Fluorine-18 complex useful for positron-emission tomography

Abstract

The present invention relates to a complex of 18F and of a compound of the following formula (I), wherein 18F is coordinated to M.

Description

[0001] FLUORINE- 18 COMPLEX USEFUL FOR POSITRON-EMISSION TOMOGRAPHY

[0002] Field of the invention

[0003] The present invention relates to a fluorine-18 (18F) complex and its use in positron- emission tomography (PET) imaging methods.

[0004] Technical background

[0005] Positron-emission tomography (PET) is a functional imaging technique that allows to probe biological processes in vivo. PET imaging has notably been used to successfully diagnose and monitor the progression of diseases.

[0006] PET is based on the detection of positron emission by radionuclides that have been administered as tracers to an individual to be imaged. The emitted positron travels a certain distance (positron range), depending on its energy, and undergoes inelastic collisions until the kinetic energy is so low that the timely overlap with an electron and annihilation is possible.

[0007] PET radionuclides are typically generated via nuclear reactions within a cyclotron or by specific decay mechanisms from a generator. The resulting radionuclides are incorporated through a variety of available radiochemical reactions into molecules, thus yielding the desired tracers. After having been administered the tracer, the individual is subsequently scanned to quantify the radioactive signal which provides information on the annihilation site, and thereby, the tracer localization at different organs.

[0008] Among the various existing positron-emitting nuclides fluorine-18 (18F) is the most widely used radionuclide for PET due to its unique advantages over other PET nuclides. These advantages include:

[0009] - a low positron range below 2.4 mm which allows the generation of high spatial resolution images,

[0010] - a clear positron emission profile comprising 97% positron emission and 3% electron capture (EC), and

[0011] - an optimal physical half-live of 109.8 min that enables off-site use in satellite facilities without a cyclotron.

[0012] Fluorine atom is typically not a constituent of most biomolecules and hence18F is frequently introduced into tracers via creation of a C-F bond via nucleophilic substitution (Rong et al. (2023) Nature Communications 14:3257-3279). However, nucleophilic substitution is generally a multi-step reaction which takes about an hour, thereby leading to a substantial loss of positron emission capacity of thus obtained18F -containing tracers prior to their administration. Besides, this reaction is generally conducted at high temperatures and low pH which is disadvantageous when working with bioconjugates bearing thermally and pH sensitive biomolecules.

[0013] Accordingly, the need for methods to introduce fluorine-18 into biomolecules under mild conditions, ideally in a single step, has led to attempts to depart from conventional C-F bond formation as a basis for18F bioconjugate synthesis, toward metal fluorine bonds (San ef al. (2024) « Radiomarquages au fluor- 18 : en quete de methodes douces pour le developpement de radiotraceurs TEP plus specifiques » Actualite chimique)

[0014] In this regard, Gallium features prominently in the search for suitable metallic fluoride-binding sites, with a range of Ga(lll) complexes with both macrocyclic and acyclic chelates being explored as alternatives. However, the use of metal complexes as binding sites for [18F]fluoride has not yet led to the ideal mild, aqueous one-step18F labeling and / or stable18F radiotracer (Blower ef al. (2021 ) Advances in Inorganic Chemistry 78:1-35).

[0015] It is therefore an object of the present invention to provide metal complexes that can efficiently and rapidly bind to18F under mild conditions, in particular at room temperature in a pH range adapted to avoid denaturation of biomolecule, and in predominantly aqueous conditions, to yield a stable18F radiotracer in vivo.

[0016] Summary of the invention

[0017] The present invention follows from the finding, by the present inventors, that complexes such as Ga18F-L' could efficiently and rapidly incorporat1e8F under mild and aqueous conditions, thereby providing a stable18F radiotracer in solution.

[0018] Ga18F-U The present invention thus relates to a complex of F, in particular18F, and of a compound of the following formula (I): wherein:

[0019] E represents CH, N or NO;

[0020] M represents a cation selected from the list consisting of Al3+, Ga3+, ln3+, Sc3+, Y3+, La3+, Ce3+, Pr3+, Nd3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+, Yb3+, Lu3+and Fe3+, and is coordinated to at least one of the N atoms linked to Q1, Q2and Q3and to E when E represents N or NO;

[0021] D, G and J, identical or different, represent a group selected from the list consisting of CH, N, C-OH, C-O-L and C-L, wherein L represents a linker group, optionally linked to a biological targeting moiety (BTM);

[0022] R1, R2, R3, R4, R5, R6, R1 1and R12, identical or different, represent a group selected from the list consisting of -H, -OH, -(CH2)n-CH3, -(CH2)n-OH and -L, wherein n represents an integer from 0 to 5,

[0023] R7, R8, R9and R10, identical or different, represent a group selected from the list consisting of -H, =O, -(CH2)P-CH3, -(CH2)P-OH and -L, wherein p represents an integer from 0 to 3, provided that when one of R7or R8and / or one of R9or R10represents =O then the other one is absent, i.e. represents no group;

[0024] Q1, Q2and Q3, identical or different, represent a group selected from the list consisting of -H, -OH, -(CH2)k-CH3, -(CH2)k-OH, -CHQ4X and -L, wherein k represents an integer from 0 to 3, Q4represents H or a C1-3 alkyl group, and X represents a group selected from the list consisting of -COO-, -PO(OH) (O_) and -Q5-PO(OH), wherein Q5represents Ar or a C1-3 alkyl group, provided that at least two of Q1, Q2and Q3represent -CHQ4X and are coordinated to M; provided that the compound of formula (I) comprises at least one L, wherein F, in particular18F, is coordinated to M. The present invention also relates to the complex as defined above wherein F is18F and the compound is linked to a biological targeting moiety (BTM), for use as an in vivo PET scan diagnostic agent.

[0025] The present invention also relates to the use of a complex as defined above, wherein F is18F and the compound is linked to a biological targeting moiety (BTM), for generating a PET scan image of a body or a body part of an individual, wherein the complex has been administered previously to the individual.

