CUCURBITURIL DERIVATIVES AS OXYGEN CARRIERS

Abstract

The present invention relates to a compound of the following formula (I) for use as a physiological gas carrier, said gas being O.sub.2, N.sub.2, NO, NO.sub.2, H.sub.2, He and/or CO.sub.2, notably O.sub.2. The invention also relates to a non-therapeutic use of a compound of the general formula (I) for binding, carrying and/or releasing a gas, said gas being O.sub.2, N.sub.2, NO, NO.sub.2, H.sub.2, He and/or CO.sub.2, notably O.sub.2.

##STR00001##

Claims

1. A method, comprising encapsulating a physiological gas in the inner cavity of a compound of the following general formula (I): ##STR00012## or a salt, a solvate or a stereoisomer thereof, wherein: n is equal to 5 or 6, X.sub.1 and X.sub.2 represent, independently of each other, an oxygen or a sulfur atom, and R.sub.1 and R.sub.2 each represent, independently of each other, a hydrogen or halogen atom, a OR.sub.3, SR.sub.4, NR.sub.5R.sub.6, S(O)R7, SO.sub.2R.sub.8, OCOR.sub.9, CO.sub.2R.sub.10, CONR.sub.11R.sub.12, CO.sub.2R.sub.13, OP(O)(OR.sub.14).sub.2, NO.sub.2 or CN group, preferably a hydrogen atom or a OR.sub.3, SR.sub.4, NR.sub.5R.sub.6 group, wherein R.sub.3 to R.sub.14 each represent, independently of each other, a hydrogen atom or a (C.sub.1-C.sub.6)alkyl group, with the proviso that at least two R.sub.1, two R.sub.2 or one R.sub.1 and one R.sub.2 do not represent a hydrogen atom, wherein said physiological gas is O.sub.2, N.sub.2, NO, NO.sub.2, H.sub.2, He and/or CO.sub.2.

2. The method according to claim 1, wherein n is equal to 5.

3. The method according to claim 1, wherein X.sub.1=X.sub.2, and X.sub.1 and X.sub.2 represent an oxygen atom.

4. The method according to claim 1, wherein each R.sub.1 and each R.sub.2 are the same.

5. The method according to claim 2, wherein said compound is CB[5]OH.sub.10, corresponding to the following formula: ##STR00013## or a salt or a solvate thereof

6. The method according to claim 1, wherein said compound is covalently linked to one or several hydrophilic polymeric side-chains, such as polyethyleneglycol (PEG) or hydroxyethyl starch (HES).

7-13. (canceled)

14. A complex of a compound and a gas, wherein the compound has the following general formula (I): ##STR00014## or a salt, a solvate or a stereoisomer thereof, wherein: n is equal to 5 or 6, X.sub.1 and X.sub.2 represent, independently of each other, an oxygen or a sulfur atom, and R.sub.1 and R.sub.2 each represent, independently of each other, a hydrogen or halogen atom, a OR.sub.3, SR.sub.4, NR.sub.5R.sub.6, S(O)R.sub.7, SO.sub.2R.sub.8, OCOR.sub.9, CO.sub.2R.sub.10, CONR.sub.11R.sub.12, CO.sub.2RA.sub.13, OP(O)(OR.sub.14).sub.2, NO.sub.2 or CN group, preferably a hydrogen atom or a OR.sub.3, SR.sub.4 NR.sub.5R.sub.6 group, wherein R.sub.3 to R.sub.14 each represent, independently of each other, a hydrogen atom or a (C.sub.1-C.sub.6)alkyl group, with the proviso that at least two R.sub.1, two R.sub.2 or one R.sub.1 and one R.sub.2 do not represent a hydrogen atom, wherein said gas is O.sub.2, N.sub.2, NO, NO.sub.2, H.sub.2, He and/or CO.sub.2, and wherein said gas is encapsulated in the inner cavity of the compound of formula (I).

