Providing a precursor compound of retinoids in the vitamin A5 pathway and its use

Abstract

The present invention relates to the field of retinoid X receptor (RXR) signaling and a novel vitamin A pathway, known as the vitamin A5 pathway. Compounds that facilitate the provision of (R)-9-cis-13,14-dihydro-retinoic acid and endogenous RXR ligands, as well as uses and preparation methods, are described. The compounds of the present invention can be used for pharmaceutical and nutritional applications.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of November 17, 2017, application number 201780083885.8, and name “Providing precursor compounds of retinoids for vitamin A5 pathway and their uses”. Technical Field

[0002] The present invention relates to the field of retinoid X receptor (RXR) signaling and a novel vitamin A pathway, known as the vitamin A5 pathway. Compounds useful for providing (R)-9-cis-13,14-dihydro-retinoic acid and endogenous RXR ligands, their uses, and preparation methods are also provided. The compounds of the present invention can be used for pharmaceutical and nutritional applications.

[0003] More specifically, the precursors of 9cDHRA are, in particular, 9-cis-13,14-dihydroretinol (9CDHROL, vitamin A5) and 9-cis-13,14-dihydro-β-carotene (9CDHBC, provitamin A5), which are novel types of retinoid precursors and carotenoid RXR ligand precursors, respectively. It has surprisingly been discovered herein that these precursors can be metabolized directly or indirectly to 9CDHRA. The present invention also relates to the preventive / pharmaceutical uses of these compounds, in particular for the treatment of depressive-like behavior in chronically stressed animal models of depression and various other diseases that affect RXR-mediated signaling or for which RXR-mediated signaling is a therapeutic target. These diseases include neurodegenerative and metabolic diseases, skin and immune dysfunction (including inflammation), cardiovascular disease, and lifestyle applications such as memory enhancement effects. Background Art

[0004] Compared to vitamins C and D, vitamin A was the first compound associated with deficiency symptoms. Later, the active lipid was named "vitamin A." In 1931, Karrer et al. identified this fat-soluble nutritional derivative in cod liver oil (Karrer et al., 1931a; Karrer et al., 1931b). Paul Karrer, who elucidated the structure of retinol (i.e., vitamin A1), was awarded the Nobel Prize in Chemistry in 1937 for his work on the basic vitamin A. Simultaneously, in 1937-38, Edisbury et al. (Edisbury et al., 1937) and Gilliam et al. (Gilliam et al., 1938) discovered a dietary factor primarily found in marine fish. They used the term vitamin A2 to designate this second type of vitamin A because it exhibited an absorption spectrum different from retinol due to the presence of an additional double bond at the C3-C4 position of the ring.

[0005] In the 1980s, the molecular role of vitamin A was further expanded, primarily by the teams of Pierre Chambon and Ronald Evans, by identifying all-trans-retinoic acid (ATRA) as the bioactive mediator of a large number of the effects of vitamin A. They identified ATRA as a nutrient-derived lipid hormone, whereby its binding to RARs (retinoic acid receptors) was used to mediate transcriptional activity (Giguere et al., 1987), and RARs themselves as new members of the nuclear hormone receptor superfamily (Petkovich et al., 1987). In addition to RARs, retinoid-X receptors (RXRs) (Kliewer et al., 1992; Leid et al., 1992; Mangelsdorf et al., 1990; Evans 2014) have also been identified as mediators of important functions and as essential heterodimer-binding partners for a variety of nuclear hormone receptors. In 1992, 9-cis-retinoic acid (9CRA) was identified as a putative "endogenous" ligand for RXR (Heyman et al., 1992; Levin et al., 1992). At the same time, Vitamin A2 derivative All-trans-3,4-didehydro-retinoic acid (ATDDRA; Vitamin A2-acid) It was identified endogenously in humans (Vahlquist et al., 1982) and was later shown to exhibit activity similar to ATRA in activating RAR-mediated gene transcription (Torma et al., 1994). However, despite being a potent RXR ligand, 9CRA has only been strictly identified endogenously after administration of highly pharmacological (toxic) retinoids or after artificial nutritional intervention with vitamin A-rich foods (Arnhold et al., 1996; Schmidt et al., 2002; Ulven et al., 2001). In addition, “vitamin A” derivatives have been found in invertebrates, namely 3-hydroxyretinal (“vitamin A3”) in arthropods and 4-hydroxyretinal (“vitamin A4”) in some crustaceans (Babino et al., 2016).

[0006] Retinol (vitamin A1) plays an important role in vision, especially night vision, normal bone and tooth development, reproduction, and the health of the skin and mucous membranes (the mucus-secreting layers that line areas of the body, such as the respiratory tract). Vitamin A also acts as an antioxidant, a protective chemical in the body that may reduce the risk of certain cancers.

[0007] With regard to the medical uses of these compounds, all-trans-retinoic acid (ATRA, also known as tretinoin) is the active agent in several pharmaceutical preparations and is used in particular for cosmetic and topical applications against, for example, acne, and for acute promyelocytic leukemia.

[0008] The isomer 9-cis-retinoic acid (9CRA) is also available as a drug called alitretinoin. An oral formulation of 9CRA (alitretinoin) is marketed under the brand name Toctino.

[0009] Isotretinoin (The active agent is 13-cis-retinoic acid) is primarily indicated for the treatment of severe cystic acne vulgaris and is also indicated for the treatment of skin lesions of AIDS-related Kaposi's sarcoma.

[0010] The compound, marketed under the brand name Toctino, has been given prescription rights in the UK for oral administration for chronic hand eczema; however guidelines recommend that it be prescribed only in severe cases.

[0011] Thus, in contrast to the multifaceted effects of retinoids, only a few of them are used as drugs for relatively few specific diseases.

[0012] All of these variants are present in the drug in the acid form and contain a 13,14 double bond.

[0013] Palczewski et al. reported the endogenous presence of 13,14-dihydroretinoids in which the 13,14 double bond is hydrogenated, and all-trans-13,14-dihydroretinoic acid (ATDHRA) was identified in cell-based assays as a low-affinity ligand for RAR and a weaker activator than ATRA of RAR-controlled genes (Moise, 2004; Moise et al., 2005, Moise et al., 2009).

[0014] In addition to RAR, another type of receptor, retinoid X receptor, which forms heterodimers with RAR, plays an important role in nuclear receptor signal transduction [DA Mangelsdorf, RM Evans, Cell 1995, 83, 841-850]. The above-mentioned 9-cis-retinoic acid (9CRA) is RXR It is a potent active agent, a mediator of important functions, and an essential heterodimeric binding partner for a variety of nuclear hormone receptors.

[0015] In addition to 9CRA, a second class of derivatives that have been found to activate RXR-mediated signaling is various fatty acids, such as phytanic acid (PHYA), docosahexaenoic acid (DHA), and oleic acid (de Urquiza et al., 2000; Goldstein et al., 2003; Kitareewan et al., 1996). However, some findings suggest that the endogenous levels of these derivatives are too low to bind to RXR and trigger transcriptional activation.

[0016] Recently, the endogenous presence of 9-cis-13,14-dihydroretinoic acid (9CDHRA) and its all-trans isomer has been confirmed in several mouse organs (liver, serum, and brain) using a combined LC-MS-MS and UV analytical setup and comparison with synthetic standards. The amounts measured are believed to be sufficient to maintain RXR-dependent activity. Indeed, 9CDHRA was found to exhibit biological activities similar to those of synthetic RXR agonists and may regulate the transcriptional activity of several nuclear receptor signaling pathways through corresponding permissive heterodimers (Rühl et al., 2015).

[0017] However, the metabolic pathways leading to this compound remain unclear, and further work is needed to characterize this ligand and determine its possible multiple roles in biological systems (de Lera et al., 2016). In summary, to date, research on vitamin A has established the fundamental principles linking diet to activation of vitamin A and lipid hormone receptors, and further regulates signaling that leads to various (patho)physiological pathways. Unfortunately, the endogenous presence and nutritional relevance of RXR ligands, and in particular the role of 9CRA in this regard, has proven to be highly controversial.

[0018] The present inventors have now identified a series of compounds comprising members of an independent novel vitamin A pathway (vitamin A5), most notably 9-cis-13,14-dihydroretinol (9cDHROL), which are precursors of current endogenous RXR ligands. They have also surprisingly found that these precursors produce unexpectedly high increases in 9cDHROL in the brain (e.g., better than in the liver) and are useful vehicles for targeted brain signaling of RXR, and have also demonstrated their systemic production. Furthermore, starting with 9cDHROL, very high levels of 9cDHROL have been produced in tissues, and particularly in the brain.

[0019] These findings make the compounds of the present invention good candidates as pharmaceuticals and nutraceuticals.

[0020] Summary of the invention

[0021] The present invention relates to a compound of general formula (I)

[0022]

[0023] wherein R is selected from COOR1 and CH2OR2 and a group of formula (A), wherein

[0024] R1 is a group that is removed by hydrolysis in mammalian tissues or organs to produce (R) 9-cis-13,14-dihydroisoretinoic acid ester and a biologically acceptable and tolerable compound, and / or

[0025] R2 is H or acyl C(O)R3, wherein R3 is a group that is removed by hydrolysis in mammalian tissues or organs to produce (R)9-cis-13,14-dihydro-retinol and a biologically acceptable and tolerable compound, and / or

[0026] R is a group of formula A

[0027]

[0028] Wherein Q1 is substituted or unsubstituted C 6-10 alkenyl or cycloalkenyl, preferably substituted or unsubstituted trimethylcycloalkenyl or more preferably substituted or unsubstituted 2,6,6-trimethylcyclohexenyl, even more preferably unsubstituted 2,6,6-trimethylcyclohex-1-en-1-yl or 2,6,6-trimethylcyclohex-2-en-1-yl. If a trimethylcyclohexenyl group, such as 2,6,6-trimethylcyclohexenyl, is substituted, it is preferably substituted by hydroxy or by oxygen, preferably by oxygen;

[0029] wherein upon administration, the compound is converted to 9-cis-13,14-dihydro-retinol in mammalian tissues, organs or cells.

[0030] Very preferably, the group of formula A is a group of formula a

[0031]

[0032] Wherein, the compound is 9-cis-13,14-dihydro-β-β-carotene (9CDHBC).

[0033] Upon administration, the compound is converted to the R-configuration 9-cis-13,14-dihydroretinoic acid in mammalian tissues, organs or cells. Mammalian tissues, organs or cells are understood herein to mean at least one or at least one type of tissue or organ or cell culture or cell population, or a plurality of tissues or organs or cell cultures or cell populations including instances where different steps of the conversion occur in different tissues or organs or cell types.

[0034] Preferably, the compound is for use in the treatment of a mammalian subject, preferably a human subject. Preferably, the compound is for use in the treatment of a disease, wherein the disease is associated with the treatment of a retinoid X subject.

[0035] Optionally, in particular wherein the invention relates to compounds themselves R1 is different from ethyl, in particular within the above definition.

[0036] The enantiomeric configuration of the compound is shown in the formula. Preferably, the compound or any composition comprising the compound is enriched in the enantiomer (usually indicated as (R)) or preferred enantiomer as defined herein.

[0037] The present invention also relates to a compound of general formula (I)

[0038]

[0039] wherein R is selected from COOR1 and CH2OR2 and a group of formula (A), wherein

[0040] R1 is C 1-25 Alkyl or C 2-25 Alkenyl and / or

[0041] R2 is H or acyl C(O)R3, wherein R3 is C 1-25 Alkyl or C 2-25 Alkenyl and / or

[0042] R is a group of formula A

[0043]

[0044] wherein Q1 is a substituted or unsubstituted trimethyl-cycloalkenyl group forming a tetraterpene-derived compound of formula A, or more preferably, Q1 is a substituted or unsubstituted 2,6,6-trimethylcyclohexenyl group, even more preferably an unsubstituted 2,6,6-trimethylcyclohex-1-en-1-yl group or a 2,6,6-trimethylcyclohex-2-en-1-yl group. If the trimethylcyclohexenyl group, such as 2,6,6-trimethylcyclohexenyl, is substituted, it is preferably substituted by hydroxy or by oxygen, preferably by oxygen;

[0045] wherein upon administration, the compound is converted to 9-cis-13,14-dihydro-retinol in mammalian tissues, organs or cells.

[0046] Very preferably, the group of formula A is a group of formula a

[0047]

[0048] Wherein, the compound is 9-cis-13,14-dihydro-β-β-carotene (9CDHBC).

[0049] Once administered, the compound can be converted to the R-configuration 9-cis-13,14-dihydroretinoic acid in mammalian tissues or organs.

[0050] In a preferred embodiment, in the compound of the present invention of general formula I, R1 or R2 is as defined above, or the compound is a 9-cis-carotenoid compound, the 9-cis-carotenoid compound is a 9-cis-13,14-dihydro-β-carotene derivative or 9-cis-13,14-dihydro-β-carotene, preferably 9-cis-13,14-dihydro-β-carotene, as a precursor compound, wherein, upon administration, the compound is converted into R-configuration 9-cis-13,14-dihydroretinoic acid in mammalian tissues, organs or cells.

[0051] In the case of R1 or R2, the preferred choice of alkyl or alkenyl chain length is as defined herein.

[0052] Preferably, the compounds are for use in the treatment of a mammal, preferably a human subject, as defined herein.

[0053] Optionally, in particular, the invention relates to the compound itself wherein R1 is different from ethyl. "The invention relates to the compound itself" means that the compound is claimed as a product and is not limited to medical uses or purposes, or diagnostic uses or purposes, on the human or animal body. Furthermore, it means that the compound is not claimed as part of a use or method in a use or method claim.

[0054] The enantiomeric configuration of the compound is shown in the formula. Preferably, the compound or any composition comprising the compound is enriched in the enantiomer (usually indicated as (R)) or preferred enantiomer as defined herein.

[0055] In the first aspect, the compound is a compound having the general formula (3)

[0056]

[0057] Wherein R2 is H or acyl C(O)R3, wherein R3 is selected from

[0058] C 1-25 Alkyl or C 1-23 Alkyl, preferably C 1-8 Alkyl or C 1-6 Alkyl and C 9-23 Alkyl, more preferably C 1-4 Alkyl and C 11-21 Alkyl; and

[0059] C 2-25 Alkenyl, preferably C 2-8 Alkenyl or C2-6 Alkenyl and C 2-23 Alkenyl, more preferably C 13-23 Alkenyl.

[0060] In a preferred embodiment, R3 is C 1-8 Alkyl, preferably C 1-6 Alkyl, more preferably C 1-4 alkyl.

[0061] In a preferred embodiment, R3 is C 9-23 Alkyl, preferably C 11-21 Alkyl, more preferably C 13-19 In a preferred embodiment, R3 is C 2-8 Alkenyl, preferably C 2-6 Alkenyl, more preferably C 2-4 Alkenyl.

[0062] In a preferred embodiment, R3 is C 9-25 Alkenyl, preferably C 11-23 Alkenyl, more preferably C 13-23 Alkenyl.

[0063] In one embodiment, R2 is H and the compound is (R)(R)9-cis-13,14-dihydro-retinol.

[0064] In another embodiment, R2 is an acyl group C(O)R3, and R3 is a compound as defined above, wherein R3 is a group that is removed by hydrolysis of the ester in mammalian tissues or organs to produce the corresponding alcohol (R) 9-cis-13,14-dihydro-retinol. Thus, the ester is converted to an alcohol and a biologically tolerable and / or acceptable compound. Upon administration, the alcohol compound is converted to the R-configuration 9-cis-13,14-dihydroretinoic acid in mammalian tissues or organs. This refers to the compound having such an ability, i.e., depending on whether the compound is actually administered, "is" refers to "being capable of."