[0026] The present invention also relates to a method of imaging an individual, comprising generating an image using PET of a body or body part of the individual, to which a complex as defined above, wherein F is18F and the compound is linked to a biological targeting moiety (BTM), has distributed, wherein the complex has been administered previously to the individual.

[0027] The present invention also relates to a pharmaceutical, imaging or diagnostic composition comprising a complex as defined above, wherein F is18F and the compound is linked to a biological targeting moiety (BTM), optionally in association with at least one biocompatible carrier.

[0028] The present invention also relates method of preparation of the complex as defined above, wherein F is18F, which comprises:

[0029] - reacting a compound of formula (I), wherein M is bound to R13, with a18F fluoride anion, wherein R13represents -OH, -OCH3, -OCH2-CH3, -CO-CH3, -CO-CH2-CH3, preferably -OH, or

[0030] - reacting a complex of19F and a compound of formula (I) wherein19F is coordinated to M, with a18F fluoride anion, or

[0031] - reacting a complex of18F and M, wherein M is as previously defined, with a compound of the following formula (I’): wherein D, E, G, J, Q1, Q2, Q3, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R" and R12are as previously defined.

[0032] The present invention also relates to a kit or cassette comprising the non- radioactive reagents necessary to carry out the method as defined above, including:

[0033] - a compound of formula (I), wherein M is bound to R13, wherein R13represents -OH, - OCH3, -OCH2CH3, -CO-CH3, -CO-CH2-CH3, preferably -OH, or

[0034] - a complex of,9F and a compound of formula (I) wherein,9F is coordinated to M, or

[0035] - a compound of formula (I’).

[0036] The present invention also relates to a compound of formula (I) as defined above, wherein M is bound to R13, wherein R13represents -OH, -OCH3, -OCH2-CH3, -CO- CH3, or -CO-CH2-CH3, preferably -OH.

[0037] The present invention also relates to a compound of formula (I).

[0038] The present invention also relates to a compound of formula (I’).

[0039] Description of the invention

[0040] Definitions

[0041] As a preliminary remark, it should be noted that the term "consisting of" means "constituted by", i.e. when an object "consists of" an element or several elements, the object cannot include other elements than those mentioned. In contrast, the term "comprising" means "including", "containing" or "encompassing", i.e. when an object "comprises" an element or elements, other elements than those mentioned can also be included in the object. In other words, when an object "comprises" an element or elements, it consists of the element(s) and possibly of other elements than these.

[0042] As should be clear to a person of skill in the art, for clarity reasons the coordinate bonds involving M and neighbouring doublet carrying atoms, such the N atoms linked to Q1, Q2and Q3, E when E represents N or NO as well as Q1, Q2and Q3, are not represented in the formulae shown herein, except in the Examples where the coordinate bonds are represented.

[0043] As intended herein, "Ar" represents a C5-12 arylene group or a C3-12 heteroarylene group.

[0044] As intended herein, the expression "biological targeting moiety" (BTM) relates to a compound which, after administration, is taken up selectively or localises at a particular site of the mammalian body in vivo. Such sites may for example be implicated in a particular disease state or be indicative of how an organ or metabolic process is functioning.

[0045] As intended herein, the BTM according to the invention includes pre-targeting moieties. The so-called pre-targeting strategy is well known to the person of skill in the art. It is notably described in Verhoeven ef al. (2019) Pharmaceutics 11(9):434. According to this strategy a non-radioactive biological targeting compound, such as. an antibody, which is taken up selectively or localises at a particular site of the mammalian body in vivo is first administered. A radioactive tracer comprising a pre- targeting moiety which can bind to the biological targeting compound in vivo is then administered. The advantage of the strategy lies in the fact that the biological targeting compound being non-radioactive it is possible to wait the necessary time for it to accumulate in sufficient amount at the targeted site. The expression “pre- targeting moiety” thus relates to a moiety which can bind to a biological targeting compound in vivo.

[0046] Compound of formula (I) or (I')

[0047] Preferably, the above-defined compound of formula (I) is of the following formula (1-1 ): wherein:

[0048] - R7and R9, identical or different, represents =O or-H, and

[0049] - D represents C-O-L, J represents C-H, and Q3represents -H, -OH, -(CH2)k-CH3, -(CH2)k- OH wherein k represents an integer from 0 to 3, or

[0050] - D represents C-H, J represents C-L or C-O-L, and G3represents -H, -OH, -(CH2)k-CH3, - (CH2)k-OH wherein k represents an integer from 0 to 3, or

[0051] - D represents C-H, J represents C-H, and Q3represents L, wherein L is as defined above. Preferably, the above-defined compound of formula (I’) is of the following formula (1-1 wherein:

[0052] - R7and R9, identical or different, represents =O or -H, and

[0053] - D represents C-O-L, J represents C-H, and Q3represents -H, -OH, -(CH2)k-CH3, -(CH2)k- OH wherein k represents an integer from 0 to 3, or

[0054] - D represents C-H, J represents C-L or C-O-L, and G3represents -H, -OH, -(CH2)k-CH3, - (CH2)k-OH wherein k represents an integer from 0 to 3, or

[0055] - D represents C-H, J represents C-H, and Q3represents L, wherein L is as defined above.

