AZA-CRYPTOPHANES, PROCESSES FOR PREPARATION THEREOF, AND THEIR USES

Abstract

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.

Claims

1. Compound of following formula (I): ##STR00050## Wherein: X.sub.1, X.sub.2 and X.sub.3 are independently selected from CH.sub.2 and NR, at least one of X.sub.1, X.sub.2 and X.sub.3 representing NR; R is chosen from: H; a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; a LZ group; an ad hoc protecting group, in particular a benzyl, p-methoxybenzyl, 2-tetrahydropyranyl, a carbamate, for example Boc, Fmoc, Troc, Cbz, a silyl, in particular tert-butyldimethylsilyl, trimethylsilyl, triisopropylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or 2-(trimethylsilyl)ethoxymethyl, or an acyl group, in particular an acetyl; Y.sub.1, Y.sub.2 and Y.sub.3 are independently selected from: H, a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; a OC.sub.1-C.sub.6 alkyl; a OC.sub.2-C.sub.6 alkenyl; a O-LZ group; OH or a OP group, wherein P is an ad hoc protecting group, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert-butyldimethylsilyl, trimethylsilyl, triisopropylsilyl, triethylsilyl, tert-butyldiphenylsilyl; 2-(trimethylsilyl)ethoxymethyl; allyl; methoxymethyl; tetrahydropyran or an acyl group, in particular an acetyl; Z.sub.1, Z.sub.2 and Z.sub.3 are independently selected from: H, a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; a OC.sub.1-C.sub.6 alkyl; a OC.sub.2-C.sub.6 alkenyl; a O-LZ group; OH or a OP group, wherein P is an ad hoc protecting group, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert-butyldimethylsilyl, trimethylsilyl, triisopropylsilyl, triethylsilyl, tert-butyldiphenylsilyl; 2-(trimethylsilyl)ethoxymethyl; allyl; methoxymethyl; tetrahydropyran 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.

2. Compound according to claim 1, wherein: X.sub.1 is NR, and X.sub.2 and X.sub.3 are CH.sub.2; or X.sub.1 and X.sub.2 are independently selected from NR groups, and X.sub.3 is CH.sub.2; or X.sub.1, X.sub.2 and X.sub.3 are independently selected from NR groups; and/or Y.sub.1, Y.sub.2 and Y.sub.3 are H; and/or Z.sub.1, Z.sub.2 and Z.sub.3 are H, or a OC.sub.1-C.sub.6 alkyl, in particular OMe; and/or n1=n2=n3=0 or 1, in particular 1.

3. Compound according to claim 1, wherein: X.sub.1 is NR with R being LZ, X.sub.2 and X.sub.3 being in particular CH.sub.2 or NH; and/or Y.sub.1 is O-LZ, Y.sub.2 and Y.sub.3 being in particular H; and/or Z.sub.1 is O-LZ, Z.sub.2 and Z.sub.3 being in particular a OC.sub.1-C.sub.6 alkyl, more particularly OMe.

4. Compound according to claim 1, wherein L is (W).sub.i-L.sub.1-(F.sub.1-L.sub.2).sub.j(F.sub.2).sub.k and/or L is (W).sub.i-L.sub.1-(F.sub.1-L.sub.2).sub.j-(F.sub.2).sub.k, wherein: W and W are independently chosen from C(?O), C(?O)O, C(?O)NH, C(?S)O; L.sub.1, L.sub.2, L.sub.1 and L.sub.2 are independently is a C.sub.1-C.sub.12 alkane diyl, a C.sub.2-C.sub.12 alkene diyl or a C.sub.2-C.sub.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; F.sub.1, F.sub.2, F.sub.1 and F.sub.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)?NNH, NHC(?O)C(H)?NNH, ester, amide, amine, CH.sub.2, ether, thioether, imine, succinimide, thio-succinimide, oxime, hydrazone, hydrazonamide, C(?O)CH.sub.2NH, NHCH.sub.2C(?O), triazole functions or groups; i, j, and k are independently 0 or 1.

5. Compound according to claim 4, wherein: 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, Z being more particularly a halogen, in particular Cl, Br, I, F, more particularly Br, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, azido, epoxide, C(?O)H, hemiacetal, C(?O)R.sub.c, acetal, SR.sub.a, NH.sub.2 or NHC(?O)CH.sub.2Hal, wherein Hal is a halogen, in particular Cl, Br, I, F, more particularly Br, N-maleimide; or 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.

