DESCRIPTION

Technical Field

The invention belongs to the field of chelation of radium in physiological media.

More specifically, it relates to a diaza-18-crown-6 derivative which is able to form stable chelates with radium ions (Ra ²⁺ ) in physiological media and which is functionalized with a moiety for a covalent bonding reaction. This moiety allows, for example, covalently bonding the diaza-18-crown-6 derivative to a biological cell-targeting moiety in view of preparing a radiopharmaceutical or grafting the diaza-18-crown-6 derivative onto a solid support in view of preparing a stationary phase for chromatography column.

It also relates to a conjugate comprising the diaza-18-crown-6 derivative covalently bonded to a biological cell-targeting moiety and, notably, to a cancer cell- targeting moiety.

It further relates to a chelate comprising radium and, notably, radium-223, radium-224 or radium-225, chelated by the conjugate.

It still further relates to uses of the diaza-18-crown-6 derivative, the conjugate and the chelate.

Background of the Invention

Radium-223 is the first a-emitter radionuclide which was approved by the Food and Drug Administration (FDA) for a-radiotherapy applications.

Radium-223 is currently used as an aqueous injectable solution containing radium- 223 chloride ( ²²³ RaCl ₂ ), which is commercially available as Xofigo™, for treating castration- resistant prostate cancers with symptomatic bone metastases and no known visceral metastases. The targeting of the cancer cells by ²²³ RaCl2 relies on the inherent chemistry of radium which is similar to that of calcium and which leads radium-223 to accumulate in bone mineralization sites. In order to broaden the uses of radium in a-radiotherapy, it is needed to have chelating agents which are able to form stable chelates with Ra ²⁺ ions in physiological media and which further comprise a moiety allowing these chelates to be selectively delivered to disease-specific sites. It is known that diaza-18-crown-6 derivatives bearing two pendent arms are able to form chelates in aqueous media with many metal ions such as barium, radium, lead, bismuth, lanthanide and actinide ions.

Particular attention has been given to a diaza-18-crown-6 derivative in which each of the pendent arms is a picolinic acid (i.e. a pyridyl-2 carboxylic acid). This diaza-18-crown- 6 derivative, which is known as Macropa, has been proposed notably to extract lanthanides from aqueous media (see ES-A-2340 120), to remove or inhibit barium sulfate scale (see WO-A-2019/232294) but also to prepare radiolabeled conjugates useful in targeted a- radiotherapy of cancers (see WO-A-2020/106886).

Considering that it should be useful to further expand the range of radium chelating agents and, notably, of radium chelating agents able to be used in a-radiotherapy, the inventors set themselves the goal of providing new compounds which are able to chelate Ra ²⁺ ions efficiently and in a stable manner in physiological media and which can further be covalently bonded to a biological cell-targeting moiety and, in particular, to a cancer cell-targeting moiety. Summary of the Invention

The invention sets out precisely to propose such a compound.

This compound is a diaza-18-crown-6 derivative of formula (I): wherein: B is a moiety for a covalent bonding reaction; the two A are identical to each other and are a group of formula (II): where: n is 0, 1, 2, 3 or 4; and

X ¹ , X ² , X ³ , X ⁴ and X ⁵ , which may be identical or different, are a nitrogen atom, a -CH group or a -CR group wherein R is a -P(O)(OR ¹ )(OR ² ), -P(O)R ¹ (OR ² ), -P(O)R ¹ R ² , -P(S)(SR ¹ )(SR ² ), -P(S)R ¹ (SR ² ) or -P(S)R ¹ R ² group in which R ¹ and R ² , which may be identical or different, are a hydrogen atom, or a straight-chain or branched alkyl group having 1 to 4 carbon atoms, with the proviso that no more than two of X ¹ , X ² , X ³ , X ⁴ and X ⁵ are a nitrogen atom and no more than one of X ¹ , X ² , X ³ , X ⁴ and X ⁵ is a -CR group.

By "moiety for o covalent bonding reaction", is meant any moiety comprising at least one reactive group allowing a covalent bonding, by a chemical reaction, of the diaza- 18-crown-6 derivative to another (organic, inorganic or organic-inorganic) compound or material which itself comprises at least one reactive group. The moiety for a covalent bonding reaction may notably be a moiety comprising at least one reactive group allowing a covalently bonding, by a chemical reaction, of the diaza-18-crown-6 derivative, directly or through a spacer arm, to a biological cell-targeting moiety.

