THEIR USES

The present invention concerns aza-cryptophanes, processes for preparation thereof, and their uses, in particular as in vivo diagnostic tools when complexing a hyperpolarized noble element, or in nanoemulsions.

Cryptophanes are cage molecules capable of encapsulating atoms or small molecules. These molecules consist of two cyclotriveratrylene units linked together by ether bridges of different lengths. The use of these cage molecules is of particular interest in the field of MRI imaging, using hyperpolarized xenon (Xe-HP) as a contrast agent.

Indeed, xenon is a promising contrast agent in the field of molecular MRI imaging, thanks to its hyperpolarization and HyperCEST techniques, this atom being detected with high contrast and in very low quantities.

Cryptophanes are the best hosts for this gas described to date.

However, Xe-HP MRI imaging using this type of in vivo bioprobe is still lacking, in particular due to the poor solubility of this type of cage in biocompatible media.

In addition, as this gas is non-specific, it may be advantageously vectorized to selectively reach a given biological target.

But such a vectorization requires beforehand the synthesis of monofunctionalised cryptophane, in order to attach the linker to this latter. This type of precursor is obtained most of the time from the symmetrical cryptophane, via a step of monodeprotection, leading to the precursor with a very poor yield.

Accordingly, it is an object of the present invention to provide alternative to conventional cryptophanes. Inventors have found that compounds of formula (I) of the invention have enhanced solubility compared to conventional cryptophanes.

Another aim of the present invention is to provide compounds that are easily and selectively functionalized.

The presence of one or more nitrogen atoms on the cages of the invention enable an easy and effective functionalization, and also if need bimodal functionalization with more than one target compounds.

The enhanced solubility of the aza-cryptophanes has also enabled to prepare nanoemulsions thanks to said aza-cryptophanes, which is another aim of the present invention.

Thus, in one aspect, the present invention relates to a compound of following formula (I): wherein: X ₁ , X ₂ and X ₃ are independently selected from –CH ₂ - and –NR-, at least one of X ₁ , X ₂ and X ₃ representing –NR-; R is chosen from: - H; - a C ₁ -C ₆ alkyl; - a C ₂ -C ₆ alkenyl; - a C ₂ -C ₆ alkynyl; - a LZ group; - an ad hoc protecting group, in particular a benzyl, p-methoxybenzyl, 2- tetrahydropyranyl (THP), a carbamate, for example Boc, Fmoc, Troc, Cbz, a silyl, in particular tert-butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), or 2- (trimethylsilyl)ethoxymethyl (SEM), or an acyl group, in particular an acetyl; Y1, Y ₂ and Y ₃ are independently selected from: - H, - a C ₁ -C ₆ alkyl; - a C ₂ -C ₆ alkenyl; - a C ₂ -C ₆ alkynyl; - a O-C ₁ -C ₆ alkyl; - a O- C ₂ -C ₆ alkenyl; - a O-L’Z’ group; - OH or a O-P’ group, wherein P’ is an ad hoc protecting group, in particular a benzyl; p- methoxybenzyl; silyl, in particular tert-butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert- butyldiphenylsilyl (TBDPS); 2-(trimethylsilyl)ethoxymethyl (SEM); allyl ; methoxymethyl (MOM) ; tetrahydropyran (THP) or an acyl group, in particular an acetyl; Z ₁ , Z ₂ and Z ₃ are independently selected from: - H, - a C ₁ -C ₆ alkyl; - a C ₂ -C ₆ alkenyl; - a C ₂ -C ₆ alkynyl; - a O-C ₁ -C ₆ alkyl; - a O-C ₂ -C ₆ alkenyl; - a O-L’’Z’’ group; - OH or a O-P’’ group, wherein P’’ is an ad hoc protecting group, in particular a benzyl; p-methoxybenzyl; silyl, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert- butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS); 2-(trimethylsilyl)ethoxymethyl (SEM); allyl ; methoxymethyl (MOM) ; tetrahydropyran (THP) or an acyl group, in particular an acetyl; n1, n2 and n3 are independently chosen from 0, 1, 2 and 3; L, L’ and L’’ are independently from each other a linker; Z, Z’ and Z’’ are independently from each other: - a terminal reactive function or group, in particular able to form a covalent bond with a peptide, a protein, an antibody or a fragment thereof, a carrier, a solid support, a multivalent scaffold; an anchor; a dye or a fluorescent residue; or - a peptide, a protein, an antibody or a fragment thereof, a carrier, a solid support, a multivalent scaffold; an anchor; a dye or a fluorescent residue; or a pharmaceutically acceptable salt thereof. It was surprising to obtain the N-cryptophanes of the invention. Indeed, the precursors of the N-cryptophanes, namely the N-CTV units (cyclotriveratrylene), are in a saddle conformation (figure 3). However, it is known that the cryptophanes of the prior art are necessarily obtained from CTV units in crown conformation, and that the CTV units of the prior art are in a crown conformation that is much more stable than the corresponding saddle conformation. In a particular embodiment, R is chosen from: - H; - a LZ group; - an ad hoc protecting group, in particular a benzyl, p-methoxybenzyl, 2- tetrahydropyranyl (THP), a carbamate, for example Boc, Fmoc, Troc, Cbz, a silyl, in particular tert-butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), or 2- (trimethylsilyl)ethoxymethyl (SEM), or an acyl group, in particular an acetyl. In a particular embodiment, Y ₁ , Y ₂ and Y ₃ are independently selected from: - H, - a O-C ₁ -C ₆ alkyl; - a O-L’Z’ group; - OH or a O-P’ group, wherein P’ is an ad hoc protecting group, in particular a benzyl; p- methoxybenzyl; silyl, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert- butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS); 2-(trimethylsilyl)ethoxymethyl (SEM); allyl ; methoxymethyl (MOM) ; tetrahydropyran (THP) or an acyl group, in particular an acetyl. In a particular embodiment, Z ₁ , Z ₂ and Z ₃ are independently selected from: - H, - a O-C ₁ -C ₆ alkyl; - a O-L’’Z’’ group; - OH or a O-P’’ group, wherein P’’ is an ad hoc protecting group, in particular a benzyl; p-methoxybenzyl; silyl, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert- butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS); 2-(trimethylsilyl)ethoxymethyl (SEM); allyl ; methoxymethyl (MOM) ; tetrahydropyran (THP) or an acyl group, in particular an acetyl. In a particular embodiment, Y ₁ , Y ₂ and Y ₃ are H. In a particular embodiment, Z ₁ , Z ₂ and Z ₃ are H, or a O-C ₁ -C ₆ alkyl, in particular OMe. In a particular embodiment, X ₁ is –NR-, and X ₂ and X ₃ are –CH ₂ -. In a particular embodiment, X ₁ and X ₂ are independently selected from –NR- groups, and X ₃ is –CH ₂ -. In a particular embodiment, X ₁ , X ₂ and X ₃ are independently selected from –NR- groups. In a particular embodiment, X ₁ is –NH- or -N(LZ)-, and X ₂ and X ₃ are –CH ₂ -. In a particular embodiment, X ₁ and X ₂ are independently selected from –NH- and -N(LZ)- groups, with notably X ₁ being -N(LZ)- and X ₂ being –NH-, and X ₃ is –CH ₂ - . In a particular embodiment, X ₁ , X ₂ and X ₃ are independently selected from –NH- and - N(LZ)- groups, with notably X ₁ being -N(LZ)- and X ₂ and X ₃ being –NH-, or X ₁ and X ₂ chosen from -N(LZ)- groups, and X ₃ being –NH-. In a particular embodiment, n1, n2 and n3 are independently chosen from 0, 1 and 2. In a particular embodiment, n1, n2 and n3 are such as n1 = n2 = n3. In a more particular embodiment, n1 = n2 = n3 = 0. In a more particular embodiment, n1 = n2 = n3 = 1. In a more particular embodiment, n1 = n2 = n3 = 2. In another particular embodiment, n1, n2 and n3 are not such as n1 = n2 = n3. In a more particular embodiment, n1 = n2 ≠ n3; or n1 ≠ n2 = n3; or n1 = n3 ≠ n2; or n1 ≠ n2 ≠ n3. In an even more particular embodiment, n1, n2 and n3 are such as: - n1 = n2 = 0 and n3= 1; or - n1=0 and n2 = n3 = 1; or - n1 = n2 = 1 and n3= 2. In a particular embodiment, X ₁ is –NR- with R being LZ, X ₂ and X ₃ being in particular –CH ₂ - or –NH-. In a particular embodiment, Y1 is O-L’Z’, Y ₂ and Y ₃ being in particular H. In a particular embodiment, Z ₁ is O-L’’Z’’, Z ₂ and Z ₃ being in particular a O-C ₁ -C ₆ alkyl, more particularly OMe. In a particular embodiment, X ₁ , X ₂ , X ₃ , Y1, Y ₂ , Y ₃ , Z ₁ , Z ₂ , Z ₃ , are such that the compound of formula (I) of the invention bears no LZ, L’Z’ or L’’Z’’ group. In a particular embodiment, X ₁ , X ₂ , X ₃ , Y ₁ , Y ₂ , Y ₃ , Z ₁ , Z ₂ , Z ₃ , are such that the compound of formula (I) of the invention bears one LZ, L’Z’ or L’’Z’’ group. In a particular embodiment, X ₁ , X ₂ , X ₃ , Y1, Y ₂ , Y ₃ , Z ₁ , Z ₂ , Z ₃ , are such that the compound of formula (I) of the invention bears two LZ groups; or one LZ group and one L’Z’ or L’’Z’’ group. In a particular embodiment, L is (W)i-L1-(F1-L2)j-(F2)k- and/or L’ is (W’)i-L’1-(F’1-L’2)j- (F’ ₂ ) _k -, wherein: - W and W’ are independently chosen from –C(=O)-, -C(=O)O-, -C(=O)NH-,-C(=S)-O- , notably –C(=O)- ; - L1, L2, L’1 and L’2 are independently is a C ₁ -C ₁₂ alkane diyl, a C ₂ -C ₁₂ alkene diyl or a C ₂ -C ₁₂ alkyne diyl chain optionally interrupted by one or more heteroatoms, notably selected from an oxygen atom, a sulphur atom or a nitrogen atom, said nitrogen and sulphur atoms being optionally oxidized, for example -(CH ₂ )mO-(CH ₂ -CH ₂ -O)m’-, wherein m and m’ are independently from 1 to 6; - F ₁ , F ₂ , F’ ₁ and F’ ₂ are independently chosen from –C(=O)-, -C(=O)-C(=O)-, -C(=O)- C(=O)-NH-, -NHC(=O)-C(=O)-, -NHC(=O)-C(=O)-NH-, -C(=O)-C(H)=N-NH-, -NH- C(=O)-C(H)=N-NH-, ester, amide, amine, -CH ₂ -, ether, thioether, imine, succinimide, thio-succinimide, oxime, hydrazone, hydrazonamide, -C(=O)CH ₂ -NH-, -NH-CH ₂ - C(=O)-, triazole functions or groups; - i, j, and k are independently 0 or 1. In a particular embodiment, k is 0, Z being in particular a terminal reactive function or group, in particular able to form a covalent bond with a peptide, a protein, a carrier, a solid support, a multivalent scaffold; an anchor; a dye or a fluorescent residue. In a particular embodiment, k is 1, Z being in particular a peptide, a protein, a carrier, a solid support, a multivalent scaffold; an anchor; a dye or a fluorescent residue. In a particular embodiment, Z is a terminal reactive function or group, more particularly a halogen, in particular Cl, Br, I, F, more particularly Br, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, azido, epoxide, C(=O)H, hemiacetal, C(=O)R _c , acetal, SR _a , NH ₂ or NHC(=O)CH ₂ Hal, wherein Hal is a halogen, in particular Cl, Br, I, F, more particularly Br, N-maleimide. In a particular embodiment, LZ and/or L’Z’ and/or L’’Z’’ is chosen from –CH ₂ -C≡CH, -(CH ₂ ) _k -triazole, wherein k is from 1 to 6, and –C(=O)–(CH ₂ ) _n -Hal, wherein n is from 1 to 6, in particular 3 and Hal is halogen, in particular Cl, Br, I, F, more particula rly Br. In a particular embodiment, the compound of the invention is chosen from the following formulae: ,