6. Compound according to claim 1, chosen from the following formulae: ##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058##

7. Process of preparation of a compound of formula (I) according to claim 1, wherein n1, n2 and n3 are independently chosen from 1, 2 and 3, comprising a step (i) of contacting a compound of following formula (II) with formic acid, P.sub.2O.sub.5, HClO.sub.4, or Sc(OTf).sub.3: ##STR00059## wherein: X.sub.1, X.sub.2 and X.sub.3 are independently selected from CH.sub.2 and NR, at least one of X.sub.1, X.sub.2 and X.sub.3 representing NR; R is chosen from: H; a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; an ad hoc protecting group, in particular a benzyl, p-methoxybenzyl, 2-tetrahydropyranyl, a carbamate, for example Boc, Fmoc, Troc, Cbz, a silyl, in particular tert-butyldimethylsilyl, trimethylsilyl, triisopropyl silyl, triethylsilyl, tert-butyldiphenylsilyl, or 2-(trimethylsilyl)ethoxymethyl, or an acyl group, in particular an acetyl; Y.sub.1, Y.sub.2 and Y.sub.3 are independently selected from: H, a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; a OC.sub.1-C.sub.6 alkyl; a OC.sub.2-C.sub.6 alkenyl; optionally a O-LZ group; a OP group, wherein P is an ad hoc protecting group, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert-butyldimethylsilyl, trimethylsilyl, triisopropylsilyl, triethylsilyl, tert-butyldiphenylsilyl; 2-(trimethylsilyl)ethoxymethyl allyl; methoxymethyl; tetrahydropyran or an acyl group, in particular an acetyl; Z.sub.1, Z.sub.2 and Z.sub.3 are independently selected from: H, a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; a OC.sub.1-C.sub.6 alkyl; a OC.sub.2-C.sub.6 alkenyl; optionally a O-LZ group; a OP group, wherein P is an ad hoc protecting group, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert-butyldimethylsilyl, trimethylsilyl, triisopropylsilyl, triethylsilyl, tert-butyldiphenylsilyl; 2-(trimethylsilyl)ethoxymethyl; allyl; methoxymethyl; tetrahydropyran 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.

8. Process according to claim 7, wherein 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.sub.2CO.sub.3: ##STR00060## wherein LG is a leaving group, in particular chosen from halogens, more particularly I, Br, Cl, notably I, and O-tosyl, the compound of formula (III) being in particular obtained by contacting a compound of following formula (V) with BBr.sub.3: ##STR00061## the compound of formula (V), when X.sub.1 is CH.sub.2 or NR, X.sub.2 is NR and X.sub.3 is CH.sub.2, being more particularly obtained by contacting a compound of following formula (VI) with HClO.sub.4: ##STR00062## wherein X.sub.1 is CH.sub.2 or NR, and X.sub.2 is NR, the compound of formula (V), when X.sub.1 and X.sub.2 are independently chosen from NR groups, R being not H, and X.sub.3 is NH, being more particularly obtained from a compound of following formula (VII), in particular by Buchwald amination ##STR00063## wherein X.sub.1 and X.sub.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.

9. Process according to claim 7: of a compound of formula (I) wherein X.sub.1, X.sub.2 and/or X.sub.3, in particular X.sub.1 and/or X.sub.2, is N(-LZ), said process comprising after step (i) a step of contacting a compound of formula (I) wherein X.sub.1, X.sub.2 and/or X.sub.3, in particular X.sub.1 and/or X.sub.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; or of a compound of formula (I) wherein Y.sub.1, Y.sub.2 and/or Y.sub.3, in particular Y.sub.1 and/or Y.sub.2, is O-LZ, said process comprising after step (i) a deprotection step of a compound of formula (I) with Y.sub.1, Y.sub.2 and/or Y.sub.3, in particular Y.sub.1 and/or Y.sub.2, being OP, followed by the contacting the obtained compound of formula (I) wherein Y.sub.1, Y.sub.2 and/or Y.sub.3, in particular Y.sub.1 and/or Y.sub.2, is OH 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; of a compound of formula (I) wherein Z.sub.1, Z.sub.2 and/or Z.sub.3, in particular Z.sub.1 and/or Z.sub.2, is O-LZ, said process comprising after step (i): a deprotection step of a compound of formula (I) with Z.sub.1, Z.sub.2 and/or Z.sub.3, in particular Z.sub.1 and/or Z.sub.2, being OP, or a dealkylation step of a compound of formula (I) with Z.sub.1, Z.sub.2 and/or Z.sub.3, in particular Z.sub.1 and/or Z.sub.2, being OC.sub.1-C.sub.6 alkyl, notably OMe, followed by the contacting the obtained compound of formula (I) wherein Z.sub.1, Z.sub.2 and/or Z.sub.3, in particular Z.sub.1 and/or Z.sub.2, is OH 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.