By "straight-chain or branched alkyl group having 1 to 4 carbon atoms”, is meant any alkyl group chosen from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl groups.

According to the invention, X ¹ is preferably a nitrogen atom, one of X ² , X ³ , X ⁴ and X ⁵ is preferably a -CR group, in which case three of X ² , X ³ , X ⁴ and X ⁵ are a -CH group.

More preferably, X ² is a -CR group while X ³ , X ⁴ and X ⁵ are a -CH group.

Also, according to the invention, the R of the -CR group is preferably a -P(O)(OR ¹ )(OR ² ), -P(O)R ¹ (OR ² ) or -P(O) R ¹ R ² group. Advantageously, n is 1. Diaza-18-crown-6 derivatives having such preferred features are in particular the diaza-18-crown-6 derivatives of formula (III): in which B, R ¹ and R ² are as defined above.

Preferably, R ¹ and R ² are identical to each other and are a hydrogen atom, a methyl group or an ethyl group. More preferably, R ¹ and R ² are a hydrogen atom.

Hence, a diaza-18-crown-6-derivative which is particularly preferred is the diaza- 18-crown-6-derivative of formula (IV):

According to the invention, the moiety B is preferably a saturated or unsaturated, acyclic hydrocarbon group comprising 1 to 12 carbon atoms with optionally one or more heteroatoms, and comprising a reactive group chosen from the carboxyl, acyl halide, acid anhydride, hydroxyl, aldehyde, azide, ketone, thiol, amine, amide, halogeno, ester, ether, vinyl, alkynyl (for example, propargyl), imide, maleimide and oxime groups.

By "saturated or unsaturated, acyclic hydrocarbon group comprising 1 to 12 carbon atoms and optionally one or more heteroatoms" , is meant any saturated, linear or branched, hydrocarbon group, i.e. any alkyl group, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms or any unsaturated, linear or branched, hydrocarbon group, i.e. any alkenyl or alkynyl group, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, the chain of which may be interrupted by one or more heteroatoms or be substituted by one or more heteroatoms.

By "heteroatom" , is meant any atom other than carbon and hydrogen atoms, the heteroatom being typically a nitrogen, oxygen or sulphur atom.

The reactive group of the moiety B is preferably a terminal carboxyl group (-COOH) or amine group (-NH2).

Hence, the moiety B may notably be a group of formula:

* -(CH ₂ ) _p -NH2, where p is an integer from 1 to 10 and, preferably, from 1 to 6,

* -(CH ₂ ) _q -COOH, where q is an integer from 1 to 10 and, preferably, from 1 to 6,

* -(CH ₂ )r-O-(CH ₂ )s-NH ₂ , where r and s, identical or different, are integers from 1 to 5 and, preferably, equal to 1, 2 or 3,

* -(CH ₂ )r-O-(CH ₂ )s-COOH, where r and s, identical or different, are integers from 1 to 5 and, preferably, equal to 1, 2 or 3,

* -(CH ₂ ) _r -NH-CO-(CH ₂ )s-NH2, where r and s, identical or different, are integers from 1 to 5 and, preferably, equal to 1, 2 or 3, or

* -(CH ₂ ) _r -NH-CO-(CH ₂ )s-COOH, where r and s, identical or different, are integers from 1 to 5 and, preferably, equal to 1, 2 or 3.

Examples of diaza-18-crown-6 derivatives comprising such a moiety B are those of formulae (VII) and (VIII) which are shown in Figures 2 and 3.

According to the invention, the diaza-18-crown-6 derivative may be used for preparing a stationary phase for chromatography column.

Such a stationary phase may be prepared by grafting the diaza-18-crown-6 derivative by means of the moiety B onto a solid support which may be either an inorganic support such as, for example, silica or alumina particles, silica gel, porous glass, or an organic support such as a polymer (polyacrylamide, polyvinyl, poly(meth)acrylate, agarose for example) or still an inorganic-organic support. As known perse, the grafting is possibly made after creation on the solid support of reactive sites able to react with the reactive group of the moiety B. However, the diaza-18-crown-6 derivative is preferably used for preparing a conjugate comprising a biological cell-targeting moiety useful, after chelation of radium, as a radiopharmaceutical.

Such a conjugate may be represented by formula (V): wherein: the two A are as defined hereinabove;

L is a linker;

C is a biological cell-targeting moiety.

