Patent ID: 12215067

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The chemical compounds object of the present invention do not exist in the chemical databases of carotenoids and di-apo-carotenoids. They are synthesized according to processes that can be industrialized, i.e. with a minimum of synthesis steps and an optimum yield. They have a better pharmacokinetic profile and a better tropism for the eye than those of norbixin. Some of these compounds moreover have a photo-protective activity of the RPE superior than that of norbixin.

Description of Syntheses and General Diagrams

The chemical compounds having general formula (I):

can be prepared by application or adaptation of any method known per se to a person skilled in the art and/or within the scope of the latter, which have been able to be described, or by application or adaptation of methods described in the following procedures.

In the description that follows the various groups refer to the definitions given hereinabove, i.e.:

COR is a secondary or tertiary amide, such that —R is chosen from:

-M; —NH—(CH2)n-M and —NH—(CH2)n—C(CH3)(CH3)-M, -M being chosen froma)

wherein,R1is chosen from an oxygen atom, a sulfur atom, a >CH2, >CH—O—(CH2)n—CH3, >CH—(CH2)n—O—(CH2)n—CH3, >CH—(CH2)n—OH, >CH—COOH, >C(OH)phenyl or >NH group;R3is chosen from a hydrogen atom, a C1-C6alkyl, —OH or C1-C6—O-alkyl group;R4is chosen from a hydrogen atom, a C1-C6alkyl, —OH or C1-C6—O-alkyl group;n is an integer comprised between 0 and 6;—NH—(CH2)n—W, W being a hydrogen atom, or a —OH group, a —O—(CH2)n—CH3group, or a group chosen fromi)

wherein,R1is chosen from an oxygen atom, a sulfur atom, a >CH2, >CH—O—(CH2)n—CH3, >CH—(CH2)n—OH, >CH—COOH, >C(OH)phenyl or >NH group;R3is chosen from a hydrogen atom, a C1-C6alkyl, —OH or C1-C6—O-alkyl group;R4is chosen from a hydrogen atom, a C1-C6alkyl, —OH or C1-C6—O-alkyl group;R5is chosen from a —CH3, —OH, —O—(CH2)n—CH3, —(CH2)n—OH or —COOH group;n is an integer comprised between 0 and 6.

In the framework of the present invention the term “C1-C6alkyl group” means any alkyl group with 1 to 6 carbon atoms, linear or branched, in particular, the methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl groups. Advantageously it is a methyl, ethyl, iso-propyl or t-butyl group, in particular a methyl or ethyl group, more particularly a methyl group.

Diagram A for the synthesis of the compound 1-[2-(2-furyl)ethanamido](2E,4E,6E,8E,10E,12E,14E,16Z,18E)-4,8,13,17-tetramethylicosa-2,4,6,8,10,12,14,16,18-nonaenedioate (called compound C21):

Formation of Amide (First Method)

Hydrolysis of Methyl Ester

Diagram B for the synthesis of the compound 1-[(E)-4-hydroxycyclohexamido](2E,4E,6E,8E,10E,12E,14E,16Z,18E)-4,8,13,17-tetramethylicosa-2,4,6,8,10,12,14,16,18-nonaenedioate (called compound C19:

Formation of Amide (Second Method)

DMF=dimethylformamide
TEA=triethylamine
DIA=diisopropylamine
THF=tetrahydrofuran
CDI=carbonyldiimidazole
HBTU=(1H-benzotriazol-1-yloxy)(dimethylamino)-N,N-dimethylmethaniminium hexafluorophosphate

EXAMPLES

Equipment and Methods

The nuclear magnetic resonance (NMR) spectra of the proton (1H) are carried out on a Bruker Avance DPX500 device (500, 0.7 MHz). The chemical shifts (δ) are measured in parts per million (ppm). The spectra are calibrated on the chemical shift of the deuterated solvent used. The coupling constants (J) are expressed in Hertz (Hz) and the multiplicity is represented in the following way, singlet (s), doublet (d), doublet of doublet (dd), triplet (t), triplet of doublet (td), quadruplet (q), multiplet (m). The mass spectra (MS) are carried out with an Agilent Technologies MSD spectrometer, G1946A type, the samples are ionized by an “Atmospheric pressure chemical ionization” (APCI) source.