10. Process of preparation of a compound of formula (I) according to claim 1, 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.sub.2Cl and a base, in particular Cs.sub.2CO.sub.3: ##STR00064## Wherein Z.sub.1, Z.sub.2 and Z.sub.3 are independently selected from: H, a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; a OC.sub.1-C.sub.6 alkyl; a OC.sub.2-C.sub.6 alkenyl; optionally a O-LZ group; a OP group, wherein P is an ad hoc protecting group, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert-butyldimethylsilyl, trimethylsilyl, triisopropylsilyl, triethylsilyl, tert-butyldiphenylsilyl; 2-(trimethylsilyl)ethoxymethyl; allyl; methoxymethyl; tetrahydropyran or an acyl group, in particular an acetyl; or a step (i) of contacting a compound of formula (III), with ClCH.sub.2SMe, then SOCl.sub.2, and then with a compound of following formula (VII), in presence of a base, in particular Cs.sub.2CO.sub.3.

11. Compound of one of the following formulae: ##STR00065## wherein: X.sub.1, X.sub.2 and X.sub.3 are independently selected from CH.sub.2 and NR, at least one of X.sub.1, X.sub.2 and X.sub.3 representing NR; R is chosen from: H; a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; an ad hoc protecting group, in particular a benzyl, p-methoxybenzyl, 2-tetrahydropyranyl, a carbamate, for example Boc, Fmoc, Troc, Cbz, a silyl, in particular tert-butyldimethylsilyl, trimethylsilyl, triisopropyl silyl, triethylsilyl, tert-butyldiphenylsilyl, or 2-(trimethylsilyl)ethoxymethyl, or an acyl group, in particular an acetyl; Y.sub.1, Y.sub.2 and Y.sub.3 are independently selected from: H, a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; a OC.sub.1-C.sub.6 alkyl; a OC.sub.2-C.sub.6 alkenyl; optionally a O-LZ group; a OP group, wherein P is an ad hoc protecting group, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert-butyldimethylsilyl, trimethylsilyl, triisopropylsilyl, triethylsilyl, tert-butyldiphenylsilyl; 2-(trimethylsilyl)ethoxymethyl; allyl; methoxymethyl; tetrahydropyran or an acyl group, in particular an acetyl; Z.sub.1, Z.sub.2 and Z.sub.3 are independently selected from: H, a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; a OC.sub.1-C.sub.6 alkyl; a OC.sub.2-C.sub.6 alkenyl; optionally a O-LZ group; a OP group, wherein P is an ad hoc protecting group, in particular a benzyl; p-methoxybenzyl; silyl, in particular tert-butyldimethylsilyl, trimethylsilyl, triisopropylsilyl, triethylsily, tert-butyldiphenylsilyl; 2-(trimethylsilyl)ethoxymethyl; allyl; methoxymethyl; tetrahydropyran or an acyl group, in particular an acetyl; n1, n2 and n3 are independently chosen from 0, 1, 2 and 3.

12. Complex constituted of or comprising a compound of formula (I) according to claim 1 complexed with a hyperpolarized noble element, in particular Xe, more particularly .sup.129Xe.

13. Compound of formula (I) according to claim 1, complexed with a hyperpolarized noble element, in particular Xe, more particularly .sup.129Xe for its use as an in vivo diagnostic tool, in particular in NMR spectroscopy or in MRI applications.

14. Nanoemulsion oil-in-water comprising: An oil; An aqueous phase; Optionally, a surfactant; Optionally, a co-surfactant and/or a solubilizier; and A compound of following formula (Ia): ##STR00066## Wherein: X.sub.1, X.sub.2 and X.sub.3 are independently selected from CH.sub.2 and NR, at least one of X.sub.1, X.sub.2 and X.sub.3 representing NR; R is chosen from: H; a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; an ad hoc protecting group, in particular a benzyl, p-methoxybenzyl, 2-tetrahydropyranyl, a carbamate, for example Boc, Fmoc, Troc, Cbz, a silyl, in particular tert-butyldimethylsilyl, trimethylsilyl, triisopropylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or 2-(trimethylsilyl)ethoxymethyl, or an acyl group, in particular an acetyl; Y.sub.1, Y.sub.2 and Y.sub.3 are independently selected from: H, a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; a OC.sub.1-C.sub.6 alkyl; a OC.sub.2-C.sub.6 alkenyl; OH; Z.sub.1, Z.sub.2 and Z.sub.3 are independently selected from: H, a C.sub.1-C.sub.6 alkyl; a C.sub.2-C.sub.6 alkenyl; a C.sub.2-C.sub.6 alkynyl; a OC.sub.1-C.sub.6 alkyl; a OC.sub.2-C.sub.6 alkenyl; OH; n1, n2 and n3 are independently chosen from 0, 1, 2 and 3, said compound of formula (Ia) being optionally complexed with a hyperpolarized noble element, in particular Xe, more particularly .sup.129Xe.

