CZ301772B6 - Alpha-cyclodextrin dimer, process for its preparation and its use - Google Patents

Abstract

The present invention relates to an alpha-cyclodextrin dimer of the general formula V coupled by disulfide bonds in positions C6eI-C6eI' and C6eIV-C6eIV', further to process for its preparation and its use for completing organic molecules and their re-detachment by the action of reducing thiols. This dimer of the general formula V can be particularly useful for targeted transport of medicaments with suitable structure.

Description

Technical field

This invention relates to cyclodextrin dimer, in which two alpha-cyclodextrin macrocycles disulfide bonds at positions 6 and ^{L l} C6 C6 C6 ^{IV IV,} method of preparation, the ability to complex organic molecules inside the cavity and re-dimer is released with reducing agents , especially L-glutathione.

BACKGROUND OF THE INVENTION

Cyclodextrins (alpha, beta and gamma) are used in a number of industries for the complexation of organic molecules. In the pharmaceutical industry, cyclodextrin complexes are used to increase the solubility of substances that are very poorly soluble in aqueous media, and to gradually release the complexed drug after administration to the patient. The average stability constant of the cyclodextrin complex with the substance molecule, which by its dimensions corresponds to the dimensions of the inner cavity, is K = Kr ^{, 5 ± 1} ' ¹ M ^-1 (Houk K .. N .; Leach AG; Kim SP; Zhang X. Angew. Chem .

Int. Ed. 2003, 42, 4872). These complexes are completely dissociated very quickly under physiologically relevant conditions (less than millimolar concentrations), which prevents the development of their other applications, for example, as carriers for targeted drug delivery. Therefore, considerable efforts have been made to increase the inner surface of the cavity of cyclodextrins by dimerizing them and thereby stabilizing the guest molecule in the cavity by a stronger hydrophobic interaction (Slíwa W .; Girek T .; Koziol 1, J. Curr. Org. Chem. 2004, 8,

1445,). In a number of published works, the authors use one connector of different lengths for dimerization of alpha- or beta-cyclodextrin cavities. Although sporadically these experiments have suggested that dimers can indeed bind organic molecules with a binding constant up to several orders of magnitude higher than native cyclodextrins, in many other cases of conformation the flexibility of a single linkage causes both cavities not to cooperate when complexing the guest molecule, creating complex with cyclodextrin stoichiometry - guest 1: 2 with low stability constant. Attempts to solve this deficiency have led to the introduction of another bridge that connects adjacent glucose units. This, however, allows two stereoisomers to be formed, one of which is capable of cooperatively binding the guest in the cavity, for spherical reasons, is not preferred, and has not been isolated at all in the described cases, or has been produced only in minority (Breslow R .; ChungS. J). Am Chem Soc.

1990, 112, 9659). Therefore, it is desirable to find a method allowing easy synthetic access to rigidly coupled cyclodextrin dimers and to investigate their ability to complex organic molecules. Furthermore, it is advantageous to design linkers between cyclodextrin macrocycles so that they can later be cleaved by an external stimulus, which would destabilize the entire complex and release the guest molecule into the environment.

- 1 GB 301772 B6

SUMMARY OF THE INVENTION

The above-mentioned drawbacks of the described method of technology virtually eliminate the compound

V according to the invention, in which two alpha-cyclodextrin macrocycles disulfide bonds at positions ¹ Co-C6 and C6 ^{IV IV.}

Another object of the invention is a method for producing a compound of formula V, in which the starting material is 2 ^I, 2 ^II, 2 ^III, 2 ^l, 2 ^V, 2 ^{VI, 3} ', 3 ^n, 3 ^ni, 3 ^IV, 3 ^V, 3 ^l O ^{Li 6 1I, 6, 6-yl} -hexadekakis T7 benzyRx-cyclodextrin of the formula I.

This is the action of a brominating agent, preferably using carbon tetrabromide and triphenylphosphine in a suitable solvent, preferably dimethylformamide, is converted to ² ', 2 ^II, 2 ^1ii, 2 ^{l, 2,} 2, ^l, 3 ^I, 3 ^II> 3 ^I 3 ^{l, 3,} 3 -dodekakis ^{yl-O-benzyl-6 l,} 6-dibromo ^l 6 ^l, 6-dideoxy acyklodextrin ^IV of structural formula II.