By "biological cell-targeting moiety", is meant any moiety able to specifically recognize and bind to biological cells. Such a moiety may be a biomolecule (i.e. a molecule which is naturally present in living beings) or with a synthetic molecule which mimics structurally and/or functionally a biomolecule.

Thus, the targeting moiety for biological cells may notably comprise or be constituted by a monoclonal antibody, a monoclonal antibody fragment, a protein, a peptide, a carbohydrate, an aptamer, a vitamin, a hormone, a neurotransmitter, a steroid, a vesicle such as a liposome, a growth factor, an agonist or antagonist ligand of a cell receptor such as a membrane receptor.

According to the invention, the diaza-18-crown-6-ether derivative may be directly bonded to the moiety C, in which case L is the rest of the moiety B of the diaza-18-crown- 6 derivative. By "rest of the moiety B", is meant the atom group belonging to the moiety B which remains on the diaza-18-crown-6-ether derivative after being bonded to the moiety C.

However, the diaza-18-crown-6-ether derivative may also be bonded to the moiety C via a spacer arm to which the moiety C is already bonded or will be later bonded, in which case L is the atom group formed by the rest of the moiety B together with the rest of the spacer arm. Hereto, by "rest of the spacer arm", is meant the atom group belonging to the spacer arm which remains after being bonded, on the one hand, to the moiety B and, on the other hand, to the moiety C.

The spacer arm may be chosen from a large number of compounds, as long as they have at least two reactive groups capable of chemically reacting with, on the one hand, the reactive group of the moiety B and, on the other hand, a reactive group belonging to the moiety C, and do not adversely affect the solubility of the conjugate in aqueous media and, notably, in physiological media.

Thus, for example, the spacer arm may notably be a polyethylene glycol such as, for example, PEG2, PEG4, PEG ₆ or PEG12, an oligopeptide (preferably of no more than 10 amino acids), a polyamine, a polyol, an amino-alkanoic acid such as 4-aminobutanoic acid, 5-aminopentanoic acid or 6-aminohexanoic acid, an alkanedioic acid such as butanedioic acid, pentanedioic acid or hexanedioic acid, a group comprising at least one cycle (which may be a cycloalkyl such as a cyclopentyl or cyclohexyl, a saturated or unsaturated heterocycle such as a piperidinyl, pyridinyl, phenyl or triazolyl cycle) together with two reactive functions such as 4-amino-l-carboxymethylpiperidine or 4-(2aminoethyl)-l- carboxymethyl piperazine, and combinations thereof.

If the conjugate is intended to be used, after chelation of radium, as a radiopharmaceutical for treating a prostate cancer and, in particular, a castration-resistant cancer prostate by targeted a-radiotherapy, then the targeting moiety for biological cells is preferably:

- either a bombesin receptor (GRPR) antagonist peptide such as, for example, one of those which are disclosed in WO-A-2009/109332 and notably the peptide known as JMV594 (of formula: DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2);

- or a ligand of the prostate specific membrane antigen (PSMA) such as, for example, one of those which are disclosed in WO-A-2009/026177 and notably one of those which are urea of two amino acids.

After chelation of radium by the conjugate as defined hereinabove, a radium chelate is obtained. Without being bound by any theory, an example of a preferred radium chelate may be the chelate of formula (VI):

According to the invention, the chelated radium is preferably radium-223, radium- 224 or radium-225, in which case the radium chelate may be used as a radiopharmaceutical and, notably, for treating a cancer by targeted a-radiotherapy, which cancer is preferably a prostate cancer and, most preferably, a castration-resistant prostate cancer.

Other characteristics and advantages of the invention will become betterapparent on reading the complement to the description that follows. Obviously, this complement to the description is only given to illustrate the object of the invention and does not constitute in any case a limitation of said object.

Brief Description of the Figures

Figure 1 illustrates a general synthesis route of the diaza-18-crown-6 derivatives of formula (I). Figure 2 illustrates a first example of adaptation of the general synthesis route of

Figure 1 to the synthesis of a particular diaza-18-crown-6 derivative of formula (I).

Figure 3 illustrates a second example of adaptation of the general synthesis route of Figure 1 to the synthesis of another particular diaza-18-crown-6 derivative of formula (I).