By way of illustrating examples of the invention, the molecules shown in table 1 were synthesized.

TABLE 1NoChemical structureChemical nameC11-[2- methoxyethanamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC21-[1,4-oxazinamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC31-[piperidinamido] (2E,4E,6E,8E,10E, 12E,14E,16Z,18E)- 4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC41-[2-hydroxyethanamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC51[1,4-oxazepanamido] (2E,4E,6E,8E,10E,  12E,14E,16Z,18E)- 4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC61-thiomorpholinamido- (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC71-pyrrolidinamido- (2E,4E,6E,8E,10E,  12E,14E,16Z,18E)- 4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC81-[2- morpholinopropanamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC91-[(S)-3- hydroxypyrrolidinamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC101-[2- morpholinoethanamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC111-[(R)-3- hydroxypyrrolidinamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)- 4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC121-[4- hydroxypiperidinamido] (2E,4E,6E,8E,10E,12E, 14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC131-[2-methyl-2- (4-morpholinyl) propylamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC141-[4- hydroxymethyl- piperidinamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)- 4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC151-(Z)-3,5- dimethylmorpholinamido] (2E,4E,6E,8E,10E,12E, 14E,16Z,18E)- 4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC161-[4- hydroxyphenyl- ethanamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC171-[4-benzyl-4- hydroxypiperidinamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC181-[4- carboxypiperidinamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC191-[(E)-4- hydroxycyclohexamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC201-[3- methoxypiperidinamido] (2E,4E,6E,8E,10E,12E,  14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC211-[2-(2-furyl) ethanamido] (2E,4E,6E,8E,10E,12E, 14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC221-[4-n- propoxypiperidinamido] (2E,4E,6E,8E,10E,12E, 14E,16Z,18E)- 4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC231-4- ethylmethoxy- piperidinamido] (2E,4E,6E,8E,10E,12E, 14E,16Z,18E)-4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioateC241-4- methoxypiperidinamido] (2E,4E,6E,8E,10E,12E, 14E,16Z,18E)- 4,8,13,17- tetramethylicosa- 2,4,6,8,10,12,14,16,18- nonaenedioate

Example 1 (Diagram A)

Preparation of the Compound C21

Step 1 (Typical Coupling Method of an Amine with Carbonyidiimidazole (CDI)): Preparation of the Methyl Ester of C21

The chemical reactions were carried out under argon atmosphere. The reagents and the products were protected from light with aluminum paper during all the steps of the reaction and during the step of extracting and removing solvents. The products were stored at 4° C. The bixin (1 g; 2.54 mmol) is dissolved in 10 mL of dry dimethylformamide (DMF) and triethylamine (1.06 mL; 7.61 mmol) then CDI (0.823 g; 5.07 mmol) are added. After two hours, 2-aminoethylfuran (845 mg; 7.61 mmol) was added and the reaction mixture was stirred for 18 h at 20° C. Hydrochloric acid (HCl, 1 N; 20 mL) is added and the resulting suspension is centrifuged. Following the removal of the supernatant, the pellet containing the methyl ester was resuspended twice in the presence of water. Following another centrifugation, the pellet (1.24 g) was used directly in the step of hydrolysis.

Step 2 (Typical Procedure of the Hydrolysis): Conversion of the Methyl Ester of C21 into C21

The chemical reactions were carried out under argon atmosphere and protected from light with aluminum paper. The products are stored at 4° C. The methyl ester of C-21 is dissolved in 15 mL of tetrahydrofuran (THF) and 13 mL of MeOH. Sodium hydroxide (NaOH, 1 N; 15.3 mL) is added and the reaction mixture is stirred for 20 h at 20° C. Hydrochloric acid (HCl; 1 N; 16 mL) was added progressively. The suspension obtained is centrifuged and the supernatant is removed. The pellet is then resuspended and mixed twice with water then centrifuged again and the supernatant is removed. The paste obtained, containing water, is transferred to a pyriform flask in the presence of water and acetonitrile, and lyophilized to give 1.04 g of an orangish to red colored powder.