-2CL JW1 / ií DO

In the next step, the protecting groups in Π are removed, preferably by catalytic hydrogenation in the presence of palladium on activated carbon as catalyst and hydrogen. The result is a derivative of 6 ', 6 ^N dibromo-6', 6 ^IV dideoxy-Ot "cyclodextrin ΙΠ structural formula.

ΓΠ derivative by reaction with thiourea or potassium thioacetate and subsequent basic hydrolysis, preferably sodium hydroxide in water - ethanol affords 6 \ 6 ^iv -dideoxy10 6 ^[6 -disulfanyl ^{LV-cyclodextrin} IV.

The derivative IV is then oxidized in basic medium, preferably under air oxidation conditions in 15 aqueous ammonium acetate at pH 9, thereby converting it to the dimer of structural formula V.

It is a further object of the present invention to determine the ability of dimer V to very tightly bind substances in aqueous solutions having dimensions corresponding to the dimensions of the inner cavity. They are, for example, general substances

As well as unsaturated linear hydrocarbons having carbon numbers of five or more and derivatives thereof, cyclic hydrocarbons and derivatives thereof, and heterocyclic compounds including derivatives thereof.

R ¹ - (CHjX -R ³ R ', R ² = OH, COOH, NH ₂

VI n = 8-20

Another object of the invention is to cleave dimer V and its complexes with other substances by treatment with reducing agents, preferably L-glutathione, to form the disulfanyl derivative IV (or other mixed disulfides) and release the complexed molecule to the environment. In this way, it is also possible to regulate the release of the complexed molecule by an external stimulus.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of ^2, 2) 2 ^l 2 ^{l, 2,} 2, ^l, 3 ', 3> 3 ^l 3 ^{l, 3,} 3 -dodekakis ^{yl-O-benzyl-6 l} 6 ^l dibromo-6 ^{1,6 lv} -dideoxy-α-cyclodextrin (II)

In a Schlenk tube, the compound of formula I (2 g, 0.83 mmol), triphenylphosphine (1.15 g, 4.38 mmol) and tetrabromomethane (1.44 g, 4.34 mmol) were gradually dissolved under stirring under argon. anhydrous DMF (24.4 mL). The reaction mixture was heated at 75 ° C for 12 hours and then quenched by cooling to room temperature and dropwise addition of methanol (0.5 mL).

The solvents were evaporated on a vacuum evaporator. The residue was taken up in a mixture of chloroform (500 ml) and water (300 ml). The organic phase was washed with water (4 x 300 mL), then dried over anhydrous sodium sulfate, filtered and evaporated in a vacuum evaporator. The crude product was purified by column chromatography (150 g silica gel, isocratic elution with toluene / acetone 99: 1). The obtained product of formula II was obtained as a foam (1.81 g, 86%) after evaporation of the solvents and drying under reduced pressure.

Example 2

Preparation of 6-β-dibromo-β-β-dideoxy-α-cyclodextrin (ΙΠ)

In a mixture of DMF and ethanol (40 mL, 1: 1) was dissolved the compound of formula II (500 mg, 0.20 mmol) and Pd / C catalyst (10% w / w, 125 mg) was added. After bubbling with argon, the mixture was placed in a glass vessel in an autoclave and stirred for 4 hours at 40 atmospheres of hydrogen (i.e.

4,053 MPa). Then the catalyst was separated by centrifugation. On a vacuum evaporator, the mixture was evaporated to dryness, then dissolved in a mixture of ethanol and water (20 mL, 1: 1) and filtered through a reverse phase silica gel column (3 g). Evaporation of the solvents on a vacuum evaporator gave the product of formula III (205 mg, 93%).

