Use of 3-deoxyanthocyanidins for treating occular diseases

Abstract

Disclosed is a method for the treatment, prevention and/or stabilisation of ARMD, Stargardt disease, pigmentary retinopathy and/or diabetic retinopathy, including the application of a 3-deoxyanthocyanidin of formula (I) in which R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11 and X.sup. are as defined, with the condition that at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4 or R.sup.5 is a hydroxyl and at least one of R.sup.8, R.sup.9, R.sup.10 or R.sup.11 is a hydroxyl.

Claims

1. A method for treating and/or stabilising AMD, Stargardt disease, pigmentary retinopathy and/or diabetic retinopathy, comprising the administration to a patient in need thereof of an effective amount of a compound of Formula I ##STR00025## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 each independently is a group selected from hydrogen, halo, hydroxyl, alkoxy, aryloxy, heteroaryloxy, acyloxy, alkyl, aryl, aralkyl, alkylaryl, alkenyl, nitro, nitrile, amino, with the condition that at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4 or R.sup.5 is a hydroxyl; R.sup.6 is hydrogen; R.sup.7 is a group selected from hydrogen, halo, hydroxyl, alkoxy, aryloxy, heteroaryloxy, acyloxy, alkyl, aryl, aralkyl, alkylaryl, alkenyl, nitro, nitrile, amino; R.sup.8, R.sup.9, R.sup.10 and R.sup.11 each independently is a group selected from hydrogen, halo, hydroxyl, alkoxy, aryloxy, heteroaryloxy, acyloxy, alkyl, aryl, aralkyl, alkylaryl, alkenyl, nitro, nitrile, amino, with the condition that at least one of R.sup.8, R.sup.9, R.sup.10 or R.sup.11 is a hydroxyl; and X.sup. is an anion selected from: anion derived from a mineral acid; anion derived from an organic acid; or an anion derived from a sulphate or sulphonate group.

2. The method according to claim 1, wherein the anion derived from a mineral acid is selected from a bromide, chloride, borotetrafluoride and perchloride anion; and wherein the anion derived from an organic acid is selected from an acetate, borate, citrate, tartrate, bisulphate, sulphate and phosphate anion.

3. The method according to claim 1, wherein the compound is of Formula Ia ##STR00026## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.8, R.sup.10 and X.sup. are such as defined in claim 1.

4. The method according to claim 1, wherein: R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 each independently is a group selected from hydrogen, hydroxyl and alkoxy, with the condition that at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4 or R.sup.5 is a hydroxyl; and R.sup.8 and R.sup.10 each independently is a group selected from hydrogen, hydroxyl and alkoxy, with the condition that at least one of R.sup.8 or R.sup.10 is a hydroxyl.

5. The method according to claim 1, wherein the compound is of Formula Ib ##STR00027## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.8 and X.sup. are such as defined in claim 1.

6. The method according to claim 1, wherein R.sup.8 is a hydrogen atom.

7. The method according to claim 1, wherein the compound is selected from: 2,7-dihydroxy-4-methoxy-flavylium chloride; 2,3,7-trihydroxy-4-methoxy-flavylium chloride; 3,7-dihydroxy-4-methoxy-flavylium chloride; 4,5,7-trihydroxy-flavylium chloride; 3,5,7-trihydroxy-4-methoxy-flavylium chloride; 3,4,5,5,7-pentadroxy-flavylium chloride; and 3,4,5,7-tetrahydroxy-flavylium chloride.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a scheme that summarises the main steps of visual cycle.

(2) FIGS. 2 and 3 are graphs showing the electroretinograms of mice one week after the induction of the phototoxicity. The wave A (FIG. 2) shows the electrical activity of the photoreceptors, and the wave B (FIG. 3) shows that of the cells of the inner retina. The data were analysed statistically by an analysis of the variance followed by a Dunnett test. *p<0.05, **p<0.01, ***p<0.001.

(3) FIG. 4 is a graph showing the number of layers of photoreceptors according to the distance from the optical nerve after intravitreous injection of diosmetinidin.

EXAMPLES

(4) This invention shall be better understood when reading the following examples which show the invention in a non-limiting manner.