15. (canceled)

16. The complex according to claim 14, wherein said gas is O.sub.2.

17. The complex according to claim 14, wherein n is equal to 5.

18. The complex according to claim 14, wherein X.sub.1=X.sub.2, and X.sub.1 and X.sub.2 represent an oxygen atom.

19. The complex according to claim 14, wherein each R.sub.1 and each R.sub.2 are the same.

20. The complex according to claim 14, wherein said compound is CB[5]OH.sub.10, corresponding to the following formula: ##STR00015## or a salt or a solvate thereof.

21. The complex according to claim 14, wherein: said compound is CB[5]OH.sub.10, corresponding to the following formula: ##STR00016## or a salt or a solvate thereof, and said gas is O.sub.2.

22. A cosmetic composition comprising a complex according to claim 16 and at least one cosmetically acceptable excipient.

23. A composition consisting in a physiologically acceptable medium comprising a complex according to claim 16.

24. The method according to claim 1, further comprising releasing the encapsulated physiological gas.

25. The method according to claim 1, wherein said physiological gas is O.sub.2.

26. The method according to claim 25, further comprising incorporating the compound comprising O.sub.2 encapsulated in its inner cavity in a medium and placing a biological material to be preserved and/or protected in the medium comprising said compound.

27. The method according to claim 26, wherein the biological material is a cell, a tissue, a body fluid, an organ or a microorganism.

28. The method according to claim 25, further comprising administering to a patient in need thereof an effective dose of the compound comprising O.sub.2 encapsulated in its inner cavity.

Description

FIGURES

[0155] FIGS. 1a and 1b represent the .sup.1H NMR spectra obtained at 20° C. for CB[5]OH.sub.10, depending on whether O.sub.2 and NaCI are present or not: [0156] SP1: without NaCI and O.sub.2; [0157] SP2: with 02 under 1 bar, no NaCI ; and [0158] SP3: with NaCI and O.sub.2 at the pressure of 1 bar.

[0159] FIG. 2 displays the .sup.1H NMR spectra obtained at 37° C. for CB[5]OH.sub.10 in function of the oxygen partial pressure, said spectra being focused on the H.sub.ax signal.

[0160] FIG. 3 represents the R.sub.1 measured for H.sub.ax at 37° C. in function of the oxygen partial pressure.

[0161] FIGS. 4a and 4b represent the .sup.1H NMR spectra SP1 and SP2 obtained at 20° C. for CB, depending on whether O.sub.2 is present or not.

[0162] FIGS. 5a and 5b represent the .sup.1H NMR spectra obtained at 20° C. for CB[5]Me.sub.10, depending on whether O.sub.2 and NaCI are present or not: [0163] SP1: without NaCI and O.sub.2; [0164] SP2: with O.sub.2 under 1 bar, no NaCI; and [0165] SP3: with NaCI and O.sub.2 at the pressure of 1 bar.

[0166] The examples that follow illustrate the invention without limiting its scope in any way.

EXAMPLES

[0167] The following abbreviations have been used: [0168] ax. : axial [0169] ca. : circa [0170] eq. : equatorial [0171] Me : Methyl (CH.sub.3) [0172] NMR : Nuclear Magnetic Resonance [0173] TSP-d.sub.4: 3-(Trimethylsilyl)propionic-2,2,3,3-d.sub.4 acid sodium salt

I-Materials and Methods

[0174] I-1. Studied Compounds

[0175] The ability of CB[5], CB[5]Me.sub.10 and CB[5]OH.sub.10 to fix O.sub.2 has been studied. Said compounds are of the following formula (IV), wherein R1 corresponds to H, CH.sub.3 and OH respectively.

##STR00011##

[0176] I-2. Synthesis

[0177] CB[5]H.sub.10 was synthetized following the method described in the literature (J. Am. Chem. Soc. 2003, 125, 10186) with a minor modification when treating the manipulation: the volume of solvent is half reduced under reduced pressure and precipitated with acetone. The filtrate is concentrated and agitated during 12 hours with a Amberlyst A21 resin (Flucka 20-50 mesh), and then filtrated on a sinter filter (P4 Porosity) and concentrated under vacuum to lead to CB[5]OH.sub.10.