The present invention also relates to a process of preparation of a compound of formula (I) as defined above, wherein n1, n2 and n3 are independently chosen from 1, 2 and 3, in particular from 1 and 2, comprising a step (i) of contacting a compound of following formula (II) with formic acid, P ₂ O ₅ , HClO ₄ , or Sc(OTf) ₃ :

wherein : X ₁ , X ₂ and X ₃ are independently selected from –CH ₂ - and –NR-, at least one of X ₁ , X ₂ and X ₃ representing –NR-; R is chosen from: - H; - a C ₁ -C ₆ alkyl; - a C ₂ -C ₆ alkenyl; - a C ₂ -C ₆ alkynyl; - an ad hoc protecting group, in particular a benzyl, p-methoxybenzyl, 2- (trimethylsilyl)ethoxymethyl (SEM), 2-tetrahydropyranyl (THP), a carbamate, for example Boc, Fmoc, Troc, Cbz, a silyl, in particular tert-butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert- butyldiphenylsilyl (TBDPS), or 2-(trimethylsilyl)ethoxymethyl (SEM), or an acyl group, in particular an acetyl; Y1, Y ₂ and Y ₃ are independently selected from: - H, - a C ₁ -C ₆ alkyl; - a C ₂ -C ₆ alkenyl; - a C ₂ -C ₆ alkynyl; - a O-C ₁ -C ₆ alkyl; - a O-C ₂ -C ₆ alkenyl; - optionally a O-L’Z’ group; - OH or a O-P’’ group, wherein P’’ is an ad hoc protecting group, in particular a benzyl; p-methoxybenzyl; silyl, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert- butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS); 2-(trimethylsilyl)ethoxymethyl (SEM); allyl ; methoxymethyl (MOM) ; tetrahydropyran (THP) or an acyl group, in particular an acetyl; Z ₁ , Z ₂ and Z ₃ are independently selected from: - H, - a C ₁ -C ₆ alkyl; - a C ₂ -C ₆ alkenyl; - a C ₂ -C ₆ alkynyl; - a O-C ₁ -C ₆ alkyl; - a O-C ₂ -C ₆ alkenyl; - optionally a O-L’’Z’’ group; - OH or a O-P’’ group, wherein P’’ is an ad hoc protecting group, in particular a benzyl; p-methoxybenzyl; silyl, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert- butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS); 2-(trimethylsilyl)ethoxymethyl (SEM); allyl ; methoxymethyl (MOM) ; tetrahydropyran (THP) or an acyl group, in particular an acetyl; L’ and L’’ are independently from each other a linker; Z’ and Z’’ are independently from each other: - a terminal reactive function or group, in particular able to form a covalent bond with a peptide, a protein, an antibody or a fragment thereof, a carrier, a solid support, a multivalent scaffold; an anchor; a dye or a fluorescent residue; or - a peptide, a protein, an antibody or a fragment thereof, a carrier, a solid support, a multivalent scaffold; an anchor; a dye or a fluorescent residue; n1, n2 and n3 are independently chosen from 0, 1, 2 and 3. The –OH groups of compound of formula (II) can be protected by an ad hoc protecting group known from the skilled in the art, for example a THP group, and deprotected prior to the reaction yielding compound (II) as defined above. In a particular embodiment, the compound of formula (II) is obtained by contacting a compound of following formula (III) with a compound of following formula (IV), in presence of a base, in particular a carbonate, more particularly Cs ₂ CO ₃ : wherein LG is a leaving group, in particular chosen from halogens, more particularly I, Br, Cl, notably I, and O-tosyl. The –OH group of compound (IV) can be protected by an ad hoc protecting group known from the skilled in the art, for example a THP group. When Z ₁ =Z ₂ =Z ₃ and n1=n2=n3, the compound of formula (IV) corresponds to a single compound of following formula: . When this is not the case, the compound of formula (IV) is a mixture of compounds as follows: , said compounds being in particular introduced as a mixture or sequentially for the reaction with the compound of formula (III). In a particular embodiment, the compound of formula (III) is obtained by contacting a compound of following formula (V) with BBr ₃ : In a particular embodiment, the compound of formula (V), when X ₁ is –CH ₂ - or –NR-, X ₂ is –NR- and X ₃ is –CH ₂ -, is obtained by contacting a compound of following formula (VI) with HClO ₄ : wherein X ₁ is –CH ₂ - or –NR-, and X ₂ is –NR-. In a particular embodiment, the compound of formula (V), when X ₁ and X ₂ are independently chosen from –NR- groups, R being not H, is obtained by from a compound of following formula (VII), in particular by Buchwald amination: wherein X ₁ and X ₂ are independently chosen from –NR- groups, R being not H, in particular – NP- groups, with P as defined above, and X is an halogen atom, in particular Br, I or Cl. In a particular embodiment, the invention concerns a process of the invention of preparation of a compound of formula (I) wherein X ₁ , X ₂ and/or X ₃ , in particular X ₁ and/or X ₂ , is –N(-LZ)-, said process comprising after step (i) a step of contacting a compound of formula (I) wherein X ₁ , X ₂ and/or X ₃ , in particular X ₁ and/or X ₂ , is –NH- with a compound of formula LG-LZ, wherein LG is a leaving group, LG being in particular a halogen, more particularly Cl, Br, I, F, preferably Br, or a O-tosyl group. In a particular embodiment, the invention concerns a process of the invention of preparation of a compound of formula (I) wherein Y ₁ , Y ₂ and/or Y ₃ , in particular Y ₁ and/or Y ₂ , is –O-L’Z’, said process comprising after step (i) a deprotection step of a compound of formula (I) with Y1, Y ₂ and/or Y ₃ , in particular Y1 and/or Y ₂ , being –OP’, followed by the contacting the obtained compound of formula (I) wherein Y1, Y ₂ and/or Y ₃ , in particular Y1 and/or Y ₂ , is –OH with a compound of formula LG’-L’Z’, wherein LG’ is a leaving group, LG’ being in particular a halogen, more particularly Cl, Br, I, F, preferably Br, or a O-tosyl group. In a particular embodiment, the invention concerns a process of the invention of preparation of a compound of formula (I) wherein Z ₁ , Z ₂ and/or Z ₃ , in particular Z ₁ and/or Z ₂ , is –O-L’’Z’’, said process comprising after step (i): - a deprotection step of a compound of formula (I) with Z ₁ , Z ₂ and/or Z ₃ , in particular Z ₁ and/or Z ₂ , being –OP’’, or - a dealkylation step of a compound of formula (I) with Z ₁ , Z ₂ and/or Z ₃ , in particular Z ₁ and/or Z ₂ , being –O-C ₁ -C ₆ alkyl, notably OMe, in particular in presence of iodotrimethylsilane, hydrobromic acid, boron tribromide or aluminium chloride. followed by the contacting the obtained compound of formula (I) wherein Z ₁ , Z ₂ and/or Z ₃ , in particular Z ₁ and/or Z ₂ , is –OH with a compound of formula LG’’-L’’Z’’, wherein LG’’ is a leaving group, LG’’ being in particular a halogen, more particularly Cl, Br, I, F, preferably Br, or a O-tosyl group. In a particular embodiment, the invention concerns a process of the invention of preparation of a compound of formula (I) as defined above, wherein n1, n2 and n3 are 0, comprising a step (i’) of contacting a compound of formula (III) with a compound of following formula (VII), in presence of BrCH ₂ Cl and a base, in particular Cs ₂ CO ₃ : wherein Z ₁ , Z ₂ and Z ₃ are independently selected from: - H, - a C ₁ -C ₆ alkyl; - a C ₂ -C ₆ alkenyl; - a C ₂ -C ₆ alkynyl; - a O-C ₁ -C ₆ alkyl; - a O-C ₂ -C ₆ alkenyl; - optionally a O-L’Z’ group; - a O-P’’ group, wherein P’’ is an ad hoc protecting group, in particular a benzyl; p- methoxybenzyl; silyl, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert- butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS); 2-(trimethylsilyl)ethoxymethyl (SEM); allyl ; methoxymethyl (MOM) ; tetrahydropyran (THP) or an acyl group, in particular an acetyl. In a particular embodiment, the invention concerns a process of the invention of 30 preparation of a compound of formula (I) as defined above, wherein n1, n2 and n3 are 0, comprising a step (i’) of contacting a compound of formula (III), with ClCH ₂ SMe, then SOCl ₂ , and then with a compound of following formula (VII), in presence of a base, in particular Cs ₂ CO ₃ . In another aspect, the invention concerns a compound of one of the following formulae: In another aspect, the invention concerns a complex constituted of or comprising a compound of formula (I) complexed with a hyperpolarized noble element, in particular Xe, more particularly ¹²⁹ Xe. By “hyperpolarized” is in particular meant a certain noble gas, the polarization of the nuclei of which being artificially enhanced over the natural levels at thermal equilibrium. Techniques that enable this hyperpolarization are well known for the skilled in the art. In a particular embodiment, the invention concerns a compound of formula (I) as defined above or a complex as defined above for its use as an in vivo diagnostic tool, in particular in NMR spectroscopy or in MRI applications. In another aspect, the invention concerns an oil-in-water nanoemulsion comprising: - An oil; - An aqueous phase; - Optionally, a surfactant; - Optionally, a co-surfactant and/or a solubilizier; and - A compound of following formula (Ia): Wherein: X1, X2 and X3 are independently selected from –CH2- and –NR-, at least one of X1, X2 and X3 representing –NR-; R is chosen from: - H; - a C ₁ -C ₆ alkyl; - a C ₂ -C ₆ alkenyl; - a C ₂ -C ₆ alkynyl; - an ad hoc protecting group, in particular a benzyl, p-methoxybenzyl, 2- tetrahydropyranyl (THP), a carbamate, for example Boc, Fmoc, Troc, Cbz, a silyl, in particular tert-butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), or 2- (trimethylsilyl)ethoxymethyl (SEM), or an acyl group, in particular an acetyl; Y ₁ , Y ₂ and Y ₃ are independently selected from: - H, - a C ₁ -C ₆ alkyl; - a C ₂ -C ₆ alkenyl; - a C ₂ -C ₆ alkynyl; - a O-C ₁ -C ₆ alkyl; - a O-C ₂ -C ₆ alkenyl; - OH; Z ₁ , Z ₂ and Z ₃ are independently selected from: - H, - a C ₁ -C ₆ alkyl; - a C ₂ -C ₆ alkenyl; - a C ₂ -C ₆ alkynyl; - a O-C ₁ -C ₆ alkyl; a O-C ₂ -C ₆ alkenyl;

