Capped cyclodextrin-hydrophobic moiety conjugate, cyclodextrin supramolecular polymer, and cyclodextrin-siRNA complex and method of synthesis thereof

Abstract

The invention relates to a capped cyclodextrin-hydrophobic moiety conjugate, to a supramolecular polymer formed of capped cyclodextrin-hydrophobic moiety conjugates according to the invention and to a siRNA-cyclodextrin complex comprising a supramolecular polymer according to the invention. The invention also relates to a method for manufacturing the capped cyclodextrin-hydrophobic moiety conjugate, the supramolecular polymer, the siRNA-cyclodextrin complex according to the invention. The capped cyclodextrin-hydrophobic moiety conjugate of the invention comprises a capped cyclodextrin group and at least one hydrophobic moiety bound by a first linker to one of the carbon atoms of the cap. The invention can be used for various applications in particular in the pharmaceutical field.

Claims

1. A capped cyclodextrin-hydrophobic moiety conjugate comprising a cyclodextrin moiety and at least one hydrophobic moiety, wherein: the cyclodextrin moiety is capped on its primary rim by a cap binding a first carbon atom, previously bearing a hydroxyl group, of a first glucopyranose unit to a second carbon, previously bearing an hydroxyl group, of a second and different glucopyranose unit, said first and second glucopyranose units being preferably separated from each other by at least one, and the hydrophobic moiety is bound by a first linker to one of the carbon atoms of the cap, and the hydrophobic moiety is selected from the group consisting of an adamantane group, a C.sub.2-C.sub.13 alkyl group optionally containing at least one heteroatom, a C.sub.5-C.sub.6 aromatic group optionally containing at least one heteroatom and a C.sub.3-C.sub.8 non-aromatic cycle optionally containing at least one heteroatom.

2. The capped cyclodextrin-hydrophobic moiety conjugate according to claim 1, wherein the hydrophobic moiety is selected from the group consisting of an adamantane group and a phenyl group.

3. The capped cyclodextrin-hydrophobic moiety conjugate of claim 1, wherein: said cap and said first linker, independently from each other, form, together with the carbon atoms to which they are bound, a chain having from 2 to 20 links, said chain comprising at least one heteroatom chosen in the group consisting of N, O, S and P and/or at least one functional group selected from the group consisting of a ketone group, an amine group, an ether group, an amide group, an ester group, a cyano group, and optionally comprising a non: aromatic or aromatic cyclic or heterocyclic group having from 3 to 8 links.

4. The capped cyclodextrin-hydrophobic moiety conjugate of claim 1, wherein the cyclodextrin moiety is an - or -cyclodextrin moiety.

5. The capped cyclodextrin-hydrophobic moiety conjugate of claim 1, wherein the cap binds the carbon atoms, previously bearing a hydroxyl group, in position 6 of the glucopyranose units, the glucopyranose units being in position A and D of the primary rim of the cyclodextrin moiety.

6. The capped cyclodextrin-hydrophobic moiety conjugate according to claim 1, wherein the cap comprises a first heteroatom and wherein the hydrophobic moiety is bound to this first heteroatom.

7. The capped cyclodextrin-hydrophobic moiety conjugate according to claim 6, wherein the cap comprises a second heteroatom which either form a positively charged link of the cap or to which at least one positively charged group is bound, the positively charged group being selected from the group consisting of a primary amine group, a secondary amine group, a guanidinium group and combination thereof.

8. The capped cyclodextrin-hydrophobic moiety conjugate of claim 7, wherein said first and second heteroatom are nitrogen atoms.

9. The capped cyclodextrin-hydrophobic moiety conjugate according to claim 1 having one of the following formulae I-1 to I-26: ##STR00021## ##STR00022## in which R is an amine group, an arginine group, a guanidine group, or a linear or branched chain comprising between 1 and 20 atoms for the main chain, these atoms being nitrogen atoms and/or oxygen atoms and/or carbon atoms, such a chain optionally comprising from 1 to 30 amine groups or guanidine groups, and in which x is equal to the number of amine group(s) plus the number of guanidine group(s) in R; ##STR00023## in which R is an amine group, an arginine group, a guanidine group, or a linear or branched chain comprising between 1 and 20 atoms for the main chain, these atoms being nitrogen atoms and/or -oxygen atoms and/or carbon atoms, such a chain optionally comprising from 1 to 30 amine groups or guanidine groups, and in which x is equal to the number of amine group(s) plus the number of guanidine group(s) in R; ##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028## wherein in formulas I-4 to I-11, Ada designates an adamantane group and in formulas I-12 to I-17, Ad designates an adamantane group.

10. The capped cyclodextrin-hydrophobic moiety conjugate according to claim 1 having the following formula I-1: ##STR00029##

11. A supramolecular polymer consisting of capped cyclodextrin-hydrophobic moiety conjugates according to claim 1 bridged to each other by inclusion of the hydrophobic moiety of each capped cyclodextrin-hydrophobic moiety conjugate into a cavity of another capped cyclodextrin-hydrophobic moiety conjugate.