[0056] Preferably, in the above formulae (I), (I’), (1-1 ) and (1-1 ’), L is a group of formula -(A)m-Y wherein

[0057] - Y represents a group selected from the list consisting of -H, -N=C=S, -N=C=O, - NH(C=S)-BTM, and -NH(C=O)-BTM,

[0058] - each A is independently selected from the list consisting of -CR2-, -CR=CR-, -C=C-, - CR2CO2-, -CO2CR2- , -NRCO-, -CONR-, -CONHCR2NH(C=S)NH-, -CONHCR2NH(C=O)NH- , -CR=N-O-, -NR(C=O)NR-, -NR(C=S)NR-, -SO2NR-, -NRSO2-, -CR2OCR2-, -CR2SCR2-, - CR2NRCR2-, -CR2NH(C=S)NH-, -CR2NH(C=O)NH-, a C4-8 eye Io heteroalkylene group, a C4-8 cycloalkylene group, -Ar-, -NR-Ar-, -O-Ar-, -Ar-(CO)-, an amino acid, a sugar or a polyethyleneglycol (PEG) building block, wherein: each R is independently selected from the list consisting of H, C1-6 alkyl, C2-4 alkenyl, C2-4 alkynyCl, 1-4 alkoxyalkyCl,1-4 hydroxyalkyl, Ar-NH(C=S)NH, Ar- NH(C=O)NH, C1-4 alkylphenyl-NH(C=S)NCH1,-4 alkylphenyl-NH(C=O)NH, m is an integer from 1 to 20, each Ar is independently a C5-12 arylene group, or a C3-12 heteroarylene group. Preferably, in the above formulae (I), (I’), (1-1 ) and (1-1 ’), L is selected from the list consisting of:

[0059] As should be clear to the person skilled in the art -NCS represents -N=C=S Preferably, M represents Ga3+. Preferably, the compound of formula (I) is represented by a formula selected from the list consisting of:

[0060]

[0061] Preferably, the compound of formula (I) or (I’) according to the invention is linked to a biological targeting moiety (BTM).

[0062] Preferably, the biological targeting moiety (BTM) is selected from the list consisting of a pre-targeting moiety, an amino-acid, a polypeptide, in particular comprising from 3 to 100 amino-acid residues, a peptoid or peptidic mimetic, an enzyme substrate, agonist or inhibitor, a ligand of a biological receptor, an antibody, an antibody fragment, a scFv, a VHH and an aptamer.

[0063] By way of example, the pre-targeting moiety can be avidin, streptavidin, biotin, a single-stranded nucleic acid, an antibody, an antibody fragment, a scFv, a VHH, an aptamer, a click-chemistry unit, such as frans-cyclooctene or tetrazine.

[0064] In an embodiment of the invention, in the compound of formula (I) according to the invention, M is bound to R13, wherein R13represents -OH, -OCH3, -OCH2-CH3, -CO- CH3, or -CO-CH2-CH3. In that case, the compound of formula (I) of the invention wherein M is bound to R13is useful as a non-radioactive precursor or synthesis intermediate which can yield the complex of the invention wherein F is by i1o8Fnic exchange.

[0065] Complex

[0066] In a preferred embodiment of the invention, in the compound of formula (I), M is coordinated to18.F

[0067] As will be clear to the person of skill in the art, the complex of the invention wherein F is18iFs useful as a tracer in PET imaging methods, i.e. as a PET agent.

[0068] In another embodiment of the invention, in the compound of formula (I), M is coordinated to,9F. In that case, the complex of the invention wherein F is,9F is useful as a non- radioactive precursor or synthesis intermediate which can yield the complex of the invention wherein F is b18yF isotopic exchange.

[0069] Use - Method of imaging

[0070] As should be clear to the person of skill in the art, in the use or the method of imaging according to the invention, the step of administering the complex according to the invention or a composition comprising it to the individual is not part of the use or the method of imaging of the invention. It has been administered before implementing the use or the method of imaging according to the invention. As such, the use or the method of imaging of the invention does not comprise a step of treatment of the human or animal body by surgery.

[0071] Besides, as should also be clear to the person of skill in the art, the use or the method of imaging according to the invention does not comprise a step of diagnosing a disease. A s such, the use or the method of imaging of the invention is not a diagnostic method practiced on the human or animal body.

[0072] The individual according to the invention can be human or an animal, in particular a mammal.

[0073] Preferably, the complex according to the invention is included in a pharmaceutical, imaging or diagnostic composition.

[0074] Pharmaceutical, imaging or diagnostic composition

[0075] As should be clear to the person of skill in the art the pharmaceutical, imaging or diagnostic composition is in a form suitable for human or animal administration.

[0076] By the phrase "in a form suitable for human or animal administration" is meant a composition which is sterile, pyrogen- free, lacks compounds which produce toxic or adverse effects, and is formulated at a biocompatible pH (approximately pH 4.0 to 10.5). Such compositions also contain only biologically compatible excipients, and are preferably isotonic.

[0077] As intended herein, a "biocompatible carrier'1is a fluid, especially a liquid, in which the imaging agent can be suspended or preferably dissolved, such that the composition is physiologically tolerable, i.e. can be administered to the mammalian body without toxicity or undue discomfort. The biocompatible carrier is suitably an injectable carrier liquid such as sterile, pyrogen-free water for injection; an aqueous solution such os saline (which may advantageously be balanced so that the final product for injection is isotonic); an aqueous buffer solution comprising a biocompatible buffering agent; an aqueous solution of one or more tonicity-adjusting substances (e.g. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or other non-ionic polyol materials (e.g. poly ethyleneglycols, propylene glycols and the like). Preferably the biocompatible carrier is pyrogen-free water for injection, isotonic saline or phosphate buffer.

[0078] The pharmaceutical, imaging or diagnostic composition according to the invention may contain additional optional excipients such as: an antimicrobial preservative, pH-adjusting agent, filler, radioprotectant, solubilizer or osmolality adjusting agent. By the term "radioprotectant" is meant a compound which inhibits degradation reactions, such as redox processes, by trapping highly reactive free radicals, such as oxygen-containing free radicals arising from the radiolysis of water. The radioprotectants of the present invention may notably be suitably chosen from ascorbic acid, 4-aminibenzoic acid, gentisic acid and salts thereof with a biocompatible cation.