[0065] Preferably, the compounds are used in the treatment of mammalian subjects, preferably human subjects.

[0066] The enantiomeric configuration of the compound is shown in the formula. Preferably, the compound or any composition comprising the compound is enriched in the enantiomer (usually indicated as (R)) or preferred enantiomer as defined herein.

[0067] In a preferred embodiment of this aspect of the invention, the compound has the general formula (4)

[0068]

[0069] Wherein, R3 is as defined in formula (3), or R3 is selected from

[0070] -C 1-4 Alkyl, preferably methyl, ethyl, propyl or isopropyl,

[0071] -C 11-21 Alkyl, preferably C 13-19 Alkyl and

[0072] -C 11-23 alkyl.

[0073] Preferably, the alkenyl group is a polyunsaturated C 13-23 Alkenyl.

[0074] In a preferred embodiment, upon administration, the compound is converted to the R-configuration of 9-cis-13,14-dihydroretinol in mammalian tissues or organs.

[0075] More preferably, the mammalian tissue is a nervous tissue, or the tissue or organ is a tissue or organ of the central nervous system or the peripheral nervous system. The mammalian cells are preferably neurons (nerve cells), preferably or especially oligodendrocytes.

[0076] In another preferred embodiment, the mammalian tissue is blood. In another preferred embodiment, the mammalian tissue is liver.

[0077] In another aspect of the present invention, the compound has the general formula (2)

[0078]

[0079] wherein R1 is selected from

[0080] C 1-25 Alkyl or C 1-23 Alkyl, preferably C 1-6 Alkyl and C 9-23 Alkyl, more preferably C 1-4 Alkyl and C 11-21 Alkyl; and

[0081] C 2-25 Alkenyl or C 2-24 Alkenyl, preferably C 2-6 Alkenyl and C 9-23 Alkenyl, more preferably C 13-23 Alkenyl.

[0082] Optionally, in particular wherein the invention relates to compounds themselves R1 is different from ethyl.

[0083] The enantiomeric configuration of the compound is shown in the formula. Preferably, the compound or any composition comprising the compound is enriched in the enantiomer (usually indicated as (R)) or preferred enantiomer as defined herein.

[0084] In a preferred embodiment, R1 is selected from C 1-8 Alkyl and C 9-23 Alkyl, more preferably C 1-4 Alkyl and C 11-21 Alkyl; and

[0085] C 2-25 Alkenyl or C 2-24 Alkenyl, preferably C 2-6 Alkenyl and C 9-23 Alkenyl, more preferably C 13-23 Alkenyl.

[0086] In a preferred embodiment, R1 is C 1-8 Alkyl, preferably C 1-6 Alkyl, more preferably C 1-4 alkyl.

[0087] In a preferred embodiment, R1 is C 2-8 Alkenyl, preferably C 2-6 Alkenyl, more preferably C 2-4 Alkenyl.

[0088] In a preferred embodiment, R1 is selected from methyl, ethyl, propyl or isopropyl, optionally methyl, propyl or isopropyl.

[0089] In another aspect, the present invention also relates to a compound of formula (5)

[0090]

[0091] Wherein Q1 is substituted or unsubstituted C 6-10 alkenyl or cycloalkenyl, preferably substituted or unsubstituted trimethylcycloalkenyl or more preferably substituted or unsubstituted 2,6,6-trimethylcyclohexenyl, even more preferably unsubstituted 2,6,6-trimethylcyclohex-1-en-1-yl or 2,6,6-trimethylcyclohex-2-en-1-yl. If a trimethylcyclohexenyl group such as 2,6,6-trimethylcyclohexenyl is substituted, it is preferably substituted by hydroxy or by oxygen, preferably by oxygen;

[0092] wherein upon administration, the compound is converted to 9-cis-13,14-dihydro-retinol in mammalian tissues, organs or cells.

[0093] Very preferably, the group of formula A is a group of formula a

[0094]

[0095] In a preferred embodiment, the present invention also relates to a 9-cis-carotenoid compound, which is a 9-cis-13,14-dihydro-β-carotene derivative or 9-cis-13,14-dihydro-β-carotene, such as 9-cis-13,14-dihydro-β-β-carotene or 9-cis-13,14-dihydro-β-α-carotene, preferably 9-cis-13,14-dihydro-β-β-carotene, as a precursor compound, wherein, upon administration, the compound is converted into R-configuration 9-cis-13,14-dihydroretinoic acid in mammalian tissues, organs or cells. In particular, the 9-cis-carotenoid compound is a biologically acceptable and tolerable compound.

[0096] In yet another aspect, the present invention relates to a pharmaceutical composition comprising a compound of formula (3) as defined above, said composition further comprising one or more pharmaceutically acceptable carriers, such as additives and / or excipients.

[0097] Preferably, the present invention relates to a pharmaceutical composition comprising a compound of formula (4) as defined above, said composition further comprising one or more pharmaceutically acceptable carriers, such as additives and / or excipients.

[0098] Preferably, the present invention relates to a pharmaceutical composition comprising a compound of the 9-cis-carotenoid as defined above and / or a compound of the general formula (5), in particular a compound of 9-cis-13,14-dihydro-β-carotene, as a precursor compound, said composition further comprising one or more pharmaceutically acceptable carriers, such as additives and / or excipients.

[0099] The present invention also relates to a nutritional composition comprising a compound of formula (3) or preferably a compound of formula (4), or a compound of formula (5) or a 9-cis-carotenoid compound, preferably a 9-cis-13,14-dihydro-β-carotene compound as defined above as a precursor compound, said composition further comprising one or more nutritionally acceptable additives and / or excipients. Preferably, said composition is a dietary supplement, a functional food, a medical food or a food with a health claim. Preferably, said nutritional composition comprises said compound in an amount or concentration which is higher than the amount and concentration present in a natural food matrix; and / or said nutritional composition comprises an additional amount of said compound, preferably an additional isolated or artificially prepared compound.

[0100] The present invention also relates to a novel chemical synthesis method of 9-cis-13,14-dihydroretinol and its ester form via 9-cis-13,14-dihydroisoretinoic acid alkyl esters. The method comprises:

[0101] i) reducing 9-cis-13,14-dihydroisotretinoin to 9-cis-13,14-dihydroretinol, and optionally

[0102] ii) Alkylation of 9-cis-13,14-dihydroretinol to produce the corresponding ester form. Examples of such esters are given below.

[0103] In a preferred embodiment, the reduction is carried out over a hydride catalyst, such as diisobutylaluminum hydride (DIBAL-H) ​​in an organic solvent, preferably a small heterocycle such as tetrahydrofuran (THF).

[0104] The esterification of the alcohol is preferably carried out by means of an alkyl and hydride reagent. Preferably, a basic nitrogen containing monocyclic heterocycle is used, such as pyridine and / or derivatives. In a very preferred embodiment, the reduction is carried out in the presence of dimethylaminopyridine (DMAP).

[0105] Preferred organic solvents include CH2Cl2, chloroform, CCl4 and the like.

[0106] Other esters of 9-cis-13,14-dihydroretinoic acid and 9-cis-13,14-dihydroretinol (eg, palmitate, etc.) can be readily prepared using the same sequence.

[0107] In yet another embodiment, the present invention also relates to a novel chemical synthesis of a compound of formula (5) as defined above, preferably a 9-cis-carotenoid compound, wherein the 9-cis-carotenoid is a 9-cis-13,14-dihydro-β-carotene derivative, preferably 9-cis-13,14-dihydro-β-β-carotene, said method comprising

[0108] --Convert the compound of general formula (3) into the corresponding aldehyde of formula (6) as an intermediate

[0109]

[0110] Wherein, R2 is H or OH-protecting group;

[0111]

[0112] - reacting the aldehyde intermediate with a Wittig reagent compound (7)

[0113]

[0114] Wherein Q1 is substituted or unsubstituted C 6-10 alkenyl or cycloalkenyl, preferably substituted or unsubstituted trimethyl-cycloalkenyl, or more preferably substituted or unsubstituted 2,6,6-trimethylcyclohexenyl, even more preferably unsubstituted 2,6,6-trimethylcyclohex-1-en-1-yl or 2,6,6-trimethylcyclohex-2-en-1-yl. If a trimethylcyclohexenyl group such as 2,6,6-trimethylcyclohexenyl is substituted, it is preferably substituted by hydroxy or by oxygen, preferably by oxygen;

[0115] And R is the formation of phosphorus-containing internal The ylide group is used to obtain the desired compound according to formula (5), wherein the phosphorus-containing internal The group of the salt is preferably a phosphorus group, preferably a triphenylphosphine group.

[0116] In a preferred embodiment, 9-cis-13,14-dihydro-β-β-carotene is prepared wherein the compound of formula (7) is a compound of formula (8)

[0117]

[0118] R is the formation of phosphorus-containing internal The group of the salt is used to obtain 9-cis-13,14-dihydro-β-β-carotene. The group of the salt is preferably a phosphorus group, preferably a triphenyl-phosphorus group.

[0119] In a further aspect, the present invention also relates to the use of a compound of formula (3), or preferably a compound of formula (4), or a compound of formula (5), or a 9-cis-carotenoid, or a specifically selected compound, as defined above, as a food ingredient or food supplement. Preferably, the food ingredient is an ingredient of a functional food or a medical food.

[0120] In one embodiment of the invention, a compound of formula (3), or preferably a compound of formula (4) or a compound of formula (5) or a 9-cis-carotenoid or a specifically selected compound as defined above is used in the treatment of a mammalian subject.

[0121] In a preferred embodiment, the compound is in the form of a pharmaceutical composition.

[0122] In another preferred embodiment, the compound is in the form of a nutritional composition, which requires authorization for sale for the indications as provided herein.

[0123] In yet another aspect, the present invention relates to a pharmaceutical composition comprising a compound of formula (2) as defined above, further comprising one or more pharmaceutically acceptable carriers, such as additives and / or excipients.

[0124] The present invention also relates to a nutritional composition comprising a compound of formula (2) as defined above, said compound further comprising one or more nutritionally acceptable additives and / or excipients. Preferably, said composition is a dietary supplement, a functional food, a medical food or a food with a health claim.

[0125] In yet another aspect, the present invention also relates to the use of the compound of formula (2) as defined above as a food ingredient. Preferably, the food ingredient is an ingredient of a functional food or a medical food.

[0126] In one embodiment of the invention, the compound of formula (2) as defined above is used for treatment of a mammalian subject.

[0127] In a preferred embodiment, the compound is in the form of a pharmaceutical composition.

[0128] In another preferred embodiment, the compound is in the form of a nutritional composition, which requires authorization for sale for the indications as provided herein.

[0129] In a preferred embodiment, the compound or pharmaceutical composition or nutritional composition is used for the prevention and / or treatment of RXR-signaling mediated dysfunctions.

[0130] In a preferred embodiment, the compound, pharmaceutical composition, or nutritional composition is used to prevent and / or treat diseases associated with retinoid X receptor signaling or diseases caused by impaired retinoid X receptor signaling. Preferably, the disease can be treated, prevented, or alleviated by a selective retinoid X receptor ligand.

[0131] In a preferred embodiment, the compound, pharmaceutical composition or nutritional composition is used for the prevention and / or treatment of diseases associated with the central nervous system and diseases associated with the peripheral nervous system.

[0132] In a preferred embodiment, the compound, pharmaceutical composition or nutritional composition is used for the prevention and / or treatment of mental illness.

[0133] In a preferred embodiment, the compound, pharmaceutical composition or nutritional composition is used for the prevention and / or treatment of memory disorders, or for enhancing memory performance, wherein preferably the memory refers to working memory.

[0134] In a preferred embodiment, the compound, pharmaceutical composition or nutritional composition is used for the prevention and / or treatment of impaired cognitive function or impaired learning ability.

[0135] In a preferred embodiment, the compound, pharmaceutical composition or nutritional composition is used for the prevention and / or treatment of depression.

[0136] In a preferred embodiment, the compound, pharmaceutical composition or nutritional composition is used for the prevention and / or treatment of neurodegenerative diseases.

[0137] In a preferred embodiment, the compound, pharmaceutical composition or nutritional composition is used for the prevention and / or treatment of a neurodegenerative disease selected from Alzheimer's disease, Parkinson's disease, mild cognitive impairment (MCI), Parkinson's disease with mild cognitive impairment, Huntington's disease, dementia with Lewy bodies (DLB), amyotrophic lateral sclerosis (ALS) and other neurodegeneration-related dementias caused by brain changes due to aging, disease or trauma; or spinal cord injury and ataxia, disseminated sclerosis and multiple sclerosis or other neurological conditions, preferably selected from Alzheimer's disease and Parkinson's disease.

[0138] In the above medical indication embodiment, the compound is preferably selected from the general compound (2), (3) or (4) as defined above. In the above medical indication embodiment, the compound is preferably selected from the general compound (3) as defined above, or preferably the compound (4).

[0139] The present invention also relates to the use of any of the above compounds in the preparation of a pharmaceutical formulation or medicament as defined herein or above.

[0140] The present invention also relates to the use of any of the above compounds in the preparation of a nutritional formulation as defined herein or above, preferably a medical food or a food with a health claim.

[0141] The present invention also relates to a method for treating a disease as defined above, wherein a compound of the present invention or a pharmaceutical composition or nutritional composition of the present invention is administered in an effective dose to a mammalian subject, preferably a human in need thereof. In a preferred embodiment, administration is regular, for example daily.

[0142] The present invention also relates to the use of any compound as defined herein for maintaining health, preferably as a food ingredient, dietary supplement, for example as a vitamin or a precursor thereof; or as a nutritional ingredient. In a preferred embodiment, the present invention relates to the use of any compound as defined herein for maintaining health to prevent a condition as defined herein. In an embodiment, the use is for maintaining normal vision, maintaining normal skin and mucous membranes, and maintaining normal hair.

[0143] More preferably, the use is for maintaining normal brain function, or maintaining mental health, or maintaining mental performance, or maintaining normal psychological function or cognitive function, or for contribution to the function of the nervous system. In an embodiment, these uses are defined in the health claim.

[0144] The present invention also relates to a method for maintaining a healthy state of a subject as defined above, wherein a compound of the present invention or a nutritional composition of the present invention is administered to a mammalian subject, preferably a human in need thereof, as part of a diet. In a preferred embodiment, administration is regular, for example daily.

[0145] It is proposed herein that non-selective precursors cannot be used according to the present invention for the treatment and prevention of RXR-signaling mediated dysfunctions, as explained herein in conjunction with RXR and RAR receptors.

[0146] The present invention also relates to the use of a compound according to any one of the general formulae (3), (4) and / or (5) as defined above as a dietary supplement, such as vitamin A5 (9CDHROL) or a precursor thereof. In a particular embodiment, the precursor is 9CDHBC. In another particular embodiment, the precursor is selected from 9CDHROL esters.

[0147] The present invention also relates to the use of a compound according to any one of the general formulae (3), (4) and / or (5) as defined above as a dietary supplement, such as a 9CDHRA precursor and a RXR-ligand. In a particular embodiment, the 9CDHRA precursor is selected from 9CDHBC, 9CDHROL, 9CDHROL-esters and 9CDHRA-esters.

[0148] Preferably, the precursor is administered intravenously, topically or orally.The precursor may be administered systemically or topically.

[0149] These precursors can be used for selective metabolic conversion into 9CDHRA, an endogenous RXR-ligand for achieving RXR-mediated signaling.

[0150] In a specific embodiment, the present invention relates to the use of 9CDHBC for preventing, treating or ameliorating any of the conditions defined above. In a preferred embodiment, the condition is selected from the group consisting of: (a) affective disorders associated with stress, including but not limited to psychiatric disorders such as depression and schizophrenia; and (b) memory impairment associated with dementia, particularly Alzheimer's disease.