Example 3

Preparation of 6 ^L , 6 ^{L in} -dideoxy-6 ^L , 6 ^IV- disulfanyl-α-cyclodextrin (IV)

In a Schlenk tube, the compound of formula III (509 mg, 0.463 mmol) and thiourea (543 mg, 7.13 mmol) were dissolved in a mixture of ethanol and water (15 mL, 7: 3) with stirring. The mixture was aerated three times in a vacuum-argon cycle and then heated to 75 ° C with stirring. After 12 hours, the mixture was cooled to 0-5 ° C under argon. A 10% aqueous sodium hydroxide solution was then added dropwise to the reaction mixture via septum

-4CL JUi / X DO (3.0 mL, 7.5 mmol). The reaction mixture was degassed three times by the argon-cycle vacuum and the Schlenk tube was heated at 75 ° C for 12 hours. The mixture was then allowed to cool to room temperature and sodium borohydride (36 mg, 0.95 mmol) was added. After half an hour, the mixture was acidified with 1M hydrochloric acid to pH 5 and filtered through a reverse phase silica gel column (3 g). The column was washed with 3: 7 methanol / water. On a vacuum evaporator, the filtrate was evaporated to a quarter volume and purified by column chromatography on an HPLC column (250 x 21 mm, reverse phase silica gel) with gradient elution (methanol / water / trifluoroacetic acid 10: 89.9: 0.1 to 25: 74.9: 0.1). The combined fractions were evaporated in a vacuum evaporator to give a white product 10 (262 mg, 56%).

Example 4

PREPARATION 6 \ 6 ^dideoxy-l O \ ^6, -disulfanyl- <i-cyclodextrin (IV) (variant using potassium thioacetate as a reagent)

In a Schlenk tube, ΠΙ (509 mg, 0.463 mmol) and potassium thioacetate (577 mg, 5.06 mmol) were dissolved in a mixture of ethanol and water (15 mL, 7: 3) with stirring. The mixture was vented three times in a vacuum-argon cycle and then heated to 75 ° C with stirring. After 12 hours, the mixture was cooled to 0-5 ° C under argon. A 10% aqueous sodium hydroxide solution (3.0 mL, 7.5 mmol) was then added dropwise to the reaction mixture over the septum. The reaction mixture was degassed three times by the argon-cycle vacuum and the Schlenk tube was heated at 75 ° C for 12 hours. The mixture was then allowed to cool to room temperature and sodium borohydride (36 mg, 0.95 mmol) was added. After half an hour, the mixture was acidified to pH 5 with 1M hydrochloric acid and filtered through a reverse phase silica gel column (3 g). The column was washed with a 3: 7 mixture of methanol and water. On a vacuum evaporator, the filtrate was evaporated to a quarter volume and purified by column chromatography on an HPLC column (250 x 21 mm, reverse phase silica gel) with gradient elution (methanol / water / trifluoroacetic acid 10: 89.9: 0.1 to 25: 74.9: 0.1). Evaporation of the combined fractions containing the desired compound afforded the product of formula IV as a white powder (337 mg, 72%).

Example 5

Preparation of α-cyclodextrin (V) dimer

The derivative of formula IV (134 mg, 0.133 mmol) was dissolved in a mixture of 0.05 M aqueous ammonium acetate solution (25 mL, pH 9) and acetonitrile (2 mL). The reaction mixture was stirred at room temperature for 48 hours. The precipitated product precipitate was separated from the solution by filtration through a frit and then dried under reduced pressure at 40 ° C. The filtrate was evaporated to half volume on a vacuum evaporator and loaded onto a column (250 χ 21 mm, reverse phase silica gel). Subsequent gradient elution (from acetonitrile / water 5: 95 to 10: 90) after evaporation of the fractions gave a second crop of product.

Portions of the product obtained by filtration of the reaction mixture and the filtrate chromatography were combined and recrystallized from boiling water. After 24 hours, the solution was filtered through a frit and the obtained crystalline product was dried under reduced pressure. The material obtained (124 mg, 93%) is a colorless crystalline solid (m.p. > 200 ° C, with decomposition).

