Inositol derivatives for use in pathological crystallization

Abstract

The present invention relates to inositol derivatives covalently modified with one, two or three solubility functions, particularly polyethylene glycol moieties, for use in therapy or prevention of conditions related to pathological calcium crystallization, such as cardiovascular calcifications, nephrocalcinosis, calcinosis cutis, chondrocalcinosis and kidney stones.

Claims

1. A method for treatment of a condition related to pathological calcium crystallization, comprising administering to a subject in need thereof a compound described by a general formula (II) ##STR00021## wherein one or two or three X are R.sup.1 and the remaining X independently from each other are selected from OPO.sub.3.sup.2, OPSO.sub.2.sup.2, and OSO.sub.3.sup.; and each R.sup.1 independently of any other R.sup.1 is a polyethylene glycol or a polyglycerola.

2. The method for treatment of a condition related to pathological calcium crystallization according to claim 1, wherein R.sup.1 has a molar mass between 100 g/mol and 3000 g/mol.

3. The method for treatment of a condition related to pathological calcium crystallization according to claim 1, wherein the compound is characterized by a general formula III a, III b, III c or III d: ##STR00022## wherein each X (independently) and R.sup.1 have the meaning outlined above.

4. The method for treatment of a condition related to pathological calcium crystallization according to claim 1, wherein more than one R.sup.1 is present and each R.sup.1 is the same as any other R.sup.1.

5. The method for treatment of a condition related to pathological calcium crystallization according to claim 1, wherein the compound is characterized by a general formula (IV a), (IV b), (IV c), (IV d), (V a) or (V b) ##STR00023## wherein each X (independently) and R.sup.1 have the meaning outlined above.

6. The method for treatment of a condition related to pathological calcium crystallization according to claim 1, wherein one or two or three X are R.sup.1 and the remaining X are all OPO.sub.3.sup.2 or all OPSO.sub.2.sup.2 or all OSO.sub.3.sup..

7. The method for treatment of a condition related to pathological calcium crystallization according to claim 1, wherein one or two or three X are R.sup.1 and the remaining X are OPO.sub.3.sup.2; and R.sup.1 is a polyethylene glycol and has a molar mass between 100 g/mol and 3000 g/mol.

8. The method for treatment of a condition related to pathological calcium crystallization according to claim 1, wherein one or two or three X are R.sup.1 and the remaining X are OPSO.sub.2.sup.2; and R.sup.1 is a polyethylene glycol and has a molar mass between 100 g/mol and 3000 g/mol.

9. The method for treatment of a condition related to pathological calcium crystallization according to claim 1, wherein two X are R.sup.1.

10. The method according to claim 1, wherein: one X is R.sup.1 and of the remaining X three X are OSO.sub.3.sup. and two X are OPSO.sub.2.sup.2, or three X are OSO.sub.3.sup. and two X are OPO.sub.3.sup.2, and R.sup.1 is a polyethylene glycol and has a molar mass between 100 g/mol and 3000 g/mol.

11. The method for treatment of a condition related to pathological calcium crystallization according to claim 1, wherein R.sup.1 is a polyethylene glycol characterized by a formula R.sup.3(OCH.sub.2CH.sub.2).sub.n or R.sup.3(OCH.sub.2CH.sub.2).sub.nO and R.sup.3 is hydrogen, methyl or ethyl.

12. The method for treatment of a condition related to pathological calcium crystallization according to claim 1, wherein the compound is described by a general formula (III k), (III l), (III m), (III n), (IV e) or (V c) ##STR00024## wherein n has a value from 2 to 200.

13. The method for treatment of a condition related to pathological calcium crystallization according to claim 1, wherein the compound is described by any one of formulae (III o), (III p), (III q), (III r), (III s), (III t), (III u), (III v), (III w), (III x), (III y), (III z), (IV f), (IV g), (IV h), (IV i), (IV j), (IV k), (V d), (V e), (V f), (V g), (V h) or (V i) ##STR00025## ##STR00026## ##STR00027##

14. The method according to claim 1, wherein said polyethylene glycol is a monodisperse polyethylene glycol.

15. The method according to claim 1, wherein the compound is described by any one of formulae (III o), (III p), (III q), (III u), (III v) or (III w) ##STR00028##

16. A compound described by a general formula (II) ##STR00029## wherein two X are R.sup.1 and the remaining X independently from each other X are selected from OPO.sub.3.sup.2, OPSO.sub.2.sup.2, and OSO.sub.3.sup.; and each R.sup.1 independently of any other R.sup.1 is a polyethylene glycol or a polyglycerol.

17. The compound according to claim 16, wherein the compound is described by any one of formulae ##STR00030## wherein each X (independently) and R.sup.1 have the meaning outlined above.