12. The supramolecular polymer according to claim 11, wherein the capped cyclodextrin-hydrophobic moiety conjugates have the following formula I-1: ##STR00030##

13. A siRNA-cyclodextrin complex comprising a supramolecular polymer according to claim 11.

14. A method for manufacturing a cyclodextrin-adamantane conjugate according to claim 7 having the following formula I-1: ##STR00031## the method comprising the following steps: a) benzylation of the hydroxyl groups of an - or -cyclodextrin, thereby obtaining a perbenzylated - or cyclodextrin, b) debenzylation of the benzyl groups in position A and D of the primary rim of the perbenzylated - or -cyclodextrin obtained in step a), by regioselective reduction, thereby obtaining a perbenzylated diol - or -cyclodextrin, c) oxidation of the hydroxyl groups to aldehyde in position A and D of the primary rim of the perbenzylated diol - or -cyclodextrin obtained in step b), by Swern oxidation, thereby obtaining a perbenzylated dialdehyde - or -cyclodextrin, d) double reductive amination with putrescine of the compound obtained in step c) thereby obtaining a capped perbenzylated - or -cyclodextrin in which the cap has the formula NH(CH.sub.2).sub.4NH, each N atom of which this cap being bound to each of the carbon atoms previously functionalized with an aldehyde group of the compound obtained in step c), e) reductive amination in presence of an adamantane group having a carbonyl functionality, of the capped perbenzylated - or -cyclodextrin obtained in step d), thereby obtaining a mixture of: a capped perbenzylated - or -cyclodextrin which is functionalized with one adamantane group, and a capped perbenzylated - or -cyclodextrin functionalized with two adamantane groups, f) purification of the mixture of compounds obtained in step e) by chromatography on a silica gel column, thereby obtaining the desired capped perbenzylated - or -cyclodextrin-adamantane conjugate functionalized with one adamantane group, g) debenzylation of the perbenzylated cyclodextrin-adamantane conjugate obtained in step f) by catalytic hydrogenation in water/THF in presence of trifluoroacetic acid, thereby obtaining a trifluoroacetate salt of the desired cyclodextrin-adamantane conjugate, and optionally, h) exchange of the trifluoroacetate ions with hydrochloride ions.

15. A method for manufacturing a supramolecular polymer according to claim 11 comprising a step of solubilization of a capped cyclodextrin-hydrophobic moiety conjugates in water or in an aqueous solution, the capped cyclodextrin-hydrophobic moiety conjugate comprising a cyclodextrin moiety and at least one hydrophobic moiety, wherein: the cyclodextrin moiety is capped on its primary rim by a cap binding a first carbon atom, previously bearing a hydroxyl group, of a first glucopyranose unit to a second carbon, previously bearing an hydroxyl group, of a second and different glucopyranose unit, said first and second glucopyranose units being preferably separated from each other by at least one, and the hydrophobic moiety is bound by a first linker to one of the carbon atoms of the cap, and the hydrophobic moiety is selected from the group consisting of an adamantane group, a C.sub.2-C.sub.13 alkyl group optionally containing at least one heteroatom, a C.sub.5-C.sub.6 aromatic group optionally containing at least one heteroatom, and a C.sub.3-C.sub.8 non-aromatic cycle optionally containing at least one heteroatom.

16. A method for manufacturing a siRNA-cyclodextrin complex according to claim 13 comprising the following steps: a) solubilization of capped cyclodextrin-hydrophobic moiety conjugates according to claim 1 in Dubelcco's Modified Eagle medium (DMEM), Stromal Vascular Fraction (SVF) 10% without antibiotics, b) incubation of the solution obtained in step a) during 5 minutes at a temperature comprised between 15 C. and 35 C., c) solubilizing SiRNAs in an Opti-MEM medium SVF, d) incubation of the solution obtained in step c) during 5 minutes at a temperature comprised between 15 C. and 35 C., and e) mixing the incubated solutions obtained in steps b) and d).

17. The method of claim 15, wherein the capped cyclodextrin-hydrophobic moiety conjugate has one of the following formulae I-1 to I-26: ##STR00032## ##STR00033## in which R is an amine group, an arginine group, a guanidine group, or a linear or branched chain comprising between 1 and 20 atoms for the main chain, these atoms being nitrogen atoms and/or oxygen atoms and/or carbon atoms, such a chain optionally comprising from 1 to 30 amine groups or guanidine groups, and in which x is equal to the number of amine group(s) plus the number of guanidine group(s) in R; ##STR00034## in which R is an amine group, an arginine group, a guanidine group, or a linear or branched chain comprising between 1 and 20 atoms for the main chain, these atoms being nitrogen atoms and/or oxygen atoms and/or carbon atoms, such a chain optionally comprising from 1 to 30 amine groups or guanidine groups, and in which x is equal to the number of amine group(s) plus the number of guanidine group(s) in R; ##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039## wherein in formulas I-4 to I-11, Ada designates an adamantane group and in formulas I-12 to I-17, Ad designates an adamantane group.

18. The method of claim 15, wherein the capped cyclodextrin-hydrophobic moiety conjugate has the following formula I-1: ##STR00040##

19. The capped cyclodextrin-hydrophobic moiety conjugate of claim 1, wherein said cap and said first linker, independently from each other, form, together with the carbon atoms to which they are bound, a chain having from 2 to 12 links.