[0079] Method of preparation, kit and cassette

[0080] In the method of preparation according to the invention the fluo1r8idFe anion can be provided under any form liable to yield a fluoride anion in the reaction mixture. In particular, the18fFluoride anion can be provided in the form of a fluor1i8dFe salt, more particularly a salt of an alkaline metal. Preferably, the f18luForide anion is provided or added as Na18F.

[0081] As should be clear to the person of skill in the art, the compound of formula (I) wherein M is bound to R13, the complex of,9F and a compound of formula (I) or the compound of formula (I’) is a precursor or a synthesis intermediate of the complex according to the invention wherein F is .18F

[0082] Preferably, the method of preparation according to the invention is carried out using an automated synthesizer.

[0083] As intended herein, the expression "automated synthesizer" relates an automated module based on the principle of unit operations as notably described by Satyamurthy et al (1999) Clin. Positr. Imag. 2(5):233-253. The expression ‘‘unit operations” means that complex processes are reduced to a series of simple operations or reactions, which can be applied to a range of materials. Automated synthesizers are commercially available from a range of suppliers as notably mentioned by Satyamurthy et al. op. cit.

[0084] The automated synthesizer preferably comprises a cassette.

[0085] By the term "cassette" is meant a piece of apparatus designed to fit removably and interchangeably onto an automated synthesize, in such a way that mechanical movement of moving parts of the synthesizer controls the operation of the cassette from outside the cassette, i.e. externally. The cassette preferably comprises all the reagents, reaction vessels and apparatus necessary to carry out the preparation of a batch of a complex according to the invention wherein F is .18F

[0086] Preferably, the precursor, the kit or cassette according to the invention is provided in sterile and / or lyophilized form. As should be clear to the person of skill in the art the precursor, kit or cassette is preferably non-radioactive.

[0087] The invention will be further described by the following non-limiting figures and examples.

[0088] Description of the figures

[0089] Figure 1 and figure 2 show the stability of Ga18F(L) obtained with method 3 of Example 8. Figure 1 : crude reaction mixture at 20 min (“to"); Figure 2: at 60 min.

[0090] Figure 3 and figure 4 represent the LC-UV-MS analysis (Figure 3: UV chromatogram, Figure 4: MS spectra at tR = 5.6 - 6.0 min) of the decayed radiofluorination assay from method 3 of Example 8; crude reaction mixture analysed after 13 days at room temperature.

[0091] EXAMPLES

[0092] General

[0093] Synthesis of the macrocyclic pyclen derivatives

[0094] • Synthesis of the triamine synthon 3.

[0095] 2-Nitro[N(2{2[(2-nitrophenyl)sulfonylamino]ethylamino})ethyl] benzenesulfonamide SI was prepared as previously reported (Devreux et al. (2019) Eur. J. Inorg. Chem. 2019(29):3354-3365). tert-Butyl bis[2-(2-nitrophenylsulfonamido)ethyl]carbamate S2 was prepared according to reported protocols with slight modifications (Siaugue et al. (2001 ) Tetrahedron 57(22):47l 3-47l 8). Disulfonamido compound SI ( 19.87 g, 41.97 mmol) was suspended in tetra hydrofuran (1 15 mL) and di-tert-butyl dicarbonate was added portion wise (10.08 g, 46.17 mmol, 1.1 eq.). The reaction was stirred I day at room temperature (r.t.). The mixture was concentrated to dryness. The resulting crude oily material was purified by chromatography on silica gel (cyclohexane / AcOEt 5 / 5 to 4 / 6 v / v) and led to the desired product S2 as a yellow oil that crystallized on standing (21.49 g, 37.47 mmol). Yield 89%. 1H NMR (CDCI3) δ (ppm): conform to reported data in CDCI3 or DMSO-d6 (Siaugue et al. (2001 ) Tetrahedron 57(22):47l 3-47l 8; Ikeda et al. (2021 ) Bioorg. Med. Chem. Letters 32:127713). tert-Butyl 2-[2-(tert-butoxycarbonyl-{2-[(2-tert-butoxy-2- oxoethyl)amino]ethyl}amino)ethylamino] acetate 3 was prepared according to reported protocols with slight modifications (Devreux et al. (2021 ) Inorg. Chem. 60(6):3604- 3619). A suspension of the previously prepared disulfamido compound S2 (16.76 g, 29.21 mmol) with potassium carbonate (10.10 g, 73.1 mmol, 2.5 eq.) in acetonitrile (175 mL) was refluxed for 15 minutes. Tert-Butyl bromoacetate (1 1 mL, 75.0 mmol, 2.5 eq.) was added in one portion. The reaction was monitored by mass spectrometry and / or TLC (cyclohexane / AcOEt 1 / 1 v / v, UV detection). After 5 hours, total conversion of both the starting amino compound S2, and of the mono-N- alkylated intermediate S3a was observed leading to the di-N-alkylated disulfonamide S3b. The reaction mixture was cooled, and a second portion of potassium carbonate (12.12 g, 87.7 mmol, 3.0 eq.) was added, followed by thiophenol (8.7 mL, 85.0 mmol, 2.9 eq.) in one portion. The reaction was monitored by mass spectrometry and / or TLC (cyclohexane / AcOEt 1 / 1 v / v, UV detection; CH2Cl2 / MeOH 97.5 / 2.5 v / v, UV and ninhydrin stain detection). After 1 .5 hours, total conversion of the intermediate compounds S3b and S4a was obtained. Warming was stopped and the reaction mixture was maintained in the hot bath that cooled to room temperature overnight. Insoluble matter was filtered off through a Celite® pad, and the organic filtrate was concentrated to dryness. The resulting canary yellow colored oil was purified by chromatography on silica gel (CH2Cl2 / MeOH 100 / 0 to 95 / 5 v / v) and led to the desired product 3 as a yellow oil that crystallizes in the freezer (12.12 g, 28.1 mmol). Yield 96%.

[0096] • Synthesis of dibromide synthons

[0097] Dibromide synthons prepared herein.

