4,6-DI-(O-THIOPHOSPHATE)-INOSITOL-1,2,3,5-TETRA-O-SULFATE FOR C. DIFFICILE INFECTION

Abstract

The present invention relates to inositol bisthiophosphates-tetrakissulfates, particularly for use in treating symptoms associated with Clostridium difficile infection.

Claims

1. A compound characterized by the general formula (I) ##STR00004## wherein two out of six X are OPSO.sub.2.sup.2 and the remaining X are OSO.sub.3.sup..

2. The compound according to claim 1, characterized by the formula (IIa) or (IIb) ##STR00005##

3. A compound according to claim 1 for use in the therapy or prevention of a disease.

4. A compound according to claim 1 for use in the therapy or prevention of C. difficile infection or in the prevention or therapy of symptoms associated with C. difficile infection.

5. A dosage form comprising the compound according to claim 1.

6. A compound according to claim 2 for use in the therapy or prevention of a disease.

7. A compound according to claim 2 for use in the therapy or prevention of C. difficile infection or in the prevention or therapy of symptoms associated with C. difficile infection.

8. A dosage form comprising the compound according to claim 2.

Description

SHORT DESCRIPTION OF THE FIGURES

[0021] FIG. 1 shows the extent of cleavage of TcdB in the presence and absence of Ca.sup.2+ (10 mM) for IP6, IP2S4 and activator compound IT2S4 (IIa).

[0022] FIG. 2 shows .sup.1H-NMR and .sup.31P-NMR of compound (IIa).

[0023] FIG. 3 shows the extent of TcdB cleavage in presence of 10 mM CaCl.sub.2 for inositol hexaphosphate (IP6), inositol hexasulphate (IS6), and two mixed phosphate-sulfate compounds.

EXAMPLES

Example 1: Synthesis of Compound (IIa)

[0024] The synthesis followed the sequence depicted in the scheme below:

##STR00003##

[0025] Phosphorylation

[0026] The known 2-tertbutyldimethylsilyl inositol orthoformate was co-evaporated 3 with toluene and dissolved in dichlormethane (DCM). 1H-tetrazole (4 eq.) followed by phosphoramidite (8 eq.) were added to the reaction and stirred overnight. Pyridine, followed by crushed sulphur flakes (20 eq.) were added to the reaction and stirred overnight. The resulting crude mixture was diluted with DCM and washed with saturated NaHCO.sub.3, dried with Na.sub.2SO.sub.4, filtered and concentrated. The product was purified by flash chromatography with DCM in toluene.

[0027] .sup.1H-NMR (400 MHz; CDCl.sub.3): 7.35-7.29 (m, 4H), 7.15 (dd, J=6.6, 2.1 Hz, 2H), 7.07-7.04 (m, 2H), 5.54 (d, J=1.1 Hz, 1H), 5.45-5.41 (m, 2H), 5.30-4.97 (m, 8H), 4.51-4.49 (m, 1H), 4.33-4.32 (m, 2H), 4.27 (d, J=1.3 Hz, 1H), 0.93 (s, 9H), 0.13 (s, 6H);

[0028] .sup.31P-NMR (162 MHz; CDCl.sub.3): 70.1

[0029] Deprotection

[0030] The following deprotection conditions are in analogy to the synthesis published in the Journal of the American Chemical Society [JACS 2005, 127, 5288].

[0031] Starting material (50 mg) was treated with thiophenol (300 l), m-cresol (300 l), trifluoroacetic acid (1.8 ml). Then added TMSBrOH slowly (360 l). Stirred 2 h at room temperature. Evaporated twice from toluene. Diluted with DCM, and ca. 5 ml water.

[0032] Neutralized with 1N NaOH. Poured aqueous layer (slightly cloudy) directly on SolEx C18 cartridge (Thermofisher, 1 g, 6 ml). Eluted with water. In some cases some aromatic impurities were found but would precipitate over time in water and could be filtered-off.

[0033] .sup.1H-NMR (500 MHz; D.sub.2O): 4.36 (q, J=9.6 Hz, 2H), 4.02 (t, J=2.7 Hz, 1H), 3.64 (dd, J=9.7, 2.8 Hz, 2H), 3.50 (t, J=9.3 Hz, 1H).