[0151] In a specific embodiment, 9CDHBC is used for memory improvement.

[0152] In a particular embodiment, the present invention relates to the use of 9CDHROL as defined herein or any ester thereof for the prevention, treatment or amelioration of any condition as defined above. In a preferred embodiment, the condition is selected from the group consisting of: (a) affective disorders associated with stress, including but not limited to psychiatric disorders such as depression and schizophrenia; (b) memory impairment associated with dementia, in particular Alzheimer's disease.

[0153] In a specific embodiment, 9CDHROL or any ester thereof is used for memory improvement.

[0154] In a particular embodiment, the present invention relates to the use of 9CBC as a carotenoid precursor for 9CDHBC.

[0155] In a specific embodiment, the present invention relates to the use of 9CBC as a food supplement to increase 9CDHBC levels in humans in non-therapeutic applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0156] Figure 1 :9CDHROL treatment improved working memory performance in the Delayed Non-Match to Place Task (DNMTP). Figure 1 The effect of 9CDHROL treatment on working memory performance in the delayed mismatch to place task (DNMTP) is shown. (a) Learning curves of wild-type C57BL6N male mice. The increasing learning curve of wild-type C57BL6N male mice (n=5) illustrates the acquisition of a working memory task. Memory performance scores for increasing inter-trial intervals (ITI) are shown in the gray area and correspond to an ITI of 3 min, with mice exhibiting memory deficits and the entire group reaching a mean ITI of 13 min (denoted as gr13). (b) Correct choice 7-9 hours after treatment with 9CDHROL and vehicle when tested at an average ITI of gr13 min. When tested at an average ITI of gr13 min, 9CDHROL (but not vehicle treatment) increased working memory performance 7-9 hours after treatment. Statistical differences determined using one or two group t-tests are represented as: *: p < 0.05 when compared with the chance level of 50% power; and #: p < 0.05 when compared with the power of the last day of the acquisition phase (ten days), respectively.

[0157] Figure 2 : 9CDHROL standard mixture (top chromatogram) and endogenous levels in vehicle-treated mouse liver (bottom chromatogram).

[0158] Figure 3 : 9CDHROL in vehicle-treated mice representing endogenous levels (blue or dark gray) and 9CDHROL in 9CDHROL-treated mice (red or light gray).

[0159] Figure 4 : 9CDHRA levels representing endogenous levels after vehicle treatment (blue or dark gray) and 9CDHRA levels after 9CDHROL-treatment (red or light gray) in mice.

[0160] Figure 5 : 9CDHRA levels representing endogenous levels after vehicle treatment (blue or dark gray) and 9CDHROL-treated 9CDHRA levels (red or light gray) in mouse liver. Top panel: normal range (set at maximum); bottom panel: expanded range of the maximum range of endogenous retinoids.

[0161] Figure 6 : 9CDHRA control treatment (endogenous levels) and 9CDHRA after retinoid supplementation to dendrites. Upper panel: endogenous (blue or dark gray), 9CDHROL (orange or light gray), and 9CDHROL-acetate (pink); and lower panel: 9CDHRA-acetate (pink or medium gray).

[0162] Figure 7 : Overview of putative nutritional precursors for the new vitamin A5 cluster precursors. Figure 7 Putative metabolic pathways for vitamin 5. Retinoids not identified as endogenous retinoids are labeled in blue. Abbreviations: 9-cis-13,14-dihydro-retinol (9CDHROL), 9-cis-13,14-dihydro-retinal (9CDHRAL), 9-cis-13,14-dihydro-retinoic acid (9CDHRA), all-trans-13,14-dihydro-retinal (ATDHRAL), all-trans-13,14-dihydro-retinol (ATDHROL), all-trans-13,14-dihydro-retinoic acid (ATDHRA).

[0163] Figure 8Increasing doses of (R)-9CDHRA reversed working memory deficits in Rbp1- / - mice and exhibited pro-mnesic activity in the DNMTP task in WT mice (n=8 / group) when tested at the minimum ITI, which was 6 min for Rbp1- / - and 12 min for WT mice. ttt: ATRA is cytotoxic at a concentration of 10-5 M. *: p < 0.05. #: p < 0.05; ##: p < 0.01 compared to vehicle treatment in the same group; $: p < 0.05; $$: p < 0.01; One-group t-tests were used for comparisons with performance at 50% chance. All error bars represent SEM.

[0164] Figure 9 R-9CDHRA exhibits RXR agonist-like activity in vivo. Reversal of behavioral deficits following treatment with UVI2108 and R-9CDHRA.

[0165] Increasing doses of R-9CDHRA (0.1, 1, 2 mg / kg) reduced despair behavior in Rbp1- / - mice in the forced swim test (n=26 for the vehicle group; n=6-8 for each of the remaining groups); Statistical differences shown by PLSD Fisher test are indicated as: ***, p<0.001 for comparison with vehicle-treated WT controls in each group. All error bars represent SEM.

[0166] Figure 10a and Figure 10b : R-9CDHRA shows antidepressant effects in the chronic social defeat stress model.

[0167] Figure 10a Social defeat stress significantly increased immobility time in the forced swim test in recipient mice (vehicle; n=12) compared to non-stressed controls (control; n=7). 9cDHRA treatment reduced this immobility time in stressed mice in a dose-dependent manner (n=8, 1 mg / kg 9cDHRA; n=6, 3 mg / kg 9cDHRA) and to a similar extent as the synthetic RXR agonist UVI2108 (n=12).

[0168] Figure 10b(b) Social defeat stress-induced sucrose deficiency was prevented by treatment with UVI2108 or 9cDHRA. Statistical differences indicated by the PLSD Fisher test are: *: p < 0.1 compared to control mice; #: p < 0.1 compared to vehicle-treated stressed mice; $$: p < 0.01, $: p < 0.1, equivalent to 50% of sucrose consumption compared to no sucrose preference. All error bars represent the SEM.

[0169] Figure 11 : Measurement of retinol in cell culture after administration of retinol: standard ROL (black line), ATROL (blue line), and 9CROL (red line).

[0170] Figure 12 : Measurement of DH-RA in cell culture after retinol administration: standard ROL (black line), ATROL (blue line), and 9CROL (red line).

[0171] Figure 13 : RA measurement in cell culture after retinol administration: standard ROL (black line), ATROL (blue line), and 9CROL (red line).

[0172] Figure 14A Figure 2: 9CDHROL standard mixture (top chromatogram) and endogenous levels in mouse brain (middle chromatogram) and endogenous levels in mouse brain after 9CDHROL-treatment (bottom chromatogram). The two y-axis scales for brain samples are exactly the same, while the y-axis for the standard is dissimilar and is fitted at the highest point of the relevant peak.

[0173] Figure 14B : 9CDHROL (top chromatogram) and 9CDHROL / ATDHROL standard mixture (bottom chromatogram) in human serum, the two y-axis scales are dissimilar and are fitted at the highest point of the relevant peak.

[0174] Figure 14C : 9CDHROL in human food chain / cow liver (top chromatogram) and 9CDHROL / ATDHROL standard mixture (bottom chromatogram), the two y-axis scales are dissimilar and are fitted at the highest point of the relevant peak.

[0175] Figure 14D : After administration of CTRL, 9CBC, 9CDHBC, and 9CDHROL, 9CDHBC and 9CBC are converted to 9CDHROL in a human oligodendrocyte cell line in vitro. The Y-axis scales of the top three chromatograms are similar and fit at the highest point of the relevant peak, while the bottom chromatogram has a larger scale.

[0176] Figure 15: 9CDHBC in the human food chain, standard 9CDHBC eluting at 25.0 min (top panel) and peaches extracted from a can (bottom panel) with a co-eluting peak at 25.0 min and comparable UV / VIS spectra (data not shown).

[0177] Figure 16A and Figure 16B For the internal transformation of carotenoids.

[0178] Figure 16A : The administered 9CBC was converted into 9CDHBC in a human oligodendrocyte cell line in vitro. A peak with a retention time of 25.1 min was present after 9CDHBC treatment, and this peak was present at a lower level after 9CBC treatment.

[0179] Figure 16B ATBC is not converted into 9CBC or 9CDHBC in human oligodendrocyte cell lines in vitro. ATBC elutes at 27.2 min (detectable at 411 nm with a UVmax of 450 nm) and is present at very high levels after ATBC treatment (the second figure from the top on the right). 9CBC exists as a major shoulder peak after 9CBC treatment and is undetectable after CTRL or optional treatment.

[0180] Figure 17A : 9CDHROL, 9CDHROL-ester and 9CDHRA-ester were converted to 9CDHRA in a human oligodendrocyte cell line in vitro. Slight differences in retention times of the derivatives were observed due to the use of different HPLC systems and extraction methods, retinoid gradients as described in the Materials and Methods section.

[0181] Figure 17B : Conversion of 9CDHBC to 9CDHRA: Standard levels of 9CDHRA and ATDHRA in mouse liver following control treatment / endogenous (top chromatogram) and after 9CDHBC supplementation (bottom chromatogram), using the same y-axis scale.

[0182] Figure 17C : Conversion of 9CDHROL to 9CDHRA: Standard levels of 9CDHRA and ATDHRA in serum, brain, and liver after control treatment / endogenous (top chromatogram) and after 9CDHROL supplementation (bottom chromatogram) in mice, using the same y-axis scale size for each relevant organ (serum, brain, liver).

[0183] Figure 18: Antidepressant activity of 9CDHROL and 9CDHBC in a chronic social defeat stress model. (a) Social defeat stress significantly increased immobility time in the forced swim test in mice receiving vehicle (vehicle; n=6) compared to non-stressed control mice (control; n=6). Similar to 9CDHBC (n=4), 9CDHROL treatment (n=5) reduced this immobility in stressed mice. (b) Treatment with 9CDHROL or 9CDHBC prevented the lack of sucrose preference caused by social defeat stress. Statistical differences shown by t-test are represented as: **, p<0.01 when compared to control mice; #, p<0.05 when compared to vehicle-treated stressed mice; $, p<0.05 compared to 50% of the value of sucrose consumption in the absence of sucrose preference. All error bars represent standard error of measurement (SEM).

[0184] definition

[0185] It should be understood that the present invention is not limited to the specific examples and embodiments provided herein, and includes alternatives within the skill of those skilled in the art. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting.

[0186] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0187] Typically, the compounds according to the invention relate to enantiomers (pure enantiomers or mixtures, provided that they are obtainable by the process of the invention), hydrates and solvates thereof, solid forms thereof and mixtures of said forms.

[0188] An "enantiomer" is one of a pair of optical isomeric compounds that are different compounds and are mirror images of each other.

[0189] In the present context, "enantiomers" are preferably understood to mean compounds of the invention obtainable by enantioselective methods, preferably enantioselective preparation methods, very preferably enantiomerically or optically pure compounds. "Enantiomerically" or "optically pure" are compounds in which the molecules have the same chirality (to the extent obtained by the methods of the invention). Preferably, enantiomerically or optically pure means optically pure, more preferably having an optical purity of at least 90%, preferably at least 95%, more preferably at least 98% or 99%. Very preferably, "enantiomerically" or "optically pure" molecules have the same chirality within the limits of detection.

[0190] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, which is saturated and has up to 25 (preferably 23, 21, or 19) carbon atoms. In certain embodiments, an alkyl group may contain from 1 to 25 carbon atoms (referred to as C 1-25 alkyl), or preferably, it may be C 13-25 Alkyl, C 13-23 Alkyl, C 13-21 Alkyl, C 13-19 Alkyl or C 13-17 Alkyl; alternatively, it can be a short chain alkyl, for example, C 1-3 Alkyl, C 1-4 Alkyl, C 1-5 Alkyl, C 1-6 Alkyl, C 1-7 Alkyl or C 1-8 Alkyl; or alternatively in some embodiments, it can be C 8-17 Alkyl, C 8-15 Alkyl or C 8-13 Alkyl. The alkyl group is attached to the rest of the molecule by a single covalent bond. The alkyl group is preferably an unbranched straight hydrocarbon chain. Alternatively, it may contain branched hydrocarbon chains, however, the branched side chains are typically methyl, ethyl or propyl groups, preferably methyl or ethyl groups. The alkyl chain may be optionally substituted with one or more of the following substituents: halogen (including -F, Br, Cl or I), cyano, nitro, oxo, C 1-3 Preferably, the alkyl group is unsubstituted.

[0191] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is unsaturated, i.e., contains at least one double bond (i.e., C=C), and has at least 2 and up to 25 (preferably 23, 21, or 19) carbon atoms. In certain embodiments, an alkyl group may contain from 2 to 25 carbon atoms (referred to as a C 2-25 alkyl); mutatis mutandis, the chain length may be the same as that of the alkyl, however the shortest chain contains at least 2 carbon atoms. With regard to branching and substitution, the same applies to the case of alkyl chains.

[0192] The esters of (R)9-cis-13,14-dihydro-retinol of the present invention can be fatty acid esters. Here, the term "fatty acid" refers to a carboxylic acid with a long aliphatic (non-aromatic) chain, which can be saturated (alkyl) or unsaturated (alkenyl). Preferably, the alkyl chain in the fatty acid used in the present invention is a straight or branched open-chain compound. Typically, the fatty acid alkyl chain contains at least 11 and up to 25, preferably 23, 22 or 21 carbon atoms.

[0193] The compounds of the present invention also have pharmaceutical (drug) and nutritional uses.

[0194] "Pharmaceutical composition" refers to a composition used to treat the human or animal body to restore or maintain health, comprising (one or more) active agents and one or more additional substances used as carriers. The term "carrier" refers to a diluent, adjuvant, filler, excipient, stabilizer, or vehicle with which a formulated pharmaceutical agent is administered.

[0195] "Nutrient" refers to a food that provides health benefits in addition to its basic nutritional value. Nutraceuticals have physiological benefits or provide prevention of physiological disorders or discomfort. Nutritional compositions comprise the composition of the present invention and at least one additional substance, such as a nutritional carrier or food component.

[0196] The term "dietary supplement" refers to a nutraceutical, such as a nutritional composition intended to provide nutrients that may not otherwise be consumed in adequate amounts.

[0197] "Functional food" is also a nutraceutical, such as a nutritional composition, and refers to any improved food or food ingredient that provides a benefit or offers protection against a physiological disorder or discomfort; beyond the traditional nutrients it contains.

[0198] A health claim defines the health benefits of a nutraceutical and is approved by regulatory authorities under national or equivalent law (similar to the instructions for a drug). A health claim should be included on food labels and when the food is sold.

[0199] As used herein, the word "comprising" means containing, i.e., allowing for the presence of other entities or components. The term can be limited to "consisting essentially of," which means including the listed essential components or ingredients and optionally other non-essential components or ingredients; or limited to "consisting of," which means excluding any additional components.

[0200] "Treatment" may include prophylaxis and / or treatment.

[0201] "Preventing" or "prevention" of the development of a disease or condition means at least reducing the likelihood of developing or being susceptible to the disease or condition, or preferably preventing at least one clinical symptom of the disease or condition from developing or developing in a patient who may be exposed to the disease or is susceptible to the disease but who has not yet developed or exhibited symptoms of the disease.

[0202] In some embodiments, "treating" or "treatment" of any disease or disorder refers to the amelioration of at least one disease, disorder, or condition, or preferably the reduction in the progression of the disease or disorder or at least one clinical symptom thereof. In certain embodiments, "treating" refers to ameliorating at least one physical parameter of the disease. In certain embodiments, "treating" refers to physically or physiologically inhibiting a disease or disorder or condition, and in certain embodiments, "treating" refers to preventing or delaying the onset of a disease or disorder.