20. The capped cyclodextrin-hydrophobic moiety conjugate according to claim 9, wherein in formulae I-6 and I-7, R is selected from the group consisting of an amine group, an arginine group, a guanidine group, and one of the following groups: ##STR00041## and in which x is equal to the number of amine group(s) plus the number of guanidine group(s) in R.

21. The method of claim 17, wherein in formulae I-6 and I-7, R is selected from the group consisting of an amine group, an arginine group, a guanidine group, and one of the following groups: ##STR00042## and in which x is equal to the number of amine group(s) plus the number of guanidine group(s) in R.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to prove several essential points of the invention such as the inclusion of the hydrophobic moiety (such as an Adamantane group) and the formation of the supramolecular polymer from the capped cyclodextrin-adamantane conjugates according to the invention, some experiments were carried out and their results are given below as examples of how to carry out the invention.

(2) These examples will be described with reference to the annexed figures in which:

(3) FIG. 1 shows the ROESY NMR spectrum of the supramolecular polymer obtained in example 3,

(4) FIG. 2 shows the ROESY spectrum of a cyclodextrin group functionalized with an adamantane group but in which the cyclodextrin group is not capped,

(5) FIG. 3 shows the agarose electrophoresis gel of an increasing N/P ratio of the supramolecular polymer obtained in example 3 in the presence of siRNA as described in example 7,

(6) FIG. 4 shows the viability of the cells and the transfection efficiency after treatment by an increasing N/P ratio of the supramolecular polymer obtained in example 3 in the presence of siRNA as described in example 7,

(7) FIG. 5 shows a synthesis scheme for synthesis of a conjugate in which the cyclodextrin group is a -cyclodextrin group in accordance with example 1,

(8) FIG. 6 shows a capped perbenzylated -cyclodextrin obtained in step d of example 1, and

(9) FIG. 7 shows a perbenzylated cyclodextrin-adamantane conjugate obtained in step e of example 1.

DETAILED DESCRIPTION

(10) These examples are only illustrative, and not limitative, of the invention.

(11) The reactants were purchased from commercial sources and used without further purification.

(12) Tetrahydrofurane (THF) was freshly distilled by standard methods on sodium/benzophenone.

(13) To characterize the compounds obtained during the synthesis of the compounds of the invention, NMR was used.

(14) NMR spectra were recorded on a Bruker Am-400 MHz or a Bruker Avance 600 MHz using CDCl.sub.3, DMSO-d6, and D.sub.2O as solvents.

(15) Assignments of the signals were done using Correlation Spectroscopy (COSY), Nuclear Overhauser Spectroscopy (NOESY), Heteronuclear Single Quantum Coherence Spectroscopy (HSQC), Heteronuclear Multiple Bound Correlation (HMBC), Total Correlation Spectroscopy (TOCSY), Transverse Rotating-frame Overhauser Enhancement Spectroscopy (T-ROESY).

(16) Diffusion ordered Spectroscopy (DOSY) NMR diffusion measurements were carried out by using the Longitudinal EDdy BiPolar gradient pulse (LEDBPGP) sequence.

(17) Sixteen spectra were acquired with gradient pulse (delta) of 4 ms ranging in strength from 0.28 to 5.26 g/mm for BBFO 5 mm NMR probe.

(18) A diffusion delay (delta) from 50 to 150 ms was used and the diffusion coefficients (D) were calculated with topspin 3.0.

Example 1

(19) Synthesis of a Conjugate According to the Invention in which the Cyclodextrin Group is a -Cyclodextrin Group.

(20) In the following of the present text CD designates a cyclodextrin group.

(21) Step a): Synthesis of Perbenzylated Cyclodextrin: Compound (2): CD Per Bn.

(22) ##STR00012##

(23) Protocol

(24) CD (compound 1) was lyophilized before use. Sodium hydride (21.4 g, 535 mmol, w/w 60% in oil) was added portionwise to a stirred solution of CD (10.4 g, 9.16 mmol) in DMSO (200 ml) at room temperature (rt), under N.sub.2. (chloromethyl)benzene (52 ml, 448 mmol) was then added carefully with vigorous mechanic stirring. Reaction was stirred overnight.

(25) The mixture was carefully hydrolysed with MeOH (40 ml) and diluted in water (200 ml). The solution was extracted with Et.sub.2O (3200 ml). The combined organic layers were washed with brine (2200 ml), dried under MgSO.sub.4 and concentrated. The resulting crude product was purified with a silica gel chromatographic column and eluted with Cyclohexane/AcOEt (95:5 then 3:1) to afford to compound (2) CD per Bn (26.6 g, 96%).

(26) The structure of the product was confirmed by comparison with the literature.sup.12. .sup.12 Chem. Eur. J. 2004. 10, 2960-2971.

(27) Step b): Synthesis of the Perbenzylated Diol -Cyclodextrin: Compound (3): CD Diol Per Bn.

(28) ##STR00013##

(29) Protocol:

(30) Compound (2) CD per Bn (15.2 g, 5.02 mmol) was solubilized in Toluene (35 ml). Diisobutylaluminum hydride (66 ml, 93 mmol) was added at rt. The mixture was heated at 60 C. under N.sub.2 flux during 1h30. The mixture was then poured carefully into an ice/water erlenmeyer (500 ml). EtOAc (500 ml) and HCl (300 ml) were then added. Solution was stirred overnight. The solution was extracted with 3300 ml d'AcOEt, washed with brine, dried with Mg504 and concentrated. The resulting crude product was purified with a silica gel column (400 ml) and eluted with Cyclohane/AcOEt (9:1 then 3:1) to afford to the compound (3) CD diol per Bn (10.4 g, 74%).