-5GB 301772 B6

Example 6

Dimem V complex with 1.10 decanediol

Dimer (V) (20 mg, 0.01 mmol) was dissolved in water (10 mL), followed by addition of 1.10-decanediol (1.74 mg, 0.01 mmol) and heating to boiling with stirring. The reaction mixture was then allowed to cool to room temperature with stirring. The resulting solution was evaporated and dried under reduced pressure to isolate the complex of both (21.7 mg).

Example 7

Determination of the binding constant of the dimer V complex with 1.10 decanediol

In a Microcal® titration microcalorimeter, a 10 ^-5 M solution of 1.10 decanediol in 0.01 M phosphate buffer at pH 6.4 was titrated with a 10 ⁴ M dimer V solution in the same buffer. Deconvolution of the binding isotherm yielded a binding constant of the complex of both substances of 4.2 x 10 ⁶ M ^-1 .

Example 8

Cleavage of dimer V by L-glutathione

Dimer of formula V dissolved in a solution of 10 mM phosphate buffer in 1 mM deuterium oxide (0.25 mL, 2.5x10 ⁷ mol) at pH 7 was mixed with 4 mM L-glutathione solution (0.25 mL) under an inert argon atmosphere. , 1.0x10 ⁶ mol). The mixture was allowed to react for 48 hours under argon. It was then analyzed by 1 H NMR spectroscopy. By integrating the * HNMR signals, the proportion of the components in the mixture was calculated, which showed that 91% of the dimer was cleaved by glutathione.

Claims (10)

PATENT CLAIMS An alpha-cyclodextrin dimer of formula V ^c = 10 ° OH HO _H2 , -OH 2. Apply the preparation of alpha-cyclodextrin dimer of formula V according to claim 1, characterized 10 in that the starting compound 2 ^', 2 ^u, 2 ^{nl, 2,, 2,} 2'^'l, 3, ^1, 3.3 "', 3 ^{IV> 3, 3'} .6 ^II 6 ^{'n 6> VI} -hexadekakis 6-O-benzyl-cyclodextrin of formula I 15 is converted to a bromine reagent in a suitable solvent 2 ^I, 2 ^n, 2 ^III, 2 ^IV, 2 ^V, 2 ^VI 3 ^{I, N} 3, 3 ^ni, 3 ^IV, 3 ^V, 3 ^VI-C -dodekakís> ^O-benzyl-I, 6 ^IV -dibr0m 'O ^i, 6 ^l -did _E0 XY acyklodextrin formula II from which they are in the next deprotection step, thereby forming OLO ^{OLO-dibromo-15 * 17-dideoxy} acyklodextrin III Which is reacted with thiourea or potassium thioacetate followed by basic hydrolysis to yield 6-β-dideoxy-β-β-disulfanyl-α-cyclodextrin IV which is then oxidized in basic medium to convert it into a basic medium. dimer of formula V 3. A process according to claim 2 wherein the brominating agent is tetrabromomethane with triphenylphosphine. Process according to either of Claims 2 and 3, characterized in that dimethylformamide is used as the solvent. A process according to any one of claims 2 to 4, characterized in that catalytic hydrogenation is used to remove the protecting group. -8jui // x no Process according to any one of claim 5, characterized in that catalytic hydrogenation in the presence of palladium on activated carbon catalyst and hydrogen is used as catalytic hydrogenation. Process according to any one of claims 2 to 6, characterized in that the basic hydrolysis is carried out with sodium hydroxide in a water-ethanol mixture. Process according to any one of claims 2 to 7, characterized in that air oxidation in aqueous ammonium acetate solution at pH 9 is also used as the oxidation in basic medium. A process according to any one of claims 2 to 8, wherein the compound of formula ΙΠ is reacted with potassium thioacetate followed by basic hydrolysis 15 the product IV. Use of dimer V as defined in claim 1 for the complexation of organic substance molecules, in particular drugs. A method for cleaving the alpha-cyclodextrin dimer of formula V as defined in claim 1, characterized in that the dimer complex of formula V containing an organic substance in the cavity is treated with reducing thiols, thereby releasing the complexed substance from the dimer cavity. 25. The cleavage process according to claim 11, wherein the reducing thiol is L-glutathione.