[0203] Specifically, in the case of treatment of central or peripheral nervous system diseases, especially in the case of degenerative disorders or in the case of mood disorders, as the case may be, it may include slowing the rate of degeneration or decline; attenuating degeneration; improving the physical or mental health of the subject; improving or maintaining memory and / or cognitive function; restoring and / or improving alertness and the ability to concentrate; or in some cases, preventing the onset of dementia.

[0204] Thus, for purposes of the above definitions, the terms "prevention" and "treatment" may overlap as follows, insofar as the latter includes preventing or delaying the onset of the disease or disorder.

[0205] The subject is any animal subject or a human subject, particularly a vertebrate subject, more particularly a warm-blooded subject or a mammalian subject.

[0206] As used herein, a "mammalian subject" can be any mammal, preferably a laboratory animal, a pet, livestock, or domestic animal. A mammalian subject can be, for example, a rodent, a primate, an ape, or a human. Preferably, the mammal is any vertebrate of the class Mammalia that has a cortex (brain region), hair, and mammary glands.

[0207] The compounds and pharmaceutical compositions according to the present invention are useful in methods of treatment.

[0208] The compounds and pharmaceutical compositions according to the present invention are useful in methods of treating diseases involving retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Preferably, the disease involves retinoid X receptors for which the ligands of the present invention are selective. Such diseases may be caused by impaired RXR signaling.

[0209] The compounds and pharmaceutical compositions according to the present invention are useful in methods of treatment, in particular in the treatment of mood disorders / diseases.

[0210] The term "CNS-related disease" refers to a disease or disorder that affects the central nervous system (CNS), i.e., the brain or spinal cord, leading to a neurological or psychiatric disorder. Preferably, the disease is associated with the RXR complex of RAR or other nuclear receptors. The causes of CNS diseases can be, for example, trauma, infection, degeneration, autoimmune disorders, structural defects, tumors, and stroke. Here, we focus on neurodegenerative diseases, mood disorders, schizophrenia, and autism.

[0211] The term "peripheral nervous system related disease" refers to a disease affecting the peripheral nervous system, preferably, the disease is associated with the RXR complex of RAR or other nuclear receptors. However, the ligands of the present invention are preferably selective for RXR.

[0212] The term "mental disorder" especially includes obsessive-compulsive disorder, post-traumatic stress disorder, anxiety disorder, irrational phobia, schizophrenia, schizoaffective disorder, depression, mania, bipolar disorder (bipolar disorder), emotional indifference, mental disorder, phobia, amnesia and eating disorders (e.g., bulimia, anorexia) etc. In one embodiment, mental disorders include obsessive-compulsive disorder, post-traumatic stress disorder, irrational phobia, schizophrenia, schizoaffective disorder, depression, mania, bipolar disorder (bipolar disorder), emotional indifference, mental disorder, phobia, amnesia and eating disorders (e.g., bulimia, anorexia). In another embodiment, mental disorders include obsessive-compulsive disorder, schizophrenia, schizoaffective disorder, depression, mania, bipolar disorder (bipolar disorder), emotional indifference, mental disorder and phobia. In another embodiment, mental disorders include obsessive-compulsive disorder, schizophrenia, schizoaffective disorder, depression, mania and bipolar disorder (bipolar disorder). Preferably or in particular, the term "mental disorder" as used herein refers to and will be understood by the skilled person as a "mental disorder" as described in sections F06-F50 of the 10th revision of the WHO International Statistical Classification of Diseases and Related Health Problems. In an embodiment, neurodegenerative diseases are excluded from the scope of mental diseases or mental disorders.

[0213] "Working memory or synonym short-time memory" refers to the acquisition, storage, memory, and recall of information over short time intervals, from minutes to hours, optionally days, and used to modify a subject's behavior.

[0214] "Memory loss" refers to a decrease in the ability to acquire, store, remember, and / or recall information, including past experiences, knowledge, and thoughts.

[0215] The terms "depression" or "depressive disorder" or "affective disorder" refer to the medical field as understood by skilled practitioners. "Affective disorder" refers to a disruption in the emotional tone or mood state experienced by an individual over an extended period of time. Affective disorders include major depressive disorder (i.e., unipolar depression), mania, dysphoria, bipolar disorder, dysthymia, cyclothymia, and the like. See, for example, the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM IV). "Major depressive disorder," "major depressive disorder," or "unipolar depression" refers to a mood disorder that includes any of the following symptoms: persistent sadness, anxiety, or a "blank" mood; feelings of hopelessness or pessimism; feelings of guilt, worthlessness, or helplessness; loss of interest or pleasure in hobbies and activities, including sex, that were once enjoyed; decreased energy, fatigue, or "being slowed down"; difficulty concentrating, remembering, or making decisions; insomnia, early awakening, or oversleeping; loss of appetite and / or weight, or overeating and weight gain; thoughts of death, suicide, or attempted suicide; restlessness or irritability; or persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders, and chronic pain. Various subtypes of depression are described, for example, in DSM IV.

[0216] As used herein, the term "neurodegenerative disease or disorder" refers to a disease or disorder characterized by progressive dysfunction of the nervous system, or progressive loss of structure or function of nervous tissue (preferably neurons), including neuronal death. "Neurodegenerative disease or disorder" is preferably selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, frontotemporal lobar degeneration associated with TDP-43 protein (FTLD-TDP), dementia with Lewy bodies (DLB), vascular dementia, amyotrophic lateral sclerosis (ALS), mild cognitive impairment, Parkinson's disease with MCI, and other neurodegenerative-related dementias due to brain changes caused by aging, disease or trauma; or spinal cord injury and ataxia, disseminated sclerosis and multiple sclerosis, or other neurological disorders.

[0217] The compounds and pharmaceutical compositions according to the present invention are useful in methods of treatment, in particular in the treatment of Alzheimer's disease.

[0218] The term "Alzheimer's disease" refers to the most common form of dementia that is clinically recognized. Memory loss is also a significant component of the disease. DETAILED DESCRIPTION

[0219] Vitamin A is a substance that can completely reverse vitamin A deficiency (IUPAC-IUB, 1982; Moore, 1929; Rühl, 2007). In this document, vitamin A1 is used as a term to include retinol, retinol esters, and retinal, as well as provitamin A derivatives such as β-carotene, α-carotene, and β-cryptoxanthin. Similarly, vitamin A2 derivatives (3,4-dihydro-retinoids and the carotenoid anhydroxanthophylls) found primarily in poultry and fish fall within this category (Cama et al., 1952; Moise et al., 2007). The human vitamin A3 and A4 clusters, which are associated with visual pigments and have not yet been linked to vertebrates, are also considered crucial (Babino et al., 2016).

[0220] In adult animals, RXR heterodimers, primarily RXR-VDR, RXR-PPAR, RXR-FXR, and RXR-LXR heterodimers, as well as RXR-RAR, regulate homeostatic lipid metabolism and inflammation (reviewed in (Chawla et al., 2001; Desvergne, 2007; Evans and Magelsdorf, 2014; Magelsdorf and Evans, 1995; Magelsdorf et al., 1995; Shulman and Magelsdorf, 2005)). Alterations in signaling mediated by this receptor lead to severe metabolic and immune diseases (reviewed in (Szanto et al., 2004a) and (Desvergne, 2007)). Some of these physiological effects are dependent on RXR-mediated processes, such as inflammatory responses and lipid signaling, and therefore they are relevant to the various pathophysiological effects identified in adult vitamin A-deficient animals (Nunez et al., 2010; Stephensen et al., 2007; Wan et al., 2003).

[0221] Endogenous RXR ligands serve as the main switch for achieving RXR heterodimer-mediated signal transduction in mammalian organisms. The various effects observed in animals deficient in vitamin A are similar to those found in RXR-KO animals (Kastner et al., 1997a; Kastner et al., 1997b). The present inventors believe that in addition to the effects of non-nuclear hormone receptors mediated in vision, RAR-, particularly RXR-mediated pathways are the main pathways for vitamin A activity, and these are dependent on endogenous RARs and / or RXR ligands.

[0222] It is widely accepted that ATRA is the endogenous relevant RAR ligand, but other ligands have also been described (Moses et al., 2005; Ruhl et al., 2015). 9-cis-Retinoid can be converted to all-trans-retinoid and serves as an additional precursor of the endogenous RAR-ligand ATRA.

[0223] In 1996, Shirley MA et al. (Shirley MA et al. 1996) found that in rats, 9-cis-RA was reduced to 13,14-dihydro-9-cis-RA, and the latter combined with taurine to form a new metabolite, but they regarded this as the initial step leading to β-oxidation.

[0224] Recently, 9-cis-13,14-dihydroretinoic acid (9CDHRA) has been identified as an endogenous retinoid in mice. The inventors have concluded that 9CDHRA is an endogenous ligand for the retinoid X receptor (RXR), a nuclear hormone receptor involved in the regulation of various physiologically relevant pathways in the brain, lipid metabolism, inflammation, differentiation, proliferation, and cell cycle. RXR-mediated signaling has been reported to be dysregulated in a variety of diseases, ranging from neurological dysfunction and cardiovascular disease to skin / immune-related disorders, including RXR signaling-related disorders of lipid homeostasis and inflammation. Due to reduced ligand levels, the inventors have also identified the endogenous RXR ligand, 9CDHRA, as a signaling lipid molecule potentially associated with RXR dysregulation. Thus, a new retinoid pathway, termed the retinoid 5 pathway, has been identified.

[0225] The present invention relates to physiological and nutritional precursors of 9CDHRA. The main precursors claimed are 9-cis-13,14-dihydroretinol (9CDHROL), i.e., vitamin A5 and its esters, 9-cis-13,14-dihydroretinol esters (9CDHROL-esters) and 9-cis-13,14-dihydroretinoic acid esters (9CDHRA-esters). In addition to these molecules, novel carotenoids, such as 9-cis-13,14-dihydro-β-carotene (9CDHBC), serve as precursors of provitamin A (5). Further upstream precursors of 9CDHBC, such as 9-cis-β-carotene (9CBC), are contemplated.

[0226] The aim of this study was to discover and synthesize these additional derivatives by organic chemical synthesis and to test representative examples of these precursors in an animal model for depression in a mouse model.

[0227] In a set of experiments described in the Examples, the present inventors have developed a new chemical synthesis of 9-cis-13,14-dihydroretinol and 9-cis-13,14-dihydroretinol acetate from ethyl 9-cis-13,14-dihydroisotretinoin. Other esters of 9-cis-13,14-dihydroretinoic acid and 9-cis-13,14-dihydroretinol (e.g., palmitate, etc.) can be readily prepared using essentially the same method.

[0228] New synthetic methods for 9-cis-13,14-dihydro-β-carotene and synthetic 9-cis-β-carotene have also been developed.

[0229] Alternatively, chemoenzymatic synthesis of long-chain retinyl esters is also an option. Some of these syntheses utilize retinol as a starting material (O'Connor et al., Ausl. Chem. 1992, 45, 641; Maugard et al., Biolechnol. Prog. 2002, 18, 424). Some authors have used retinyl esters such as retinyl acetate as starting materials for the biocatalytic preparation of long-chain retinyl esters (see, for example, unexamined Japanese patent application JP 62-248495, 1987). U.S. Pat. No. 7,566,795 B2 describes an improved method for preparing long-chain esters of retinol from short-chain retinyl esters and appropriate long-chain acids or esters by chemoenzymatic treatment in the presence of an enzyme and an organic solvent, optionally in the presence of at least one molecular sieve and / or at least one ion exchange resin, to form retinyl esters.

[0230] Several patent applications relate to precursors of various vitamin A forms and variants thereof.

[0231] As for the acid form of the ester, WO 95 / 04018 A1 relates to the preparation of 9-cis retinoic acid ester. This compound includes double bonds between carbon atoms 11 and 12, and between carbon atoms 13 and 14, and can have a cis or trans configuration. Although this method can be used herein, the compound It must not be a 13,14 dihydro derivative WO 95 / 32946 A1 also relates to the preparation of 9-cis-retinoic acid esters. The compound must contain a double bond between carbons 11-12 and 13-14, which may exist as cis and trans isomers.

[0232] Regarding retinol esters, WO2013134867 A1 describes the use of 9-cis or 11-cis retinyl esters for the treatment of retinitis pigmentosa (see claim 17). The specification mentions 9-cis-13,14-dihydroretinoic acid as a metabolite, but there is no suggestion of metabolism to 9-cis-13,14-dihydroretinol derivatives. (“Pharmacokinetic analysis showed that the major metabolites were 9-cis-retinoate or 9-cis-retinyl palmitate and 13,14-dihydro-9-cis-retinoic acid. At 4 hours after administration, the concentrations of these compounds were higher than those of 9-cis-retinyl acetate and 9-cis-retinol. See page 70, penultimate paragraph.)

[0233] WO 2011 / 034551 A2 describes pharmaceutical preparations containing one or more 9-cis-retinyl esters (acetate, palmitate, etc.) in a lipid carrier and their use in ophthalmology. This specification does not mention the use of 9-cis-13,14-dihydroretinol derivatives.

[0234] Therefore, there is apparently no literature describing the medical or, in particular, nutritional use of 9-cis-13,14-dihydroretinol or its ester derivatives. Furthermore, we found no data related to ester derivatives of 9-cis-13,14-dihydroretinol.

[0235] RXR-ligand binding is a particularly important mechanism responsible for vitamin A-mediated effects.

[0236] The RXR-precursor pathway is distinct from the RAR-precursor pathway that leads to ATRA and selective RAR-mediated signaling. Alternatively, as demonstrated by ATBC, provitamin A1 carotenoids cannot be converted to 9-cis-derivatives in human tissues, thereby eliminating precursors for RXR-selective ligands. The vitamin A1 ethanol, all-trans-retinol, is a weaker and non-isomerically selectively converted to 9CDHROL, and thus is a weaker precursor and lacks a selective precursor for RXR-selective ligands. ATROL primarily serves as a multifunctional precursor that is stored as a retinyl ester and is tightly controlled by binding proteins in serum and cells for general homeostasis and enables the synthesis of RAR- and RXR-ligands, as well as further RAR- and RXR-mediated signaling, without RXR-selectivity.

[0237] It is contemplated herein that 9-cis-13,14-dihydroretinoids may be considered specific and / or selective precursors of RXR ligands. Based on the above information, potential RXR-ligands derived from the vitamin A1 and A2 pathways appear to have no or only very limited endogenous relevance. However, the inventors have shown that this new signaling pathway associated with RXR activation should also be included as an important criterion for general vitamin A function ( Figure 7 ). In the figure, the presence of 9CDHRAL and ATDHRAL has not yet been confirmed. This pathway appears to be specific to the vitamin A pathway, as the similar alcohol forms 9CROL and ATROL are essentially not converted to the corresponding acids in cell culture (e.g., in oligodendrocytes). In summary, since non-endogenously related 9CRAs derived from the vitamin A1 pathway should be excluded as endogenously related RXR-activators, then 9-cis-13,14-dihydroretinoids and their nutritional precursors represent a new vitamin A signaling pathway, which has been termed the vitamin A5 pathway.

[0238] The present inventors have demonstrated the metabolic transformation of compounds of the vitamin A5 pathway in in vitro cell systems and mammalian supplementation experiments, identifying them as endogenous derivatives in mice and humans, and identifying these derivatives directly or indirectly in the human food chain. Furthermore, they have investigated and surprisingly found that 9CDHROL and 9CDHBC exhibit antidepressant activity in a social stress defeat protocol, a RXR-mediated process. Therefore, the present invention relates to novel methods for treating depression by administering RXR-ligands or vitamin A5 (9CDHROLs) and RXR ligand pre-proforms or vitamin A5 precursors (provitamin A5 derivatives), as well as 9CDHBC.