Detailed Description of the Invention I - General synthesis route of the diaza-18-crown-6 derivatives of formula (I):

The diaza-18-crown-6 derivatives of formula (I) may be obtained by the general synthesis route illustrated in Figure 1. As shown in Figure 1, at a first step denoted A, 1,2-ethanediol, denoted 1 and substituted with a moiety B which has been previously protected by an appropriate protective group and which is therefore denoted B _pr , is subjected to a reaction with a monohalogenoacetic acid of formula: HalCH ₂ COOH in which Hal is, for example, a chlorine, bromine or fluorine atom, in the presence of a strong base such as potassium tert- butoxide in tert-butanol, to obtain compound 2.

At a second step denoted B in Figure 1, compound 2 is subjected to a reaction with an oxalyl halide of formula: (COHal) ₂ in which Hal is, for example, a chorine, bromine or fluorine atom, in the presence of a weak base such as pyridine, in an apolar solvent such as toluene, to obtain compound 3.

At a third step denoted C in Figure 1, compound 3 is subjected to a reaction with 2,2'-(ethylenedioxy)bis(ethylamine) of formula: NH ₂ (CH ₂ ) ₂ O(CH ₂ ) ₂ O(CH ₂ ) ₂ NH ₂ , in the presence of a strong base such as triethylamine (NEt ₃ ), in an apolar solvent such as toluene, to obtain compound 4.

At a fourth step denoted D in Figure 1, compound 4 is subjected to a reaction with a strong reducing agent, such as a tetrahydroaluminate of formula: MAIH4 in which M is, for example, sodium, potassium or lithium, in a polar solvent such as tetrahydrofurane (THF) to obtain compound 5.

At a fifth step denoted E in Figure 1, compound 5 is subjected to a reaction with a halide of formula: in which Hal is, for example, a chlorine, bromine or fluorine atom, in the presence of a carbonate of formula: M ₂ CO ₃ in which M is, for example, sodium or potassium, in a polar solvent such as acetonitrile, to obtain compound 6.

At a last step denoted F in Figure 1, the moiety B is deprotected to obtain a diaza- 18-crown-6 derivative of formula (I).

II - Examples of adaptation of the general synthesis route to the synthesis of particular diaza-18-crown-6 derivatives of formula (I): As obvious for one skilled in the art, the general synthesis route shown in Figure 1 may be adapted notably depending on the meaning of the moiety B and the meanings of R ¹ and/or R ² in the -CR group of the diaza-18-crown-6 derivative of formula (I).

As a first example, Figure 2 illustrates an adaptation of the general synthesis route shown in Figure 1 to the synthesis of the diaza-18-crown-6 derivative of formula (VII): which corresponds to the diaza-18-crown-6 derivative of formula (IV) wherein the moiety B is a -CH ₂ -O-(CH ₂ ) ₂ -COOH group.

As shown in Figure 2, this synthesis comprises 9 steps, denoted a) to i). Step a):

Step a) corresponds to step A of Figure 1.

To a solution of potassium tert- butoxide ( ^t BuOK) in tert-butanol was added 3-(allyl)oxy-l, 2-propanediol. The solution was stirred at room temperature for 1 hour before a solution of chloroacetic acid (CICH ₂ COOH) in tert-butanol was added dropwise over an hour. The mixture was refluxed for 18 hours before the reaction was cooled to room temperature and the solvent was removed under reduced pressure. The residue was dissolved in H ₂ O and extracted with diethyl ether (Et ₂ O). The aqueous layer was acidified with hydrochloric acid (HCI) and extracted with ethyl acetate (EtOAc) and the combined organic layers were washed with brine, dried over magnesium sulfate (MgSO ₄ ), filtered and the solvent was removed under reduced pressure to give the crude compound as a yellow oil. The crude oil was purified by vacuum distillation to give 2,2'-((3-(allyloxy)propane-l,2- diyl)bis(oxy))diacetic acid.

Step b):

Step b) corresponds to step B of Figure 1. To a solution of 2,2 ^, -((3-(allyloxy)propane-l,2-diyl)bis(oxy))diacetic acid in toluene were added oxalyl chloride ((COCI) ₂ ) and pyridine. The mixture was stirred at room temperature for 18 hours. The mixture was filtered and the solvent was removed under reduced pressure. The excess oxalyl chloride was removed by co-evaporation with toluene to give 2,2'-((3-(allyloxy)propane-l,2-diyl)bis(oxy))diacetyl chloride.

Step c):

Step c) corresponds to step C of Figure 1.