(31) The structure of the product was confirmed by comparison with the literature.sup.13. .sup.13 Eur. J. Org. Chem. 2010, 1510-1516.

(32) Step c): Synthesis of a Perbenzylated Dialdehyde -Cyclodextrin: Compound (4): Dialdehyde Per Bn.

(33) ##STR00014##

(34) Protocol:

(35) Oxalyl dichloride (3 ml, 35.5 mmol) was dissolved in DCM (30 ml) under Argon at 78 C. (methylsulfinyl)methane (5 ml, 70.4 mmol) was diluted in DCM (30 ml) and added over 30 min to the solution. Solution was stirred for 30 minutes. CD diol per Bn (compound 3) (10.5 g, 3.69 mmol) was dissolved in DCM (40 ml) and added slowly to the solution. Reaction was stirred for 1h30. Triethylamine (5.9 ml, 42.8 mmol) was added and the solution was stirred overnight and warmed slowly to rt. Solution was quenched with water (300 ml). The mixture was diluted in DCM, and layers were separated. Aqueous layer was extracted with DCM (3200 ml). The combined organic layers were washed with water (2300 ml), dried under Mg504, filtrated and concentrated. The resulting crude product was purified with a silica gel column and eluted with Cyclohexane/AcOEt (4:1) to afford to the compound (4) CD dialdehyde per Bn (9.2 g, 88%).

(36) The structure was not characterized by NMR but was confirmed by the reactivity of compound (4) in the following step.

(37) Step d): Synthesis of a Capped Perbenzylated -Cyclodextrin: Compound (5): CD Capped (C4) Diamine Per Bn.

(38) ##STR00015##

(39) Protocol:

(40) CD dialdehyde per Bn (compound 4) (9.2 g, 3.24 mmol) was solubilized in DCM (100 ml). Butane-1,4-diamine (0.4 ml, 3.99 mmol) and Sodium triacetoxyborohydride (3.5 g, 16.51 mmol) were then added at rt. The solution was stirred during 1h30. EtOAc (200 ml) and NaHCO.sub.3 (200 ml) were added. Layers are separated, aqueous layer is extracted with AcOEt (2200 ml). Organic layers were combined and washed with NaHCO.sub.3 and NaCl, dried with Mg504 and concentrated. The resulting crude product was purified with a silica gel column (700 ml) and eluted with Cyclohexane/AcOEt (4:1 then 6:4 with Et.sub.3N) to afford compound (5) CD bridge (C4) diamine per Bn (7.76 g, 83%).

(41) RMN (600 MHz, CDCl.sub.3):

(42) TABLE-US-00001 Cycle /G H-1 5.77 3.58 .sup.H.sub.4 C-1 97.88 H-2 3.55 C-2 78.05 H-3 4.10 C-3 81.00 H-4 4.00 3.99 .sup.H.sub.1 C-4 81.49 H-5 3.81 C-5 71.95 H-6 3.70 3.57 C-6 69.13 Cycle/C H-1 5.32 4.06 .sup.H.sup.4 C-1 98.40 H-2 3.47 C-2 79.07 H-3 3.99 C-3 81.64 H-4 3.91 3.92 .sup.H.sub.1 C-4 80.83 H-5 3.80 C-5 71.64 H-6 3.56 3.90 C-6 69.28 Cycle/D H-1 5.20 3.27 .sup.H.sub.4 C-1 99.29 H-2 3.51 C-2 78.96 H-3 4.11 C-3 81.31 H-4 4.06 4.06 .sup.H.sub.1 C-4 77.28 H-5 3.99 C-5 71.46 H-6 3.63 4.09 C-6 68.85 Cycle/B H-1 4.96 3.92 .sup.H.sub.4 C-1 99.56 H-2 3.44 C-2 78.63 H-3 3.96 C-3 80.52 ou H-4 3.92 3.91 .sup.H.sub.1 C-4 81.27 H-5 3.85 C-5 72.03 ou H-6 4.21 3.46 C-6 68.65 Cycle/F H-1 4.89 3.99 .sup.H.sub.4 C-1 99.70 H-2 3.41 C-2 78.79 H-3 3.98 C-3 81.70 ou H-4 3.81 3.81 .sup.H.sub.1 C-4 81.39 H-5 3.83 C-5 72.05 ou H-6 4.02 3.42 C-6 68.54 Cycle H-1 4.73 3.91 .sup.H.sub.4 /A C-1 98.13 H-2 3.31 C-2 80.23 H-3 3.98 C-3 81.70 ou H-4 3.58 3.58 .sup.H.sub.1 C-4 76.11 H-5 4.07 C-5 69.59 H-6 2.79 2.66 C-6 51.36 Cycle H-1 4.69 3.81 .sup.H.sub.4 /E C-1 98.92 H-2 3.27 C-2 79.80 H-3 3.96 C-3 80.52 ou H-4 3.27 3.27 .sup.H.sub.1 C-4 81.91 H-5 3.91 C-5 69.72 H-6 3.04 2.50 C-6 52.70