[0239] As an example of illustrating the effects of RXR signaling, depression has been selected as a diverse group of psychiatric disorders with an unclear etiology among RXR-signaling disorders. It is characterized by some core affective symptoms including lack of pleasure and despair, as well as less specific cognitive symptoms such as insufficient working memory. It has been shown that reduced bioavailability of 9CDHRA in mice carrying a null mutation of cellular retinol binding protein (Rbp1) leads to depressive-like behavior, while drug treatment with 9CDHRA restores normal behavior in these mice. For depression, the administration of selective RAR-ligands does not lead to any improvement, and the disorder appears to be a disorder associated with selective RXR-signaling. The treatment using 9CDHRA precursors as disclosed herein is directly relevant to the study of depression because, similar to synthetic RXR ligands, 9CDHRA and 9CDHRA-precursors are effective in preventing depressive-like behavior in chronic stress animal models.

[0240] Specifically, the inventors have determined that 9CDHROL, a precursor of 9CDHRA, can also positively affect the memory performance of mice when administered orally to mice. Furthermore, they have determined that 9CDHROL is a precursor of 9CDHRA when administered orally to mice.

[0241] This is a surprising finding, since the analogs 9-cis-retinol (9CROL) and all-trans-retinol (ATROL) are not or only weakly converted to 9-cis-retinoic acid (9CRA) and all-trans-retinoic acid (ATRA) in vitro (see Figure 13 ), and therefore they are not good precursors to the corresponding acid forms.

[0242] Similarly, the analogs 9-cis-retinol (9CROL) and all-trans-retinol (ATROL) are not or only weakly converted to 9-cis-13,14-dihydroretinoic acid (9CDHRA) and all-trans-13,14-dihydroretinoic acid (ATDHRA) in vitro (see Figure 13 ), so they are not good precursors of active RAR ligands and RXR ligands.

[0243] Thus, the present inventors have discovered an unexpected new vitamin A pathway by showing that the 9CDHRA precursor 9CDHROL and its esters are specific precursors of active RAR ligands and RXR ligands.

[0244] The present results allow the conclusion that 9CDHROL is a novel endogenous retinoid in mice and that derivatives that produce 9CDHROL upon administration in animals or humans can be considered as this novel source of vitamin A.

[0245] The present inventors have also determined that 9CDHROL-ester and 9CDHRA-ester can replace 9CDHROL as precursors of 9CDHRA.

[0246] Therefore, 9CDHROL and 9CDHROL esters as well as 9CDHRA esters are physiological and nutritional precursors of 9CDHRA and can be used as alternative treatments to achieve higher 9CDHRA levels in (specific) tissues.

[0247] Additional animal studies in a panel of psychiatric disease models provided further evidence that R-9CDHRA exhibits RXR agonist-like activity in vivo and reverses behavioral deficits in mice.

[0248] Together with the evidence that 9CDHRA-esters and 9CDHROL and their esters increase 9CDHRA-levels in the brain and oligodendrocytes in a surprising manner, the reversal of behavioral deficits in Rbp1- / - mice, the improvement of cognitive performance in wild-type mice, and the reversal of antidepressant effects in a chronic social defeat stress model are shown, further supporting the utility of the compounds of the present invention in a wide range of diseases of the central and peripheral nervous systems. Rbp1- / - mice were used because they show the same type of deficits as RXRg- / -, indicating that RXRg signaling is downregulated. The normalization of their memory is evidence that these mice lack RXR ligands rather than the receptors themselves (or receptor functionality). RXRg- / - mice were used as negative controls to show that 9cDHRA acts on RXRg to improve memory—thus, it cannot improve memory in the absence of RXRg. This result was confirmed in wild-type animals.

[0249] RXR-ligands through 9CDHRA lead to selective RXR-activation pathways and RXR-LXR, RXR-NR4A, RXR-VDR, RXR-FXR and RXR-PPAR-mediated signaling. These RXR-mediated signaling pathways can be modified only by RXR-ligands and not by RAR-ligands.

[0250] The present inventors have discovered the endogenous RXR ligand 9CDHRA and its potent precursor 9CDHROL (vitamin A5) as well as upstream precursors. This vitamin A5 / provitamin A5 pathway involves the pharmaceutical or nutritional or dermal topical administration of the selective RXR-precursors we claim.

[0251] These vitamin A5 / provitamin A5 compounds are used to prevent and treat diseases where RXR-mediated signaling is enhanced, such as various neurodegenerative diseases, and other diseases of the skin and cardiovascular system, including RXR-mediated alterations in lipid homeostasis and immune regulation, such as atherosclerosis, obesity, and diabetes. These diseases associated with dysfunctional RXR-mediated signaling can be prevented and / or treated with our RXR-selective precursors.

[0252] As an example used in this study, depression is considered to be an RXR-mediated signaling dysfunction, and 9CDHRA and its precursors 9CDHROL and 9CDHBC are effective treatment / prevention strategies. However, many other diseases have been reported in the art, and the RXR-KO phenotype is mainly found in neurodegenerative diseases and cardiovascular system dysfunctions, such as obesity, diabetes, atherosclerosis, and appetite regulation.

[0253] We claim that we have discovered an important physiological switching mechanism in the human body that can be selectively switched by a) a selective physiological ligand (9CDHRA) or b) selectively by a nutritional precursor present in food (which can also be administered as the supplements / drugs 9CDHROL and 9CDHBC). This switch enables RXR-mediated signaling, thereby preventing RXR-dependent dysfunction and diseases.

[0254] It was also surprisingly discovered that the selective 9CDHRA-precursor 9CDHBC is a provitamin A5 carotenoid to effect RXR-mediated signaling.

[0255] As shown herein, only 9CDHBC, but not 9CBC, is an excellent precursor for 9CDHROL (vitamin A5). In in vivo supplementation experiments, when orally administered to mice, the carotenoid 9CDHBC was a good precursor for 9CDHRA in mouse serum, liver, and brain. In humans, carotenoids are transported (but not in mice) and stored in the body, which does not occur in mice. 9CDHBC, but not 9CBC and especially not ATBC, has been shown to be an excellent precursor for 9CDHROL and 9CDHRA.

[0256] Both 9CBC (known) and 9CDHBC are present in the human food chain (peaches and, in addition, broccoli are shown), and all precursors in the food chain are present in algae. Plankton, primarily consisting of algae, is the starting material for all food on our planet: starting with plankton / algae, fish, large aquatic animals, and later terrestrial animals, and finally humans, as the end point of the global food chain, selectively assessing relevance from a human perspective.

[0257] According to the present invention, a functional food is claimed, which comprises added isolated vitamin A5 or any precursor thereof such as 9CDHBC or other compounds or compounds for use according to the present invention. Preferably, the functional food according to the present invention is a processed food and is preferably a canned food.

[0258] Surprisingly, the selective RXR precursor ligands of the present invention are more effective than non-selective compounds. For example, the known endogenous retinoid retinol, ATROL, is only a weak and non-isomerically selective precursor of 9CDHROL and has no 9CDHRA precursor in human oligodendrocytes in vitro. In supplemented mice, it was only weakly and partially insignificantly converted to 9CDHRA: after oral supplementation, a factor of 2 was detected in serum (not significant), a factor of 2.7 in liver (significant), and a factor of 7 in mouse brain (significant), thus confirming that the precursor substance has no or only inefficient potential as a selective RXR precursor ligand. For comparison, when the same amounts of these retinoids were administered to supplemented mice, as were our newly claimed vitamin A5 alcohols, 9CDHROL was excellently and efficiently converted to 9CDHRA in vitro in human oligodendrocyte cultures (a factor of 98 in serum, a factor of 76 in liver, and a factor of 1294 in mouse brain, all significant). As shown herein, 9CDHROL has been confirmed to be present in human serum and in the human food chain when detected in commercially available bovine liver.

[0259] Therefore, 9CDHROL is an excellent physiological and nutritional precursor of 9CDHRA and has been demonstrated in mouse behavioral studies to be an alternative treatment to selectively achieve higher 9CDHRA levels and selective RXR-mediated signaling. As shown in in vitro and in vivo supplementation experiments, 9CDHROL is very efficiently converted to 9CDHRA. Esters of 9CDHROL (9CDHROL-esters) and esters of 9CDHRA (9CDHRA-esters) are also excellent and more stable derivatives and can be administered alternatively, resulting in even higher conversion rates to 9CDHRA. We claim that 9CDHROL belongs to a new family of selective vitamin A compounds that function as selective RXR-ligand precursors, designated the vitamin A5 family.

[0260] 9CDHBC is a novel endogenous derivative identified in large quantities at various levels in the human food chain. We therefore claim that 9CDHBC is a novel selective provitamin, termed provitamin A5, that acts as a selective precursor of the selective RXR ligand 9CDHRA.

[0261] We also claim that 9CBC is a pre-precursor of RXR-ligand and pro-pro-vitamin A5. Alternatively, 9CBC can be used as a food ingredient or nutritional derivative with very weak and non-selective derivatives for the prevention of VA5-deficiency and for general VA5-supplementation to prevent RXR-dependent dysfunctions in our organism, represented herein by depression as a selective RXR-ligand / RXR-mediated neurodegenerative disease.

[0262] To date, 13,14-dihydro-carotenoids have not been reported, with the exception of acyclic carotenoids such as lycopene, phytoene, and phytoene. Formal hydrogenation of the 13,14-double bond can be caused by the action of retinol-saturase (RETSAT) on the unsaturated precursor retinol or by alternative metabolism of the classically known retinoids (Moses et al., 2004). All-trans-13,14-dihydro-retinoic acid has been identified by the inventors' research team at high concentrations in the serum, liver, and brain of young, wild-type, non-vitamin A-supplemented mice (96 ng / ml, 352 ng / mg, and 38 ng / g, respectively).

[0263] In conclusion, the pathway from 9-cis-dihydro-carotenoids, preferably 9-cis-13,14-dihydro-carotenoids, is physiologically relevant to the synthesis of endogenous 9CDHRA starting from dietary carotenoids. Therefore, 9CDHROL and 9CDHBC are expected to be nutritionally and endogenously relevant precursors of the endogenous RXR-ligand 9CDHRA.

[0264] Therefore, the present invention also relates to the use of 9-cis-dihydro-carotenoid (9CDHBC) for the purposes disclosed herein.

[0265] Experiments have shown that the compounds of the present invention can provide 9cDHRA and can play a role in maintaining health, especially the health of the central nervous system or peripheral nervous system, and / or maintaining health against the diseases mentioned herein, or preventing or treating the diseases. In particular, the diseases are central nervous system or peripheral nervous system diseases as listed herein.

[0266] Certain aspects and embodiments of the present invention are described in detail below and then illustrated by the examples which form a part hereof; however, the skilled artisan will appreciate that other variations and embodiments are within reach and can be made based on the specific and general teachings provided herein.

[0267] Preparation of the compounds of the present invention

[0268] Starting compounds are either commercially available or can be synthesized according to standard methods.

[0269] Preparation of 9-cis-13,14-dihydroretinoic acid and its esters

[0270] In the embodiment given in the examples, the preparation of the ethyl ester (R)-4 of (R)-9-cis-13,14-dihydroretinoic acid [(R)-1] is based on the Suzuki coupling of an optically pure enantiopure trienyliodide 3 and a boronic acid 2 (see Scheme 1 in Example 1). The synthesis of 3 begins with a (Z)-stannyl dienol 5, which is converted to a benzothiazolyl allyl sulfide 6 by a Mitsunobu reaction with the corresponding thiol and subsequently oxidized to the sulfone 7 using H2O2 and a peroxomolybdate(VI) reagent. Julia-Kocienski olefination is performed using a slight excess of base in an excess of an optically pure enantiopure aldehyde (R)-8, which is the ethyl ester. As expected from previous findings regarding the stereochemistry of reactions of allyl sulfones with aldehydes, the olefin of the newly formed trialkenyl ester (R)-9 possesses Z-geometry. Treatment of the precursor stannane with a solution of iodine in CHCl generates the iodide (R)-3 via Sn-I exchange, which then undergoes iodine-promoted isomerization of the 9Z,11Z diene to the desired 9Z,11E geometry. The geometry can be confirmed by NOE experiments.

[0271] Suzuki reaction of freshly prepared boronic acid 2 and dienyl iodide (R)-3, followed by immediate workup, afforded (R)-9-cis-13,14-dihydroisoretinoic acid ethyl ester (R)-4. Saponification of (R)-4 provided the desired carboxylic acid (R)-1 with no detectable loss of stereochemical integrity. (See Reaction Scheme 1)

[0272] Following the general protocol, enantiomer (S)-1 was also prepared with similar efficiency.

[0273] The preparation of the compounds of the present invention is described in more detail in Example 1.

[0274] Alternative 9-cis-13,14-dihydroretinoic acid esters can be prepared using the corresponding ester variants of the (R)-8 compound.

[0275] Additionally, the carboxylic acid 9-cis-13,14-dihydroretinoic acid can be esterified with other alkyl- or alkenyl-containing alcohols using standard procedures, such as treatment of both compounds with a mixture of dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP).

[0276] Furthermore, other esters, other than the ethyl ester, can be prepared starting from the corresponding aldehyde (R)-8.

[0277] Preparation of 9-cis-13,14-dihydroretinol and its 9-cis-13,14-dihydroretinyl ester

[0278] As described herein, 9-cis-13,14-dihydroretinol was prepared by DIBAL-H (diisobutylaluminum hydride) reduction of the ethyl ester 13,14-dihydroisoretinoate.

[0279] Ester reduction can also be accomplished using lithium aluminum hydride (LiAlH4) in tetrahydrofuran (THF) or diethyl ether, or using lithium borohydride and an alcohol (ethanol, methanol) in THF / diethyl ether as solvent.

[0280] 9-cis-13,14-dihydroretinol acetate was prepared from 9-cis-13,14-dihydroretinol by adding excess Ac2O and pyridine in the presence of DMAP. The obtained ester was isolated and purified.

[0281] This synthetic method maintains the enantiomeric configurational optical purity.

[0282] By this method, alternative esters can be prepared using a carboxylic anhydride reagent R2O (R is a carboxylic acid), or using the corresponding chloride derived from the carboxylic acid, or using activated carboxylic acids prepared, for example, with DCC and DMAP.

[0283] Esters of 9-cis-13,14-dihydroretinol can be prepared by transesterification of the ester, thereby replacing the acyl group.

[0284] Alternatively, retinyl esters can be prepared from 9-cis-13,14-dihydroretinol using a suitable esterification reagent.

[0285] Another method may be a selective enzymatic reaction.

[0286] For example, lecithin: retinol acyltransferase (LRAT) catalyzes the transesterification of long-chain fatty acyl moieties (primarily palmitic, stearic, oleic, and linoleic acids) present at the sn-1 position of bilayer membrane phosphatidylcholine to retinol to form retinyl esters (O'Byrne, Sheila M. et al., 2013). Lipases form another group of enzymes suitable for synthesizing esters such as fatty acid esters (Kai ZJ 2011, Yin Chunhua 2006). Alternatively, a combined chemical-enzymatic method can be used (Liu ZQ 2015).

[0287] These enzymes can be prepared by recombinant technology and used to selectively prepare the desired ester. Alternatively, the enzyme can be cloned into a suitable host cell, overexpressed in an activated form, and the recombinant cell can be used to prepare the desired ester.

[0288] Certain preferred esters of the present invention

[0289] Preferred 9-cis-13,14-dihydroretinoic acid esters

[0290] Preferred 9-cis-13,14-dihydroretinoic acid esters are generally lower alkyl esters of 9-cis-13,14-dihydroretinoic acid such as methyl, ethyl or propyl esters. Esters of 9-cis-13,14-dihydroretinoic acid with long-chain fatty alcohols are less preferred than esters of 9-cis-13,14-dihydroretinol with long-chain fatty acids.