[0178] CB[5]Me.sub.10 was synthetized following the method described in the literature (Angew. Chem. Int. Ed. 1992, 31, 1475).

[0179] CB[5] as bought from Sigma Aldrich.

[0180] I-3. .sup.1H NMR

[0181] Samples Preparation: [0182] CB[5]OH.sub.10: 0.319 mM solution in D20 (and NaCl when mentioned); [0183] CB[5]: 0.501 mM solution in D20 (and NaCl when mentioned); and [0184] CB[5]Me.sub.10: 0.472 mM solution in D20 (and NaCl when mentioned).

[0185] The concentrations were measured in separate samples by adding a known volume to a known solution of TSP-d.sub.4.

[0186] Samples containing O.sub.2 to the pressure of ca. 1 bar where prepared by addition of pure 02 at a pressure slightly higher than atmospheric pressure using a syringe, then equilibration of the solution and quick release towards the ambient air.

[0187] In fact, the experience is conducted on a 156 m altitude and the medium pressure is 0.982 bar.

[0188] An experience involving a sample containing O.sub.2 to the pressure of 5 bar has been conducted: 02 has been added by condensation of a known quantity of O.sub.2 in a previously degassed tube, using a bath of liquid nitrogen.

[0189] Measurement of T.sub.1:

[0190] The longitudinal relaxation time T1 is measured by the standard inversion-recovery method (180° -t-90° acq.), followed by phasing of the spectrum and a baseline correction in Topspin®. The data is then exported to Excel. The signals are integrated and the intensity is adjusted according to the inversion delay based on a 3-parameter model:

S32 S.sub.0(1−2Axe.sup.−R .sup.1.sup.1)

[0191] wherein the A factor takes into account the imperfect inversion of the magnetization, and R.sub.1 is the relaxation rate, reverse of T.sub.1.

[0192] Adjustment of the Complexing Constant K:

[0193] The adjustment of the complexing constant K is obtained by adjusting the 1:1 complexation model as follows:

[00001]$R_1=\begin{array}{c}{R_{1.Math..Math.\mathrm{empty}}\left[\mathrm{compound}\right]}_{\mathrm{empty}}+R_{1.Math..Math.\mathrm{complex}}\left[\mathrm{complex}\right]\\ {\left[\mathrm{compound}\right]}_{\mathrm{total}}\end{array}$

[0194] By definition:

[00002]$K.Math.=\frac{\left[\mathrm{compound}\right]}{{\left[O_2\right]\left[\mathrm{compound}\right]}_{\mathrm{empty}}}$

[0195] with:

└complex┘+└compound└.sub.empty=┘compound└.sub.total

[0196] thus, leading to:

[00003]$R_1-R_{1.Math..Math.\mathrm{complex}}-\frac{1}{1+{\mathrm{sKP}}_{O_2}}.Math.\left(R_{1.Math..Math.\mathrm{empty}}-R_{1.Math..Math.\mathrm{complex}}\right)$

[0197] wherein:

[0198] R.sub.1 empty, which corresponds to R.sub.1 of the nucleus considered in the absence of oxygen, is obtained with a fairly good accuracy (10%).

[0199] R.sub.1 complex, which corresponds to R.sub.1 of the nucleus when a complex of the tested compound and O.sub.2 encapsulated in its inner cavity (or cage) is formed, is an adjustable parameter. It is not known precisely because, in view of the results, a pressure much higher than the experimental pressures (maximum 5 bar) would be required to saturate the cages almost completely.

[0200] s, which is the solubility of the oxygen in a 9 g/I NaCI solution at 37° C., is obtained by intrapolation of tabulated data. Indeed, the concentration of O.sub.2 in solution is proportional to P.sub.O2, the O.sub.2 pressure above the solution: Henry's law is very well verified in the experimental pressure range.