OH; nl, n2 and n3 are independently chosen from 0, 1, 2 and 3, said compound of formula (I) being optionally complexed with a hyperpolarized noble element, in particular Xe, more particularly ¹²⁹ Xe.

The oil-in-water nanoemulsions of the invention may in particular be formed by spontaneous emulsification.

The term "nanoemulsion" refers in particular to oil-in-water dispersions comprising nanometer scale oil phase droplets in an aqueous phase.

The diameter of the nanoemulsion oil phase droplets is in particular from 20 to 300 nm, preferably from 25 to 100 nm.

Size distributions can be determined for example by the method of dynamic light scattering.

The oil-in-water nanoemulsions of the invention are preferably made from excipients, which are Generally Recognized As Safe (GRAS) ingredients.

In a particular embodiment, the oil is a natural oil, more particularly a vegetable oil or an animal oil; a synthetic oil; or a mixture thereof. For example, the synthetic oil can be medium chain triglycerides such as Labrafac® WL1349.

In a particular embodiment, the aqueous phase consists of or comprises water or an aqueous buffer. Such an aqueous buffer may be useful to control the pH and the osmolarity of the preparation.

The surfactant may be selected as well known by the skilled in the art. In a particular embodiment, the surfactant is chosen from anionic surfactants, nonionic surfactants, cationic surfactants, amphoteric surfactants and (extended) surfactants containing a non-ionic spacerarm central extension and an ionic or nonionic polar group. For example, the surfactant can be a PEGlygated non-ionic surfactant such as hydroxystearate PEG 15 (Kolliphor® HS15).

The co- surfactant may be selected as defined above for the surfactant.

The solubilizier may be selected as well known by the skilled in the art. It can be selected from glycerides, or can be a solvent such as dimethyl sulfoxide, ethanol or a derivate, and in particular diethylene glycol monoethyl ether (Transcutol® HP).

The co-surfactant and/or a solubilizier may be useful to optimize if needed the stability and the drug-loading of the nanodroplets.

Definitions

The following terms and expressions contained herein are defined as follows: As used herein, a range of values in the form “x-y” or “x to y”, or “x through y”, include integers x, y, and the integers therebetween. For example, the phrases “1-6”, or “1 to 6” or “1 through 6” are intended to include the integers 1, 2, 3, 4, 5, and 6. Preferred embodiments include each individual integer in the range, as well as any subcombination of integers. For example, preferred integers for “1-6” can include 1, 2, 3, 4, 5, 6, 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2- 5, 2-6, etc.

As used herein, the term “alkyl” refers to a straight-chain, or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, neopentyl, 1 -ethylpropyl, 3 -methylpentyl, 2,2-dimethylbutyl, 2,3- dimethylbutyl, hexyl, etc. The alkyl moiety of alkyl-containing groups, such as aralkyl or O- alkyl groups, has the same meaning as alkyl defined above. Lower alkyl groups, which are preferred, are alkyl groups as defined above which contain 1 to 4 carbons. A designation such as “C1-C4 alkyl” refers to an alkyl radical containing from 1 to 4 carbon atoms.

As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.

All other terms used in the description of the present invention have their meanings as is well known in the art.

In another aspect, the present invention is directed to pharmaceutically acceptable salts of the compounds described above. As used herein, “pharmaceutically acceptable salts” includes salts of compounds of the present invention derived from the combination of such compounds with non-toxic acid.

Acid addition salts include inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, nitric and phosphoric acid, as well as organic acids such as acetic, citric, propionic, tartaric, glutamic, salicylic, oxalic, methanesulfonic, para-toluenesulfonic, succinic, and benzoic acid, and related inorganic and organic acids.

In addition to pharmaceutically-acceptable salts, other salts are included in the invention. They may serve as intermediates in the purification of the compounds, in the preparation of other salts, or in the identification and characterization of the compounds or intermediates.

The pharmaceutically acceptable salts of compounds of the present invention can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, ethyl acetate and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent. Such solvates are within the scope of the present invention.

It is recognized that compounds of the present invention may exist in various stereoisomeric forms. As such, the compounds of the present invention include both diastereomers and enantiomers. The compounds are normally prepared as racemates and can conveniently be used as such, but individual enantiomers can be isolated or synthesized by conventional techniques if so desired. Such racemates and individual enantiomers and mixtures thereof form part of the present invention.

It is well known in the art how to prepare and isolate such optically active forms. Specific stereoisomers can be prepared by stereospecific synthesis using enantiomerically pure or enantiomerically enriched starting materials. The specific stereoisomers of either starting materials or products can be resolved and recovered by techniques known in the art, such as resolution of racemic forms, normal, reverse-phase, and chiral chromatography, recrystallization, enzymatic resolution, or fractional recrystallization of addition salts formed by reagents used for that purpose. Useful methods of resolving and recovering specific stereoisomers described in Eliel, E. L.; Wilen, S.H. Stereochemistry of Organic Compounds', Wiley: New York, 1994, and Jacques, J, et al. Enantiomers, Racemates, and Resolutions', Wiley: New York, 1981, each incorporated by reference herein in their entireties.

In particular, compounds of formula (II), (III) and (V) of the present invention may be obtained as mixtures of enantiomers, and the corresponding individual enantiomers can for example be isolated by chiral chromatography.

Synthesis

The compounds of the present invention may be prepared in a number of methods well known to those skilled in the art, including, but not limited to those described below, or through modifications of these methods by applying standard techniques known to those skilled in the art of organic synthesis. The appropriate modifications and substitutions will be readily apparent and well known or readily obtainable from the scientific literature to those skilled in the art. In particular, such methods can be found in R.C. Larock, Comprehensive Organic Transformations, Wiley-VCH Publishers, 1999.

All processes disclosed in association with the present invention are contemplated to be practiced on any scale, including milligram, gram, multigram, kilogram, multikilogram or commercial industrial scale. It will be appreciated that the compounds of the present invention may contain one or more asymmetrically substituted carbon atoms, and may be isolated in optically active or racemic forms. Thus, all chiral, diastereomeric, racemic forms, isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. It is well-known in the art how to prepare and isolate such optically active forms. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic forms, normal, reverse-phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from chiral starting materials or by deliberate synthesis of target chiral centers.

Compounds of the present invention may be prepared by a variety of synthetic routes. The reagents and starting materials are commercially available, or readily synthesized by well- known techniques by one of ordinary skill in the arts. All substituents, unless otherwise indicated, are as previously defined.

In the reactions described in the present specification, it may be necessary to protect reactive functional groups, for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Conventional protecting groups may be used in accordance with standard practice, for examples see T.W. Greene and P. G. M. Wuts in Protective Groups in Organic Chemistry, 3 ^rd ed., John Wiley and Sons, 1999; J. F. W. McOmie in Protective Groups in Organic Chemistry, Plenum Press, 1973.

DRAWINGS

[Fig. 1] illustrates the ¹²⁹ Xe NMR spectrum (5 bars, CDCI ₃ ) of cryptophane-lN 12.

[Fig. 2] illustrates the 3D structure of cryptophane-lN 12 determined by RX crystallography. [Fig. 3] shows two views of the structure of N-CTV compound 7 obtained by X-ray diffraction. [Fig. 4] presents the UV absorbance spectra of NE loaded with IN Cr over time at 4°C (A) and at RT (B).

[Fig. 5] is related to haemolysis tests involving nanoemulsions of the invention according to example 9.

[Fig. 6] presents the UV absorbance spectra of Cr A loaded NE (not part of the invention) over time at 4°C, according to example 9, last paragraph.

[Fig. 7] illustrates the stability of nanoemulsions of the invention according to example 9 in biomimetic medium at 37°C by UV spectrophotometry every hour during 3h. EXAMPLES

General experimental procedure

All reagents and solvents were obtained from commercial suppliers and were used without further purification. Purification was carried out according to standard laboratory methods. Solvents for purification purposes were used as obtained from suppliers without further purification. NMR spectra were recorded at 400 MHz (Bruker Advance III 400 MHz) for 1 H NMR and at 100 or 125MHz for 13C NMR in chloroform-d with chemical shift (6) given in parts per million (ppm) relative to TMS as internal standard and recorded at 295K. The following abbreviations are used to describe peak splitting patterns when appropriate: br=broad, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, dd=doublet of doublet, dt=doublet of triplet. Coupling constants J are reported in hertz units (Hz). Chromatography was carried out on a column using silica gel 60 Merck (0.063-0.200 mm) as the stationary phase. Melting points were measured on a Stuart Automatic Melting Point SMP-50 apparatus. High resolution mass spectra (HRMS) were obtained by electronic impact (HRMS/EI), or by electrospray (HRMS/ ESI) on a Bruker maXis mass spectrometer.