[0034] .sup.31P-NMR (203 MHz; D.sub.2O): 45.7

[0035] Sulfation

[0036] The sulfation reaction of the thiophosphate has to be performed carefully because the thiophosphate is eventually converted to the phosphate under the reaction conditions. We thus monitored the sulfation carefully and saw that the reaction was complete after ca. 30 min. and that no decomposition could be observed in this time. Thus, sulphurtrioxide dimethylformamide (SO.sub.3-DMF) complex (12 eq.) was added to a suspension of inositol phosphate in DMF and the reaction was stirred 35 min. The reaction was quenched by adding 1N NaOH, until ca. pH 8 followed by ca. 3 ml methanol (MeOH) to precipitate salts. The solid was purified by Sephadex LH-20 column, eluting with water.

[0037] .sup.1H-NMR (500 MHz; D.sub.2O): 5.06 (s, 1H), 5.04-4.98 (m, 4H), 4.79-4.76 (m, 1H).

[0038] .sup.31P-NMR (203 MHz; D.sub.2O): 44.5

[0039] .sup.1H-NMR and .sup.31P-NMR results are shown in FIG. 2.

Example 2: Comparison of Cleavage Efficiency

[0040] IP6, activator compound (IIa) and IP2S4 were compared with regard to the extent of cleavage of TcdB (FIG. 1). The compound to be tested was added at 1 mM to 150 ng toxin B in presence or absence of 10 mM Ca.sup.2+ in 100 mM Tris pH7.4 and incubated for 3 h at 37 C. Cleaved protein fragments were separated by SDS-PAGE and visualized by silver staining. The extent of cleavage was quantified from protein band intensities using the ImageJ software package. Signals were normalized to cleavage of positive and negative controls. The results show that the di-thiophosphate-tetra-sulfate inositols are surprisingly superior even in comparison to di-phosphate-tetra-sulfate inositols, which in turn are significantly superior to the inositolhexasulfate and inositol hexaphosphate previously published (FIG. 3 and comparative example 3).

Example 3 (Comparative): P2S4 Inositol, IP6 and IS6 Cleavage Efficiency

[0041] Samples were prepared and processed as described in example 2. Error bars show s.d.; Asterisk indicates statistical difference compared to IP2S4 (P<0.05); n=3.

[0042] Methods:

[0043] Analogue Solubility Measurements by ICP-MS.

[0044] 100 mM solutions of inositol hexakisphosphate (IP6) analogues with or without 10 mM CaCl.sub.2 were prepared in 10 mM tris pH 7.4 and incubated with agitation for 2 h at 37 C. The solutions were immediately filtered through 0.2 mm nylon filters equilibrated to 37 C. The phosphorous content in each filtrate was determined by inductively coupled plasma-mass spectrometry (ICP-MS). The values obtained were divided by the number of phosphates in each IP6 analogue to determine the concentration of the compound in the solutions.

[0045] Free Calcium Ion Quantification.

[0046] A fresh 1 mM solution of murexide (Merck, Germany) was prepared in 10 mM tris pH 7.4. For each IP6 analogue, samples containing 0.5 mM analogue, murexide and CaCl.sub.2 in 50 mL 10 mM tris were prepared in triplicate. After 5 min incubation at room temperature, the samples were centrifuged at 20000 g for 2 min and the upper 40 mL of the supernatant transferred to a 384-well plate. Samples without IP6 analogue containing CaCl.sub.2 ranging from 1 mM to 20 mM, and 20 mM were also prepared and used for calibrating each experiment. The absorbance was measured at 474 nm and 544 nm and the data analyzed as reported by Ohnishi. [Anal. Biochem. 85, 165 (1978)] The experiment was repeated in triplicate.

[0047] Cleavage Assays with Holotoxin.

[0048] 1 mM IP6 analogues were equilibrated with 10 mM CaCl.sub.2 in 100 mM tris pH 7.4 for 15 min at 37 C. before addition of 150 ng TcdB (TgcBiomics, Germany) in a total volume of 20 mL. A negative control (no IP6, 10 mM CaCl.sub.2) and a positive control (1 mM IP6) were also included on every gel. The reaction mixtures were incubated for 3 h at 37 C. and then placed on ice. Laemmli sample buffer (5) was added to stop the reactions and 10 mM EDTA was added to the samples containing CaCl.sub.2 before heating at 95 C. for 3 minutes. The cleavage products were visualized by SDS-PAGE (using 15-well 8% acrylamide Precise Tris-Glycine gels, ThermoScientific, USA) followed by silver staining according to a modified Vorum protocol [Proteomics 1, 1359 (2001)] with the thiosulphate sensitization step extended to 10 min. The linearity of the staining protocol was verified with serial dilutions of TcdB starting at 160 ng/lane down to 20 ng/lane. The band intensities were quantified as described for the cleavage assays with recombinant toxin. The experiment was done in triplicate.