II-Results

[0201] II-1. CB[5]OH.sub.10

[0202] Attribution:

[0203] When considering the .sup.1H NMR spectra obtained at 20° C. displayed on FIGS. 1a and 1b, the attribution is obvious by massifs. Indeed, the 10 methylenes are equivalent. H.sub.ax (see formula (IV)) resonates at ca. 5.40 ppm, and H.sub.eq. at ca. 4.58 ppm.

[0204] Besides, each signal is composed of 2 doublets: [0205] one depends on the presence or not of O.sub.2 (circles), whose paramagnetism has a considerable influence on the NMR signals nearby; the linewidth and the value of T.sub.1 vary greatly. [0206] for the other one (black circles), the NMR characteristics are invariable by the addition of O.sub.2. It is therefore hypothesized that the cage-molecules corresponding to the black circles contain a molecule of high affinity, which might be N.sub.2 or a solvent. The encapsulation of said molecule could be prevented by working under an higher oxygen partial pressure, by using an oxygen/helium mixture, or by removing said molecule by purification.

[0207] The circles correspond to a weighted average between “empty” molecules (i.e. containing no molecules other than solvent), and complexes of CB[5]OH.sub.10 and O.sub.2. Indeed, as previously stated, a pressure much higher than the experimental pressures (maximum 5 bar) would be required to saturate the cages almost completely.

[0208] The fact that the encapsulation of O.sub.2 is favoured under higher oxygen partial pressure is evidenced by FIG. 2, which represents the .sup.1H NMR spectra obtained at 37° C. in function of said pressure of samples containing NaCI at physiological concentrations.

[0209] Therefore, CB.sub.5OH.sub.10 significantly encapsulates O.sub.2 at 37° C. even in the presence of NaCI at physiological concentrations.

[0210] Determination of K via R.sub.1: [0211] With respect to the H.sub.ax signal of interest: [0212] T.sub.1empty, measured using the degassed sample, is equal to 2.1 s.sup.−1. [0213] T.sub.1, and hence R.sub.1, was measured for various pressures of oxygen. [0214] The results obtained are displayed on FIG. 3. [0215] The adjustment gives K=3000 M.sup.−1. The sum of the square of the deviations to the model is doubled for K=1170 and 6380 M.sup.−1, giving an estimate of the measurement error. [0216] R.sub.1 was measured for the proton H.sub.ax of CB[5]OH.sub.10 in the presence and the absence of NaCI 9 g/I and of O.sub.2 1 bar at two different temperatures.

TABLE-US-00001 R.sub.1 CB.sub.5OH.sub.10 (s.sup.−1) Without O.sub.2 With O.sub.2 Without NaCl, 21° C. 2.2 18.5 With NaCl, 37° C. 2.1 20.8

[0217] The slightly different temperature softly influences the R.sub.1. [0218] Conclusion: NaCI does not compete with O.sub.2 for complexation.

[0219] II-2. CB[5]

[0220] Besides of the two doublets corresponding to H.sub.ax and H.sub.eq (ca. 5.69 ppm and 4.35 ppm respectively), the spectra of CB[5] displayed on FIGS. 4a and 4b show a singlet at 5.5 ppm that corresponds to R.sub.1=H.

[0221] The fact that the spectra SP1 and SP2 are identical proves that O.sub.2 is not significantly encapsulated by CB[5].

[0222] II-3. CB[5]Me.sub.10

[0223] The characteristic signal of the methyl groups of CB[5]Me.sub.10 which resonates at 1.8 ppm is not represented on the spectra displayed on FIGS. 5a and 5b.

[0224] The comparison of spectra SP1, SP2 and SP3 shows that O.sub.2 is encapsulated by CB[5]Me.sub.10 in the absence of NaCI, but is no longer so significantly in the presence of NaCI. The main ions present in the blood are therefore too strong competitors of O.sub.2 for the encapsulation in CB[5]Meio.

[0225] III-Conclusion

[0226] Therefore, the results displayed above show that CB[5]OH.sub.10 exhibits a remarkably strong affinity for oxygen, notably in comparison with CB or CB[5]Me.sub.10.