Description

DRAWINGS

[0194] FIG. 1 illustrates the .sup.129Xe NMR spectrum (5 bars, CDCl.sub.3) of cryptophane-1N 12.

[0195] FIG. 2 illustrates the 3D structure of cryptophane-1N 12 determined by RX crystallography.

[0196] FIG. 3 shows two views of the structure of N-CTV compound 7 obtained by X-ray diffraction.

[0197] FIG. 4 presents the UV absorbance spectra of NE loaded with 1N Cr over time at 4? C. (A) and at RT (B).

[0198] FIG. 5 is related to haemolysis tests involving nanoemulsions of the invention according to example 9.

[0199] 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.

[0200] 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 3 h.

EXAMPLES

General Experimental Procedure

[0201] 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 1H NMR and at 100 or 125 MHz for 13C NMR in chloroform-d with chemical shift (?) 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 TosylhydrazonesGeneral Procedure A

[0202] 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 2 h30. 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 CouplingGeneral Procedure B

[0203] 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 2 h30. 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 MgSO.sub.4, 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 1N-cryptophane of the Invention

N-[(1E)-(2-bromo-4-methoxyphenyl)methylidene]-4-methylbenzene-1-sulfonohydrazide 1

[0204] ##STR00017##

[0205] 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%).

[0206] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0207] .sup.13C NMR (100 MHz, CDCl.sub.3): ? 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.

[0208] Melting point: 149? C.

1-Bromo-3-methoxy-6-(3-methoxybenzyl)-benzene 2

[0209] ##STR00018##

[0210] 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%).

[0211] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0212] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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.

[0213] HRMS-ESI calculated for C.sub.15H.sub.15BrO.sub.4 [M.sup.+.], 306.0255 found 306.0253.

(2-Amino-5-methoxyphenyl)methanol 3

[0214] ##STR00019##

[0215] Obtained according to the procedure described by Wales et al. (Org. Lett. 2019, 21 (12), 4703-4708).

2-[(Tert-butyldimethylsilyl)oxymethyl]-4-methoxyaniline 4

[0216] ##STR00020##

[0217] 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 MgSO.sub.4 and concentrated under vacuum. Purification over silica gel (cyclohexane/Et.sub.2O 9/1 to 8/2) afforded the product as a red oil (1.37 g, 60%).

[0218] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0219] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=152.4, 139.5, 126.9, 116.9, 114.5, 113.8, 64.7, 55.9, 26.0 (3C), 18.4, ?5.1 (2C).

[0220] HRMS-ESI calculated for C.sub.14H.sub.25NO.sub.2Si [M.sup.+. ] 267.1655, found 267.1657.

2-(((Tert-butyldimethylsilyl)oxy)methyl)-4-methoxy-N-(5-methoxy-2-(3-methoxybenzyl)phenyl)aniline 5

[0221] ##STR00021##

[0222] 2 (1 g, 3.26 mmol), 4 (872 mg, 3.26 mmol), Pd(OAc).sub.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 3 h. 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.sub.2O 95/5) afforded the product as a yellow oil. (1.4 g, 87%).

[0223] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0224] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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).

[0225] HRMS-EST calculated for C.sub.29H.sub.39NO.sub.4 [M.sup.+.] 493.2648, found 493.2647.

(5-methoxy-2-((5-methoxy-2-(3-methoxybenzyl)phenyl)amino)phenyl)methanol 6

[0226] ##STR00022##

[0227] 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.sub.3, extracted with ethyl acetate, washed with brine, dried over MgSO.sub.4 and concentrated under vacuum. Purification over silica gel (Cyclohexane/Et.sub.2O 7/3 to 4/6) afforded the product as a light yellow oil. (1.034 g, 96%).

[0228] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0229] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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.

[0230] HRMS-ESI calculated for C.sub.23H.sub.26NO.sub.4 [M+H.sup.+] 380.1862, found 380.1843.

2,7,12-trimethoxy-10,15-dihydro-5H-tribenzo[b,e,h]azonine 7

[0231] ##STR00023##

[0232] Perchloric acid (60%, 0.160 mL) was added dropwise to a solution of 6 (100 mg, 0.264 mmol) in acetonitrile (53 mL) at 0? C. and then the solution was heated to 60? C. for 1 h. The reaction was quenched with water, extracted with ethyl acetate, washed with brine, dried over MgSO.sub.4 and concentrated under vacuum. Purification over silica gel (cyclohexane/Et.sub.2O 8/2 to 7/3) afforded the product as a white powder (85 mg, 89%).