General procedures

Synthesis of tosylhydrazones - General procedure A

In a round bottom flask, para-toluene sulfonylhydrazide (1.05 equiv.) was solubilized in methanol (4 mL/g). Substituted benzaldehyde (1.00 equiv.) was added to the solution, the resulting mixture was stirred vigorously for 2h30. The resulting mixture was evaporated to dryness, dissolved in a small portion of diethyl ether. The solid was filtered, and the expected product was obtained as a white powder.

Synthesis for the Barluenga Boronic Coupling - General procedure B

In a round bottom flask, substituted para-toluene sulfonylhydrazone (1.00 equiv.) was solubilized in dioxane (15 mL/mmol). Potassium carbonate (4 equiv.) and boronic acid (1.5 equiv.) were added to the solution, and the reaction mixture was heated at reflux for 2h30. The resulting mixture was allowed to reach room temperature, an aqueous saturated solution of sodium bicarbonate was added (15 mL/ mmol), and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over MgSO4, filtered and evaporated under vacuum. The crude mixture was purified using silica chromatography and a mixture of cyclohexane and ethyl acetate as eluent (95/5), affording the expected product.

Example 1: Synthesis of IN-cryptophane of the invention N'-[(1E)-(2-bromo-4-methoxyphenyl)methylidene]-4-methylbenze ne-1-sulfonohydrazide 1 Prepared from 2-bromo-4-methoxybenzaldehyde (10.00 g, 46.53 mmol), following general procedure A, the expected compound was obtained as a white powder (17.39 g, 97%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 8.06 (s, 1H), 7.90 – 7.79 (m, 4H), 7.32 (d, J = 8.0 Hz, 2H), 7.03 (d, J = 2.5 Hz, 1H), 6.85 (m, 1H), 3.81 (s, 3H), 2.41 (s, 3H). ¹³ C NMR (100 MHz, CDCl3): δ = 161.7, 146.4, 144.5, 135.4, 129.9, 128.8, 128.1, 125.0, 124.8, 117.7, 114.6, 55.8, 21.8. Melting point: 149 °C 1-Bromo-3-methoxy-6-(3-methoxybenzyl)-benzene 2 Prepared from tosylhydrazone 1 (17.4 g, 45.4 mmol) and 3-methoxyphenylboronic acid (8.97 g, 59 mmol), following general procedure B, the expected compound was obtained as a light yellow oil (11.7 g, 84 %). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.21 (t, J = 7.8 Hz, 1H), 7.13 (d, J = 2.6 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H), 6.81-6.72 (m, 4H), 4.03 (s, 2H), 3.78 (s, 3H), 3.77 (s, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 159.8, 158.7, 141.8, 132.3, 131.5, 129.5, 125.0, 121.4, 118.0, 114.8, 113.8, 111.5, 55.7, 55.3, 41.0. HRMS-ESI calculated for C15H15BrO4 [M ^+. ], 306.0255 found 306.0253 (2-Amino-5-methoxyphenyl)methanol 3 Obtained according to the procedure described by Wales et al. (Org. Lett.2019, 21 (12), 4703– 4708). 2-[(Tert‐butyldimethylsilyl)oxymethyl]-4-methoxyaniline 4 3 (1.3 g, 8.55 mmol), TBDMSCl (1.6 g, 12.8 mmol) and imidazole (872 mg, 12.8 mmol) were dissolved in dichloromethane (20 mL) and the solution was stirred at room temperature. After completion, the reaction was quenched water, extracted with ethyl acetate, washed with brine, dried over MgSO4 and concentrated under vacuum. Purification over silica gel (cyclohexane/Et ₂ O 9/1 to 8/2) afforded the product as a red oil (1.37 g, 60%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 6.70 – 6.66 (m, 2H), 6.65 – 6.59 (m, 1H), 4.65 (s, 2H), 3.86 (s, 2H), 3.74 (s, 3H), 0.91 (s, 9H), 0.08 (s, 6H). ¹³ C NMR (100 MHz, CDCl3): δ = 152.4, 139.5, 126.9, 116.9, 114.5, 113.8, 64.7, 55.9, 26.0 (3C),18.4, -5.1 (2C). HRMS-ESI calculated for C ₁₄ H ₂₅ NO ₂ Si [M ^+. ] 267.1655, found 267.1657 2-(((Tert-butyldimethylsilyl)oxy)methyl)-4-methoxy-N-(5-meth oxy-2-(3- methoxybenzyl)phenyl)aniline 5 2 (1 g, 3.26 mmol), 4 (872 mg, 3.26 mmol), Pd(OAc)2 (74 mg, 0.326 mmol), Binap (406 mg, 0.652 mmol), cesium carbonate (3.7 g, 11.4 mmol) were added in toluene (30 mL) under nitrogen atmosphere and heated to reflux for 3h. The mixture was filtrated through a pad of celite using ethyl acetate as the eluent. The solvent was removed under vacuum and purification over silica gel (Cyclohexane/Et ₂ O 95/5) afforded the product as a yellow oil. (1.4 g, 87%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.21 (t, J = 8.0 Hz, 1H), 7.05 (d, J = 8.7 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 3.0 Hz, 1H), 6.81 – 6.73 (m, 4H), 6.38 (dt, J = 8.2, 2.6 Hz, 2H), 5.67 (s, 1H), 4.44 (s, 2H), 3.89 (s, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 3.69 (s, 3H), 0.89 – 0.86 (m, 9H), 0.03 – -0.01 (m, 6H). ¹³ C NMR (100 MHz, CDCl3): δ = 159.9, 159.5, 155.6, 144.8, 141.7, 134.9, 133.7, 131.5, 129.7, 123.4, 121.1, 120.0, 114.5, 113.6, 113.3, 111.8, 104.4, 101.5, 62.9, 55.7, 55.3, 55.2, 37.4, 26.0 (3C), 18.4, -5.1 (2C). HRMS-ESI calculated for C ₂₉ H ₃₉ NO ₄ [M ^+. ] 493.2648, found 493.2647 (5-methoxy-2-((5-methoxy-2-(3-methoxybenzyl)phenyl)amino)phe nyl)methanol 6 TBAF (1M in THF, 5.7 mL, 5.7 mmol) was added at 0°C to a solution of 5 (1.4 g, 2.84 mmol) in THF (25 mL) and the mixture was stirred at room temperature until completion of the reaction. The solution was then quenched with saturated NaHCO ₃ , extracted with ethyl acetate, washed with brine, dried over MgSO4 and concentrated under vacuum. Purification over silica gel (Cyclohexane/Et ₂ O 7/3 to 4/6) afforded the product as a light yellow oil. (1.034 g, 96%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.25 – 7.19 (m, 1H), 7.12 (d, J = 8.6 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.79 (ddd, J = 8.6, 7.6, 2.6 Hz, 5H), 6.41 (dt, J = 8.1, 2.5 Hz, 2H), 5.82 (s, 1H), 4.32 (s, 2H), 3.94 (s, 2H), 3.77 (s, 3H), 3.75 (s, 3H), 3.71 (s, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 159.9, 159.5, 155.6, 144.9, 141.8, 134.3, 134.1, 131.9, 129.6, 124.0, 120.9, 119.4, 114.8, 114.4, 114.0, 111.7, 104.0, 101.2, 63.4, 55.6, 55.3, 55.2, 37.7. HRMS-ESI calculated for C23H26NO4 [M+H ⁺ ] 380.1862, found 380.1843 2,7,12-trimethoxy-10,15-dihydro-5H-tribenzo[b,e,h]azonine 7 Perchloric acid (60%, 0.160 mL) was added dropwise to a solution of 6 (100 mg, 0.264 mmol) in acetonitrile (53mL) at 0°C and then the solution was heated to 60°C for 1h. The reaction was quenched with water, extracted with ethyl acetate, washed with brine, dried over MgSO4 and concentrated under vacuum. Purification over silica gel (cyclohexane/Et ₂ O 8/2 to 7/3) afforded the product as a white powder (85 mg, 89%). ¹ H NMR (400 MHz, CDCl3): δ = 7.06 (d, J = 8.1 Hz, 1H), 6.87 – 6.81 (m, 2H), 6.72 – 6.64 (m, 3H), 6.50 (s, 1H), 6.34 (d, J = 8.4 Hz, 1H), 6.30 (s, 1H), 5.71 (s, 1H), 3.83 (s, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 3.69 (s, 5H). ¹³ C NMR (100 MHz, CDCl3): δ = 159.0, 158.4 (2C), 155.4, 144.9, 141.4, 136.2, 134.0, 131.8, 130.9 (2C), 122.1, 116.9, 116.6, 113.1, 110.62, 106.29, 103.29, 55.51, 55.32, 55.30, 35.61, 35.47 HRMS-ESI calculated for C ₂₃ H ₂₄ NO ₃ [M+H ⁺ ] 362.1756, found 362.1755 Melting point: 166-168 °C The conformation of compound 7 has been determined by X-ray diffraction. Compound 7 is of saddle conformation (figure 3). 10,15-dihydro-5H-tribenzo[b,e,h]azonine-2,7,12-triol 8 BBr ₃ (1M in DCM, 12.5 mL, 12.5 mmol) was added to a solution of 7 (450 mg, 1.25 mmol) in dichloromethane at 0°C and the mixture was stirred at room temperature until completion of reaction. The reaction was quenched at 0°C with water, extracted with ethyl acetate, washed with brine, dried over MgSO ₄ and concentrated under vacuum. Purification over silica gel (Et ₂ O/cyclohexane 7/3) afforded the product as a light pink powder (383 mg, 96%). ¹ H NMR (400 MHz, Acetone D-6): δ = 8.08 (s, 1H), 8.00 (s, 1H), 7.80 (s, 1H), 7.00 (d, J = 8.2 Hz, 2H), 6.83 (d, J = 8.5 Hz, 1H), 6.71 (d, J = 8.0 Hz, 2H), 6.62 (dd, J = 8.4, 2.7 Hz, 2H), 6.47 – 6.38 (m, 2H), 6.27 – 6.20 (m, 1H), 3.81 (s, 2H), 3.64 (s, 2H). ¹³ C NMR (100 MHz, CDCl3): δ = 155.9, 155.4, 152.0, 145.5, 141.3, 135.4, 133.2, 131.4, 130.5, 130.3, 121.9, 117.4, 116.8, 115.7, 113.8, 111.9, 106.6, 103.7, 34.5, 34.5. Melting point: 210-212 °C [4-(2-Bromoethoxy)-3-methoxyphenyl] methanol 9 Obtained according to the procedure described by Spence et al. (J. Am. Chem. Soc. 2004, 126 (46), 15287–15294). [4-(2-Iodoethoxy)-3-methoxyphenyl]methanol 10 Obtained according to the procedure described by Spence et al. (J. Am. Chem. Soc. 2004, 126 (46), 15287–15294). (((((10,15-Dihydro-5H-tribenzo[b,e,h]azonine-2,7,12-triyl)tr is(oxy))tris(ethane-2,1- diyl))tris(oxy))tris(3-methoxybenzene-4,1-diyl))trimethanol 11 10 (866 mg, 2.82 mmol) and cesium carbonate (916 mg, 2.82 mmol) was added to a solution of 8 (150 mg, 0.47 mmol) in dry DMF (10 mL) placed under nitrogen atmosphere. The mixture was stirred overnight at 80°C, and allowed to reach room temperature. The reaction was quenched with water and extracted with ethyl acetate. The organic layers were combined, washed with brine, dried over MgSO ₄ and concentrated under vacuum. Purification over silica gel (AcOEt/MeOH, 100/0 to 95/5) afforded the product as a white solid (275 mg, 68%). ¹ H NMR (400 MHz, CDCl3): δ = 6.98 (dd, J = 16.8, 6.2 Hz, 1H), 6.93 – 6.75 (m, 11H), 6.73 (d, J = 2.5 Hz, 1H), 6.70 – 6.65 (m, 2H), 6.51 (s, 1H), 6.34 (s, 1H), 6.32 (s, 1H), 4.63 – 4.55 (m, 6H), 4.37 – 4.28 (m, 6H), 4.25 (t, J = 5.1 Hz, 4H), 4.18 (t, J = 4.8 Hz, 2H), 3.85 – 3.81 (m, 6H), 3.80 (s, 3H),3.78 (s, 2H), 3.64 (s, J = 12.0 Hz, 2H). ¹³ C NMR (100 MHz, CDCl3): δ = 157.97, 157.42, 154.26, 149.85, 149.68, 147.64, 147.59, 147.52, 144.84, 141.38, 136.50, 134.83, 134.7, 134.68, 133.90, 132.15, 131.47, 130.90, 121.97, 119.65, 119.46, 119.41, 118.27, 117.88, 117.30, 114.29, 114.12, 114.09, 113.80, 111.52, 111.15, 111.13, 111.05, 110.83, 106.90, 104.32, 71.47, 67.92, 66.66, 66.46, 66.42, 65.23, 65.21, 65.18, 61.26, 55.99, 55.93, 55.83, 35.52, 35.41. HRMS-ESI calculated for C ₅₀ H ₅₃ NO ₁₂ Na [M+Na ⁺ ] 882.3465, found 882.3474 Melting point: 140-142 °C Cryptophane-1N 12 Formic acid (300 mL) was added to a solution of 11 (270 mg, 0.313 mmol) in chloroform (15 mL) and the reaction mixture was heated for 3h at 55°C. The solvent was removed under vacuum and purification over silica gel (DCM/Et2O 95/5 to 80/20) to afford the product as a light pink powder (182 mg, 72%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.13 (d, J = 8.6 Hz, 1H), 7.05 (dd, J = 8.4, 6.1 Hz, 2H), 6.80 (dd, J = 4.6, 2.7 Hz, 2H), 6.72 (d, J = 2.6 Hz, 1H), 6.69 (s, 1H), 6.67 – 6.63 (m, 4H), 6.59 (s, 1H), 6.38 (dd, J = 8.7, 2.9 Hz, 1H), 6.33 (dt, J = 8.4, 2.4 Hz, 2H), 4.64 – 4.56 (m, 5H), 4.42 – 4.30 (m, 3H), 4.29 – 4.13 (m, 6H), 4.11 – 3.93 (m, 3H), 3.84 (s, 3H), 3.80 (s, 3H), 3.76 (s, 3H), 3.47 – 3.35 (m, 5H). ¹³ C NMR (100 MHz, CDCl3): δ = 157.8, 156.3, 155.0, 148.7, 148.7, 148.5, 147.4, 147.3, 147.3, 147.2, 140.9, 139.3, 139.0, 133.0, 132.9, 132.7, 132.4, 131.8, 131.8, 131.4, 130.8, 130.5, 129.4, 126.8, 120.0, 119.7, 117.3, 116.8, 116.2, 114.9, 114.9, 114.5, 114.4, 112.3, 111.4, 110.4, 67.3, 67.0, 66.5, 65.9, 65.6, 65.2, 56.7, 56.4, 56.3, 36.4, 36.3, 35.1, 35.0, 31.1. ¹²⁹ Xe NMR (5 bars, CDCl ₃ ): illustrated in figure 1. In fact, two signals were observed at at 278 K, corresponding to the xenon signals in the solvent and in the cryptophane respectively. It can be concluded that the 1N cryptophanes of the invention successfully encapsulate xenon. RX: illustrated in figure 2. HRMS-ESI calculated for C ₅₀ H ₄₇ NO ₉ Na [M+Na ⁺ ] 828.3149, found 828.3162. Melting point: 257-259 °C. In addition, the procedure described above has been scaled up. This made it possible to isolate, in addition to the anti cryptophane described above, the following syn compound: Cryptophane-1N Syn Formic acid (470 mL) was added to a solution of 11 (452 mg, 0.494 mmol) in chloroform (18 mL), in four different batches and the reaction were heated for 3 h at 55 °C. The mixture was evaporated to dryness and purification of the crude residue over silica gel (DCM/Et2O 95/5 to 80/20) afforded the product (first spot) as a withe powder (365 mg, 23%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.14 (d, J = 9.5 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H), 6.88 (s, 1H), 6.80 (d, J = 2.8 Hz, 2H), 6.71 (m, 7H), 6.62 (d, J = 2.6 Hz, 1H), 6.54 (dd, J = 8.4, 2.5 Hz, 1H), 5.24 (s, 1H), 4.61 (m, 5H), 4.37 – 4.20 (m, 8H), 3.99 – 3.86 (m, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 3.77 (s, 3H), 3.70 – 3.60 (m, 2H), 3.49 – 3.39 (m, 5H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 158.5, 157.0, 155.2, 150.3, 149.9, 149.8, 147.0, 146.9, 146.8, 146.7, 140.6, 139.3, 138.9, 134.9, 134.6, 134.4, 132.1, 131.8, 131.7, 131.4, 131.3, 130.3, 129.8, 126.6, 122.5, 120.5, 120.3, 119.5, 119.0, 114.7, 114.1, 114.0, 113.9, 113.7, 113.3, 111.8, 72.0, 71.3, 71.0, 66.4, 66.3, 66.0, 56.3, 56.1, 56.1, 53.5, 36.4, 36.3, 35.2, 35.1. Melting point 205-207 °C HRMS-ESI calculated for C50H47NO9Na [M+Na ⁺ ] 828.3149, found 828.3162. Crystallographic data of both anti and syn cryptophanes 1N were obtained by slow evaporation in dichloromethane at 4°C. The anti compound has a cavity size of 137.0 Å ³ and the syn compound has a cavity size of 121.4 Å ³ , both larger than the size of the prior art cryptophane C (114.5 Å ³ ). Anti cryptophanes 1N crystals were also obtained by slow evaporation in anisole, which is too large to enter the cage. This enabled to visualise the conformation of the cryptophane 1N and the size of its cavity without caged compound. The size of the empty cavity is between 85.4 and 86.8 Å ³ . Example 2: Alkylation of the nitrogen of 1N-cryptophane Cryptophane 13 NaH (7.5 mg, 0.186 mmol) was added to a solution of 12 (100 mg, 0.124 mmol) in dry DMF (3 mL) at 0°C and the mixture was stirred for 30 min under nitrogen