[0098] 2, 6-Bis(bromomethyl) pyridine 4a was synthesized according to the protocol of Dioury et al. (2009) Tetrahedron 65(36):7573-7579.

[0099] Ethyl 2-[{2,6-bis(bromomethyl)pyridin-3-yl}oxy]acetate 4b was prepared according to the previously reported protocol by Devreux et al. (2019) Eur. J. Inorg. Chem. 2019(291.3354-3365. • Macrocyclisation: general procedure Scheme 2. Synthetic pathway of the pyclen derivatives: a) Na2COs, CHsCN, reflux, 1 h.

[0100] The dibromide 4b (1.1-1 .2 eq.) was added in one portion to a stirred suspension of the triamino synthon 3 with Na2CO3 (4 eq.) in refluxing acetonitrile (0.01 M). The reaction was monitored by mass spectrometry and / or TLC. After completion of the reaction, the carbonate was filtered off, and the clear filtrate was concentrated under reduced pressure.

[0101] • Post-macrocycling functionalizations

[0102] H2(PC2A-C4) R = OCH2CONH(CH2)3CH3

[0103] Scheme 3. Synthetic pathway of the pyclen derivatives: post-macrocycling functionalization, a) ethylenediamine, r.t., I h; b) CSCI2, DIPEA, CH2CI2, r.t., 30 min; c) i-butylamine, CH2CI2, r.t., 1.5 h; d) HCIanh, Et2O, r.t.

[0104] Example 1 Synthesis of tert- Butyl

[0105] 3,9-bis(2-tert-butoxy-2-oxoethyl)-12-(2-ethoxy-2-oxo-ethoxy)-3,6,9,15tetraazabicycl o[9.3.1]pentadeca-l(15),l l,13-triene-6-carboxylate 5b (Scheme 3. R = OCH2CC>2Et).

[0106] Formula: C32H52N4O9

[0107] Mwt: 636.78 g / mol

[0108] Monoisotopic Mwt: 650.400299

[0109] Done with pyridinic dibromide 4b (467 mg, 1.27 mmol, 1.1 eq.; prepared according to the reported protocol by Devreux et al. (2019) Eur. J. Inorg. Chem. 2019(29) :3354- 3365), triamine 3 (500 mg, 1.16 mmol), and Na2CO3 (500 mg, 4.73 mmol, 4 eq.) in acetonitrile (120 mL); refluxed 1 hour. Macrocyclic compound 5b was obtained as a yellow oil (864 mg) and was used as such in the next aminolysis step.

[0110] Example 2

[0111] Synthesis of tert-Butyl 12-[2-(2-amino-ethylamino)-2-oxo-ethoxy]-3,9-bis(2-tert- butoxy-2-oxoethyl)-3,6,9,l 5-tetrazabicyclo[9.3.1 ]pentadeca-l (15), 11 ,13-triene-6- carboxylate 6. Formula: C32H54N4O8

[0112] Mwt: 650.81 g / mol

[0113] Monoisotopic Mwt: 650.400299

[0114] Done with the previously prepared macrocycle 5b (848 mg, 1.16 mmol), ethylenediamine (3.4 mL, 51 .72 mmol, 45 eq.); stirred at room temperature for 1 hour. The solution was diluted in dichloromethane (25 mL) and washed with water (1 x 5 mL). Purification was done by chromatography on silica gel (CH2Cl2 / MeOH / NH3 7N in MeOH 100 / 0 / 0 to 90 / 5 / 5).

[0115] Macrocyclic compound 6 obtained as a yellow solid (547 mg, 0.841 mmol, 73%).

[0116] Example 3

[0117] Synthesis of tert-Butyl 3,9-bis(2-tert-butoxy-2-oxoethyl)-12-{[(2- isothiocyanatoethyl)carbamoyl]methoxy}-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca- 1(15),1 l,13-triene-6-carboxylate 7.

[0118] Formula: C33H52N4O8S

[0119] Mwt: 692.87 g / mol

[0120] Monoisotopic Mwt: 692.356714

[0121] To the previously prepared macrocycle 6 (1.031 g, 1.58 mmol) solubilized in dichloromethane (30 mL), diisoproylethylamine (DIPEA, 825 pL, 4.85 mmol, 3 eq.) was added. A 0.4 M solution of thiophosgene (5.95 mL, 2.40 mmol, 1.5 eq.) in dried dichloromethane was then added, and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. After 30 min, completion of the reaction was reached. The solution was concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (100% AcOEt) to provide the desired product 7 (874 mg, 1 .26 mmol, 80%) as a brown solid. Example 4

[0122] Synthesis of tert-butyl 3,9-bis(2-tert-butoxy-2-oxo-ethyl)-12-[2-[2-

[0123] (butylcarbamothioylamino)ethylamino]-2-oxo-ethoxy]-3,6,9,15- tetrazabicyclo[9.3.1]pentadeca-l(15),ll,13-triene-6-carboxylate 8.

[0124] Formula: C37H43N7O8S

[0125] Mwt: 766.00 g / mol

[0126] Monoisotopic Mwt: 765.445948

[0127] To the previously prepared macrocycle 7 (240 mg, 0.346 mmol) solubilized in dichloromethane (5 mL), / -butylamine (85 pL, 0.860 mmol, 2.5 eq.) was added and the solution was stirred at room temperature. The reaction was monitored by TLC. After 1 h30, completion of the reaction was reached. The solution was concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (CH2Cl2 / MeOH 100 / 0 to 95 / 5 v / v) to provide the desired product 8 (262 mg, 0.347 mmol, 99%) as an orange solid.