18. The compound according to claim 16, described by the formula ##STR00031## wherein n has a value from 2 to 200, particularly n is 2 or n is 7 to 50, more particularly n is 2, 7 to 12 or 40 to 50, even more particularly n is 2, 7, 9, 11 or 45.

19. The compound according to claim 16, described by any one of formulae ##STR00032##

20. A compound described by any one of formulae ##STR00033##

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows calciprotein particle (CPP) maturation time in human serum according to the test described in Pasch et al. JASN 2012. The y-axis indicates the half-maximal transition time (T.sub.50) in minutes of primary CPPs to secondary CPPs. IP6: myo-inositol hexakisphosphate (Biosynth); IS6: myo-inositol hexakissulfate (Sigma); IP2S4: 4,6-di-(O-phosphate)-myo-inositol 1,2,3,5-tetra-O-sulfate; IT2S4: 4,6-di-(O-thiophosphate)-myo-inositol 1,2,3,5-tetra-O-sulfate; IP5-PEG: 2-PEG(2000)-myo-inositol pentakisphosphate.

(2) FIG. 2 shows the result of a determination of calciprotein particle (CPP) maturation time in human serum according to a similar test as in FIG. 1 (with the test compounds added to the reaction mixture last).

EXAMPLES

(3) TABLE-US-00001 TABLE 1 Compounds No. of PEG M.sub.w of PEG M.sub.w of Substituents other than Compound Formula tails (monodisperse) compound PEG INS-2001 III o 1 100 672.11 5 OPO.sub.3.sup.2 INS-2031 III p 1 320 892.37 5 OPO.sub.3.sup.2 INS-2041 III q 1 500 1068.59 5 OPO.sub.3.sup.2 INS-2042 1 550 (polydisp.) 1112.64 5 OPO.sub.3.sup.2 INS-2101 III r 1 100 752.41 5 OPSO.sub.2.sup.2 INS-2131 III s 1 320 972.68 5 OPSO.sub.2.sup.2 INS-2141 III t 1 500 1148.89 5 OPSO.sub.2.sup.2 INS-4001 III u 1 100 675.37 2 OPO.sub.3.sup.2, 3 OSO.sup.3 INS-4031 III v 1 320 895.63 2 OPO.sub.3.sup.2, 3 OSO.sup.3 INS-4041 III w 1 500 1071.84 2 OPO.sub.3.sup.2, 3 OSO.sup.3 INS-4101 III x 1 100 707.49 2 OPSO.sub.2.sup.2, 3 OSO.sup.3 INS-4131 III y 1 320 927.75 2 OPSO.sub.2.sup.2, 3 OSO.sup.3 INS-4141 III z 1 500 1103.96 2 OPSO.sub.2.sup.2, 3 OSO.sup.3 INS-3001 IV f 2 100 696.28 4 OPO.sub.3.sup.2 INS-3031 IV g 2 320 1136.81 4 OPO.sub.3.sup.2 INS-3041 IV h 2 500 1489.23 4 OPO.sub.3.sup.2 INS-3101 IV i 2 100 760.52 4 OPSO.sub.2.sup.2 INS-3131 IV j 2 320 1201.05 4 OPSO.sub.2.sup.2 INS-3141 IV k 2 500 1553.48 4 OPSO.sub.2.sup.2 INS-5001 V d 3 100 720.45 3 OPO.sub.3.sup.2 INS-5031 V e 3 320 1381.24 3 OPO.sub.3.sup.2 INS-5041 V f 3 500 1909.88 3 OPO.sub.3.sup.2 INS-5101 V g 3 100 768.63 3 OPSO.sub.2.sup.2 INS-5131 V h 3 320 1429.42 3 OPSO.sub.2.sup.2 INS-5141 V i 3 500 1958.06 3 OPSO.sub.2.sup.2

(4) Calcification Assay

(5) The inventors performed an in vitro assay that measures the propensity for calcification of human serum and has been clinically validated as a predictor of all-cause mortality in CKD patients and renal transplant recipients (as described in Pasch, Journal of the American Society of Nephrology 23, 1744-1752, 2012). The experiment was carried out by mixing a calcium solution, human pooled serum, the test compound at the final concentration indicated and a phosphate solution, and the transition time of primary to secondary CPPs was measured at 37 C. using a nephelometer for up to 600 minutes.

(6) The data of FIG. 1 show that the compounds IP2S4 and IT2S4 are more active than IP6. Furthermore, compound 2-PEG-IP5 is far more active than any other compound in this assay. This result suggests a key role of the polymer moiety in preventing the transition of primary to secondary CPPs and in reducing the propensity for calcification of human serum.