atmosphere. Propargyl bromide (18 μL, 0.186 mmol) was added and the solution was stirred at 80°C for 3h. The reaction was quenched with a saturated solution of NH ₄ Cl, extracted with ethyl acetate, washed with brine, dried over MgSO4, filtered and concentrated under vacuum. Purification over silica gel (DCM/Et ₂ O 10/0 to 80/20) afforded the product as a light yellow powder (32 mg, 31%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.38 (d, J = 8.8 Hz, 1H), 7.14 (d, J = 8.5 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 2.5 Hz, 1H), 6.81 (d, J = 2.9 Hz, 1H), 6.75 (d, J = 2.6 Hz, 1H), 6.67 (s, 1H), 6.66 (d, J = 1.9 Hz, 2H), 6.63 (s, 1H), 6.61 (d, J = 3.7 Hz, 2H), 6.45 – 6.39 (m, 2H), 6.34 (dd, J = 8.4, 2.6 Hz, 1H), 5.09 (dd, J = 12.2, 10.7 Hz, 2H), 4.60 (dd, J = 13.7, 3.7 Hz, 3H), 4.40 – 4.30 (m, 3H), 4.31 – 4.15 (m, 6H), 4.11 (dd, J = 5.5, 2.4 Hz, 2H), 4.03 (m, 3H), 3.81 (s, 3H), 3.79 (s, 3H), 3.79 (s, 3H), 3.38 (d, J = 13.8 Hz, 3H), 3.30 (d, J = 13.5 Hz, 2H), 2.13 (t, J = 2.4 Hz, 1H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 157.1, 156.2, 156.1, 150.0, 148.7, 148.6, 148.5, 147.5, 147.4, 147.3, 143.81, 141.84, 141.6, 134.5, 132.87, 132.86, 132.83, 132.4, 132.1, 132.09, 132.06, 130.8, 130.6, 128.0, 119.5, 119.3, 118.1, 117.0, 116.6, 116.4, 114.7, 114.6, 114.5, 112.3, 112.1, 111.4, 80.9, 71.6, 67.1, 66.9, 66.8, 66.0, 65.8, 65.6, 65.4, 56.6, 56.5, 56.4, 47.1, 36.4, 35.0, 34.9, 15.4. HRMS-ESI calculated for C53H49NO9Na [M+Na] 866.3305, found 866.3312 Melting point: 274-276°C Another example of N-alkylation has been performed as follows: . Example 3: Synthesis of OBn-CTV1N and preparation of a O-protected aza-cryptophane 5‐(Benzyloxy)‐2‐bromo‐4‐methoxybenzaldehyde 15 Obtained according to the procedure described by Xie et al. (Bioorganic Med. Chem. 2019, 27 (13), 2764–2770). N'‐[(Z)‐[5‐(benzyloxy)‐2‐bromo‐4‐methoxyphenyl ]methylidene]‐4‐methylbenzene‐1‐ sulfonohydrazide 16 Prepared from 15 (2.5 g, 7.78 mmol), following general procedure A, the expected compound was obtained as a white powder (3.534 g, 93%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.98 (s, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.72 (s, 1H), 7.48-7.45 (m, 3H), 7.43-7.35 (m, 3H), 7.27-7.25 (m, 2H), 6.95 (s, 1H), 5.16 (s, 2H), 3.87 (s, 3H), 2.41 (s, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 152.3, 147.9, 146.3, 144.4, 136.5, 135.4, 129.8 (2C), 128.9 (2C), 128.3, 128.1 (2C), 127.6 (2C), 124.4, 116.2, 115.4, 111.4, 71.1, 56.4, 21.8. Melting Point: 175-177°C HRMS-ESI calculated for C ₂₂ H ₂₂ BrN ₂ O ₄ S [M+H ⁺ ] 489.0485, found 489.0484 1‐(Benzyloxy)‐4‐bromo‐2‐methoxy‐5‐[(3‐methox yphenyl)methyl]benzene 17 Prepared from 16 (850 mg, 1.73 mmol) and 3-methoxyphenylboronic acid (317 mg, 2.08 mmol) following general procedure B, the expected product was obtained as a white powder (600 mg, 84%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.35 – 7.28 (m, 5H), 7.21 (t, J = 7.9 Hz, 1H), 7.07 (s, 1H), 6.78 (dd, J = 8.1, 2.1 Hz, 1H), 6.69 (m, 2H), 6.67 (s, 1H), 5.05 (s, 2H), 3.97 (s, 2H), 3.87 (s, 3H), 3.77 (s, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 159.8, 148.8, 147.4, 141.4, 136.7, 132.1, 129.5, 128.6 (2C), 128.0, 127.5 (2C), 121.3, 116.5, 116.0, 115.2, 114.7, 111.6, 71.1, 56.3, 55.2, 41.3. Melting Point: 80-82°C 4‐(Benzyloxy)‐N‐(2‐{[(tert‐butyldimethylsilyl)oxy] methyl}‐4‐methoxyphenyl)‐5‐ methoxy‐2‐[(3‐methoxyphenyl)methyl]aniline 18 17 (600 mg, 1.45 mmol), 4 (388 mg, 1.45 mmol), palladium acetate (33 mg, 0.145 mmol), Binap (181 mg, 0.2 mmol), cesium carbonate (1.41 g, 4.35 mmol) were added in toluene (15 mL) under nitrogen atmosphere and heated to reflux for 3h. The mixture was filtrated through a pad of celite using ethyl acetate as the eluent. The solvent was removed under vacuum and purification over silica gel (Cyclohexane/Et ₂ O 90/10) afforded the product as an orange oil. (539 mg, 62%). ¹ H NMR (400 MHz, CDCl3): δ = 7.39 – 7.27 (m, 5H), 7.19 – 7.14 (t, J = 7.9 Hz, 1H), 6.86 (d, J = 2.9 Hz, 1H), 6.82 (d, J = 8.7 Hz, 1H), 6.76 – 6.70 (m, 2H), 6.68 (d, J = 7.5 Hz, 1H), 6.66 (s, 1H), 6.64 – 6.61 (m, 1H), 6.60 (s, 1H), 5.75 (s, 1H), 5.04 (s, 2H), 4.54 (s, 2H), 3.78 (s, 2H), 3.77 (d, 3H), 3.75 (s, 3H), 3.72 (s, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 159.9, 153.8, 149.2, 143.1, 141.9, 137.6, 137.1, 136.4, 131.0, 129.6, 128.6 (2C), 127.8, 127.7 (2C), 123.2, 121.2, 118.7, 117.9, 114.4, 114.2, 113.3, 111.8, 105.2, 72.0, 63.9, 56.2, 55.8, 55.2, 37.2, 26.0 (3C), 18.4, -5.1 (2C). (2‐{[4‐(Benzyloxy)‐5‐methoxy‐2‐[(3‐methoxyphen yl)methyl]phenyl]amino}‐5‐ methoxyphenyl)methanol 19 TBAF (1M in THF, 1.33 mL, 1.33 mmol) was added at 0°C to a solution of 18 (535 mg, 0.89 mmol) in THF (20 mL) and the mixture was stirred at room temperature until completion of the reaction. The solution was then quenched with saturated NaHCO ₃ , extracted with ethyl acetate, washed with brine, dried over MgSO4 and concentrated under vacuum. Purification over silica gel (Cyclohexane/Et2O 5/5) afforded the product as a light yellow powder. (405 mg, 94%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.44 – 7.40 (m, 2H), 7.39 – 7.34 (m, 2H), 7.31 (m, 1H), 7.18 (t, J = 7.9 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.80 (d, J = 2.8 Hz, 1H), 6.78 – 6.74 (m, 3H), 6.70 (d, J = 7.6 Hz, 1H), 6.67 (d, J = 1.8 Hz, 1H), 6.63 (s, 1H), 5.83 (s, 1H), 5.09 (s, 2H), 4.45 (s, 2H), 3.82 (s, 2H), 3.77 (s, 3H), 3.76 (s, 3H), 3.72 (s, 3H), 1.29 (s, 1H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 159.8, 153.9, 149.3, 142.7, 141.8, 137.6, 137.1, 136.6, 130.7, 129.5, 128.5 (2C), 127.8, 127.7 (2C), 122.2, 121.1, 119.5, 118.5, 115.1, 114.3, 114.1, 111.7, 104.5, 72.1, 63.9, 56.1, 55.7, 55.2, 37.6. Melting Point: 103-105°C 3-Benzyloxy-2,7,12-trimethoxy-10,15-dihydro-5H-tribenzo[b,e, h]azonine 20 Phosphorus pentoxide (37 mg, 0.13 mmol) was added to a solution of 19 (50 mg, 0.1 mmol) in acetonitrile (20 mL) at 0°C and then the solution was heated to 60°C for 1h. The reaction was quenched with water, extracted with ethyl acetate, washed with brine, dried over MgSO ₄ and concentrated under vacuum. Purification over silica gel (cyclohexane/Et ₂ O 8/2) afforded the product as a light pink powder (36 mg, 78%). ¹ H NMR (400 MHz, CDCl3): δ = 7.35 (m, 2H), 7.30 (m, 2H), 7.24 (s, 1H), 7.04 (d, J = 7.08 Hz, 1H), 6.74 (d, J = 8.7 Hz, 1H), 6.68 – 6.64 (m, 3H), 6.50 – 6.45 (m, 2H), 6.35 (s, 1H), 5.55 (s, 1H), 4.94 (s, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.79 (s, 2H), 3.68 (s, 3H), 3.67 (s, 2H). ¹³ C NMR (100 MHz, CDCl3): δ = 158.4, 154.6, 149.4, 142.7, 141.0, 138.0, 137.5 (2C), 137.0, 132.1, 131.0, 130.1, 128.5 (2C), 127.8 (2C), 120.8, 119.3, 118.0, 117.3, 116.6, 113.1, 110.6, 103.8, 72.1, 56.1, 55.5, 55.3, 35.5, 29.8. Melting point: 93-95°C The corresponding aza-cryptophane is obtained similarly to what has been described above regarding example 1. Example 3: Synthesis of 2N-cryptophane of the invention 4‐Methoxy‐N‐(3‐methoxyphenyl)‐2‐nitroaniline 21 3-bromoanisole (1.5 g, 8.02 mmol), 4-methoxy-2-nitroaniline (1.35 g, 8.02 mmol), palladium acetate (180 mg, 0.80 mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthalene (998 mg, 1.60 mmol), cesium carbonate (9.1 g, 28.07 mmol) were added in anhydrous toluene (40 mL) placed under nitrogen and heated to reflux for 3h. The mixture was filtrated through a pad of celite using ethyl acetate as eluent. The solvent was removed under vacuum and purification over silica gel (cyclohexane/Et ₂ O 95/5) afforded the product as a dark red oil. (2.13 g, 97%). ¹ H NMR (400 MHz, CDCl3): δ = 9.28 (s, 1H), 7.60 (d, J = 3.0 Hz, 1H), 7.31 – 7.24 (m, 2H), 7.06 (dd, J = 9.4, 3.0 Hz, 1H), 6.82 (dd, J = 7.9, 1.8 Hz, 1H), 6.77 (t, J = 2.2 Hz, 1H), 6.71 (dd, J = 8.1, 2.1 Hz, 1H), 3.81 (s, 3H), 3.80 (s, 3H). ¹³ C NMR (100 MHz, CDCl3): δ = 160.8, 151.4, 140.7, 137.7, 133.2, 130.5, 126.2, 118.5, 115.6, 110.5, 109.1, 107.0, 55.9, 55.5. 4‐Methoxy‐N1‐(3‐methoxyphenyl)benzene‐1,2‐diamin e 22 21 (2 g, 7.3 mmol) and SnCl ₂ (16.4 g, 73 mmol) were dissolved in a 1/1 mixture acetonitrile/water (40 mL) and the solution was stirred at 80°C overnight. After completion of the reaction, a saturated solution of potassium carbonate and a 2M solution of sodium tartrate were added, the solution was stirred for two hours and extracted three times with ethyl acetate. The organic layers were combined, washed with brine, dried over MgSO ₄ , filtered and concentrated under vacuum. The expected product was obtained without purification as a light yellow powder (1.77 g, 99%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.08 (t, J = 8.1 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.36 – 6.29 (m, 3H), 6.25 (ddd, J = 8.1, 2.1, 0.7 Hz, 1H), 6.18 (t, J = 2.3 Hz, 1H), 5.03 (s, 1H), 3.86 (s, J = 9.9 Hz, 1H), 3.78 (s, J = 4.9 Hz, 3H), 3.74 (s, J = 4.0 Hz, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 160.9, 158.8, 148.4, 144.9, 130.2, 128.8, 120.6, 106.9, 104.2, 103.7, 101.3, 100.0, 55.4, 55.2. N2‐(2‐{[(Tert‐butyldimethylsilyl)oxy]methyl}‐4‐met hoxyphenyl)‐4‐methoxy‐N1‐(3‐ methoxyphenyl)benzene‐1,2‐diamine 23 4 (1.42 g, 5.8 mmol), 22 (1.93 g, 5.8 mmol), palladium acetate (130 mg, 0.58 mmol), 2,2′- bis(diphenylphosphino)-1,1′-binaphthalene (722 mg, 1.16 mmol) and cesium carbonate (6.6 g, 20.3 mmol) were added in anhydrous toluene (50 mL) placed under nitrogen and heated to reflux for 3h. The mixture was filtrated through a pad of celite using ethyl acetate as eluent. The solvent was removed under vacuum and purification over silica gel (cyclohexane/Et ₂ O 9/1 to 8/2) afforded the product as a green oil. (1.79 g, 63%). ¹ H NMR (400 MHz, CDCl3): δ = 7.19 (d, J = 8.7 Hz, 1H), 7.07 (ddd, J = 12.6, 8.1, 4.6 Hz, 2H), 6.94 (d, J = 3.0 Hz, 1H), 6.78 (dd, J = 8.7, 3.0 Hz, 1H), 6.44 (d, J = 2.8 Hz, 1H), 6.36 – 6.29 (m, 4H), 6.25 (t, J = 2.3 Hz, 1H), 5.05 (s, 1H), 4.58 (s, 2H), 3.80 (s, J = 3.2 Hz, 3H), 3.73 (s, 3H), 3.71 (s, 3H), 0.84 (s, J = 2.9 Hz, 9H), -0.02 (s, J = 3.1 Hz, 6H). ¹³ C NMR (100 MHz, CDCl3): δ = 160.9, 158.5, 155.9, 148.3, 143.5, 135.9, 133.1, 130.1, 127.8, 124.1, 122.2, 113.8, 113.3, 107.7, 104.3, 103.8, 100.7, 100.0, 63.2, 55.6, 55.4, 55.2, 25.9 (3C), 18.4, -5.2 (2C). [5‐Methoxy‐2‐({5‐methoxy‐2‐[(3‐methoxyphenyl)a mino]phenyl}amino)phenyl]methanol 24 TBAF (1M in THF, 7.2 mL, 7.2 mmol) was added at 0°C to a solution of 23 (1.79 g, 3.62 mmol) in THF (30 mL) and the mixture was stirred at room temperature until completion of the reaction. The reaction mixture was quenched with a saturated aqueous solution of NaHCO ₃ , extracted three times with ethyl acetate. The organic layers were combined, washed with brine, dried over MgSO ₄ , filtered and concentrated under vacuum. Purification over silica gel (cyclohexane/Et ₂ O 5/5) afforded the product as a light yellow oil. (1.034 g, 96%). ¹ H NMR (400 MHz, CDCl3): δ = 7.23 (d, J = 8.6 Hz, 1H), 7.11 – 7.05 (m, 2H), 6.85 (d, J = 2.9 Hz, 1H), 6.81 (dd, J = 8.6, 3.0 Hz, 1H), 6.46 (d, J = 2.6 Hz, 2H), 6.37 – 6.28 (m, 3H), 6.23 (t, J = 2.2 Hz, 1H), 5.20 (s, 1H), 4.48 (s, 2H), 3.78 (s, 3H), 3.72 (s, 3H), 3.71 (s, 3H), 1.99 (s, 1H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 160.9, 158.7, 155.9, 148.4, 143.6, 135.0, 133.7, 130.1, 128.6, 124.2, 121.5, 114.8, 114.0, 107.2, 104.0, 103.6, 100.2, 100.0, 63.4, 55.6, 55.4, 55.2. 2,7,12-Trimethoxy-10,15-dihydro-5H-tribenzo[b,e,h][1,4]diazo nine 25 Perchloric acid (60% in water, 0.160 mL) was added dropwise to a solution of 24 (100 mg, 0.264 mmol) in acetonitrile (53mL) at 0°C and the solution was heated to 60°C for 1h. The reaction mixture was quenched with water, extracted three times with ethyl acetate. The organic layers were combined, washed with brine, dried over MgSO4, filtered and concentrated under vacuum. Purification over silica gel (cyclohexane/Et2O 9/1 to 8/2) afforded the product as a white powder (58 mg, 61%). ¹ H NMR (400 MHz, CDCl ₃ ): δ = 6.95 (dd, J = 8.3, 7.3 Hz, 2H), 6.86 (d, J = 8.6 Hz, 1H), 6.66 (dd, J = 8.5, 3.0 Hz, 1H), 6.55 (d, J = 2.9 Hz, 1H), 6.39 (td, J = 8.6, 2.7 Hz, 2H), 6.34 (d, J = 2.5 Hz, 1H), 6.31 (d, J = 2.8 Hz, 1H), 5.38 (s, 1H), 4.79 (s, 1H), 3.76 (s, 3H), 3.73 (s, 6H), 3.63 (s, 2H). ¹³ C NMR (100 MHz, CDCl3): δ = 159.1, 157.9, 156.2, 145.4, 142.9, 136.7, 136.2, 132.5, 128.4, 126.6, 124.5, 122.3, 116.7, 111.6, 107.1, 106.6, 106.1, 106.0, 55.5 (2C), 55.3, 34.9. Melting point: 166-168°C 10,15-Dihydro-5H-tribenzo[b,e,h][1,4]diazonine-2,7,12-triol 26 BBr3 (1M in DCM, 4.6 mL, 4.6 mmol) was added to a solution of 25 (475 mg, 1.3 mmol) in dichloromethane (7 mL) at 0°C and the reaction mixture was stirred at room temperature until completion of reaction. The reaction was quenched at 0°C with water, extracted three times with ethyl acetate. The organic layers were combined, washed with brine, dried over MgSO ₄ , filtered and concentrated under vacuum. Purification over silica gel (cyclohexane/Et2O 5/5 to 7/3) afforded the product as a light brown powder (295 mg, 70%). ¹ H NMR (400 MHz, Acetone-d ₆ ): δ = 7.94 (s, 1H), 7.92 (s, 1H), 7.71 (s, 1H), 6.88 (d, J = 8.1 Hz, 1H), 6.76 (dd, J = 10.6, 8.4 Hz, 2H), 6.51 (dd, J = 8.4, 2.9 Hz, 1H), 6.42 (m, 3H), 6.33 (d, J = 2.7 Hz, 1H), 6.28 (dd, J = 8.1, 2.5 Hz, 1H), 6.22 (dd, J = 8.4, 2.8 Hz, 1H), 5.49 (s, 1H), 3.58 (s, 2H). ¹³ C NMR (100 MHz, Acetone-D6): δ = 158.1, 157.1, 154.5, 148.7, 146.0, 137.8, 136.5, 133.2, 131.3, 126.6, 124.9, 123.4, 118.8, 114.4, 108.9, 108.7, 108.4, 107.4, 35.9. (((((10,15-Dihydro-5H-tribenzo[b,e,h][1,4]diazonine-2,7,12-t riyl)tris(oxy))tris(ethane- 2,1-diyl))tris(oxy))tris(3-methoxybenzene-4,1-diyl))trimetha nol 27