[0233] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0234] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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

[0235] HRMS-ESI calculated for C.sub.23H.sub.24NO.sub.3 [M+H.sup.+] 362.1756, found 362.1755.

[0236] Melting point: 166-168? C.

[0237] The conformation of compound 7 has been determined by X-ray diffraction. Compound 7 is of saddle conformation (FIG. 3).

10,15-dihydro-5H-tribenzo[b,e,h]azonine-2,7,12-triol 8

[0238] ##STR00024##

[0239] BBr.sub.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.sub.4 and concentrated under vacuum. Purification over silica gel (Et.sub.2O/cyclohexane 7/3) afforded the product as a light pink powder (383 mg, 96%).

[0240] .sup.1H NMR (400 MHz, Acetone D-6): ?=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).

[0241] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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.

[0242] Melting point: 210-212? C.

[4-(2-Bromoethoxy)-3-methoxyphenyl]methanol 9

[0243] ##STR00025##

[0244] 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

[0245] ##STR00026##

[0246] 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)tris(oxy))tris(ethane-2,1-diyl))tris(oxy))tris(3-methoxybenzene-4,1-diyl))trimethanol 11

[0247] ##STR00027##

[0248] 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.sub.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%).

[0249] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0250] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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.

[0251] HRMS-ESI calculated for C.sub.50H.sub.53NO.sub.12Na [M+Na.sup.+] 882.3465, found 882.3474.

[0252] Melting point: 140-142? C.

Cryptophane-1N 12

[0253] ##STR00028##

[0254] 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 3 h at 55? C. The solvent was removed under vacuum and purification over silica gel (DCM/Et.sub.2O 95/5 to 80/20) to afford the product as a light pink powder (182 mg, 72%).

[0255] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0256] .sup.13C NMR (100 MHz, CDCl.sub.3): ? 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.

[0257] .sup.129Xe NMR (5 bars, CDCl.sub.3): illustrated in FIG. 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.

[0258] RX: illustrated in FIG. 2.

[0259] HRMS-ESI calculated for C.sub.50H.sub.47NO.sub.9Na [M+Na.sup.+] 828.3149, found 828.3162.

[0260] Melting point: 257-259? C.

[0261] 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:

##STR00029##

Cryptophane-1N Syn

[0262] 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/Et.sub.2O 95/5 to 80/20) afforded the product (first spot) as a withe powder (365 mg, 23%).

[0263] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0264] .sup.13C NMR (100 MHz, CDCl.sub.3): ? 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.

[0265] Melting point 205-207? C.

[0266] HRMS-ESI calculated for C.sub.50H.sub.47NO.sub.9Na [M+Na.sup.+] 828.3149, found 828.3162.

[0267] 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 ?.sup.3 and the syn compound has a cavity size of 121.4 ?.sup.3, both larger than the size of the prior art cryptophane C (114.5 ?.sup.3).

[0268] 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 ?.sup.3.

Example 2: Alkylation of the Nitrogen of 1N-Cryptophane

Cryptophane 13

[0269] ##STR00030##

[0270] 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 ?L, 0.186 mmol) was added and the solution was stirred at 80? C. for 3 h. The reaction was quenched with a saturated solution of NH.sub.4Cl, extracted with ethyl acetate, washed with brine, dried over MgSO.sub.4, filtered and concentrated under vacuum. Purification over silica gel (DCM/Et.sub.2O 10/0 to 80/20) afforded the product as a light yellow powder (32 mg, 31%).

[0271] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0272] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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.

[0273] HRMS-ESI calculated for C.sub.53H.sub.49NO.sub.9Na [M+Na] 866.3305, found 866.3312.

[0274] Melting point: 274-276? C.

[0275] Another example of N-alkylation has been performed as follows:

##STR00031##

Example 3: Synthesis of OBn-CTV1N and preparation of a O-protected aza-cryptophane

5-(Benzyloxy)-2-bromo-4-methoxybenzaldehyde 15

[0276] ##STR00032##

[0277] 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

[0278] ##STR00033##

[0279] 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%).

[0280] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0281] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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.

[0282] Melting Point: 175-177? C.

[0283] HRMS-ESI calculated for C.sub.22H.sub.22BrN.sub.2O.sub.4S [M+H.sup.+] 489.0485, found 489.0484.

1-(Benzyloxy)-4-bromo-2-methoxy-5-[(3-methoxyphenyl)methyl]benzene 17

[0284] ##STR00034##

[0285] 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%).

[0286] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0287] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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.

[0288] Melting Point: 80-82? C.