Analyses of the Compound C21

LC-MS: m/z=474.3 (MH+) UV purity at 460 nm=99%.

NMR1H (500 MHz, DMSO-d6) −δ 8.11 (t, 1H), 7.54 (s, 1H), 6.19 (d, 1H), 3.42-3.38 (m, 2H), 2.78 (t, 2H).

Preparation of the Compound C19

Step 1 (Typical Procedure for the Coupling of the Amine by Using HBTU): Preparation of the Methyl Ester of C19 (Method Used for the Preparation of the Methyl Ester of C19 Only, as the Reaction Using CDI is Too Slow for this Compound)

The chemical reactions were carried out under argon atmosphere. The reagents and the products were protected from light with aluminum paper during all the steps of the reaction and during the step of extracting and removing solvents. All the products were stored at 4° C. The bixin (1.17 g; 2.97 mmol) is dissolved in 20 mL of anhydrous DMF and diisopropylamine (1.23 mL; 7.42 mmol); HBTU ((1H-benzotriazol-1-yloxy)-dimethylamino hexafluorophosphate)-N, N′-dimethylmethaniminium; 1.69 g; 4.45 mmol) is then added. After 1 h, trans-4-aminocyclohexanol (680 mg; 4.45 mmol) was added and the reactive mixture is stirred for 18 h at 20° C. Sixty milliliters of water (60 mL) is added and the precipitate was filtered and washed twice with 60 mL of water to give 1.4 g of violet solid compound which is used as is in the step of hydrolysis.

Step 2 (Typical Procedure of the Hydrolysis): Conversion of the Methyl Ester of C19 into C19

The chemical reactions were carried out under argon and protected from light with aluminum paper. The products are stored at 4° C. The 1.4 g of methyl ester of C-21 are dissolved in 10 mL of tetrahydrofuran (THF) and 10 mL of MeOH (dark red solution). Five equivalents of sodium hydroxide (NaOH, 10N; 1.43 mL) are added and the reaction mixture is stirred for 48 h at ambient temperature. In HPLC-MS, a single peak is observed and the absence of starting material. Hydrochloric acid (HCl 12N; 1.18 mL) was added. The precipitate obtained is diluted with 60 mL of water. The solution is centrifuged and the aqueous supernatant is removed. The solid pellet is then taken and mixed with water (30 mL) then centrifuged again and the supernatant is removed. This step is repeated twice until a solid paste of orange color is obtained (which forms a suspension in the presence of water). The solid paste is taken in 50 mL of water to give an orange suspension, frozen and directly lyophilized to give an orange powder (1.02 g).

Analyses of the Compound C19

LC-MS: m/z=478.2 (MH+) UV purity at 460 nm=97.3%.

NMR1H (500 MHz, DMSO-d6) −δ 47.82 (s, 1H), 4.51 (m, 1H), 3.58.-3.53 (m, 1H), 3.40-3.36 (m, 1H), 1.83-1.75 (m, 4H), 1.26-1.15 (m, 4H).

Cascade Screening and Characterization of the Biological Effects of the Chemical Compounds (C) Derived from Norbixin.

The development of the screening test was initiated from work in literature and based on the characteristics of the pathology of dry AMD. At the physiopathologic level, this disease is characterized by a progressive loss of induced vision following the degeneration of the photoreceptors and of the cells of the RPE. The cells of the RPE play a crucial role in the survival and correct operation of the photoreceptors by providing them with the necessary nutrients, by participating in the visual cycle and by removing the debris coming from external segments of photoreceptors and that result from this cycle. It is important to screen the drugs in development on their capacity to target preferably the eye while still avoiding an intraocular administration that is traumatizing for patients and having risks of local infections. To do this, a pharmacokinetic study of the chemical compounds (C) derived from norbixin object of the present invention at the plasma and ocular level was conducted so as to select the chemical compounds having an ocular AUC (“area under the curve”) that is improved in relation to norbixin.

