hour or incubated with vehicle (50 pl), before [18F]3b (50 pl) was added, followed by vortexing and incubation at 37°C for one hour. After incubation, the solution was centrifuged and the supernatant was taken up, before ice cold PBS (500 pl) was added, followed by vortexing, centrifugation and take up of the supernatant. This procedure was repeated, before aqueous sodium hydroxide (0.1 M, 200 pl) was added. After transfer to Eppendorf tubes, radioactivity was measured using a gamma counter (2470 Wizard2, PerkinElmer, Rodgau, Germany). After the measurement, the cell lysates were used for protein determination by BCA Protein Assay with bovine serum albumin standards (Pierce™ Bovine Serum Albumin, Biotylated, ThermoFisher Scientific GmbH, Dreieich, Germany).

[0105] EXAMPLE 3 - In vivo Biology All animal experiments were approved by the Animal Ethics Committee of the National Cerebral and Cardiovascular Center, Research Institute, Osaka, Japan (approval number 18019) and were conducted strictly according to the Guide for the Care and Use of Laboratory Animals36and the ARRIVE guidelines.37Anaesthesia was induced in male Wistar rats (n = 2, weighing 196-210 g, Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany) by using 5% isoflurane and maintained during the whole experiment with 2% isoflurane. PET imaging of [18F]3b was obtained according to a previously described protocol.38In brief, PET imaging in rodents was obtained using a dedicated small animal PET system (microPET Focus 120, Siemens, Germany). Shortly before the single injection of [18F]3b (30 MBq) via the tail vein, a 120-min dynamic PET scan was initiated with acquisition in list-mode format. The obtained PET images were analyzed with the public domain tool AMIDE imaging software (A Medical Imaging Data Examiner, version 1.01). For the rat biodistribution studies (n = 1), radiotracers (0.1 MBq) were administered via the tail vein. The animals were euthanized 60 minutes after radiotracer administration. The organs of interest were harvested for tissue counting with a y-counter (2480 Automatic Gamma Counter WIZARD2, PerkinElmer LAS GmbH, Rodgau, Germany).

[0106] After initial blood-pool circulation, radiotracer [18F]3b starts to be enriched in the liver and kidney followed by excretion into intestines and bladder. No uptake in CXCR2-expressing organs, e.g. spleen or bone marrow, could be observed. This unexpected behaviour could be accounted to the species difference apparent for chemokine receptors.33Similarly, Wester et al. observed that [natGa]Pentixafor, possessing high binding affinity to / ?CXCR4, does not bind to mCXCR4 or rCXCR4.34For CXCR2 ligand Navarixin, a species difference is reported showing higher affinity for cynomolgus than for mouse- or rat-derived CXCR2.8This difference is even more pronounced for imidazolylpyrimidine-based compounds as tested by de Kruijf et al. showing good binding to human CXCR2 but no affinity for CXCR2 from rhesus or cynomolgus monkey.35However, as initial studies were carried out in a healthy rat, several advantageous characteristics can be observed. Fast clearance from most of the organs can be an advantage for low background. While retention in liver, often resulting from high tracer lipophilicity, is comparably short, tracer accumulation in the bladder can be observed starting at approximately 60 min, suggesting good renal clearance, and is useful information to estimate dosage and establish clinical imaging protocols. Additionally, negligible bone uptake, a common indicator of in vivo stability due to defluorination, demonstrates high metabolic stability of compound [18F]3b, as expected for introduced pyrrolidine moiety. REFERENCES

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Claims

CLAIMS1 . A PET tracer of formula (I):whereinR1and R2are independently selected from alkyl and Hydrogen,A is -A1-A2,A1is selected from O, NH, or a direct bond,A2is selected from alkyl, aryl, heteroaryl, alkylaryl, and alkylheteroaryl,B is -B1-B2,B1is selected from sulfonyl, C(O), or a direct bond,B2is selected from heterocyclyl, NH2, or -N(alkyl)2 n is 0 or 1 ,R3is selected from18F, I, Br, Cl, and F, with the proviso that when n is 0 or R3is not18F, then A2or B2is substituted with a18F group.

2. The PET tracer according to claim 1 , whereinR1is H and R2is ethyl, n is 0 and B1is C(O).

3. The PET tracer according to claim 1 or 2, wherein A1is a direct bond,A2is selected from alkyl or alkylheteroaryl, andB2is heterocyclyl, preferably pyrrolidinyl.

4. The PET tracer according to any one of claims 1 to 3, wherein the PET tracer is of formula (II):preferably of formula (Ila)5. A PET tracer composition comprising the PET tracer according to any one of claims 1 to 4, and further comprising a pharmaceutically acceptable excipient.

6. A process for preparing the PET tracer of claim 4, comprising the steps of:(v) providing an intermediate compound of formula (III)(III), wherein R4is a leaving group selected from aryloxy, halo, and heterocyclyloxy.(vi) coupling said intermediate compound of formula (III) to a compound of formula (IV)7. The process of claim 6, wherein R4is pentafluorophenoxy.

8. The PET tracer according to any one of claims 1 to 4 or the PET tracer composition of claim 5 for use in the in vivo diagnosis of a disease.

9. The PET tracer or the PET tracer composition for use according to claim 8, wherein the disease is selected from inflammatory diseases, cancer, autoimmune diseases, cardiovascular diseases, and neutropenia.

10. The PET tracer or the PET tracer composition for use according to claim 8 or 9, wherein the disease is a CXCR2 mediated disease.11 . The PET tracer or the PET tracer composition for use according to any one of claims 8 to 10, wherein the disease is an acute or chronic inflammatory disease, preferably selected from psoriasis, rheumatoid arthritis, radiation-induced fibrotic pulmonary disease, autoimmune bullous skin disease (AIBD), chronic obstructive pulmonary disease, and ozone-induced airway inflammation.

12. The PET tracer or the PET tracer composition for use according to any one of claims 8 to 10, wherein the disease is a cancer selected from rhabdomyosarcoma, Lewis Lung Cancer (LLC), non-small cell lung cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma (RCC), colorectal cancer (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, prostate small cell neuroendocrine carcinoma (SCNC), liver cancer, glioblastoma, liver tumor, oral squamous cell carcinoma, squamous cell carcinoma, pancreatic cancer, papillary thyroid cancer, intrahepatic cholangiocellular carcinoma, hepatocellular carcinoma, bone cancer, and nasopharyngeal carcinoma.

13. The PET tracer or the PET tracer composition for use according to any one of claims 1 to 10, wherein the disease is an autoimmune disease, preferably selected from neutrophilic asthma.

14. The PET tracer or the PET tracer composition for use according to any one of claims 8 to 13, wherein the diagnosis is for imaging neutrophils.

15. Use of the PET tracer of any one of claims 1 to 4 or PET tracer composition of claim 5 for in vitro diagnosis.

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