To a stirred flask of toluene were simultaneously added dropwise a solution of 2'-((3-(allyloxy)propane-l,2-diyl)bis(oxy))diacetyl chloride in toluene and a solution of NEt ₃ and 2,2'-(ethane-1,2-diylbis(oxy))bis(ethan-l-amine) in toluene. The mixture was stirred for 18 hours before the solid was filtered and washed with toluene. The solvent was removed under reduced pressure to give the crude compound as a pale yellow oil. The crude compound was purified by column chromatography (alumina, CHCI ₃ eluent) to afford 2-((allyloxy)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctad ecane-6,17-dione. Step d):

Step d) corresponds to step D of Figure 1.

To a suspension of LiAIH ₄ in THF was added 2-((allyloxy)methyl)-1,4,10,13- tetraoxa-7,16-diazacyclo-octadecane-6,17-dione. The mixture was stirred at reflux, before sodium hydroxide was added dropwise and the mixture was stirred at room temperature. The reaction mixture was filtered and washes with hot THF before the solvent was removed under reduced pressure. The crude compound was purified by column chromatography (alumina, CHCl ₃ EtOH 25:1) to give (1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2- yl)methanol. Step e):

To a solution of (1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)methanol in THF was added K ₂ CO ₃ and the mixture was stirred at room temperature for 15 min. To the stirred mixture benzyl chloride (BnCI) was added and the reaction was stirred. The excess K ₂ CO ₃ was removed via filtration and the reaction was washed with THF before the solvent was removed under reduced pressure. The crude compound was purified by column chromatography (silica, CHCl ₃ :MeOH:NH ₄ OH 100:5:0.1) to give (7,16-dibenzyl-1,4,10,13- tetraoxa-7,16-diazacyclooctadecan-2-yl)methanol.

Step f):

To a solution of (7,16-dibenzyl-l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2 - yl)methanol in THF was added sodium hydride (NaH). The mixture was stirred at room temperature before methyl 3-bromopropionate was added. The reaction was stirred. To the crude reaction, H ₂ O was added and the product was extracted with dichloromethane. The combined organic extracts were washed with brine, dried with Na ₂ SO ₄ , filtered and the solvent was removed under reduced pressure. The crude compound was purified by column chromatography (silica/alumina, solvent system) to give methyl 3-((7, 16-dibenzyl- 1,4,10,13-tetraoxa-7,16-diazacyclo-octadecan-2-yl)methoxy)pr opanoate.

Step g):

To a flask containing of methyl 3-((7,16-dibenzyl-l,4,10,13-tetraoxa-7,16- diazacyclooctadecan-2-yl)methoxy)propanoate in MeOH was added 10% palladium on carbon (Pd/C). The flask was flushed with N ₂ and then H ₂ and the reaction mixture was stirred under an H ₂ atmosphere. The reaction mixture was filtered through celite and the washed with methanol and the solvent was removed under reduced pressure to give methyl 3-((l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)methoxy )propanoate.

Step h):

Step h) corresponds to step E of Figure 1.

To a stirred solution of methyl 3-((l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2- yl)methoxy)propanoate in acetonitrile was added K ₂ CO ₃ and diethyl(6-(chloro- methyl)pyridin-2-yl)phosphonate. The reaction mixture was stirred at reflux for 18 hours before the excess K2CO3 was removed via filtration and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (silica/alumina, CHCI3) to give methyl 3-((7,16-bis((6-(diethoxy-phosphoryl)-pyridin-2- yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl) -methoxy)-propanoate. Step i):

A solution of methyl 3-((7,16-bis((6-(diethoxyphosphoryl)pyridin-2-yl)methyl)- l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)methoxy)pro panoate in 8 M HCI was stirred. The solvent was removed under reduced pressure to give the diaza-18-crown-6 derivative of formula (VII).

As a second example, Figure 3 illustrates an adaptation of the general synthesis route shown in Figure 1 to the synthesis of the diaza-18-crown-6 derivative of formula (VIII): which corresponds to the diaza-18-crown-6 derivative of formula (IV) wherein B is a -CH ₂ - NH-CO-(CH ₂ )2-COOH group.

As shown in Figure 3, this synthesis comprises 12 steps, denoted a) to I).

Steps a) to e):

Steps a) to e) are identical to steps a) to e) as described above and shown in Figure 2 and lead to (7,16-dibenzyl-l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2 -yl)-methanol.