10 (1.67 g, 5.4 mmol) and cesium carbonate (1.76 g, 5.4 mmol) were added to a solution of 26 (290 mg, 0.905 mmol) in dry DMF (14 mL) placed under nitrogen. The mixture was stirred overnight at 80°C, and allowed to reach room temperature. The reaction was quenched with water and the product was extracted three times with ethyl acetate. The organic layers were combined, washed with brine, dried over MgSO ₄ , filtered and concentrated under vacuum. Purification over silica gel (AcOEt/MeOH, 100/0 to 96/4) afforded the product as a pink powder (485 mg, 62%). ¹ H NMR (400 MHz, DMSO): δ = 7.58 (s, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.99 – 6.94 (m, 5H), 6.86 – 6.83 (m, 3H), 6.80 (dd, J = 8.8, 3.5 Hz, 3H), 6.68 (dd, J = 8.6, 2.9 Hz, 1H), 6.53 (d, J = 2.5 Hz, 1H), 6.48 (d, J = 2.8 Hz, 1H), 6.43 – 6.39 (m, 2H), 6.36 (dd, J = 8.6, 2.8 Hz, 1H), 6.06 (s, 1H), 4.45 (m, 6H), 4.26 (m, 4H), 4.24 – 4.19 (m, 6H), 4.14 – 4.12 (m, 2H), 3.78 (s, 3H), 3.77 (s, 3H), 3.75 (s, 3H), 3.55 (s, 2H). Melting point: 99-101°C. The corresponding aza-cryptophane can been obtained as follows, similarly to what has been described above regarding example 1: Example 4: Comparison of the solubility of aza-cryptophanes of the invention VS conventional cryptophanes outside the invention Experimental solubility of cryptophane A (conventional cryptophane outside the invention): DMSO : 25 mg/mL or 27.93 µmol/mL (corresponds to the solubility limit) CHCl ₃ : not evaluated MeCN: very poorly soluble. Experimental solubility of Cryptophane-1N : DMSO : 59.5 mg/mL or 73.8 µmol/mL (solubility limit not reached) CHCl ₃ : 8.27 µmol/mL (solubility limit not reached) MeCN: 2.23 µmol/mL (solubility limit not reached) Example 5: Synthesis of a N-protected 2N-cryptophane A N-protected 2N-cryptophane has been obtained as follows: wherein P is a benzyl, p-methoxybenzyl, 2-tetrahydropyranyl (THP), a carbamate, for example Boc, Fmoc, Troc, Cbz, a silyl, in particular tert-butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropyl silyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), or 2-(trimethylsilyl)ethoxymethyl (SEM), or an acyl group, in particular an acetyl. Example 6: synthesis of an aza-cryptophane with n1= n2=n3=0 An aza-cryptophane with n1= n2=n3=0 is obtained as follows: wherein X is CH ₂ or NH or NP, wherein P is a benzyl, p-methoxybenzyl, 2-tetrahydropyranyl (THP), a carbamate, for example Boc, Fmoc, Troc, Cbz, a silyl, in particular tert- butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropylsilyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), or 2-(trimethylsilyl)ethoxymethyl (SEM), or an acyl group, in particular an acetyl. An aza-cryptophane with n1= n2=n3=0 can also be obtained as follows: wherein X is CH ₂ or NH or NP, wherein P is a benzyl, p-methoxybenzyl, 2-tetrahydropyranyl (THP), a carbamate, for example Boc, Fmoc, Troc, Cbz, a silyl, in particular tert- butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropyl silyl (TIPS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), or 2-(trimethylsilyl)ethoxymethyl (SEM), or an acyl group, in particular an acetyl. Example 7: synthesis of an 3N-cryptophane A 3N aza-cryptophane is obtained as follows:

wherein P ₁ and P ₂ are chosen from benzyl, p-methoxybenzyl, 2-tetrahydropyranyl (THP), carbamate, for example Boc, Fmoc, Troc, Cbz, silyl, in particular tert-butyldimethylsilyl (TBS or TBMS), trimethylsilyl (TMS), triisopropyl silyl (TIPS), triethylsilyl (TES), tert- butyldiphenylsilyl (TBDPS), or 2-(trimethylsilyl)ethoxymethyl (SEM), and acyl groups, in particular an acetyl. Example 8: selective dealkylation of a compound of formula (I) with Z ₁ , Z ₂ or Z ₃ being – O-C ₁ -C ₆ alkyl, notably OMe, to obtain the corresponding compound with Z ₁ , Z ₂ or Z ₃ being –OH Iodotrimethylsilane (0.6 eq.) was added in one portion to a stirred solution of cryptophane (1 eq.) in dichloromethane. The solution was stirred in the dark for 16 h under nitrogen at room temperature. Dichloromethane was added and the solution was quenched with hydrochloric acid (1M), the organic layer was washed with water, dried over MgSO4, filtered and concentrated under vacuum. Purification on alumina neutral gel of the crude mixture afforded the expected product. Example 9: Nanoemulsions comprising an aza-cryptophane of the invention Nanoemulsions comprising the IN-cryptophane 12 (CrlN) of the invention and control nanoemulsions comprising cryptophane A (CrA, outside the invention) have been preprared as follows.