(43) Step e): Synthesis of the Perbenzylated Cyclodextrin-Adamantane Conjugate: Compound (8)

(44) ##STR00016##

(45) Protocol:

(46) To a solution of CD capped (C4) diamine per Bn (compound 5) (7.76 g, 2.68 mmol) in DCM (1200 ml) and Sodium triactoxyborohydride (2.9 g, 13.68 mmol) was added very slowly 2-((1r,3r,5r,7r)-adamantan-2-yl)acetaldehyde (0.5 g, 2.80 mmol) at 0 C. for 1 h. Then the reaction is stirred overnight at rt. Et.sub.2O (200 ml) and water (200 ml) were added, layers were separated. The Aqueous layer was extracted with Et.sub.2O (2100 ml). Organic layers were combined, washed with saturated NaHCO.sub.3 and brine. The resulting crude product contained a mixture of two compounds: a capped perbenzylated - or -cyclodextrin functionalized with one adamantane group, and a capped perbenzylated - or -cyclodextrin functionalized with two adamantane group.

(47) For obtaining the desired compound (8), the crude product was purified with a silica combi flash column (200 g) and eluted with Cyclohexane/AcOEt (gradient from 95:5 to 1:1 with Et.sub.3N) to afford the expected product: CD cap (C4) diamine mono Ad (AD) per Bn (1.86 g) and the starting material (compound 5) (4.25 g), Yield=41%.

(48) Global Yield=21.4%.

(49) RMN (600 MHz, CDCl.sub.3):

(50) TABLE-US-00002 Cycle /G H-1 5.66 3.49 .sup.H.sub.1 C-1 98.28 H-2 3.48 C-2 77.08 H-3 4.04 C-3 81.01 H-4 3.94 4.85 .sup.H.sub.1 C-4 81.16 H-5 3.82 C-5 71.85 ou 72.09 H-6 4.10 3.64 C-6 69.07 Cycle/D H-1 5.26 3.21 .sup.H.sub.4 C-1 99.44 H-2 3.46 C-2 78.72 H-3 4.06 C-3 81.67 H-4 4.00 5.19 .sup.H.sub.1 C-4 78.46 H-5 3.98 C-5 71.23 H-6 3.60 4.10 C-6 68.93 Cycle/C H-1 5.19 4.00 .sup.H.sub.4 C-1 98.43 H-2 3.39 C-2 79.38 H-3 3.90 C-3 80.29 ou 80.58 H-4 3.77 4.88 .sup.H.sub.1 C-4 81.24 ou 81.75 H-5 3.72 C-5 71.87 H-6 3.81 3.53 C-6 69.38 Cycle/B H-1 4.88 3.77 .sup.H.sub.4 C-1 100.29 H-2 3.33 C-2 78.46 H-3 3.90 C-3 80.29 ou 80.58 H-4 3.78 4.64 .sup.H.sub.1 C-4 81.24 ou 81.75 H-5 3.80 C-5 71.85 ou 72.09 ou 72.18 H-6 3.46 4.12 C-6 68.93 Cycle/F H-1 4.85 3.94 .sup.H.sub.4 C-1 99.38 H-2 3.36 C-2 79.52 H-3 3.90 C-3 80.29 ou 80.58 H-4 3.76 4.81 .sup.H.sub.1 C-4 81.24 ou 81.75 H-5 3.82 C-5 71.85 ou 72.09 ou 72.18 H-6 3.54 4.30 C-6 69.62 Cycle/E H-1 4.81 3.76 .sup.H.sub.4 C-1 99.72 H-2 3.23 C-2 79.89 H-3 3.95 C-3 80.97 H-4 3.21 5.26 .sup.H.sub.1 C-4 81.91 H-5 3.73 C-5 70.36 H-6 2.48 2.39 C-6 59.44 Cycle /A H-1 4.64 3.78 .sup.H.sub.4 C-1 98.45 H-2 3.25 C-2 80.51 H-3 3.92 C-3 81.75 H-4 3.49 5.66 .sup.H.sub.1 C-4 77.47 H-5 3.97 C-5 69.99 H-6 2.53 2.80 C-6 51.85

(51) Step f): Deprotection of the Perbenzylated Cyclodextrin Conjugate (Compound (8)) and Step e)) Exchange of the Trifluoroacetate Ions with Chlorhydrate Ions: (Compound (10))

(52) ##STR00017##

(53) Protocol:

(54) Compound (8) (101 mg, 0.033 mmol) was dissolved in THF/H.sub.2O (18/6 ml) under argon atmosphere in a 100 ml round bottom flask. 2,2,2-trifluoroacetic acid (20 l, 0.260 mmol) and palladium (100 mg, 0.940 mmol) were added and the reaction mixture was stirred under H.sub.2 atmosphere for 24 h. Then the mixture was purged under nitrogen, filtered through a pad of celite. The organic solvents were evaporated under vacuum and the residue was lyophilized. The crude product was filtrated through a micro filter and purified by HPLC (0-40 min/0-40%/ACN:H.sub.2O) to afford a white amorphous powder (24 mg, 46%). The powder was solubilized in the minimum volume of water, and eluted with water through a Ion exchange column (Amberlite Cl resin). The product is then lyophilized to afford to a powder that was solubilized in the minimum volume of water, precipitated in acetone and centrifuged. Acetone was removed. The operation was carried out twice to afford to a white solid, which was dissolved in water, and freeze dried to afford the expected product (10) as a white amorphous powder (17 mg, 32%).