[0291] More preferred 9-cis-13,14-dihydroretinyl ester compounds

[0292] More preferred 9-cis-13,14-dihydroretinyl ester compounds include formate (formates), acetates, and propionates, and, although less preferred, butyrates and valerates.

[0293] Long-chain fatty acids can also form esters with 9-cis-13,14-dihydroretinol.

[0294] Long chain fatty acids (LCFAs) include fatty acids with aliphatic tails of 13 to 21 carbons.

[0295] The long chain fatty acids may be selected from saturated and unsaturated fatty acids, the latter including monounsaturated and polyunsaturated fatty acids.

[0296] saturated fatty acids

[0297] Typical esters with saturated fatty acids can be formed, for example, with the following long-chain fatty acids:

[0298] Myristic acid CH3(CH2) 12 COOH14:0

[0299] Palmitic acid CH3(CH2) 14 COOH16:0

[0300] Stearic acid CH3(CH2) 16 COOH18:0

[0301] Arachidic acid CH3(CH2) 18 COOH20:0

[0302] Although saturated fatty acids are generally claimed to be undesirable in the diet, they may nonetheless be useful components of the esters of the present invention as precursors of 9CDHROL in human tissues.

[0303] Monounsaturated fatty acids (MUFAs)

[0304] Monounsaturated fats have one carbon-carbon double bond that can occur in a variety of positions. The most common monounsaturated fats have chain lengths of 16-22 and double bonds with a preferred cis configuration.

[0305] Preferred MUFAs suitable for use in the present invention are oleic acid CH3(CH2)7(HC=CH)(CH2)7COOH and palmitoleic acid CH3(CH2)5(HC=CH)(CH2)7COOH.

[0306] Polyunsaturated fatty acids (PUFAs)

[0307] In polyunsaturated fatty acids (PUFAs), the first double bond is found between the third and fourth carbon atoms from the first carboxylic acid carbon; these are called omega-3 fatty acids. If the first double bond is between the sixth and seventh carbon atoms, they are called omega-6 fatty acids.

[0308] Preferred PUFAs suitable for use in the present invention are, for example, ω-3 and ω-6 fatty acids.

[0309] Among the PUFAs, arachidonic acid (ARA) and docosahexaenoic acid (DHA) are useful in the treatment of Alzheimer's disease (see, for example, EP1419780B1).

[0310] In this case, once hydrolyzed, PUFAs can also exert their advantages in the organ in question.

[0311] In one embodiment, essential fatty acid esters are preferred. Two essential fatty acids are linoleic acid (LA) and alpha-linolenic acid (ALA). The human body has a limited ability to convert ALA into long-chain omega-3 fatty acids—eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are also preferred.

[0312] Branched-chain fatty acids (BCFA)

[0313] Branched-chain fatty acids (BCFAs) are bioactive food components [Ran-Ressler RR et al. "Branched-chain fatty acid content of foods and estimated intake in the United States." Br J Nutr. 2014 Aug 28;112(4):565-72] and are found in milk and soy, and in some cases (e.g., phytanic acid), in ruminant fat and certain fish. Naturally occurring branched-chain fatty acids exhibit potential health properties and are preferred components of the esters of the present invention.

[0314] These preferred BCFAs are, for example, the naturally occurring 13-methyltetradecanoic acid, 15-methylpalmitic acid and phytanic acid (3,7,11,15-tetramethylhexadecanoic acid).

[0315] Fatty acids and their role in living things can be found, for example, in the following books:

[0316] (Ching Kuang Chow, "Fatty Acids in Food and Their Health Effects," 2007; Arild C et al., "Fatty Acids: Structure and Properties," ENCYCLOPEDIA OF LIFE SCIENCES 2005)

[0317] The compounds and / or pharmaceutical compositions according to the invention may be used in methods of treatment. Preferred treatments are diseases involving the nervous system.

[0318] Compounds of the invention in the nervous system and nervous tissue

[0319] In the nervous tissue or in the nervous system, the compounds of the invention may be converted into their active form. Preferably, if the compounds are administered to an animal or human subject, this conversion is preferably in the nervous system or in the nervous tissue.

[0320] In a preferred embodiment of the present invention, the compound is converted into 9CDHROL by hydrolysis. Alternatively, the compound is 9CDHROL. In the nervous system, 9CDHROL is further converted into 9CDHRA, which exerts its RXR agonist effect.

[0321] As used herein, the nervous system consists of the central nervous system (CNS) and the peripheral nervous system (PNS). The central nervous system (CNS) consists of the brain and spinal cord.

[0322] The peripheral nervous system (PNS) includes all (branches of) peripheral nerves and regulates and controls body functions and activities.

[0323] According to the present invention, 9CDHRA formed in situ exerts its RXR agonist effect in neural tissue to prevent or treat diseases of the CNS or PNS (as described in detail herein, where these diseases are discussed).

[0324] Nervous tissue (also known as nerve tissue) is the primary component of the two parts of the nervous system: the CNS and PNS. It is composed of neurons and nerve cells, which receive and transmit impulses, and glial cells, also known as (neuro)glial cells (neuroglia), which assist in the propagation of nerve impulses and provide nutrients to neurons.

[0325] Examples of glial cells are:

[0326] Microglia, astrocytes, oligodendrocytes, NG2glia, Schwann cells, satellite glial cells, enteric glia

[0327] To explain in more detail, oligodendrocytes are central nervous system structures that form myelin sheaths on the axons of neurons. Myelin sheaths are lipid-based insulators that facilitate the efficient conduction of nerve impulses down the axons. NG2 glial cells are CNS cells that serve as developmental precursors to oligodendrocytes. Schwann cells are the PNS equivalent of oligodendrocytes; they help maintain axons and form myelin sheaths in the PNS.

[0328] The experiments using oligodendrocytes, together with data reporting the role of synthetic RXR agonists in controlling remyelination, support the effects and utility of the compounds described in this patent for controlling oligodendrocyte and myelin function in the CNS and PNS, as well as on NG2 glia.

[0329] Therefore, CNS and PNS disorders that can be treated, prevented, or alleviated by RXR agonists can be treated with esters of 9CDHRA, 9CDHROL, and esters thereof.

[0330] RAR / RXR, RXR / PPAR or RXR / LXR signaling in the brain is reviewed or mentioned in, for example, the following publications: (van Neerven 2008; Shudo K et al., 2009; Skerrett R et al., 2015).

[0331] There are three retinoid X receptors (RXRs): RXR-α, RXR-β, and RXR-γ, encoded by the RXRA, RXRB, and RXRG genes, respectively. RXR-ligand binding is a particularly important mechanism responsible for vitamin A-mediated effects. RXR occupies a central position in nuclear receptor (NR) signaling, serving as a common heterodimer partner for multiple NRs. These heterodimers are termed permissive or nonpermissive, depending on whether the RXR's role is that of an active or cryptic transcriptional partner, respectively. Permissive RXR-NR heterodimers are activated by either the RXR ligand or the ligand of the partner, and synergistic effects are often observed if both partners of the heterodimer are bound to their ligands. One such heterodimer is the RXR-RAR heterodimer.

[0332] The present inventors demonstrate that 9CDHRA, as an RXR agonist, may coordinate the transcriptional activities of several nuclear receptor-signaling pathways through corresponding permissive heterodimers.

[0333] The compounds of the present invention are particularly useful as RXR agonists, and where agonists of RXRs, RXR-RARs, and RXR-PPARs or RXR-LXRs are used to maintain or restore health.

[0334] In a preferred embodiment, the compounds are useful in the treatment of impaired cognitive function and / or impaired learning ability.

[0335] The compounds and / or pharmaceutical compositions according to the present invention can be used in therapeutic methods, in particular for the treatment of memory impairment (memory loss), in particular working memory impairment (e.g., deficit or loss) or short-term memory impairment (e.g., deficit or loss such as amnesia, etc.). Memory impairment, impaired learning ability and impaired cognitive function may be associated with: psychiatric disorders, such as mild cognitive impairment, amnesia, memory and cognitive deficits in schizophrenia; and mood disorders, such as bipolar disorder and depression, stress-related disorders and anxiety disorders, such as partial and complete amnesia, dissociative amnesia.

[0336] Some psychoactive substances and drugs are known to cause memory loss, impaired learning ability and impaired cognitive function, such as tricyclic antidepressants, dopamine agonists, antihistamines, benzodiazepines, statins, beta-blockers, barbiturates, opioids, THC, alcohol, etc.

[0337] The compounds and / or pharmaceutical compositions according to the present invention are particularly useful for reversing such memory impairment caused by the above-mentioned diseases or drugs.

[0338] The compounds and / or pharmaceutical compositions according to the present invention are particularly useful for the prevention and / or treatment of impaired short-term memory. In particular, the compounds and / or pharmaceutical compositions according to the present invention are useful for improving working memory performance or reducing working memory loss.

[0339] The compounds and / or pharmaceutical compositions according to the present invention can be used to treat depression, such as mild, moderate and severe depressive episodes and the depressive phase of bipolar illness; cyclothymia or dysthymia; mixed anxiety-depression; depression or depressive episodes associated with other diseases such as schizophrenia, cancer, metabolic diseases, etc.

[0340] The compound and / or pharmaceutical composition according to the present invention can be used for preventing and / or treating neurodegenerative diseases. Preferably, neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, mild cognitive impairment (MCI), Parkinson's disease with mild cognitive impairment, Alzheimer's disease, Huntington's disease, dementia with Lewy bodies (DLB), amyotrophic lateral sclerosis (ALS) and other neurodegenerative related dementias due to aging, disease or trauma-induced brain changes; or spinal cord injury and ataxia, disseminated sclerosis and multiple sclerosis or other nervous system diseases.

[0341] According to a specific embodiment, the present invention also relates to a kit suitable for treatment by the above method.

[0342] In embodiments, these kits comprise a composition comprising a compound of the invention at a suitable dosage; and a second composition comprising a mood stabilizer, antidepressant, anxiolytic, or the like.

[0343] In another embodiment, the kit comprises a composition comprising a suitable dose of a compound of the invention; and a second composition comprising an agent active against a neurodegenerative disease as defined or listed herein.

[0344] The present invention also relates to a combination comprising the components of the kit as listed above.

[0345] Compounds of the invention can be tested in animal models (and non-animal models), such as the chronic social defeat stress model of depressive behavior, animal models of amyloidosis or Alzheimer's and Parkinson's diseases.

[0346] Nutritional uses

[0347] The compound of the present invention can be used as a nutrition or dietary supplement in a functional food composition, a dietary supplement composition or a nutritional composition. Such nutrition or dietary supplements, functional food compositions, dietary supplement compositions or nutritional compositions have the effect of preventing, reversing and / or alleviating a condition as disclosed herein. In a preferred embodiment, the condition is memory loss, particularly working or short-term memory impairment, particularly memory loss without the administration of a drug, that is, in a non-medical manner and / or with the benefit of preventing, reversing and / or alleviating learning impairment and / or cognitive decline. Non-medical treatment refers to an application purpose for which normal body function is maintained, wherein the condition defined herein is non-pathological or does not reach a pathological level. Preferably, in non-medical treatment, nutrition or dietary supplements, functional food compositions, dietary supplement compositions or nutritional compositions are used to prevent, reverse and / or alleviate memory loss, learning impairment and / or cognitive decline in a condition that is still not or cannot be considered a disease.

[0348] The nutritional composition may comprise a "nutritionally acceptable carrier," which means that the carrier is acceptable for human consumption and maintains or improves the biological properties of the active agent.

[0349] Similar to other dietary vitamin A compositions according to the present invention, the amount of vitamin A5 pathway retinoids can be measured in 9CDROL equivalents.

[0350] The composition of the present invention is suitable for use in nutritional compositions, such as dietary supplements, functional foods, medical foods, or foods with health claims.

[0351] As shown in this article, the fat-soluble form will be stored in the liver and the body's supply can be achieved from it.

[0352] preparation

[0353] In addition to the active agent, the composition comprises at least one carrier.

[0354] The carrier can be, for example, a diluent, adjuvant, excipient, stabilizer, or a carrier formulated for administration with the medicament. Pharmaceutical carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic ones such as peanut oil, soybean oil, mineral oil, and sesame oil. When the pharmaceutical composition is administered intravenously, water is a typical carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0355] In one embodiment, the medicament is prepared according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to a mammal or human. Typically, the pharmaceutical composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent.

[0356] In solid preparations, suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride; skim milk powder, glycerol, propylene, ethylene glycol, water and ethanol, etc. If necessary, the composition may also contain a small amount of a wetting agent or emulsifier such as a lipid.

[0357] The pharmaceutical or nutritional composition can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0358] In particular, in the case of longer alkyl chain esters, lipid carriers are advantageous. The compounds of the present invention are light-sensitive and oxygen-sensitive. Therefore, the selection of a suitable carrier can be based on its ability to stabilize the compounds of the present invention. For example, such formulations are described in WO2011034551A2 (Boch et al., Pharmaceutical dosage forms comprising 9-cis-retinol in a lipid carrier). Exemplary formulations and dosages are described in WO2011034551A2 and WO2013134867A1.

[0359] Suitable oral dosage forms include, for example, tablets, pills, powders, or capsules of hard or soft gelatin, methylcellulose, or other suitable materials that are readily soluble in the digestive tract. Suitable non-toxic solid carriers may be used, including, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate. (See, for example, Remington "Pharmaceutical Science," 17 Ed., Gennaro (ed.), Mack Publishing Co., Easton, Pennsylvania, 1985.)

[0360] Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, and oral routes. The agent can be administered by any convenient route, such as by infusion or bolus injection, absorption through epithelial or mucosal skin protective layers (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other functional activators.

[0361] dose

[0362] The dosage of the pharmaceutical agent can be carefully selected according to the clinical status, the condition and age of the subject, and the dosage form and the like.

[0363] For example, a human effective dose can be calculated from the animal dose, for example, as described in Chapter V (Step 2: Calculation of the human equivalent dose) and in Table 1 of the July 2005 U.S. FDA guidance. This indicates that, to give a dose value that would initiate in humans, the animal dose should be divided by a factor based on the surface area of ​​the human body, for example, by a factor of 6.2 for the kilogram dose between rats and humans, and by a factor of 12.3 for the kilogram dose between mice and humans.

[0364] Therefore, at least an upper limit can be safely given based on species-dependent animal models.

[0365] Side effects are dose dependent, and the compounds of the present invention are expected to be advantageous in this regard.

[0366] Only an exemplary embodiment is given, and the subject is used for testing. In the case of eye drops, medicament can be applied with, for example, every single amount from about 0.01mg, about 0.1mg or about 1mg to about 25mg, to about 50mg, to about 90mg. As needed, eye drops can be applied once a day or multiple times. In the case of injection, suitable dosage can be, for example, about 0.0001mg, about 0.001mg, about 0.01mg or about 0.1mg to about 10mg, to about 25mg, to about 50mg or to about 90mg medicament, once a week to four times. In other embodiments, the medicament of about 1.0 to about 30mg can be applied once a week to three times, or more times can be applied when needing more frequently, for example, once a day. Oral dose can fall into the same magnitude, for example, in the subject from about 1mg or 5mg to about 10mg, to about 25mg, to about 50mg or to about 100mg medicament, once a day or once a week to six times.