Protocol:

Nanoemulsion formulation was adapted from spontaneous nano-emulsification method previously described by Seguy el al. (Pharmaceutics. 2020, 12, 7747;). Rearding CrlN, the anhydrous phase, composed of oil (Labrafac® WL 1349), surfactant (Kolliphor® HS 15) and solubilizer (Transcutol® HP), and a solution of CrlN 12 in DMSO (160pL of 46.9 mg/mL) were heated at 70°C under gentle magnetic stirring (250 rpm) and cooled down at 25°C.

Similarly, for CrA, the anhydrous phase, composed of oil (Labrafac® WL 1349), surfactant (Kolliphor® HS 15) and solubilizer (Transcutol® HP), was heated at 70°C under gentle magnetic stirring (250 rpm). 160pL of a saturated solution of CrA in DMSO (25.0 mg/mL in DMSO), and the anhydrous phase was heated at 70°C for 15 minutes under gentle magnetic stirring, then allowed to reach 25 °C.

In both cases, when the anhydrous mixture reached this temperature, the magnetic stirring was increased from 250 rpm to 750 rpm and the aqueous phase (HPLC grade water, 25°C) was suddenly added, leading to spontaneous emulsification. After the addition of water, the stirring was maintained for 15 minutes at room temperature. Then, the formulation was filtered through 0.2 pm regenerated cellulose syringe filters (Minisart® Syringe Filter, Sartorius, Goettingen, Germany).

Physicochemical characterization of nanoemulsions:

Dynamic light scattering (DLS) was used to determine the average hydrodynamic diameter, the polydispersity index (PDI) and the diameter distribution by volume of the nanoemulsions using a Zetasizer ultra apparatus (Malvern Instruments, Worcestershire, UK) equipped with a 633 nm laser at a fixed scattering angle of 173°. The temperature of the cell was kept constant at 25 °C. The nanoemulsions were diluted 1/100 (v/v) in NaCl 1 mM in order to assure an appropriate scattered intensity on the detector before measurements. Measurements were performed in triplicate.

Zeta potential measurements:

Zeta potential analyses were realized, after filtration and 1/100 dilution in NaCl 1 mM, using a Zetasizer ultra apparatus equipped with DTS 1070 cell. All measurements were performed in triplicate at 25°C, with a dielectric constant of 78.5, a refractive index of 1.33, a viscosity of 0.8872 cP and a cell voltage of 150 V. The zeta potential was calculated from the electrophoretic mobility using the Smoluchowski equation. Calibration curve:

Cryptophanes concentration were defined using spectrophotometry (Infinite M200, Tecan, Mannedorf, Switzerland) by determining absorbance spectrum of Crytophane in solution in MeOH.

The encapsulation efficiency (EE) was determined after filtration through 0.2 pm syringe filters (Minisart® Syringe Filter, Sartorius, Goettingen, Germany) to remove unentrapped cryptophanes. Then, these samples were diluted in methanol (1/100, v/v) and the concentration of cryptophane was determined by spectrophotometry at 288 nm. The EE was determined in duplicate and calculated as follows:

The drug loading (DL) was defined as:

In vitro haemolysis assay

Haemolysis tests were adapted from the protocol initially described by Dobrovolskaia et al. (Nano Let. 2008, 8, 2180-2187). Whole human blood samples from three healthy compatible volunteers were collected in Li-heparin tubes (Etablissement Francais du Sang, EFS Hauts-de- France-Normandie, France). For overcoming any variability, the three samples were pooled and the total haemoglobin concentration was measured and adjusted to 10 mg/mE by dilution with DPBS (SDiomg/mL). An aliquot (900 pF) of the pooled whole blood was centrifuged for 15 minutes at 3,000 rpm to determine plasma free haemoglobin (PFH).

The various excipients and nanoemulsions were assayed in the final concentration range 0.05- 2.50 mg/mE. For each test, 100 pL of the excipient or nanoemulsion solutions were introduced into eppendorf tubes with 700 pL of DPBS and 100 pL of SDiomg/mL and incubated for three hours at 37°C, with constant horizontal shaking (water bath WNB-22, Memmert, Schwabach, Germany). After incubation, the samples were centrifuged (15 minutes at 3,000 rpm, 25 °C using a Universal 320R apparatus, Hettich, Bach, Switzerland) to separate the pellet containing undamaged erythrocytes from the supernatant containing the haemoglobin released during haemolysis. Haemolysis percentage was quantified by spectrophotometry (Infinite M200, Tecan, Mannedorf, Switzerland) by determining absorbance of red cyanmethaemoglobin (CMH) at 540 nm, at 25°C, after addition of Drabkin’s reagent (dissolved in deionized water in the presence of Brij® L23 0.05% (w/w)) in a 96 well plate. These measured absorbances were compared to a standard curve of human haemoglobin with satisfactory linearity (R ² > 0.99) in the concentration range studied (0.0625-1 mg/mL). The concentration of haemoglobin in the supematant was compared to that in the supernatant of an untreated blood sample with samples to obtain the percentage of the sample induced haemolysis (referred to as percent haemolysis). Before each experiment, PFH was determined and must not exceed 1% of the total haemoglobin. Each test was approved with a positive control, Triton® X-100 known to be haemolytic and a negative control, PBS. In order to overcome potential particles optical interferences with the signal emitted at or close to the assay wavelength (540 nm), false-positive and false-negative were made for each test. The results shown for the nanoemulsions were adjusted taking into account these interferences.

Haemolytic properties were evaluated according to the haemolysis percentage calculated as follows:

In this assay, an haemolysis threshold of 5% was defined. When it was crossed, the compound was considered as haemolytic. All assays were performed in triplicate.

Results

Cryptophane A:

1 batch of nanoemulsions was prepared, using a saturated solution of cryptophane A in DMSO (53.3 mg in 2.13 mL, see protocol above). The hydrodynamic diameter of the formulated nanoemulsions, the polydispersity index and the surface charge of the formed objects were characterised (see table 1 below). In addition, the UV evaluation of the batch could be carried

Table 1: Physico-chemical properties of empty or CrA-charged nanoemulsions at 4°C at storage concentration

CrlN:

1 batch of nanoemulsions was prepared, using a solution of cryptophane IN in DMSO (see protocol above). The hydrodynamic diameter of the formulated nanoemulsions, the polydispersity index and the surface charge of the formed objects were characterised (see table 2 below).

Table 2: Physico-chemical properties of empty or CrlN-charged nanoemulsions at 4°C at storage concentration

Stability of the batches over time, as a function of temperature

The hydrodynamic diameter of the formulated nanoemulsions loaded with CrlN, the polydispersity index (PDI) and the charge on the surface of the formed objects were characterised as a function of the storage temperature, over 7 days (Table 3 and Table 4).

Table 3: Physico-chemical properties of “empty” NEs (formulated with DMSO) or NEs loaded with CrlN solution, at 4°C and at storage concentration.

Table 4: Physico-chemical properties of “empty” NEs (formulated with DMSO) or NEs loaded with CrlN solution, at RT at storage concentration.

Calculation of the encapsulation efficiency A UV calibration curve for each of the compounds was carried out (see protocol above) using a stock solution of CrlN in acetonitrile (1.05 mg/mL) and a stock solution of cryptophane A in DMSO (1.85 mg/mL), solutions subsequently diluted in methanol.

The batch of nanoemulsions loaded with CrlN was diluted 100-fold in methanol. Under these conditions, it was confirmed by DLS that the nanoemulsions were destroyed. The amount of cryptophane in this sample was then determined. The concentration of cryptophane in the sample was 28 pM, i.e. an encapsulation efficiency of 60%.

Similarly, the batch of nanoemulsions loaded with CrA was diluted under the same conditions. The concentration of the sample is 11 pM, an encapsulation efficiency of 49%.

It has been thus demonstrated that, thanks to the high solubility of IN cryptophane, it is possible to load more than twice as much IN cryptophane as cryptophane A into these nanoemulsions.

Characterisation of the batches by UV spectrophotometry was carried out over time (Figure 4, A at 4°C; B at RT). The loaded batches are stable for at least 24 hours, and even for at least 3 days at 4°C.

Stability of the batches in biomimetic medium

The stability at 37°C of the batches diluted 50 fold in PBS was evaluated by UV spectrophotometry every hour during 3h (Figure 7). It has been demonstrated that the charged nanoemulsions were stable under these conditions and that there was no cage leakage during this time period.

Haemolysis test

The haemolytic properties of the batches of nanoemulsions of the invention were evaluated according to the protocol defined above (figure 5). It can be noted that the percentage of haemolysis remains below 5% when the "loaded" nanoemulsions are used below a concentration of 1 mg/mL dry matter, i.e. below 268 nM.

Comparison with CrA-loaded nanoemulsions (not part of the invention)

A batch of cryptophane A (CrA)-loaded nanoemulsions was prepared following the same protocol. The characterisation of this batch by UV spectrophotometry was carried out over time (Figure 6).

This figure clearly demonstrates that the nanoemulsions loaded with cryptophane A are less stable than the nanoemulsions loaded with cryptophane of the invention.