In addition to better distribution in the target-tissue, selecting new compounds must also be based on good photo-protective activity that limits the loss of the cells of the RPE and thus reducing the retinal degeneration observed during the AMD and other degenerative diseases such as Retinitis pigmentosa and Stargardt's disease. At the cellular level, on cultures of cells the RPE coming from pig retina, Fontaine et al. (2016) have shown that a treatment with norbixin (BIO201) protects the cells of the RPE against apoptosis following an illumination in the presence of A2E (80% survival 24 h after exposure). This same screening test according to the percentage of photoprotection was used so as to determine its modulation by and the chemical compounds derived from norbixin (C) object of the present invention in comparison with the photo-protective effect of norbixin and characterize these modulations from a statical standpoint.

Protocols

Pharmacokinetic Study Via Oral Administration of the Molecules in Mice

The pharmacokinetic study of the chemical compounds (C) following the oral administration thereof was conducted using mice C57BL/6 (January, 53940 The Genest Saint Isle, France). The chemical compounds derived from norbixin (C) were administered at a dose of 50 mg/kg of body weight. After administration, the blood was sampled at the tail at t=0.25 h; 0.5 h; 1 h; 3 h; 6 h and 8 h. The blood samples were centrifuged and the plasmas taken. The dosage of the plasma samples made it possible to determine pharmacokinetic parameters, namely Cmax, which corresponds to the maximum concentration observed after the administration of the molecule, Tmaxwhich is the time required to reach maximum concentration after administration of the molecule and the AUC: the area under the curve which corresponds to the plasma exposure (FIG.2).

In parallel, the ocular concentrations of the chemical compounds derived from norbixin (C) were dosed in the following way (FIG.1): both eyes of each mouse were taken in Precellys tubes and stored at −80° C. until the time of dosing. The eyes are then ground in the Precellys tubes with a bench homogenizer, Fast-prep (Fischer Scientific, Hampton, United States) in a mixture of organic solvents, in a first step with 500 μL of chloroform/methanol (1/1, v/v) then 500 μL of chloroform/dichloromethane (1/1, v/v). The supernatants are recovered at each step and transferred to a 96-well plate of 2 mL.

For the quantification of the chemical compounds C, a calibration curve is carried out with 8 standards (5 to 5,000 ng/mL) in the same organic solvent mixtures and transferred (100 μL) to the 96-well plate of 2 mL.

The supernatants and standards are evaporated in a EZ2 (Genevac, Ipswich, United Kingdom), without heating, then taken with 100 μL of DMSO/methanol (20:80, v/v) before being transferred to a 96-well plate of 200 μL.

The LC-MSMS analysis is carried out with a HPLC 1200 Infinity chain (Agilent Technologies, Santa-Clara, United States), a UV detector and a mass spectrometer QQQ6420 (Agilent Technologies Santa-Clara, United States). The injection volume is 5 μL. The chemical compounds C are eluted on an inverse phase column C18 (2.1*50 mm, particles 3 μm; Ace-C18-Excel, AIT) with a gradient of acetonitrile and water (containing 0.1% formic acid) and a flow rate of 0.3 mL/min. The conditions of the gradient can change according to the chemical compound C analyzed. The UV detector analyzes at 460 nm and the mass spectrometer analyzed in Mode MRM—Positive.