(55) RMN (600 MHz, D.sub.2O):

(56) TABLE-US-00003 Cycle H-1 5.20 3.79 C-1 102.26 H-2 3.75 C-2 H-3 3.96 C-3 H-4 C-4 H-5 C-5 H-6 C-6 Cycle H-1 5.17 C-1 102.87 H-2 3.75 C-2 H-3 3.94 C-3 H-4 C-4 H-5 C-5 H-6 C-6 Cycle H-1 5.14 C-1 102.26 H-2 3.72 C-2 H-3 3.97 C-3 H-4 C-4 H-5 C-5 H-6 C-6 Cycle H-1 5.13 C-1 102.00 H-2 3.68 C-2 H-3 3.99 C-3 H-4 C-4 H-5 C-5 H-6 C-6 Cycle H-1 5.102 C-1 101.68 H-2 3.71 C-2 H-3 3.93 C-3 H-4 3.45 C-4 H-5 4.05 C-5 H-6 3.06 3.08 C-6 Cycle H-1 5.09 C-1 100.83 H-2 3.68 C-2 H-3 3.95 C-3 H-4 C-4 H-5 C-5 H-6 C-6 Cycle H-1 5.09 C-1 100.08 H-2 3.74 C-2 H-3 3.91 C-3 H-4 3.54 C-4 H-5 4.10 C-5 H-6 3.33 3.33 C-6 52.55

Example 2

(57) A cyclodextrin-adamantane conjugate in which the cyclodextrin is an -cyclodextrin has been manufactured as in example 1 by only using an -cyclodextrin instead of a -cyclodextrin.

Example 3

(58) In this example, a supramolecular polymer has been obtained from the cyclodextrin-adamantane conjugates (10) obtained in example 1.

(59) The monomer of cyclodextrin was first diluted in D.sub.2O at a concentration of 15.8 mM in order to carry out the NMR DOSY experiments.

(60) Then, several dilutions of this solution have been made in order to arrive to a final concentration of 0.079 mM.

(61) Water is the solvent in which a supramolecular polymer can be formed since cyclodextrins can form inclusion complex in this solvent only.

(62) D.sub.2O has the same properties as water.

(63) Aqueous solution can also be used, of course.

(64) In the DOSY analysis one can see that the size of the formed supramolecular polymer is more important than with the monomer. The above is true for all the tested concentrations.

(65) However, from a concentration of 5 mM a clear raised of the diffusion coefficient is observed.

(66) The formation of the supramolecular polymer has been demonstrated by NMR DOSY.

(67) In this study, three compounds were studied. compound 10 obtained in example 1, which is a capped cyclodextrin-adamantane conjugate according to the invention, the cycicodextrin-adamantane conjugate described in the article Cyclodextrin-adamantane conjugates, self-inclusion and aggregation versus supramolecular polymer formation, Org. Chem. Front. 2014, 1, 703-706. This conjugate is made of a -cyclodextrin functionalized with an adamantane group. But the cyclodextrin group is not capped. It has been demonstrated in the article Cyclodextrin-adamantane conjugates, self-inclusion and aggregation versus supramolecular polymer formation that this conjugate does not form a supramolecular polymer because the adamantane self-includes in the cavity of the cyclodextrin on which it is bound, the capped cyclodextrin with the formula I-1

(68) ##STR00018##

(69) The results of the NMR DOSY study of this example are that the diffusion coefficient of the compound functionalized with an adamantane group but in which the cyclodextrin is not capped does not vary as a function of its concentration, this being the confirmation of the fact that it does not form a supramolecular polymer.

(70) In the same manner, the diffusion coefficient of the capped cyclodextrin not functionalized with an adamantane group does not vary as a function of its concentration. This confirms that this compound cannot form a supramolecular polymer because not having an adamantane group.

(71) In contrast, the NMR DOSY study of the conjugate (compound 10 of example 1) of the invention shows a variation of the diffusion coefficient as a function of its concentration.

(72) Indeed, at high concentration (15.8 mM) a diffusion coefficient higher than at low concentration (0.079 mM) is seen, confirming that the conjugate of the invention forms species having more important sizes at high concentration.

(73) The inclusion of compound 10 obtained in example 1 in the cavity of the cyclodextrin of an other compound 10 of the invention has been demonstrated by ROESY (Rotating Frame OverHause Effect Spectroscopy) NMR study.

(74) The ROESY NMR spectrum of the conjugate obtained in example 1 is shown in FIG. 1.

(75) This spectrum is difficult to read due to the presence of the CH.sub.2 of the linker(cap) under the signal of the H of the adamantane.

(76) Nevertheless, it is possible to see that the adamantane group is included in the cavity of the cyclodextrin group due to the correlation of the H-3s of the cyclodextrin and the Ha and Hb of the adamantane and even in very clear manner with the two H5s of the adamantane.