[0128] Example 5

[0129] Synthesis of 2-[12-[2-[2-(Butylcarbamothioylamino)ethylamino]-2-oxo-ethoxy]-9-

[0130] (carboxymethyl)-3,6,9,15-tetrazabicyclo[9.3.1]pentadeca-l(14),l l(15),12-trien-3- yljacetic acid 13 (LH2)

[0131] Formula: C24H39N7O4S

[0132] At pH 6.0, C24H38N7NaO6S /

[0133] C24H39N7O6S / C24H40CIN7O6S in ratio 79.1 / 20.1 / 0.7 ; calculated at pH 6.0 Mwt: 571 .32 g / mol (estimated at pH 6.0)

[0134] To the previously prepared macrocycle 8 (225 mg, 0.294 mmol) solubilized in dichloromethane (1 mL), a 2M solution of anhydrous HCI in Et2O (21 mL, 42 mmol, 143 eq.) was added in one-portion under vigorous stirring at room temperature. The resulting heterogeneous medium was stirred until completion of the reaction (mass spectrometry monitoring; at least one day). The crude residue was alkalinized from pH 0.9 to pH 6.0 with NaOHaq then purified by flash chromatography on RP18 silica gel (water / MeCN 90 / 10 v / v). Macrocyclic compound LH2 obtained at pH ~ 6 as a white powder (105 mg, 0.184 mmol, 63%).

[0135] Example 6

[0136] Synthesis of {GaF} reagent “Na2GaF5”

[0137] Formula: FsGa.NsNasO?

[0138] Mwt: 465.70 g / mol

[0139] The preparation of the water-soluble {GaF} reagent of formula referenced “Na2GaF5” was carried out according to the protocol described by Xu ef al. (US pat. appl. pub. No. 2020 / 0390104). Sodium fluoride NaF (213 mg, 5.073 mmol, 5 eq.) was solubilized in ultrapure water (100 mL) giving a solution of pH 7.3. An aqueous solution of 0.0106 M (IGF evaluation) gallium nitrate (100 mL, 1.06 mmol) was added to the previously prepared solution of NaF and the resulting mixture was stirred at room temperature for 2 hours leading to a clear solution of pH 4.6 which gallium concentration was estimated by ICP titration: 4.76 mM. The resulting lyophilisate was diluted in water to different concentrations; interestingly, concentrated solutions (0.12 ou 0.06 M) led to a turbid liquid phase.

[0140] Example 7

[0141] Synthesis of gallium complexes GaX(L) with X = OH, F

[0142] Formula: C24H3sGaN7O7S

[0143] Mwt: 638.39 g / mol

[0144] Monoisotopic Mwt: 637.180933

[0145] Formula: C24H37FGaN7O4S

[0146] Mwt: 640.39 g / mol

[0147] Monoisotopic Mwt: 639.176600 ous solution of 0.0529 M (ICP evaluation) gallium nitrate (5.2 mL,

[0148] 0.275 mmol, 1.1 eq.) was added to the previously prepared LH2 ligand isolated at pH 6 (143 mg, 0.248 mmol based on anhydrous mono-sodium form). The pH of the reaction solution dropped to pH 1.4. A 1 M aqueous solution of NaF (pH 10.6) was gradually added to adjust the pH to 4.4 (3.1 mL, 3.1 mmol, 12.5 eq.). The reaction was monitored by mass spectrometry and proved complete after less than 5 minutes at room temperature. The reaction solution was concentrated to dryness. The desired product was obtained after removing several inorganic sodium salts introduced or formed during the process (NaCI, NaF, NaNOs) by reverse phase chromatography on cyano(propyl)-modified silica gel (water / acetone 9 / 1 v / v) leading to the desired compound isolated by freeze-drying as a white solid (63 mg, 0.098 mmol based on GaF(L) anhydrous form, 40%).

[0149] Protocol 2: A solution of the previously prepared ligand LH2 (28 mg, 45.8 pmol) in ultrapure water (3.7 mL) was adjusted to pH 5.3 with an aqueous solution of NaOH. An aqueous solution of reagent “NckGaFs” previously prepared and titrated by ICP (5.61 m / Vl at pH 4.6, 8.5 mL, 47.7 pmol, 1 eq.) was added dropwise under magnetic stirring at room temperature for 5 minutes. The pH dropped to pH 3.8 and was carefully adjusted to pH 5.1 with an aqueous solution of NaOH (0.1 M, 50 pL, 50 pmol, 1.1 eq.). A monitoring by mass spectrometry indicated a total conversion to the desired gallium complex. The resulting mixture was concentrated by freeze-drying to give a pale pink solid (90 mg vs co. 30 mg anhydrous form of expected compound) corresponding to a mixture of the desired gallium complex with several inorganic sodium salts introduced or formed during the process (NaCI, NaF, NaNOs). Interestingly, 'H NMR analysis of the crude material revealed the presence of glycerol, which might have originated from various plastic devices involved in the process. An aliquot of crude compound (60 mg) was purified by reverse phase HPTLC on cyano(propyl)-modified silica gel glass plates (water / MeCN 9 / 1 v / v) and led to the desired compound as a white solid (24 mg). Final washes of the desalted product with acetonitrile (1 x 1 mL), then with acetone (1 x 1 mL) were necessary to remove residues of the modified silica absorbent (cyanopropyl and others) and led to the desired GaF(L) gallium complex as a white solid. Yield (extrapolated from the purified portion of crude material): 73% (21 mg, based on GaF(L) anhydrous form).

[0150] For X = OH

[0151] An aqueous solution of 0.0529 M (ICP evaluation) gallium nitrate (4.9 ml, 0.259 mmol, 1 eq.) was added to the previously prepared LH2 ligand isolated at pH 6 (150 mg, 0.261 mmol based on anhydrous mono-sodium form). The pH of the reaction solution dropped to pH 1 .5. A 1 M and 2 M aqueous solution of NaOH was gradually added to adjust the pH to 4.7 (0.585 mmol, 2.2 eq.). The reaction was monitored by mass spectrometry and proved complete after less than 5 minutes at room temperature. The fine solid formed was filtered out over a Celite® pad. The aqueous filtrate was then treated with Chelex®100 resin (sodium form, 1 1 mg), stirred for 24 hours and centrifuged to collect the supernatant. This operation was repeated once. The solution was concentrated to dryness. The desalting process was further completed by chromatography on reverse phase chromatography on cyano(propyl)-modified silica gel (water / acetone 9 / 1 v / v) and led to the desired product as a white solid (91 mg, 55%).