(7) The data of FIG. 2 show that the compounds INS-2031 (III p), INS-3001 (IV f) and INS-3031 (IV g) are more active than IP6. The compounds having two PEG moieties (INS-3001, INS-3031) are more active than the compounds having one PEG moiety (INS-2031). This result suggests a key role of the polymer moiety in preventing the transition of primary to secondary CPPs and in reducing the propensity for calcification of human serum.

(8) Synthesis of IT2S4 (VI a)

(9) The synthesis followed the sequence depicted in the scheme below:

(10) ##STR00015##

(11) Phosphorylation

(12) The known 2-tertbutyldimethylsilyl inositol orthoformate was co-evaporated 3 with toluene and dissolved in dichloromethane (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.

(13) .sup.1H-NMR (400 MHz; CDCl3): 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);

(14) .sup.31P-NMR (162 MHz; CDCl3): 70.1;

(15) Deprotection

(16) The following deprotection conditions are in analogy to the synthesis published in the Journal of the American Chemical Society (JACS 2005, 127, 5288).

(17) Starting material (50 mg) was treated with thiophenol (300 l), m-cresol (300 l), trifluoroacetic acid (1.8 ml). Trimethylsilyl bromide (TMSBr) was then added slowly (360 l). The mixture was stirred 2 h at room temperature. And then evaporated twice from toluene. The crude residue was diluted with DCM, and ca. 5 ml water and neutralized with 1N NaOH. The aqueous layer (slightly cloudy) was poured directly on SolEx C18 cartridge (Thermofisher, 1 g, 6 ml) and eluted with water. In some cases some aromatic impurities were found in the final product but would precipitate over time in water and could be filtered-off.

(18) .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).

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

(20) Sulfation

(21) 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.

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

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

(24) Synthesis of IP2S4 (VI c)

(25) The synthesis followed the sequence depicted in the scheme below:

(26) ##STR00016##

Hydrolysis

4,6-Di-O-phosphate-myo-inositol (2)

(27) 2-O-Tert-butyldimethylsilyl-1,3,5-orthoformate-4,6-(O-dixylylenephospho)-myo-inositol (1.00 g, 1.5 mmol, 1 eq.) in methanol/dichloromethane (MeOH/DCM) 30% (30 ml, 0.05 M) was treated with trimethylsilyl bromide (TMSBr) (11 ml, 83.8 mmol, 56 eq.) and stirred for 5 h. The reaction mixture was degased with N.sub.2 and the HBr was neutralized with 1 M NaOH solution. After 1-2 h it was concentrated to dryness. The crude was washed twice with acetone and twice with acetonitrile (ACN) to give 2 as a white solid (539 mg, quantitative yield).

(28) .sup.1H-NMR (400 MHz, MeOD): (ppm)=4.40 (q, 3JHH=9.1 Hz, 2JHP=9.1 Hz, 2H, HC4/6), 4.01 (t, J=2.6 Hz, 1H, HC2), 3.63 (dd, J=9.68, 2.76 Hz, 2H, HC1/3), 3.61 (t, J=9.27 Hz, 1H, HC5);

(29) .sup.31P-NMR (160 MHz, 1H-decoupled, MeOD): (ppm)=1.15 (PC4/6); 13CNMR (150 MHz, MeOD): (ppm)=81.28 (d, 2JCP=6.1 Hz, 2 C, C4/6), 74.12 (t, 3JCP=3.8 Hz, 1 C, C5), 73.75 (s, 1 C, C2), 72.13 (d, 3JCP=3.2 Hz, 2 C, C1/3); [m/z (ESI) (M+H)+C6H15O12P2 required 341.0033, found 341.0037].

Sulfation

1,2,3,5-Tetra-O-sulfonyl-4,6-(di-O-phosphate)-myo-inositol (1)

(30) 4,6-di-O-phosphate-myo-inositol (30 mg, 90 mol, 1 eq.) was co-evaporated with toluene (3) and dried under high vacuum for 1 h. Dry dimethylformamide (DMF) (1 ml, 0.09 M) was added and the reaction mixture was treated with SO.sub.3-Et.sub.3N (197 mg, 109 mol, 12 eq.) and TfOH (190 l, 215 mol, 24 eq.). It was heated at 45 C. and stirred overnight. The reaction mixture was neutralized by addition of Et.sub.3N (0.15 ml, 12 eq.). Immediately after the neutralization the mixture was diluted in nanopure water (2 ml) and loaded on a sephadex G10 column. 14 fractions of 3-4 ml were collected and put into the freeze-dryer overnight. Fractions 3-7 were combined to give 1 as a white solid (46.31 mol, 51%).