Photoprotection of the Cells of the RPE

Tests in vitro of photoprotection by the various chemical compounds C of the Cells of the RPE Illuminated in the Presence of A2E

The test in vitro described hereinabove and intended to study the photo-protective effect of norbixin was used so as to quantify the photo-protective effects of the various chemical compounds derived from norbixin (C) on the cells of the RPE illuminated with blue light in the presence of A2E (FIG.3). The photo-protective effect of the molecules was evaluated in a phototoxicity cellular model induced by treatment by A2E followed by an illumination with blue light. The term “blue radiation” means the radiation corresponding to the blue band of the visible light spectrum, i.e. of a wavelength comprised between 435 and 490 nm. This model uses primary cultures of RPE of adult pigs. The cell survival is quantified thanks to a test of cellular viability. At −48 h the compounds to be tested (in solution at 5 mM in the DMSO) are added to obtain the final concentrations of 1 to 20 μM) then at −19 h of the A2E (final concentration 30 μM) and the cells (time 0 h) are illuminated. 24 h after the survival of the cells is measured. The acquisition of images, as well as the processing thereof, are carried out using a fluorescence microscope controlled by the Metamorph software and a dedicated quantification program. The experiments are conducted on a 96-well microplate in quadruplicate and the experiment is reproduced at least four times. 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 controlled wells (treated by the dilution medium without A2E). This test made it possible hereinabove to reveal the photo-protective activity of norbixin (Fontaine et al. 2016).

Results

Pharmacokinetic Study of the Chemical Compounds C in Mice

Table 2 discloses the pharmacokinetic results of the chemical compounds C following the administration p.o. 50 mg/kg in D-α-Tocopheryl polyethylene glycol 1000 succinate (VitE-TPGS) 20% in a sodium bicarbonate buffer (0.1M).

TABLE 2EyePlasmaCMaxTMaxExposureCMaxTMaxExposure(ng/eye)(h)(ng · h/eye)(μg/mL)(h)(μg · h/mL)BIO2018.60.522.434.990.25100.7C110.70.526.618.730.552.66C2410.5186.5340.5176.2C35.10.519.14.050.510.37C410.20.53057.071208.6C531.40.5188.737.711299.9C63.90.523.13.950.510.81C770.518.56.890.517.29C856.81472.459.820.5173.74C953.1145237.921342.68C1064.31533.230.421108.1C1147.71238.435.911190C1250.71538.429.310.5186.3C1360.71461.127.890.5139.7C1443.21322.519.020.5162.2C1522.80.5108.517.340.546.99C167.40.58.710.51113.23C17182135.28.06132.39C1827.72307.861.650.5948.84C195.70.537.56.620.2522.8C2080.42421.935.021160.4C211.60.250.66.610.259.17C2242.12358.512.830.2537.25C2311254.75.01124.1C24961.5499.148.310.5141.9

Table 3 corresponds to the percentages of photoprotection of the cells of the RPE by the chemical compounds C in vitro: it shows the percentage of cells of RPE surviving in the presence of N-retinyl-N-retinylidene ethanolamine (A2E) and of the various chemical compounds C derived from norbixin (tested at 5, 10 or 20 μM) or of norbixin in the same concentrations after having been subjected to an illumination.

TABLE 3Eye Exposure50 mg/kg p.o.Photoprotection (%)AUC5 μM10 μM20 μM(ng · h/eye)MeanSEMeanSEMeanSEBIO20122.419.26.434.16.663.64.6C126.655.04.867.72.277.87.1C2186.533.913.722.312.949.111.2C319.111.311.368.132.145.110.3C430.016.414.036.017.767.216.4C5188.711.311.325.623.336.011.9C623.1nd60.630.319.119.1C718.55.25.252.814.8ndC8472.428.68.433.48.275.26.7C9452.022.19.432.012.763.76.1C10533.215.51.127.427.480.311.4C11238.421.17.739.316.763.48.2C12538.47.73.114.37.659.64.3C13461.18.04.00.50.38.06.5C14322.53.13.146.81.279.36.0C15108.55.44.010.05.158.55.7C168.770.26.181.71.626.85.6C17135.271.75.467.26.444.04.9C18307.827.311.743.112.463.85.9C1937.560.67.560.67.579.94.1C20421.965.65.765.65.779.05.4C210.656.77.056.77.079.61.1C22358.564.58.272.85.766.07.9C2354.738.410.459.26.873.18.3C24499.147.015.069.013.084.013.6

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