4-(Benzyloxy)-N-(2-{[(tert-butyldimethylsilyl)oxy]methyl}-4-methoxyphenyl)-5-methoxy-2-[(3-methoxyphenyl)methyl]aniline 18

[0289] ##STR00035##

[0290] 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 3 h. 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.sub.2O 90/10) afforded the product as an orange oil. (539 mg, 62%).

[0291] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0292] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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-methoxyphenyl)methyl]phenyl]amino}-5-methoxyphenyl)methanol 19

[0293] ##STR00036##

[0294] 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.sub.3, extracted with ethyl acetate, washed with brine, dried over MgSO.sub.4 and concentrated under vacuum. Purification over silica gel (Cyclohexane/Et.sub.2O 5/5) afforded the product as a light yellow powder. (405 mg, 94%).

[0295] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0296] .sup.13C NMR (100 MHz, CDCl.sub.3): ? 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.

[0297] Melting Point: 103-105? C.

3-Benzyloxy-2,7,12-trimethoxy-10,15-dihydro-5H-tribenzo[b,e,h]azonine 20

[0298] ##STR00037##

[0299] 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 1 h. The reaction was quenched with water, extracted with ethyl acetate, washed with brine, dried over MgSO.sub.4 and concentrated under vacuum. Purification over silica gel (cyclohexane/Et.sub.2O 8/2) afforded the product as a light pink powder (36 mg, 78%).

[0300] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0301] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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.

[0302] Melting point: 93-95? C.

[0303] 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

[0304] ##STR00038##

[0305] 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 3 h. 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.sub.2O 95/5) afforded the product as a dark red oil. (2.13 g, 97%).

[0306] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0307] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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-diamine 22

[0308] ##STR00039##

[0309] 21 (2 g, 7.3 mmol) and SnCl.sub.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.sub.4, filtered and concentrated under vacuum. The expected product was obtained without purification as a light yellow powder (1.77 g, 99%).

[0310] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0311] .sup.13C NMR (100 MHz, CDCl.sub.3): ? 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-methoxyphenyl)-4-methoxy-N1-(3-methoxyphenyl)benzene-1,2-diamine 23

[0312] ##STR00040##

[0313] 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 3 h. 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.sub.2O 9/1 to 8/2) afforded the product as a green oil. (1.79 g, 63%).

[0314] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0315] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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)amino]phenyl}amino)phenyl]methanol 24

[0316] ##STR00041##

[0317] 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.sub.3, extracted three times with ethyl acetate. The organic layers were combined, washed with brine, dried over MgSO.sub.4, filtered and concentrated under vacuum. Purification over silica gel (cyclohexane/Et.sub.2O 5/5) afforded the product as a light yellow oil. (1.034 g, 96%).

[0318] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0319] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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]diazonine 25

[0320] ##STR00042##

[0321] Perchloric acid (60% in water, 0.160 mL) was added dropwise to a solution of 24 (100 mg, 0.264 mmol) in acetonitrile (53 mL) at 0? C. and the solution was heated to 60? C. for 1 h. The reaction mixture was quenched with water, extracted three times with ethyl acetate. The organic layers were combined, washed with brine, dried over MgSO.sub.4, filtered and concentrated under vacuum. Purification over silica gel (cyclohexane/Et.sub.2O 9/1 to 8/2) afforded the product as a white powder (58 mg, 61%).

[0322] .sup.1H NMR (400 MHz, CDCl.sub.3): ?=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).

[0323] .sup.13C NMR (100 MHz, CDCl.sub.3): ?=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.

[0324] Melting point: 166-168? C.

10,15-Dihydro-5H-tribenzo[b,e,h][1,4]diazonine-2,7,12-triol 26

[0325] ##STR00043##

[0326] BBr.sub.3 (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.sub.4, filtered and concentrated under vacuum. Purification over silica gel (cyclohexane/Et.sub.2O 5/5 to 7/3) afforded the product as a light brown powder (295 mg, 70%).

[0327] .sup.1H NMR (400 MHz, Acetone-d.sub.6): ?=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).

[0328] .sup.13C NMR (100 MHz, Acetone-D.sub.6): ?=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-triyl)tris(oxy))tris(ethane-2,1-diyl))tris(oxy))tris(3-methoxybenzene-4,1-diyl))trimethanol 27

[0329] ##STR00044##

[0330] 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.sub.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%).

[0331] .sup.1H NMR (400 MHz, DMSO): ?=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).

[0332] Melting point: 99-101? C.

[0333] The corresponding aza-cryptophane can been obtained as follows, similarly to what has been described above regarding example 1:

##STR00045##

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)

[0334] DMSO: 25 mg/mL or 27.93 ?mol/mL (corresponds to the solubility limit)

[0335] CHCl.sub.3: not evaluated

[0336] MeCN: very poorly soluble.