[0152] Example 8

[0153] 18F Radiolabeling and stability study

[0154] Preparation of the stock solutions

[0155] A stock solution in ultrapure water of Ga(NOs)3 at concentration c 201.6 mM (ICP titration) was used. Stock solutions in ultrapure water of LH2, GaF(L), or GaOH(L) were prepared at concentration c ~ 200 mM. AcONa / H buffer solutions at c O.l M were prepared at pH 4.0 and pH 5.5.

[0156] Fluorine- 18 production

[0157] [,8F]F" was produced with,8O enriched water via the [18O(p,n)18F] nuclear reaction for 10-15 min in a cyclotron (Cyclone 18 / 9, IBA). 2.2 ml of [18F]F~ in water with an activity between 10 and 30 GBq arrived in the automated synthesizer (AllinOne, Trasis) for its purification and elution. The aqueous solution of [18F]F“ was loaded onto a QMA cartridge (Sep-Pak Accell Plus QMA Plus Light, Waters) equilibrated with ultrapure water. After the loading, sample was rinsing with 5 mL of ultrapure water to remove any dissolved impurities. [18F] F“ was then eluted with 2 mL of 0.9% NaCI in a vial that has been placed in a shield container beforehand. Radioactivity of the final solution [18F]NaF was measured with a dose calibrator (PET Dose 5 Ci, COMECER), activity concentration was approximately 10 GBq / mL. This solution was used as is, with the activity concentration naturally decayed in proportion to the time elapsed since production. For some assays, the [18F]NaF solution was diluted in 0.9% NaCI to reduce its activity concentration.

[0158] Radiolabeling

[0159] • Method I : One-pot with chelating agent.

[0160] Radiolabeling was performed by adding the chelating agent LH2 (30 pL of the stock solution, 6 pmol), Ga(NOs)3 (15 pL of the stock solution, 3.0 pmol, 0.5 eq.), AcONa / H buffer (30 pL, pH 5.5, 0.1 M) and [18F]NaF (50 pL, 271 ± 17 MBq, n = 3) in a glass test tube at room temperature for 20 min.

[0161] • Method 2: Radiolabeling by isotopic exchange.

[0162] Radiolabeling was performed by adding Ga1?F(L) (30 pL of the stock solution, 6 pmol), AcONa / H buffer (45 pL, pH 4.0, 0.1M) and [18F]NaF (50 pL, 120 MBq) in a glass test tube at room temperature for 20 min.

[0163] • Method 3: Radiolabeling with preformed gallium complexes.

[0164] Radiolabeling was performed by adding GaOH(L) (30 pL of the stock solution, 6 pmol), AcONa / H buffer (45 pL, pH 4.0, 0.1M) and [18F]NaF (50 pL of a solution naturally decayed at 78 MBq.) in a glass test tube at room temperature for 20 min, resulting pH ~ 5.2 (pH test strip).

[0165] Determination of radiochemical conversion (RCC)

[0166] The radiochemical conversion (RCC) was determined by radio-HPLC operated on a ThermoFisher® Dionex Ultimate 3000 HPLC system equipped with LPG-3400SD pump, WPS-3000SL / TSL autosampler, TCC-3000SD column oven, DAD-3000 detector and coupled with an Eckert Ziegler® Mini-Scan TLC scanner and Flow-Count detector. Data were acquired with Chromeleon™ software (version 6.8; ThermoFisher®). RCC was determined based on the relative percentage area of the peaks. method: Column Kinetex EVO Cl 8 100 x 4.6 mm x 5 pm (Phenomenex®) set at 25 °C. Eluent: Phase A: 5 mM AcONa / H buffer at pH 4.0. Phase B: acetonitrile.

[0167] Gradient: A / B: 0 -2 min: 95 / 5, 2- 7 min: 95 / 5 to 70 / 30, 7 - 9 min: 70 / 30, 9 - 12 min: 70 / 30 to 40 / 60, 12 - 13 min : 40 / 60, 13 - 17 min: 40 / 60 to 95 / 5, 17 - 20 min: 95 / 5). Flow rate: 1 mL / min. The dead volume of the system was evaluated by injection of AcONa / H buffer: tR = 0.95 min. Sample: crude reaction mixture; injection volume: 5 pL.

[0168] For this study, the assay with the highest RCC (radiolabeling from Method 3) was considered. 5 pL of the crude solution was injected into the radio-HPLC at several times measured from the introduction of [18F]NaF: 20 min (“to”) and 60 min (see Figures 1 and 2). The LC-MS-UV analysis (see Figures 3 and 4) shows a peak at 5.7 min which corresponds to the complex GaOH(L) as fluorine- 18 decays in oxygen- 18.