(31) .sup.1H-NMR (400 MHz, D.sub.2O): (ppm)=5.40 (br, 1H, HC2), 4.64-4.44 (m, 5H, HC1/3, HC5, HC4/6), 3.70 (s, 8H, internal standard dioxane), 3.15 (q, J=7.3 Hz, 6H, CH2-Et.sub.3N), 1.23 (t, J=7.3 Hz, 9H, CH3-Et.sub.3N).

(32) Synthesis of PEG-IP5 (III o, III p, III q)

(33) The synthesis followed the sequence depicted in the scheme below:

(34) ##STR00017##

(35) Inositol orthoformate was reacted with 1 eq. of PEG tosylate to the singly PEG-ylated 4- or 6-PEG inositol orthoformate. The orthoformate protection group was removed using trifluoroacetic acid and dichloromethane. The compound was reacted with phosphoramidite, 1H-tetrazole, dichloromethane and meta-chloroperoxybenzoic acid. The resulting compound was reacted with H.sub.2, MeOH and PdO to 4-PEG-IP5 or 6-PEG-IP5, respectively.

(36) Synthesis of 4,6-PEG-IP4 (IV f, IV g, IV h)

(37) The synthesis followed the sequence depicted in the scheme below:

(38) ##STR00018##

(39) Inositol orthoformate was reacted with PEG tosylate to the doubly PEG-ylated 4,6-PEG inositol orthoformate. The orthoformate protection group was removed using trifluoroacetic acid and dichloromethane. The compound was reacted with phosphoramidite, 1H-tetrazole, dichloromethane and meta-chloroperoxybenzoic acid. The resulting compound was reacted with H.sub.2, MeOH and PdO to 4,6-PEG-IP4

(40) Synthesis of 4-PEG-IP2S3 (III u, III v, III w)

(41) The synthesis followed the sequence depicted in the scheme below:

(42) The known myo-inositol orthoformate can be mono alkylated with a commercial PEG tosylate in the presence of a strong based such as sodium hydride in DMF. The reaction mixture is then quenched with water and extracted with dichloromethane. The organic layer is dried and concentrated under reduced pressure. The product can be purified by silica gel chromatography. Phosphorylation of the free hydroxyl groups is done under standard conditions using a phosphoramidite reagent followed by oxidation with meta-chloroperbenzioc acid. The product can be purified by normal or reverse phase chromatography. The orthoester and phosphate groups are then deprotected concomitantly using excess bromotrimethylsilane in a mixture of methanol and dichloromethane. The product can be purified by precipitation or reverse phase chromatography. Sulfation of the free hydroxyl group is performed by suspending the product in dry DMF and reacting with excess sulfur trioxide-DMF complex. The reaction is then quenched with water and neutralized. The final product can be precipitated out of the reaction mixture by adding methanol and purified by size-exclusion chromatography or reverse phase chromatography.

(43) ##STR00019##

(44) Synthesis of PEG-IT5, 4,6-PEG-IT4 and PEG-IT2S3

(45) The synthesis of PEG-IT5 (III r, III s, III t), 4,6-PEG-IT4 (IV i, IV j, IV k) and PEG-IT2S3 (III x, III y, III z) followed the sequences specified for PEGIP5, 4,6-PEG-IP4 and PEG-IP2S3, except that the phosphorylation was performed by addition of 1H tetrazole (4 eq.) followed by phosphoramidite (8 eq.) to the reaction and stirred overnight. Afterwards, pyridine, followed by crushed sulphur flakes (20 eq.) were added to the reaction and stirred overnight to complete the thiophosphorylation.

(46) Synthesis of 2,4,6-PEG-IP3 (V d, V e, V f)

(47) The synthesis followed the sequence depicted in the scheme below:

(48) ##STR00020##

(49) Inositol orthoformate was reacted with PEG tosylate to the triple PEG-ylated 2,4,6-PEG inositol orthoformate. The orthoformate protection group was removed using trifluoroacetic acid and dichloromethane. The compound was reacted with phosphoramidite, 1H-tetrazole, dichloromethane and meta-chloroperoxybenzoic acid. The resulting compound was reacted with H.sub.2, MeOH and PdO to 2,4,6-PEG-IP4.

(50) Synthesis of 2,4,6-PEG-IT3 (V g, V h, V i)

(51) The synthesis of 2,4,6-PEG-IT3 followed that described for 2,4,6-PEG-IP3 except that the phosphorylation was performed by addition of 1H tetrazole (4 eq.) followed by phosphoramidite (8 eq.) to the reaction and stirred overnight. Afterwards, pyridine, followed by crushed sulphur flakes (20 eq.) were added to the reaction and stirred overnight to complete the thiophosphorylation.