(77) But no correlation is seen between the H-5s and the protons of the adamantane which means that the adamantane is included in the cavity of the cyclodextrin. Because the self-inclusion of the adamantane in the cavity of the cyclodextrin to which it is bound is impossible due to the the capping of the cyclodextrin cavity, the adamantane group which is included in the cavity of the cyclodextrin group is indeed the adamantane group of an other cyclodextrin-adamantane conjugate.

(78) For reinforcing this demonstration, the ROESY NMR analysis of the cyclodextrin functionalized with an adamantane group in position A of its primary rim but not capped has been made.

(79) The ROESY spectrum of this compound is shown in FIG. 2.

(80) As can be seen in FIG. 2, in this compound the adamantane group is indeed included in the cavity of the cyclodextrin, but upside down.

(81) Indeed, the 2D spectrum obtained with this analysis clearly shows a correlation between the H-5s of the cyclodextrin and the Ha of the adamantane while the H-3s of the cyclodextrin correlate with the Hb and Hc of the adamantane.

Example 4: Preparation of the Compound of Formula I-26

(82) ##STR00019##
This compound is prepared according to the following synthesis scheme.

(83) ##STR00020##
For obtaining the compound of Formula I-26, noted 4 in the above synthesis scheme, in which R is NH.sub.2CNHNH(CH.sub.2).sub.3CHNH.sub.2, a reductive amination is first carried out on compound 1 which is a perbenzylated capped betacyclodextrin in order to obtain compound noted 2 in the above synthesis scheme. The protective group Boc of the amine group of the second linker is then removed in an acidic medium in order to obtain a NH.sub.2 group thereby obtaining compound noted 3 in the above synthesis scheme. The amine group then enables the functionalization of compound 3 via a peptidic coupling with carboxylic acids. Compound 4 is obtained by carrying out the removal of the protecting groups of Compound 4 in which R is NH.sub.2CNHNH(CH.sub.2).sub.3CHNH.sub.2 i.e. the compound of Formula I-26

Example 5: Preparation of a Cyclodextrin-siRNA Complex

(84) Compound 11 obtained in example 1 is treated in a DMEM medium (Dubelccos's modified Eagle's medium), SVF (Stromal Vascular Fraction) 10%, without antibiotics.

(85) Then it is incubated during five minutes at ambient temperature.

(86) The siRNAs are treated in a Opti-MEM medium SVF (poor in a fetal veal serum) and then incubated during 5 minutes at ambient temperature.

(87) Two amounts of siRNA have been prepared: 5 pmol and 10 pmol.

(88) Each solution of siRNA are then mixed with a solution of compound 10 and complexed during 20 minutes at ambient temperature.

(89) Different ratio nitrogen/phosphate (NIP) of cyclodextrin-siRNA have been chosen varying from 6 to 1190.

Example 6: Comparative Example: Preparation of a Lipofectamine 2000.siRNA Complexes

(90) A Lipofectamine 2000.siRNA complexes have been prepared as follows.

(91) These complexes are used as positive references of transfection of RNA.

(92) Lipofectamine 2000 (Invitrogen) treated in a DMEM, SVF 10%, medium, without antibiotics and the incubated during 5 minutes at ambient temperature.

(93) The siRNAs are treated in a Opti-MEM poor in a fetal veal serum medium and then incubated during 5 minutes at ambient temperature.

(94) Two amounts of siRNA were prepared: 5 pmol and 10 pmol.

(95) The solutions of siRNA and of Lipofectamine are then mixed and complexed during 20 minutes at ambient temperature: 50 L of Lipofectamine are combined to 50 L of siRNA.

(96) The cell line to be transfected is a cell line of human embryonic kidney 293 cells (HEK293). This cell line is cultivated with a DMEM medium to which SVF (10%) is added.

(97) The antibiotics penicillin and streptomycin as well as the antibiotic hygromycin B are also added.

(98) The cultures are made in an incubator at 37 C., 5% of CO.sub.2. The cell line HEK293 express constitutively the firefly luciferase GL3.

(99) The HEK293 cells are deposited in a 96 wells plaque 1.Math.10.sup.3 cell/wells.

(100) The siRNA which is used is the luciferase GL3 siRNA.

(101) The siRNA corresponding to the firefly luciferase issued from plasmid PGL3-basic (Promega) has been synthesized by Sigma.

(102) The sequence indicated is under the form of an RNA (19nt) to which is added 2T in 3(dTdTd for deoxyDNA).

(103) The sequences are:

(104) TABLE-US-00004 (sensestrand) 5-CUUACGCUGAGUACUUCGAdTdT-3 (antisensestrand) 5-UCGAAGUACUCAGCGUAAGdTdT-3

Example 7: KnockDown Test of the Luciferase GL3 Gene

(105) Transfection of cell line HEK293 with the siRNA directed against the firefly luciferase GL3 with as vector of transfection the compound 10 obtained in example 1 or Lipofectamine 2000 (used as a positive reference of transfection) has been carried out.

(106) The efficacy of transfection on the cells has been evaluated by a method of luminescence.

(107) One day before the transfection, the HEK293 cells are deposited in a 96 wells plaque: 1.Math.10.sup.3 cell/wells treated in 200 L of DMEM, SVF 10%, without antibiotics, in order that the cells reach a 30-50% of confluence the day after.