Example 1: Synthesis of the Compounds of the Invention

(5) Compound 1. The 2,4-dihydroxybenzaldehyde (69 mg) and the 2-hydroxy-4-methoxy-acetophenone (83 mg-1 equivalent) are brought into solution in 500 L of ethanol. 40 L of thionyl chloride are added directly into the reaction medium which quickly becomes highly coloured. After one hour of stirring, the reaction medium is evaporated and the residue is precipitated in ethyl acetate, filtered and dried in order to yield the final compound (77 mg-yield 50%).

(6) Compound 2. The 2,4-dihydroxybenzaldehyde (276 mg) and the 2,3-dihydroxy-4-methoxy-acetophenone (364 mg-1 equivalent) are brought into solution in 2 mL of ethanol and 2 mL of ethyl acetate. 500 L of thionyl chloride are added directly into the reaction medium which quickly becomes highly coloured. After one hour of stirring, the reaction medium is evaporated and the residue is precipitated in ethyl acetate, filtered and dried. The solid is brought into solution in a minimum of methanol and is precipitated by the adding of an equivalent volume of tertio-butyl-methyl-ether (TBME). The solid is filtered and dried. The procedure is repeated until the purity is correct. The final yield is 60%.

(7) The compounds obtained have purities>95% (HPLC) and their identity was confirmed by MS and NMR spectroscopy. The high-resolution mass spectrums were carried out on a LTQ Orbitrap-XL spectrometer (ThermoFisher Scientific), equipped with an NSI source (nano-ESI). The nuclear magnetic resonance spectrums (NMR) of the proton (.sup.1H) were carried out in the DMSO-d.sub.6+1% CF.sub.3COOD on a Bruker Avance DPX300 device (300.16 MHz).

(8) HRMS (Orbitrap).

(9) Compound 1. m/z 269.0809 (M)+, calc. 269.0808 for C.sub.16H.sub.13O.sub.4, =0.054 ppm.

(10) Compound 2. m/z 285.0757 (M)+, calc. 285,0757 for C.sub.16H.sub.13O.sub.5, =0.105 ppm.

(11) .sup.1H-NMR spectrums ( ppm)

(12) TABLE-US-00003 Compound 1 Compound 2 0 3-H 7.44 (dd, 8.9, 2.2 Hz) 7.42 (dd, 8.9, 2.2 Hz) 4-H 9.11 (d, 9.0 Hz) 9.09 (d, 9.0 Hz) 5-H 8.65 (d, 9.0 Hz) 8.64 (d, 8.9 Hz) 6-H 6.77 (d, 2.3 Hz) 6.94 (d, 9.4 Hz) 8-H 7.57 (d, 2.0 Hz) 7.53 (d, 1.9 Hz) 2-H 8.18 (d, 8.9 Hz) 8.16 (d, 9.0 Hz) 3-H 6.82 (dd, 9.2, 2.1 Hz) 7.92 (d, 9.3 Hz) 4-OCH.sub.3 3.92 (3H, s) 4.01 (3H, s) 5-H 8.36 (d, 9.1 Hz) 6-H

Example 2: In Vitro Assay of the Photoprotective Activity

(13) Method

(14) A cell model of phototoxicity induced by the association of a treatment by the A2E and of an illumination by blue light on primary cultures of RPE, wherein cell survival was measured, was used. This model uses primary cultures of retinal pigment epithelium of adult pigs.

(15) This model makes it possible in particular to carry out the screening of molecules aimed at the discovery of new candidates for a treatment of the dry form of AMD. This model is closer to the physiological situation than the cell lines commonly used in literature, because the cells used contain protective substances provided by the diet of the animal and are therefore not in a situation of deficiency, and their disturbance is caused by the adding of A2E into the culture medium.

(16) The cell cultivated in 96-well plates were treated for 48 hours with the compounds to be tested (in a 5 mM solution in the DMSO) in such a way as to obtain final concentrations of 5 or 20 M), of which the last 19 hours in the presence of A2E (final concentration 30 M). The pre-treated cells are then illuminated for 50 min with blue light (470 nm) provided by the 96 LED W7113PBC/H (Kingbright) with a beam angle of 16, beaming 1440 mcd (millicandela) under a current of 8.6 mA. Cell survival is measured after 24 hours.

(17) Cell survival and death are detected 24 hours after the induction of the phototoxicity by colouration of the cells with Hoechst (a nuclear marker) and with ethidium (a marker of the nuclei of dead cells). Images of each well are acquired on a fluorescent microscope equipped with a motorised stage controlled by the Metamorph software, and cell survival is quantified by a dedicated quantification program. The experiments are conducted on 96-well microplates in quadruplicate and each experiment is reproduced at least four times.