Experimental Solubility of Cryptophane-1N

[0337] DMSO: 59.5 mg/mL or 73.8 ?mol/mL (solubility limit not reached)

[0338] CHCl.sub.3: 8.27 ?mol/mL (solubility limit not reached)

[0339] MeCN: 2.23 ?mol/mL (solubility limit not reached)

Example 5: Synthesis of a N-Protected 2N-Cryptophane

[0340] A N-protected 2N-cryptophane has been obtained as follows:

##STR00046##

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

[0341] An aza-cryptophane with n1=n2=n3=0 is obtained as follows:

##STR00047##

wherein X is CH.sub.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.

[0342] An aza-cryptophane with n1=n2=n3=0 can also be obtained as follows:

##STR00048##

wherein X is CH.sub.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

[0343] A 3N aza-cryptophane is obtained as follows:

##STR00049##

wherein P.sub.1 and P.sub.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.SUB.1., Z.SUB.2 .or Z.SUB.3 .being OC.SUB.1.-C.SUB.6 .Alkyl, Notably OMe, to Obtain the Corresponding Compound with Z.SUB.1., Z.SUB.2 .or Z.SUB.3 .being OH

[0344] 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 MgSO.sub.4, 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

[0345] Nanoemulsions comprising the 1N-cryptophane 12 (Cr1N) of the invention and control nanoemulsions comprising cryptophane A (CrA, outside the invention) have been prepared as follows.

[0346] Protocol:

[0347] Nanoemulsion formulation was adapted from spontaneous nano-emulsification method previously described by S?guy et al. (Pharmaceutics. 2020, 12, 1141). Rearding Cr1N, the anhydrous phase, composed of oil (Labrafac? WL 1349), surfactant (Kolliphor? HS 15) and solubilizer (Transcutol? HP), and a solution of Cr1N 12 in DMSO (160 ?L 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). 160 ?L 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.

[0348] 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 ?m regenerated cellulose syringe filters (Minisart? Syringe Filter, Sartorius, Goettingen, Germany).

[0349] Physicochemical Characterization of Nanoemulsions:

[0350] 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.

[0351] Zeta Potential Measurements:

[0352] 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.

[0353] Calibration Curve:

[0354] Cryptophanes concentration were defined using spectrophotometry (Infinite M200, Tecan, Mannedorf, Switzerland) by determining absorbance spectrum of Crytophane in solution in MeOH.

[0355] Dosage:

[0356] The encapsulation efficiency (EE) was determined after filtration through 0.2 ?m 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:

[00001]$\begin{array}{cc}\mathrm{EE}\left(\%\right)=100?\frac{\mathrm{Quantity}\mathrm{of}\mathrm{API}\mathrm{entrapped}}{\mathrm{Total}\mathrm{quantity}\mathrm{of}\mathrm{API}\mathrm{added}}& \left[1\right]\end{array}$

[0357] The drug loading (DL) was defined as:

[00002]$\begin{array}{cc}\mathrm{DL}\left(\%\right)=100?\frac{\mathrm{Quantity}\mathrm{of}\mathrm{API}\mathrm{entrapped}}{\mathrm{Total}\mathrm{quantity}\mathrm{of}\mathrm{anhydrous}\mathrm{excipients}}& \left[2\right]\end{array}$

[0358] In Vitro Haemolysis Assay

[0359] Haemolysis tests were adapted from the protocol initially described by Dobrovolskaia et al. (Nano Lett. 2008, 8, 2180-2187). Whole human blood samples from three healthy compatible volunteers were collected in Li-heparin tubes (Etablissement Fran?ais 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/mL by dilution with DPBS (SD.sub.10 mg/mL). An aliquot (900 ?L) of the pooled whole blood was centrifuged for 15 minutes at 3,000 rpm to determine plasma free haemoglobin (PFH).

[0360] The various excipients and nanoemulsions were assayed in the final concentration range 0.05-2.50 mg/mL. For each test, 100 ?L of the excipient or nanoemulsion solutions were introduced into eppendorf tubes with 700 ?L of DPBS and 100 ?L of SD.sub.10 mg/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, B?ch, 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, M?nnedorf, 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.sup.2>0.99) in the concentration range studied (0.0625-1 mg/mL). The concentration of haemoglobin in the supernatant 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.

[0361] Haemolytic properties were evaluated according to the haemolysis percentage calculated as follows:

[00003]$\mathrm{Haemolysis}\left(\%\right)=100?\frac{{\left[\mathrm{Cyanmethaemoglobin}\right]}_{\mathrm{supernatant}}}{{\left[\mathrm{Cyanmethaemoglobin}\right]}_{\mathrm{total}}}$

[0362] 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.