(108) The day of the transfection, the 200 L/wells of medium are withdrawn.

(109) After preparation of the complex of cyclodextrin-siRNA according to the invention and of Lipofectamine 2000-siRNA, 100 L/wells of solution of siRNA-cyclodextrin of the invention or 100 L/wells of solution of siRNA Lipofectamine 2000 are deposited in the plaque.

(110) This latter is incubated at 37 C. during 24 hours until the analysis by luminescence of the KD of the gene of the luciferase (Kit One-Glo luciferase assay (Promega)).

(111) For this aim, 100 L/wells of reactants are added to the cells before the plaque be analyzed by luminescence.

(112) Toxicity Test

(113) In order to evaluate the toxicity of compound 11 of example 1 on HEK293 cells, the kit CellTiter Glo 2.0 (Promega) has been used.

(114) One day before the transfection, the cells HEK293 are deposited in a 96-wells plaque: 1.Math.10.sup.3 cell/wells and then treated with 200 L of DMEM, SVF 10%, without antibiotics, in order that the cells reach a 30-50% of confluence the day after.

(115) The day of the transfection, the cells are incubated with the complex siRNA-cyclodextrin conjugate obtained in example 1 and Lipofectamine 2000.siRNA at 37 C. during 24 hours until analysis of the cells viability (ATP activity) using a kit CellTiter Glo 2.0.

(116) For this aim 100 L/wells of CellTiter Glo 2.0. reactant are added to the cells before the plaque be analyzed by luminescence.

(117) Agarose Gel Electrophorese

(118) In order to characterize the formation of complexes between the siRNA and the compound 11 of the invention obtained in example 1, an electrophorese technique on agarose gel has been used.

(119) The molecule of siRNA and of compound of example 1 have been migrated according to N/P previously chosen.

(120) A solution of 17 L of siRNA-compound of example 1 is prepared in sterile water and incubated during 20 minutes at ambient temperature.

(121) Then, this solution is deposited on the gel with 3 L of bromophenol blue before launching the migration: 100 V, 40 min.

(122) One can conclude from these experiments that, thanks to ROESY-NMR studies, it has been shown that the capping of the cyclodextrin derivative avoid the self inclusion of the hydrophobic moiety. Secondly, thanks to DOSY-NMR studies, it has been shown that both the capping and the functionalization of the cyclodextrine derivative are essential to obtain results which are in agreement with the formation of supramolecular polymer.

(123) Third, thanks to the experiment with agarose electrophoresis gel, it has been shown that capped cyclodextrine-adamantane conjugate are able to interact and to complex siRNA. Indeed, capped cyclodextrine-adamantane complex formation was evaluated at increasing N/P ratios using agarose gel electrophoresis. (FIG. 3)

(124) N/P ratio corresponds to the amount of protonated aminated residue of the cationic species divided by the amount of phosphate residues coming from the nucleic acids..sup.14 This ratio enables to compare the efficiency of a cationic compound to be complexed with nucleic acids. .sup.14 Pharmaceutics. 2011, 3, 125-140

(125) $\mathrm{Thus},\frac{N}{P}=\frac{\begin{array}{c}\mathrm{number}\mathrm{of}\mathrm{all}\mathrm{cationic}\mathrm{compounds}.Math.\\ \mathrm{number}\mathrm{of}\mathrm{charges}\mathrm{by}\mathrm{molecule}\end{array}}{\mathrm{number}\mathrm{of}\mathrm{moles}\mathrm{of}\mathrm{siRNA}.Math.\mathrm{number}\mathrm{of}\mathrm{base}\mathrm{pairs}2}.$

(126) In the present case, the cationic compound is a conjugate according to the invention, the number of charges by molecule is the number of charge by conjugate of the invention and the number of base pairs is the number of base pairs of the siRNA.

(127) The positive charges of the amine group in the capped cyclodextrine-adamantane conjugate have the ability to associate with the negatively charged phosphate groups of the siRNA. While uncomplexed siRNA migrated freely through the gel, complete binding of siRNA with capped-cyclodextrine-adamantane conjugate appeared to occur abruptly at low N/P ratios, with no migration of siRNA. These results demonstrate that the negatively charged siRNA had been effectively complexed by the capped-cyclodextrine-adamantane conjugate.

(128) Finally, the results of transfection also showed the capacity of the capped cyclodextrine-adamantane conjugate to transfect siRNA. The in vitro transfection activity of capped cyclodextrine-adamantane vector was evaluated in HEK293 cells at a range of N/P ratios. The HEK293 cells have a constitutive expression of the firefly luciferase (GL3). Untransfected cells was considered as 100% of luciferase expression (negative control). A reduction in GL3 luciferase expression was achieved with transfection in HEK293 cells with the capped cyclodextrine-adamantane siRNA complexes (compose 11). In fact, until 50% knockdown was observed for a transfection at the N/P ratio of 1190. These transfection efficiencies are lower than that of lipofectamine 2000 (knockdown of 90%). Nevertheless, over the range of N/P ratios, transfection with capped cyclodextrine-adamantane siRNA complexes exhibited no cytotoxicity. Overall, these tests showed the ability of capped-cyclodextrine-adamantane (compose 11) to transfect siRNA in HEK293 cells with a favorable toxicity profile. (FIG. 4)