[0367] Example

[0368] Example 1: Chemical Synthesis - Synthesis of Dihydroretinoids:

[0369] To confirm whether the relevant MS-MS signals detected at 303 > 207 m / z corresponded to 9CDHRA, a stereoselective synthesis of the two enantiomers of 9-cis-13,14-dihydroretinoic acid was performed based on a palladium-catalyzed Csp2-Csp2 Suzuki coupling according to a previously described strategy (Pazos et al., 2001). Detailed descriptions of the stereocontrolled synthesis, purification, and characterization of the (R)- and (S)-enantiomers of 9-cis-13,14-dihydroretinoic acid are provided below.

[0370] The preparation of the ethyl ester (R)-4 of (R)-9-cis-13,14-dihydroretinoic acid was based on the Suzuki coupling of optically pure trienyl iodide 3 and boronic acid 2 (Scheme 1). The synthesis of 3 began with (Z)-stannyl dienol 5 (Domínguez B. et al., 2000, Pazos Y. et al., 1999), which was converted to benzothiazolyl allyl sulfide 6 by Mitsunobu reaction with the corresponding thiol and subsequently oxidized to sulfone 7 with H2O2 and peroxomolybdate(VI) reagent at -10°C (Schultz HS et al., 1963). Julian olefin synthesis (Blakemore PR. 2002; Alissa et al., 2003) was performed using a slight excess of base (NaHMDS, 1.15 equiv) and 1.7 equiv of optically pure aldehyde (R)-8 (Moses et al., 2008, Leonard J. et al., 1995). C 2009). As expected from previous findings on the stereochemistry of reactions of allyl sulfones with aldehydes (Sorg A et al., 2005, Vaz B et al., 2005), the olefin of the newly formed trialkenyl ester (R)-9 has a Z-geometry (as confirmed by NOE experiments). Treatment of the precursor stannane with a solution of iodine in CHCl generates the iodide (R)-3 via Sn-I exchange, and iodine-promoted isomerization of the 9Z,11Z diene to the desired 9Z,11E geometry (as confirmed by NOE experiments).

[0371]

[0372] Protocol 1. Reagents and conditions:

[0373] (a) PPh3, BTSH, DIAD, CH2Cl2, 2h (98%).

[0374] (b)(NH4)6Mo7O 24 .4H2O, 30% hydrogen peroxide H2O2, EtOH, -10℃, 17h (66%).

[0375] (c) NaHMDS, THF, -78°C, 30 min (93%).

[0376] (d) I2, CH2Cl2, 25℃, 30min.

[0377] (e) Pd(PPh3)4, 10% TlOH solution, THF, 25°C, 3h (42%).

[0378] (f) 2M KOH, MeOH, 80°C, 45min (84%).

[0379] Suzuki reaction of freshly prepared boronic acid 2 and dienyl iodide (R)-3 was carried out in THF at room temperature using Pd(PPh3)4 as catalyst and 10% TlOH solution as base, and then immediately examined to obtain (R)-9-cis-13,14-dihydroisoretinoic acid ethyl ester (R)-4 in 78% yield.

[0380] (From (R)-4, the corresponding carboxylic acid (R)-1 was afforded in 84% yield with no detectable loss of stereochemical integrity.

[0381] Following the general protocol, enantiomer (S)-4 was also prepared with similar efficiency.

[0382] Preparation and characterization of compounds - general procedures

[0383] Before use, the solvent was dried and distilled according to the published method. All other reagents were commercial compounds with the highest purity available. Unless otherwise stated, all reactions were carried out under an argon atmosphere, and reactions not involving aqueous reagents were carried out in oven-dried glass dishes. Analytical thin layer chromatography (TLC) was performed on an aluminum plate with Merck Kieselgel 60F254 and observed by UV irradiation (254 nm) or stained with an ethanolic solution of phosphomolybdic acid. Flash column chromatography was performed under pressure using Merck silica gel 60 (230-400 mesh). Electron impact (EI) mass spectra were obtained by operating at 70 eV on a Hewlett-Packard HP59970 instrument. Alternatively, an APEX IIIFT-ICR MS (Daltonics) equipped with a 7T active shielded magnet was used and ions were generated using an Apollo API electrospray ionization (ESI) source, with a voltage between 1800 and 2200 V (to optimize ionization efficiency) applied to the needle and a counter voltage of 450 V applied to the capillary. For ESI spectroscopy, samples were prepared by adding a spray solution of 70:29.9:0.1 (volume ratio) CH3OH / water / formic acid to the sample solution in a volume ratio of 1 to 5% to give the best signal-to-noise ratio. High-resolution mass spectra were obtained on a VG Autospec instrument. Recorded at 400 MHz in C6D6 and acetone-d6 on a Bruker AMX-400 spectrometer at ambient temperature. 1 H nuclear magnetic resonance (NMR) spectra, with residual protonated solvent as an internal reference.

[0384] (2Z,4E)-1-(Benzothiazol-2-yl)sulfanyl-5-(tri-n-butylstannyl)-3-methylpenta-2,4-diene(6)

[0385] ((2Z,4E)-1-(Benzothiazol-2-yl)sulfanyl-5-(tri-n-butylstannyl)-3-methylpenta-2,4-diene(6)).

[0386] A solution of (2Z,4E)-3-methyl-5-(tributylstannyl)penta-2,4-dien-1-ol (1.0 g, 2.58 mmol), 2-mercaptobenzothiazole (0.65 g, 3.87 mmol), and PPh3 (1.10 g, 4.21 mmol) in THF (14 mL) was stirred at 0°C for 5 min. A solution of DIAD (0.77 mL, 3.87 mmol) in THF (5 mL) was added dropwise, and the mixture was stirred at 25°C for 30 min. The solvent was removed and the residue was purified by column chromatography (C18-silica gel, acetonitrile) to provide 1.11 g (78%) of a colorless oil identified as (2Z,4E)-1-(benzothiazol-2-yl)sulfanyl-5-(tri-n-butylstannyl)-3-methylpenta-2,4-diene 6. 1 H NMR(400MHz,C6D6)δ8.04(d,J=8.2Hz,1H,CH),7.46(d,J=19.2Hz, 3 J SnH =71.2Hz,1H,CH),7.34(d,J=8.0Hz,1H,CH),7.21(ddd,J=8.3,7.3,1.2Hz,1H,CH),7.01(ddd,J=8.3,7.4,1.1Hz,1H,CH),6.66(d,J=19.2Hz, 2 J SnH =71.2Hz,1H,CH),5.65(t,J=8.1Hz,1H,CH),4.37(d,J=8.2Hz,2H,CH2),1.86(s,3 H,CH3),1.8-1.6(m,6H,CH2),1.5-1.4(m,6H,CH2),1.1-1.0(m,15H,CH3+CH2)ppm. 13C NMR(100MHz,C6D6)δ166.4(s),153.7(s),142.6(d),138.6(s),135.7(s), 132.0(d),125.9(d),124.1(d),122.1(d),121.7(d),121.0(d),30.2(t), 29.3(t,3x),27.6(t,3x),19.8(q),13.8(q,3x),9.7(t,3x)ppm; IR(NaCl)ν2955(s,CH),2923(s,CH),2870(m,CH),2850(m,CH),1460(s),1427(s)cm -1 . C 25 H 40 NS2 120 HRMS (ESI + ) m / z calculated: 538.1620; found: 538.1621.

[0387] (2Z,4E)-1-(Benzothiazol-2-yl)sulfonyl-5-(tri-n-butylstannyl)-3-methylpenta-2,4-diene (7)

[0388] ((2Z,4E)-1-(Benzothiazol-2-yl)sulfonyl-5-(tri-n-butylstannyl)-3-methylpenta-2,4-diene(7)).

[0389] To a solution of (2Z,4E)-1-(benzothiazol-2-yl)sulfanyl-5-(tri-n-butylstannyl)-3-methylpenta-2,4-diene 6 (0.48 g, 0.89 mmol) in EtOH (9 mL) was added (NH4)6Mo7O at -10 °C. 24 A solution of 4H₂O (0.44 g, 0.36 mmol) in aqueous hydrogen peroxide (35%, 7.7 mL, 89.1 mmol) was prepared. After stirring at -10°C for 17 h, the mixture was quenched with H₂O and extracted with Et₂O (3x). The combined organic layers were washed with brine (3x) and dried (Na₂SO₄), and the solvent was removed. The residue was purified by chromatography (C₁₈-silica gel, MeOH) to provide 0.33 g (66%) of a colorless oil identified as (2Z,4E)-1-(benzothiazol-2-yl)sulfonyl-5-(tri-n-butylstannyl)-3-methylpenta-2,4-diene 7. 1H NMR(400MHz,C6D6)δ8.10(d,J=8.2Hz,1H,CH),7.18(d,J=19.2Hz,1H,CH),7.16(t,J=7 .5Hz,1H,CH),7.12(t,J=8.1Hz,1H,CH),6.98(t,J=7.7Hz,1H,CH),6.59(d,J=19.2Hz, 2 J SnH =68.2Hz,1H,CH),5.43(t,J=8.0Hz,1H,CH),4.32(d,J=8.1Hz,2H,CH2),1.8-1.6(m,9H,CH3+CH2),1.6-1.4(m,6H,CH2),1.1-1.0(m,15H,CH3+CH2)ppm. 13 C NMR(100MHz,C6D6)δ167.1(s),152.9(s),143.1(s),141.7(d),136.9(s) ,134.4(d),127.3(d),127.1(d),125.0(d),122.1(d),112.0(d),53.3(t ),29.3(t,3x),27.5(t,3x),20.0(q),13.8(q,3x),9.6(t,3x)ppm; IR(NaCl)ν2955(s,CH),2923(s,CH),2850(m,CH),1467(m),1333(s),1151(s)cm -1 . C 25 H 39 NNaO2S2 120 HRMS (ESI + ) m / z calculated value: 592.1338; found: 592.1334. UV (MeOH) λ max 239nm.

[0390] (3S,4Z,6Z,8E)-Ethyl 3,7-Dimethyl-9-(tri-n-butylstannyl)nona-4,6,8-trienoate((S)-9).

[0391] A cooled (-78°C) solution of (2Z,4E)-1-(benzothiazol-2-yl)sulfonyl-5-(tri-n-butylstannyl)-3-methylpenta-2,4-diene (0.115 g, 0.20 mmol) in THF (9 mL) was treated with NaHMDS (0.23 mL, 1 M in THF, 0.23 mmol). After stirring at this temperature for 30 minutes, a solution of (S)-ethyl-3-methyl-4-oxobutanoate (0.044 g, 0.30 mmol) in THF (4.5 mL) was added, and the resulting mixture was stirred at -78°C for 1 hour and allowed to reach room temperature over 3 hours. Et2O and water were added at low temperature, and the mixture was allowed to warm to room temperature. It was then diluted with Et2O and the layers separated. The aqueous layer was extracted with Et2O (3x), and the combined organic layers were dried (Na2SO4) and the solvent removed. The residue was purified by column chromatography (C-18 silica gel, MeOH) to obtain 0.94 g (93%) of a pale yellow oil identified as (3S,4Z,6Z,8E)-ethyl-3,7-dimethyl-9-(tri-n-butylstannyl)nona-4,6,8-trienoic acid ethyl ester (S)-9. [α] 28 D +11.5° (c 1.22, MeOH). 1 H NMR(400MHz,C6D6)δ7.60(d,J=19.1Hz, 3 J SnH =65.1Hz,1H,CH),6.85(t,J=11.4Hz,1H,CH),6.64(d,J=19.2Hz, 2 J SnH =71.9Hz 1H,CH),6.58(d,J=11.9Hz,1H,CH),5.31(t,J=10.4Hz,1H,CH),4.03(q,J=7.1Hz,2H,CH2),3.5-3.3(m,1H,CH),2 .4-2.2(m,2H,CH2),2.02(s,3H,CH3),1.8-1.6(m,6H,CH2),1.6-1.4(m,6H,CH2),1.2-0.9(m,18H,CH3+CH2)ppm. 13C NMR(100MHz,C6D6)δ171.3(s),143.2(d),135.8(d),135.4(s),130.7(d),123.8(d),122.9(d),59.8(t) ,41.8(t),29.3(t,3x),29.2(d),27.5(t,3x),20.7(q),20.3(q),14.0(q),13.7(q,3x),9.6(t,3x)ppm. IR(NaCl)ν2957(s,CH),2924(s,CH),2871(m,CH),2851(m,CH),1737(s,C=O),1459(m),1160(m)cm -1 . C 25 H 46 NaO2 120 HRMS (ESI + ) m / z calculated value: 521.2416; found: 521.2408. UV (MeOH) λ max 285nm.

[0392] (3R,4Z,6Z,8E)-Ethyl 3,7-Dimethyl-9-(tri-n-butylstannyl)nona-4,6,8-trienoate((R)-9)).[α] 29 D -10.3° (c 1.25, MeOH).

[0393] (3R,4E,6Z,8E)-Ethyl 9-Iodo-3,7-dimethylnona-4,6,8-trienoate((R)-3)

[0394] To a solution of (3R,4Z,6Z,8E)-ethyl-3,7-dimethyl-9-(tri-n-butylstannyl)nona-4,6,8-trienoic acid ethyl ester (R)-9 (0.060 g, 0.121 mmol) in CHCl (5.3 mL) was added dropwise a solution of iodine (0.046 g, 0.182 mmol) in CHCl (2.8 mL), and the resulting mixture was stirred at 25° C. for 30 min. Saturated NaSO solution was added, and the reaction mixture was extracted with EtO (3×), the combined organic layers were dried (NaSO), and the solvent was removed. The residue was purified by column chromatography (silica gel, 97:3 hexanes / Et3N) to afford 0.037 g (92%) of a light yellow oil identified as (3R,4E,6Z,8E)-ethyl-9-iodo-3,7-dimethylnona-4,6,8-trienoic acid ethyl ester (R)-3. 24 D +14.8° (c 0.74, MeOH). 1 H NMR(400MHz,C6D6)δ7.65(d,J=14.5Hz,1H,CH),6.28(dd,J=14.9,11.3Hz,1H,CH),6.05( d,J=14.5Hz,1H,CH),5.64(d,J=11.1Hz,1H,CH),5.44(dd,J=15.0,7.8Hz,1H,CH),3.95(q ,J=7.1Hz,2H,CH2),2.64(dt,J=14.1,7.0Hz,1H,CH),2.15(dd,J=14.9,7.1Hz,1H,CH),2. 06(dd,J=14.9,7.3Hz,1H,CH), 0.97(t,J=7.2Hz,3H,CH3), 0.88(d,J=6.8Hz,3H,CH3)ppm. 13 C NMR(100MHz,C6D6)δ171.9(s),142.7(d),140.4(d),132.7(s),130.9(d),124 .6(d),78.5(d),60.5(t),41.8(t),34.5(d),20.4(q),19.9(q),14.7(q)ppm. IR(NaCl)ν2972(m,CH),2932(m,CH),1731(s,C=O),1666(m),1180(m)cm -1 . C 13 H 19 HRMS (ESI + ) m / z calculated value: 357.0322; found: 357.0316. UV (MeOH) λmax 275nm.

[0395] (3S,4E,6Z,8E)-Ethyl 9-Iodo-3,7-dimethylnona-4,6,8-trienoate ((S)-3)). [α] 23 D -16.7° (c 0.72, MeOH).