Step f):

To a solution of (7,16-dibenzyl-l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2 - yl)methanol in DCM were added triethylamine and methanesulfonyl chloride. The reaction mixture was stirred at room temperature for 3 hours. The product was extracted with HCI (1 M) and washed with DCM. The aqueous layer was basified with NaOH (2 M), extracted with DCM and the organic solvent was removed under reduced pressure to give (7,16- dibenzyl-l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)me thyl methane-sulfonate.

Step g):

To a solution of (7,16-dibenzyl-l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2 - yl)methyl methanesulfonate in DMF was added sodium azide. The reaction mixture was stirred at 60 °C for 2 hours before the solvent was removed under reduced pressure. The residue was redissolved in HCI (1 M) and washed with DCM. The aqueous layer was basified with NaOH (2 M), extracted with DCM and the organic solvent was removed under reduced pressure. The crude product was purified by column chromatography (silica, DCM:MeOH:NH ₄ OH 100:5:0.1) to give 2-(azidomethyl)-7,16-dibenzyl-l,4,10,13-tetraoxa- 7,16-diazacyclooctadecane.

Step h):

To a solution of 2-(azidomethyl)-7,16-dibenzyl-l,4,10,13-tetraoxa-7,16- diazacyclooctadecane in THF:H ₂ O was added PPh ₃ . The reaction mixture was stirred at 40 °C for 3 hours before the solvent was removed under reduced pressure. The residue was redissolved in HCI (1 M) and washed with DCM. The aqueous layer was basified with NaOH (2 M) and extracted with DCM and the organic solvent was removed under reduced pressure. The crude product was dissolved in THF and 4-dimethylaminopyridine and di-tert- butyl dicarbonate were added. The reaction was stirred at 45 °C for 24 hours before the solvent was removed under reduced pressure. The residue was redissolved in HCI (1 M) and washed with DCM. The aqueous layer was basified with NaOH (2 M), extracted with DCM and the organic solvent was removed under reduced pressure. The crude product was purified by column chromatography (alumina, DCM:MeOH 100:0.05) to give tert-butyl (tert-butoxycarbonyl)((7,16-dibenzyl-l,4,10,13-tetraoxa-7,16 -diazacycloocta-decan-2- yl)methyl)carbamate. Step i):

To a solution of tert-butyl (tert-butoxycarbonyl)((7,16-dibenzyl-l,4,10,13- tetraoxa-7,16-diazacyclooctadecan-2-yl)methyl)carbamate in MeOH was added Pd/C (10 %). The reaction vessel was purged with H2 and the reaction was stirred under a H2 atmosphere for 18 hours. The mixture was filtered through Celite™ and the solvent was removed under reduced pressure. The residue was redissolved in HCI (1 M) and washed with DCM. The aqueous layer was basified with NaOH (2 M), extracted with DCM and the organic solvent was removed under reduced pressure to give tert-butyl ((1,4,10,13- tetraoxa-7,16-diazacyclooctadecan-2-yl)methyl)(tert-butoxyca rbonyl)carbamate.

Step i):

To a solution of tert-butyl ((l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2- yl)methyl)(tert-butoxycarbonyl)carbamate in THF were added potassium carbonate and diethyl (6-(chloromethyl)pyridin-2-yl)phosphonate in THF. The reaction mixture was stirred at reflux for 20 hours before the solvent was removed under reduced pressure. The residue was redissolved in HCI (1 M) and washed with DCM. The aqueous layer was basified with NaOH (2 M), extracted with DCM and the organic solvent was removed under reduced pressure. The crude product was purified by column chromatography (alumina, CHCbiMeOH 100:0.05) to give tert-butyl ((7,16-bis((6-(diethoxyphosphoryl)-pyridin-2- yl)methyl)-l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl) methyl)(tert- butoxycarbonyl)carbamate.

Step k):

A solution of tert-butyl ((7,16-bis((6-(diethoxyphosphoryl)pyridin-2-yl)methyl)- l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-2-yl)methyl)(ter t-butoxycarbonyl)carbamate in HCI (6 M) was stirred at reflux for 18 hours. The solvent was removed under reduced pressure to give (((2-(aminomethyl)-l,4,10,13-tetraoxa-7,16-diazacyclooctadec ane-7,16- diyl)bis(methylene))bis(pyridine-6,2-diyl))bis(phosphonic acid).

Step I):

To a mixture of (((2-(aminomethyl)-l,4,10,13-tetraoxa-7,16-diazacyclo- octadecane-7,16-diyl)bis(methylene))bis(pyridine-6,2-diyl))b is(phosphonic acid) in DMF were added diisopropylethylamine and succinic anhydride. The reaction mixture was stirred at 75 °C for 20 hours before the solvent was removed under reduced pressure to give the diaza-18-crown-6 derivative of formula (VIII).