(18) The results are expressed in the form of a ratio representing the number of living cells in the wells treated by the molecules to be tested divided by the number of living cells in the control wells (treated with the dilution medium without A2E) and multiplied by 100. The value of the controls treated with A2E but without the molecule is 39.73.7.

(19) Results

(20) The compounds of the invention make it possible to obtain very high cell survival percentages, at 20 M as well as at 5 M (Table 2).

(21) TABLE-US-00004 TABLE 2 Cell survival: compounds of the invention. Compounds 20 M 5 M 1 89.9 4.9 NA 2 86.7 2.3 NA 3 77.0 2.7 NA 4 89.5 3.5 71 3.6 5 90.8 3.6 84.5 1.4 6 99.3 3.3 74.9 4.2 7 93.6 6.7 72 4.4 NA: not available

(22) For comparison, 3-hydroxy-anthocyanidins were tested (Table 3).

(23) TABLE-US-00005 TABLE 3 3-hydroxy-anthocyanidins tested. R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 cyanidin OH H OH H H delphinidin OH OH OH H H fisetinidin H OH H H H gossypetinidin H OH OH H OH guibourtinidin H H H H H malvidin OCH.sub.3 OCH.sub.3 OH H H peonidin OCH.sub.3 H OH H H petunidin OH OCH.sub.3 OH H H quercetagetinidin OH H OH OH H

(24) The results of cell survival in the presence of 3-hydroxy-anthocyanidins are reported in table 4.

(25) TABLE-US-00006 TABLE 4 Cell survival: 3-hydroxy-anthocyanidins. Compounds 20 M 5 M Cyanidin 92.9 1.9 56.4 8.8 Delphinidin 57.8 5.6 40.5 6.2 Fisetinidin 39.3 3.2 NA Gossypetinidin 36.5 14.4 NA Guibourtinidin 42.8 6.9 NA Malvidin 51.5 6.9 NA Peonidin 60.4 8.5 NA Petunidin 67.2 6.9 NA Quercetagetinidin 54.3 6.6 NA

(26) These results show that the presence of a hydroxyl group in position 3 of the anthocyanidins substantially reduces the effectiveness of the photoprotection. Only cyanidin has a substantial photoprotector effect at 20 M. However, at a lower concentration, its effectiveness is substantially reduced, contrary to the compounds of the invention.

(27) For comparison, 3-deoxy-anthocyanidins that do not carry at least one free hydroxyl on the cycle A and on the cycle B were also tested (Table 5).

(28) TABLE-US-00007 TABLE 5 Cell survival: 3-deoxy-anthocyanidins not carrying at least one hydroxyl on the cycle A or on the cycle B. Compounds 20 M 5 M 39.2 7.4 NA 48.7 5.5 NA 46.1 4.7 NA

(29) The percentages of cell survival obtained at 20 M are less than 50%; the concentration 5 M was therefore not tested.

(30) The results obtained clearly show that when there is not at least one hydroxyl group on each one of the cycles A and B, the efficacy of the photoprotection is much less substantial than with the compounds of the invention.

Example 3: In Vivo Assay of the Photoprotective Activity in Mice

(31) A genetically modified mouse model developed by Maeda et al. (Invest Ophthalmol. Vis. Sci., 2009, 50, 4917-4925) was used to test the photoprotective activity of the compounds of the invention.

(32) In this mouse model, two genes involved in the visual pigment cycle (ABCA4 and Rdh8, see FIG. 1) are inactivated, which results in an early accumulation of A2E in the eyes. This animal model is therefore representative of the human pathology.

(33) Mice aged 7 weeks were used to carry out the unilateral intravitreal injections of solubilised diosmetinidin (50 M) in DMSO and diluted in PBS (1.2:100), in order to obtain a concentration in the vitreous of 100-130 M. DMSO diluted in the PBS was injected into the control animals. After 24 h in the dark, the mice were subjected to an exposure to blue light (4000 lux, 1 h).

(34) The electroretinograms carried out 7 days later showed a protective effect of the diosmetinidin, of which the presence made it possible to maintain significant electrical activity (FIGS. 2 and 3) and good survival of the photoreceptors (FIG. 4).