Claims

CLAIMS1. A complex of F, in particular18F, and of a compound of the following formula (I):wherein:E represents CH, N or NO;M represents a cation selected from the list consisting of Al3+, Ga3+, ln3+, Sc3+, Y3+, La3+, Ce3+, Pr3+, Nd3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+, Yb3+, Lu3+and Fe3+, and is coordinated to at least one of the N atoms linked to Q1, Q2and Q3and to E when E represents N or NO;D, G and J, identical or different, represent a group selected from the list consisting of CH, N, C-OH, C-O-L and C-L, wherein L represents a linker group, optionally linked to a biological targeting moiety (BTM);R1, R2, R3, R4, R5, R6, R" and R12, identical or different, represent a group selected from the list consisting of -H, -OH, -(CH2)n-CH3, -(CH2)n-OH and -L, wherein n represents an integer from 0 to 5,R7, R8, R9and R10, identical or different, represent a group selected from the list consisting of -H, =O, -(CH2)P-CH3, -(CH2)P-OH and -L, wherein p represents an integer from 0 to 3, provided that when one of R7or R8and / or one of R9or R10represents =O then the other one is absent, i.e. represents no group;Q1, Q2and Q3, identical or different, represent a group selected from the list consisting of -H, -OH, -(CH2)k-CH3, -(CH2)k-OH, -CHQ4X and -L, wherein k represents an integer from 0 to 3, Q4represents H or a C1-3 alkyl group, and X represents a group selected from the list consisting of -COO", -PO(OH)(O’) and -Q5-PO(OH), wherein Q5represents Ar or a C1-3 alkyl group, provided that at least two of Q1, Q2and Q3represent -CHQ4X and are coordinated to M; provided that the compound of formula (I) comprises at least one L,wherein F, in particular18F, is coordinated to M.

2. The complex according to claim 1 , wherein the compound is of the following formula (1-1 ):wherein:- R7and R9, identical or different, represents =O or -H, and- D represents C-O-L, J represents C-H, and Q3represents -H, -OH, -(CH2)k-CH3, -(CH2)k- OH wherein k represents an integer from 0 to 3, or- D represents C-H, J represents C-L or C-O-L, and C3represents -H, -OH, -(CH2)k-CH3, - (CH2)k-OH wherein k represents an integer from 0 to 3, or- D represents C-H, J represents C-H, and C3represents L, wherein L is as defined in claim 1 .

3. The complex according to claim 1 or 2, wherein L is a group of formula -(A)m-Y wherein- Y represents a group selected from the list consisting of -H, -N=C=S, -N=C=O, - NH(C=S)-BTM, and -NH(C=O)-BTM,- each A is independently selected from the list consisting of -CR2-, -CR=CR-, -C=C-, - CR2CO2-, -CO2CR2- , -NRCO-, -CONR-, -CONHCR2NH(C=S)NH-, -CONHCR2NH(C=O)NH- , -CR=N-O-, -NR(C=O)NR-, -NR(C=S)NR-, -SO2NR-, -NRSO2-, -CR2OCR2-, -CR2SCR2-, - CR2NRCR2-, -CR2NH(C=S)NH-, -CR2NH(C=O)NH-Za C4-8 eye Io heteroalkylene group, a C4-8 cycloalkylene group, -Ar-, -NR-Ar-, -O-Ar-, -Ar-(CO)-, an amino acid, a sugar or a polyethyleneglycol (PEG) building block, wherein: each R is independently selected from the list consisting of H, C1-6 alkyl, C2-4 alkenyl, C2-4 alkynyCl, 1-4 alkoxyalkyl, C1-4 hydroxyalkyl, Ar-NH(C=S)NH, Ar- NH(C=O)NH, C1-4 alkylphenyl-NH(C=S)NCH1,-4 alkylphenyl-NH(C=O)NH,m is an integer from 1 to 20, each Ar is independently a C5-12 arylene group, or a C3-12 heteroarylene group.

4. The complex according to anyone of claims 1 to 3, wherein L is selected from the list consisting of:

5. The complex according to anyone of claims 1 to 4, wherein M represents Ga3+.

6. The complex according to anyone 1 to 5, wherein the compound is represented by a formula selected from the list consisting of:

7. The complex according to any one of claims 1 to 6, which is linked to a biological targeting moiety (BTM).

8. The complex according to any one of claims 1 to 7, wherein the biological targeting moiety (BTM) is selected from the list consisting of a pre-targeting moiety, an amino- acid, a polypeptide, a peptoid or peptidic mimetic, an enzyme substrate, agonist or inhibitor, a ligand of a biological receptor, an antibody, an antibody fragment, a scFv, a VHH and an aptamer.

9. The complex according to any one of claims 1 to 8, wherein F is18aF nd the compound is linked to a biological targeting moiety (BTM), for use as an in vivo PET scan diagnostic agent.

10. The use of a complex according to any one of claims 1 to 8, wherein F is and18F the compound is linked to a biological targeting moiety (BTM), for generating a PET scan image of a body or a body part of an individual, wherein the complex has been administered previously to the individual.

11. A method of imaging an individual, comprising generating an image, using PET, of a body or body part of the individual, to which a complex as defined in any one of claims 1 to 8, wherein F is18aFnd the compound is linked to a biological targeting moiety (BTM), has distributed, wherein the complex has been administered previously to the individual.

12. A pharmaceutical, imaging or diagnostic composition comprising a complex as defined in any one of claims 1 to 8, wherein F is an1d8F the compound is linked to a biological targeting moiety (BTM), optionally in association with at least one biocompatible carrier.

13. A method of preparation of the complex according to any one of claims 1 to 8, which comprises:- reacting a compound of formula (I), wherein M bound to R13, with a fluoride1a8Fnion, wherein R13represents -OH, -OCH3, -OCH2-CH3, -CO-CH3, -CO-CH2-CH3, or- reacting a complex of,9F and a compound of formula (I) wherein,9F is coordinated to M, with a18F fluoride anion, or- reacting a complex of18F and M, wherein M is as previously defined, with a compound of the following formula (I’):wherein D, E, G, J, Q1, Q2, Q3, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R" and R12are as previously defined.

14. A kit or cassette comprising the non-radioactive reagents necessary to carry out the method of claim 13, including:- a compound of formula (I), wherein M is bound to R13, wherein R13represents -OH, - OCH3, -OCH2CH3, -CO-CH3, -CO-CH2-CH3, or- a complex of, 9F and a compound of formula (I) wherein, 9F is coordinated to M, or- a compound of formula (I’).

15. A compound of formula (I) as defined in any one of claims 1 to 8, wherein M is bound to R13, wherein R13represents -OH, -OCH3, -OCH2-CH3, -CO-CH3, or -CO-CH2- CH3, preferably -OH

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