[0363] Results

[0364] Cryptophane A:

[0365] 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 out.

TABLE-US-00001 TABLE 1 Physico-chemical properties of empty or CrA-charged nanoemulsions at 4? C. at storage concentration ? Empty ? nanoemulsions ? Loaded ? nanoemulsions ?(nm) PDI ?(nm) PDI 3 h 57.9 ? 0.6 0.184 ? 0.015 58.3 ? 0.4 0.164 ? 0.017

[0366] Cr1N:

[0367] 1 batch of nanoemulsions was prepared, using a solution of cryptophane 1N 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-US-00002 TABLE 2 Physico-chemical properties of empty or Cr1N-charged nanoemulsions at 4? C. at storage concentration ?(nm) PDI ? (mV) 3 h 57.8 ? 0.3 0.184 ? 0.005 ?6.1 ? 0.4

[0368] Stability of the Batches Over Time, as a Function of Temperature

[0369] The hydrodynamic diameter of the formulated nanoemulsions loaded with Cr1N, 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-US-00003 TABLE 3 Physico-chemical properties of empty NEs (formulated with DMSO) or NEs loaded with Cr1N solution, at 4? C. and at storage concentration. ? Empty ? nanoemulsions ? Loaded ? nanoemulsions ?(nm) PDI ? (mV) ?(nm) PDI ? (mV) 3 h 61.8 ? 0.5 0.178 ? 0.012 ?6.9 ? 1.5 57.8 ? 0.3 0.184 ? 0.005 ?6.1 ? 0.4 24 h 63.4 ? 0.7 0.171 ? 0.007 ?4.5 ? 0.4 60.9 ? 0.2 0.191 ? 0.005 ?7.0 ? 0.2 3 days 64.1 ? 0.3 0.168 ? 0.010 ?5.1 ? 0.7 60.3 ? 1.4 0.171 ? 0.002 ?3.6 ? 0.7 7 days 65.0 ? 0.0 0.164 ? 0.003 ?5.0 ? 0.6 61.0 ? 1.1 0.168 ? 0.014 ?6.1 ? 0.4

TABLE-US-00004 TABLE 4 Physico-chemical properties of empty NEs (formulated with DMSO) or NEs loaded with Cr1N solution, at RT at storage concentration. ? Empty ? nanoemulsions ? Loaded ? nanoemulsions ?(nm) PDI ? (mV) ?(nm) PDI ? (mV) 3 h 62.4 ? 0.5 0.176 ? 0.008 ?5.0 ? 1.0 57.0 ? 0.2 0.171 ? 0.005 ?5.5 ? 1.1 24 h 66.9 ? 0.7 0.191 ? 0.003 ?3.4 ? 0.8 60.0 ? 0.4 0.169 ? 0.004 ?4.5 ? 0.2 3 days 66.4 ? 0.3 0.167 ? 0.002 ?3.8 ? 0.7 61.7 ? 0.6 0.179 ? 0.016 ?4.7 ? 0.3 7 days 67.9 ? 0.9 0.166 ? 0.011 ?6.7 ? 0.5 61.6 ? 0.5 0.156 ? 0.005 ?6.7 ? 0.4

[0370] Calculation of the Encapsulation Efficiency

[0371] A UV calibration curve for each of the compounds was carried out (see protocol above) using a stock solution of Cr1N in acetonitrile (1.05 mg/mL) and a stock solution of cryptophane A in DMSO (1.85 mg/mL), solutions subsequently diluted in methanol.

[0372] The batch of nanoemulsions loaded with Cr1N 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 ?M, i.e. an encapsulation efficiency of 60%.

[0373] Similarly, the batch of nanoemulsions loaded with CrA was diluted under the same conditions.

[0374] The concentration of the sample is 11 ?M, an encapsulation efficiency of 49%.

[0375] It has been thus demonstrated that, thanks to the high solubility of 1N cryptophane, it is possible to load more than twice as much 1N cryptophane as cryptophane A into these nanoemulsions.

[0376] Characterisation of the batches by UV spectrophotometry was carried out over time (FIG. 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.

[0377] Stability of the Batches in Biomimetic Medium

[0378] The stability at 37? C. of the batches diluted 50 fold in PBS was evaluated by UV spectrophotometry every hour during 3 h (FIG. 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.

[0379] Haemolysis Test

[0380] The haemolytic properties of the batches of nanoemulsions of the invention were evaluated according to the protocol defined above (FIG. 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.

[0381] Comparison with CrA-Loaded Nanoemulsions (not Part of the Invention)

[0382] 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 (FIG. 6).

[0383] This figure clearly demonstrates that the nanoemulsions loaded with cryptophane A are less stable than the nanoemulsions loaded with cryptophane of the invention.