III - Radium chelation properties of the diaza-18-crown-6 derivatives of formula (I):

An experimental study and a computational analysis were carried out in view of appreciating the ability of the diaza-18-crown-6 derivatives of formula (I) to chelate radium in physiological media.

Insofar as the moiety B is presumed not to be involved in the ability of the diaza- 18-crown-6 derivatives to chelate radium, the experimental study and the computational analysis were carried out using the diaza-18-crown-6 derivative of formula (IX): which differs from the diaza-18-crown-6 derivative of formula (IV) only in that moiety B is absent.

This diaza-18-crown-6 derivative was synthetized as shown in Figure 1 using 1,2-ethanediol as a starting product.

The diaza-18-crown-6 derivative of formula (IX) was compared to Macropa in the experimental study while it was compared to CyDTA (i.e. 1,2-cyclohexylenedinitrilo- tetraacetic acid), DTPA (i.e. diethylenetriaminepentaacetic acid), EDTA (i.e. ethylene- diaminetetraacetic acid), EGTA (i.e. triethyleneglycoldiaminetetraacetic acid), DOTA (i.e. 1,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid) and Macropa in the computational analysis.

In this respect, it is reminded that Macropa is of formula:

Sterile water (Aqua, B. Braun) previously demetalled by means of a self-built cartridge equipped with Chelex™ 100 sodium form resin (Sigma-Aldrich) was used in the experiments described below including the preparation of all aqueous solutions (buffer, HCI etc.).

Commercially available ²²³ RaCl ₂ , namely Xofigo™, was used as a source of radium- 223 and was therefore desalinated using a self-built cartridge equipped with 200 mg of TK100-B01-S (Triskem International) ion exchange resin. To do this, 6 mL of Xofigo™ were loaded to the cartridge. First, 15 mL of an aqueous solution of 0.01 M HCI were loaded to the cartridge and were let eluting. Then, the desalinated trapped ²²³ Ra was eluted with 300 μL portions of an aqueous solution of 0.1 M HCI. The fractions were collected and the radioactivity of each portion was measured. The total concentration of the eluted ²²³ Ra was measured as 0.5 MBq/mL with a recovery rate >90%. 0,1 mL solutions containing 5 μg, 10 mg, 25 mg, 50 μg and 100 μg of diaza-18- crown-6 derivative of formula (IX) or Macropa were prepared with sterile, demetallated water. Then, desalinated ²²³ Ra in 0.1 M HCI (0.02 MBq, 0.1 mL) and 0.2 mL of 1 M ammonium acetate buffer (pH 7.5) were successively added to each solution and the pH was adjusted to 7 by addition of 1 M NaOH (1-3 pL) if necessary. Reactions were performed for 30 minutes at room temperature.

The chelating yields were evaluated by thin layer radiochromatography. To do this, 15 μL of the prepared Ra-labeled chelator solutions were spotted onto DGA TLC sheets (Triskem International). The impregnated DGA sheets were then developed in a 0.1 M NaOH solution and monitored by means of a phosphor imager (Cyclone™ Plus Storage Phosphor System, Perkin Elmer).

The results showed that:

- a chelating yield of >99 % was obtained with the solution containing 50 mg of the diaza-18-crown-6 derivative of formula (IX) whereas a chelating yield of >99% was only obtained with the solution containing 100 μg of Macropa; and

- the specific activity of the chelate formed by the diaza-18-crown-6 derivative of formula (IX) with radium-223 was twice higher than the specific activity of the chelate formed by Macropa with radium-223 (0.24 MBq/μmol versus 0.11 MBq/μmol ).

Computational analysis:

A computational analysis based on Density Functional Theory (DFT) modelling method has been performed in order to determine the binding constant K _eq of the diaza- 18-crown-6 derivative of formula (IX) with radium.

The binding constant K _eq is related to reaction enthalpy ΔG by:

The reaction enthalpy AG (or Gibbs free energy) for the formation of the complex can be calculated by the difference of the inner enthalpy of the reaction components:

By calculation of the vibrational nodes of the structures at 298.150 Kelvin (20° Celsius) and atmospheric pressure, the inner enthalpy of each reaction part can be obtained.

The calculations have been made with Gaussianl6 software (see M. J. Frisch et a!., Gaussian 16 Revision C.01, Gaussian Inc. Wallingford CT, 2016) using the CAM-B3LYP functional (see T. Yanai et al., Chem. Phys. Lett. 2004, 393, 51-57) and the correlation- consistent basis sets by Dunning (see J.G. Hill and K.A. Peterson, J Chem. Phys. 2017, 147, 244106). The metal ions were calculated with additional diffuse and pseudo-potential functions (see I.S. Lim et. al.,J. Chem. Phys. 2006, 124, 034107). The Polarizable Continuum Model (PCM) has also been used to mimic solvent-specific effects. The metal-chelator complexes were simulated without additional water molecules. The calculation considers only the metal-chelator interaction enthalpy, while further effects are taken into account by linear correction factors. Based on linear correlation, all potential sources of systematic errors will be collected in a chelator- or metal-specific correction terms ( ^a metal, ^a chelator) and (bmetai, bcheiator) according to the following equation:

The correction factors, a _m etai, ^a chelator and correction constants ^b metal, ^b chelator, were determined by plotting the calculated binding constants as a function of literature binding constants described by T. Sekine et ol. (Bult. Chem. Soc. J. 1968, 41, 3013-3015) for Ca, Sr, Ba and Ra cations and CyDTA, DTPA, EDTA, EGTA chelators. The correction factors a and b were fitted until a slope of 1.00 was achieved for all calculated systems.

For the calculation of chelators for which no literature data are available (e.g. Ra- Macropa and Ra-diaza-18-crown-6 derivative of formula (IX)) the a-factor and b-factor, which are unknown (and no binding constant can be calculated), have been therefore estimated in taking into account the number of atoms in the chelator system.

Tables I and II below give the literature, calculated and interpolated binding constants (expressed as log K) of Ca, Sr, Ba and Ra cations with different chelators.

Table I

Table II

Literature values: T. Sekine et ol., Bult. Chem. Soc. J. 1968, 41, 3013-3015 n. a.: missing values

The current results allow an estimation of the binding constants and indicate that a stronger coordination for the diaza-18-crown-6 derivative of formula (IX) than for Macropa can be expected. Without being bound by any theory, it is inferred that the difference may be related to the denticity of the chelating systems. While Macropa denticity is limited to a denticity of n=10, that one of the diaza-18-crown-6 derivative of formula (IX) can vary between n=10-12 due to the presence of the phosphonate groups which can coordinate the cation via two or four oxygen atoms.

Calculations are underlining that the coordination sphere of Ra and Ba is fully saturated by diaza-18-crown-6 derivative of formula (IX) and is containing no additional water molecules. Therefore, no significant difference in coordination behavior is expected for the diaza-18-crown-6 derivatives of formula (VI) to (IX).

IV - Preparation of a conjugate:

The conjugate of formula (X): was obtained by coupling the diaza-18-crown-6 derivative of formula (VII) to the bombesin receptor antagonist peptide JMV594 of formula: DPhe-GIn-Trp-Ala-Val-Gly-His-Sta-Leu- NH2, via a spacer arm, namely a 4-amino-l-carboxymethyl-piperidine.

To do this, the peptide JMV594 was firstly synthesized according to standard Fmoc peptide synthesis on solid support. The amino acids were coupled one by one on a rink amide resin with HATU (1.9 eq.) and DIPEA (5 eq.).

Next, the spacer arm 4-amino-l-carboxymethylpiperidine was coupled in the same manner to the N -terminus of the resulting peptide chain.

Next, the diaza-18-crown-6 derivative of formula (VII) was coupled to the spacer arm by reaction of the carboxyl group of the diaza-18-crown-6 derivative with the amino group of the spacer arm.

In a last step, the conjugate was globally deprotected and cleaved from the resin with a cocktail containing trifluoroacetic acid, phenol, water and triisopropylsilane.

References cited

ES-A-2 340120 WO-A-2019/232294 WO-A-2020/106886 WO-A-2009/109332 M. J. Frisch et al., Gaussian 16 Revision C.01, Gaussian Inc. Wallingford CT, 2016 T. Yanai et al., Chem. Phys. Lett. 2004, 393, 51-57 J.G. Hill and K.A. Peterson, J Chem. Phys. 2017, 147, 244106 I.S. Lim et. al., J. Chem. Phys. 2006, 124, 034107

T. Sekine et al., Bult. Chem. Soc. J. 1968, 41, 3013-3015