[0396] (9Z,13R)-13,14-dihydroisoretinoate ((R)-4)). To a THF solution (2.3 mL) of (3R,4E,6Z,8E)-9-iodo-3,7-dimethylnona-4,6,8-trienoic acid ethyl ester (R)-3 (0.036 g, 0.107 mmol) was added Pd(PPh3)4 (0.013 g, 0.011 mmol). After 5 min at room temperature, 2,6,6-trimethylcyclohex-1-enylboronic acid 2 (0.027 g, 0.161 mmol) was added in one portion, followed by TlOH (10% aqueous solution, 0.75 mL, 0.407 mmol). After stirring at 25 ° C for 3 h, Et2O was added and the mixture was dried over a mixture of 4% ethanol and 1% ethanol. The reaction mixture was filtered through a short pad of 100 μl. The filtrate was washed with NaHCO₃ (saturated), the organic layer was dried (Na₂SO₄), and the solvent was removed. The residue was purified by column chromatography (silica gel, 97:3 hexane / EtOAc) to obtain 0.028 g (78%) of a light yellow oil identified as (9Z,13R)-13,14-dihydroisoretinoic acid ethyl ester (R)-4. [α] 23 D +14.2° (c 0.48, MeOH). 1H NMR(400MHz,C6D6)δ6.90(d,J=16.0Hz,1H,CH),6.71(dd,J=14.9,11.2Hz,1H,CH),6.28(d,J=16.0Hz,1H,CH),5.96 (d,J=11.1Hz,1H,CH),5.51(dd,J=15.0,7.8Hz,1H,CH),3.94(q,J=7.2Hz,2H,CH2),2.77(dt,J=14.1,7.1Hz,1H,CH) ,2.21(dd,J=14.8,7.3Hz,1H,CH),2.11(dd,J=14.8,7.2Hz,1H,CH),1.95(t,J=6.1Hz,2H,CH2),1.90(s,3H),1.79(s ,3H),1.66–1.52(m,2H),1.52–1.41(m,2H),1.11(s,6H),0.95(t,J=7.1Hz,3H,CH3),0.94(d,J=6.8Hz,3H,CH3)ppm. 13 C NMR(100MHz,C6D6) 171.4(s), 138.3(s), 137.6(d), 132.7(s), 130.6(d), 129.1(d), 129.0(s)127.9(d),124.8(d),59.8(t),41. 6(t),39.6(t),34.2(s),34.1(d),33.0(t),28.9(q,2x),21.8(q),20.4(q),20.1(q),19.5(t),14.1(q)ppm. IR(NaCl)ν2961(s,CH),2929(s,CH),2866(m,CH),1737(s,C=O),1455(m),1372(m),1167(m)cm -1 . C 22 H 35 HRMS (ESI) of O2 + ) m / z calculated: 331.2632; found: 331.2625. UV (MeOH) λ max 287nm(ε=20000L·mol -1 cm -1 ).

[0397] (9Z,13S)-Ethyl 13,14-dihydroretinoate((S)-4) [α] 22 D-15.5° (c 0.51, MeOH).

[0398] (9Z,13R)-13,14-Dihydroretinoic acid ((R)-1). To a solution of (9Z,13R)-13,14-dihydroisoretinoic acid ethyl ester (R)-4 (0.023 g, 0.069 mmol) in MeOH (4.7 mL) was added KOH (2M aqueous solution, 1.1 mL, 2.27 mmol), and the reaction mixture was stirred at 80°C for 45 min. After the reaction was cooled to room temperature, CH2Cl2 and brine were added and the layers were separated. The aqueous layer was washed with H2O (3x). The combined aqueous layers were acidified with 10% HCl and extracted with CH2Cl2 (3x). The combined organic layers were dried (Na2SO4) and the solvent removed. The residue was purified by column chromatography (silica gel, gradient from 95:5 to 90:10 CH2Cl2 / MeOH) to afford 0.017 g (84%) of a light yellow oil identified as (9Z,13R)-13,14-dihydroretinoic acid ((R)-1). [α] 22 D +7.1° (c 0.67, MeOH). 1 H NMR (400MHz, acetone-d6): δ = 6.66 (d, J = 16.0Hz, 1H), 6.60 (dd, J = 15.0, 11.2Hz, 1 H),6.18(d,J=16.0Hz,1H),5.93(d,J=11.1Hz,1H),5.65(dd,J=15.0,7.5Hz,1H), 2.73(dt,J=13.9,7.0Hz,1H),2.4–2.2(m,2H),2.1–2.0(m,1H),1.91(s,3H),1.72 (s,3H),1.7–1.6(m,2H),1.5–1.4(m,2H),1.08(d,J=6.8Hz,3H),1.03(s,6H)ppm. 13C NMR(100MHz,acetone-d6)173.5(s),138.9(s),138.8(d),133.4(s),131.1(d),129.8(s),129.7(d),128.5(d) ,125.4(d),41.8(t),40.3(t),34.9(s),34.6(d),33.6(t),29.4(q),22.1(q),20.7(q),20.6(q),20.0(t)ppm. IR(NaCl)ν2957(s,CH),2923(s,CH),2855(m,CH),1709(s,C=O),1446(m),1290(m)cm -1 . C 20 H 31 HRMS (ESI) of O2 + ) m / z calculated value: 303.2319; found: 303.2313. UV (MeOH) λ max 289nm(ε=17600L·mol -1 cm -1 ).

[0399] (9Z,13S)-13,14-Dihydroretinoic acid ((S)-1) [α] 24 D -6.9° (c 0.26, MeOH).

[0400] Example 2.1: (R)-9-cis-13,14-dihydroretinol and (R)-9-cis-13,14-dihydroretinol ethyl Synthesis of acid esters

[0401] (R)-9-cis-13,14-dihydroretinol was synthesized in 95% yield by DIBAL-H reduction of (9Z,13R)-13,14-dihydroisoretinoic acid ethyl ester ((R)-4) in THF at -78°C (Scheme 1). (R)-9-cis-13,14-dihydroretinol acetate was prepared in 86% yield by acetylation of (R)-9-cis-13,14-dihydroretinol with acetic anhydride and pyridine in the presence of dimethylaminopyridine (DMAP).

[0402]

[0403] Option 2

[0404] (3R,4E,6Z,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-ol ((R)-10)

[0405] ((3R,4E,6Z,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-ol((R)-10))

[0406] To a cooled (-78°C) solution of (3R,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trienoic acid ethyl ester [(R)-9-cis-13,14-dihydroisoretinoate, (R)-4] (32.0 mg, 0.097 mmol) in THF (1.0 mL) was added DIBAL-H (0.242 mL, 0.242 mmol, 1.0 M solution in hexanes), and the resulting mixture was stirred at -78°C for 30 min. The mixture was allowed to warm to -20°C over 2.5 h. H2O was added and the mixture was extracted with Et2O (3x). The combined organic layers were dried (Na2SO4) and the solvent removed. The residue was subjected to flash chromatography (silica gel, first neutralized with 98:2 hexanes / Et3N, then a gradient from 95:5 hexanes / EtOAc to EtOAc) to afford (3R,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-ol (26.5 mg, 95%) as a colorless oil. 1 H-NMR (400.13MHz, C6D6): δ6.93 (d, J=16.0Hz, 1H), 6.69 (dd, J=14.9, 11.1Hz, 1H), 6.29 (d ,J=16.0Hz,1H),6.01(d,J=11.1Hz,1H),5.46(dd,J=15.0,8.3Hz,1H),3.35(t,J=6.6Hz,2 H),2.27(dt,J=14.0,6.7Hz,1H),1.98–1.92(m,2H),1.93(s,3H),1.79(s,3H),1.63–1.52 (m,2H),1.51–1.43(m,2H),1.36(q,J=6.7Hz,2H),1.11(s,6H),0.91(d,J=6.7Hz,3H)ppm. 13C-NMR (100.62MHz, C6D6): δ140.0(d),138.6(s),132.5(s),130.9(d),129.6(d),129.2(s),128.1(d),124.9(d),60 .9(t),40.2(t),39.9(t),34.5(s),34.3(d),33.2(t),29.21(q),29.18(q),22.0(q),20.9(q),20.7(q),19.7(t)ppm HRMS(ESI + ):C 20 H 33 O([M+H] + ) calculated value, 289.2526; found, 289.2526. IR (NaCl): ν 3338 (m, CH), 2861 (m, CH), 1375 (m), 965 (s) cm -1 .

[0407] (3R,4E,6Z,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-yl acetate ((R)-11)

[0408] ((3R,4E,6Z,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-yl Acetate((R)-11))

[0409] To a solution of (3R,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-ol (5.7 mg, 0.020 mmol) in CHCl (0.5 mL) were added AcO (0.009 mL, 0.099 mmol), pyridine (0.008 mL, 0.099 mmol), and DMAP (0.5 mg, 0.004 mmol). The resulting mixture was stirred at 25°C for 1 h. EtO was then added, and the resulting solution was washed with a saturated aqueous solution of CuSO (2x). The organic layer was dried (NaSO) and evaporated. The residue was subjected to flash chromatography (silica gel, gradient from 85:15 hexanes / EtOAc to EtOAc) to afford (3R,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-yl acetate (5.6 mg, 86%) as a colorless oil. 1H-NMR(400.13MHz,C6D6):δ6.92(d,J)16.0Hz,1H),6.67(dd,J15.0,10.8Hz,1H),6.29( d,JH16.0Hz,1H),5.98(d,JS11.1Hz,1H),5.39(dd,J15.0,8.2Hz,1H),4.11–3.92(m,2H). ),2.16(dq,J14.4,7.2Hz,1H),1.99–1.90(m,2H),1.93(s,3H),1.79(d,J1.0Hz,3H),1 .67(s,3H),1.62–1.53(m,2H),1.50–1.41(m,4H),1.11(s,6H),0.86(d,J6.8Hz,3H)ppm. 13 C-NMR(100.62MHz,C6D6): δ170.0(s), 138.9(d), 138.6(s), 132.8(s), 130.9(d), 129.4(d), 129.3(s), 128.2(d), 125.2(d), 62.7(h); ,39.9(h),36.0(h),34.5(s),34.4(d),33.2(h),29.19(q),29.18(q),22.0(q),20.74(q),20.70(q),20.6(q),19.7(t)ppm.HRMS(ESI). + ):C 22 H 35 O2([M+H] + IR(NaCl):ν2957(s,CH),2926(s,CH),331.2632; ),2860(m,CH),1741(s,C0),1455(m),1365(m),1238(s),966(m)cm -1 。 。

[0410] Example 2.2: Synthesis of 9-cis-β,β-carotene (Scheme 3, including added numbers)

[0411] 9-cis-Retinoic acid ethyl ester (III). To a cold (0°C) solution of (E)-4-(diethoxyphosphoryl)-3-methylbut-2-enoic acid ethyl ester (II) (0.227 g, 1.10 mmol) in THF (2.0 mL) were added nBuLi (0.63 mL, 1.00 mmol, 1.6 M solution in hexanes) and DMPU (0.15 mL, 1.24 mmol). After stirring for 1 h, the reaction was cooled to -78°C and a solution of (2Z,4E)-3-methyl-5-(2,6,6-trimethylcyclohex-1-en-1-yl)penta-2,4-dienal (I) (0.100 g, 0.46 mmol) in THF (2.5 mL) was added and stirred for 2 h. Water was added and the mixture was extracted with Et2O (3x). The combined organic layers were dried (Na2SO4) and the solvent evaporated. The crude product was purified by column chromatography (silica gel, 95:5 hexanes / EtOAc) to afford 0.144 g (96%) of a yellow solid identified as 9-cis-retinoic acid ethyl ester (III).

[0412] 9-cis-Retinol (IV). To a cold (-78 ° C) solution of 9-cis-retinoic acid ethyl ester (III) (0.154 g, 0.47 mmol) in THF (2.3 mL) was added DIBAL-H (1.08 mL, 1.08 mmol, 1.0 M in toluene) and the reaction was stirred for 2 h. Water was then added and the mixture was extracted with EtOAc (3x). The combined organic layers were dried (Na2SO4) and the solvent evaporated. The crude product was purified by column chromatography (silica gel, gradient from 95:5 to 80:20 hexane / EtOAc) to obtain 0.080 g (60%) of a yellow oil, which was identified as 9-cis-retinol (IV).

[0413] 9-cis-Retinaldehyde (V). To a solution of 9-cis-retinol (IV) (65 mg, 0.23 mmol) in CH2Cl2 (9.1 mL) were added MnO2 (197 mg, 2.27 mmol) and Na2CO3 (240 mg, 2.27 mmol), and the reaction was stirred at 25°C for 1.5 h. The mixture was passed through The plate was filtered and washed with CH2Cl2. The solvent was evaporated to give 38 mg (58%) of a yellow oil which was identified as 9-cis-retinal (V).

[0414] (2E,4E,6E,8E)-Bromo-(3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)non-2,4,6,8-tetraen-1-yl)triphenylphosphine (VII). To a solution of vitamin A / all-trans-retinol (VI) (1.370 g, 4.78 mmol) in MeOH (2.7 mL) was added PPh3 (1.443 g, 5.5 mmol) and HCl solution (1.4 mL, 4 M in dioxane), and the reaction was stirred at 25°C for 2 h. The mixture was then poured into water and extracted with Et2O (2x). The aqueous layer was extracted with AcOEt (3x), the combined organic layers were dried (Na2SO4), and the solvent was evaporated. The light orange residue was triturated three times with EtOAc to afford 1.390 g (52%) of a yellow foam identified as (2E,4E,6E,8E)-bromo-(3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraen-1-yl)triphenyl-phosphorus VII. 1H NMR (400.16MHz, CDCl3): δ7.88(m,6H),7.78(m,3H),7.69(m,6H),6.56–6.42(m,1H),6.24–5.94(m,4H),5.3 9(d,J=10.6Hz,1H),4.99(m,2H),2.01(m,2H),1.92(s,3H),1.69(s,6H),1.55–1.40(m,4H),1.01(s,6H)ppm. 13C NMR (101.63MHz, CDCl3): δ143.7(s),137.7(s),137.5(s),137.4(d),134.9(d),134.8(d),132.0(d),129.5(d),128.45(d)127.5(d),1 26.4(d),118.7(s),117.8(s),114.1(d),39.6(t),34.2(s),33.0(t),28.9(q,2x),25.4(t),24.9(t),19.2(q),13.1(q),12.8(q)ppm. HRMS (ESI+): calcd. for C38H44P+([M-Cl]+), 531.3175; found, 531.3166.

[0415] 9-cis-β,β-carotene (VIII). To a solution of phosphonium salt (VII) (24.9 mg, 0.044 mmol) in THF (0.20 mL) at -78°C was added nBuLi (0.027 mL, 0.044 mmol, 1.6 M hexane solution), and the mixture was stirred for 30 minutes. A solution of 9-cis-retinal V (9.0 mg, 0.031 mmol) in THF (0.13 mL) was then added, and the mixture was stirred at -78°C for 1 hour and at 25°C for 30 minutes. Water was added, and the mixture was extracted with Et2O (3x). The combined organic layers were washed with brine (2x), dried (Na2SO4), and the solvent evaporated. The residue was purified by column chromatography (C18-silica gel, acetonitrile) to obtain 11.3 mg (67%) of a red foam, which was identified as 9-cis-β,β-carotene (VIII). The spectral data are identical to those published previously (LIT).

[0416]

[0417] Example 2.3: Synthesis of (R)-9-cis-13,14-dihydro-β,β-carotene (Scheme 4, including numbering)

[0418] (Z)-3-iodo-3-methylprop-2-en-1-ol (X). CuI (0.34 g, 1.78 mmol) was added to a solution of propargyl alcohol (IX) (1.00 g, 11.84 mmol) in Et2O (13.2 mL). At -20°C, MeMgBr solution (17.8 mL, 53.51 mmol, 3M in Et2O) was added. After stirring at this temperature for 2 h, the solution was allowed to reach 25°C and stirred for a further 12 h. A solution of I2 (9.10 g, 35.67 mmol) in Et2O (40.0 mL) was added at 0°C, and the cold water bath was removed. After stirring at 25°C for 24 h, the resulting mixture was cooled to 0°C, and ice water was added. The organic layer was washed with a saturated aqueous solution of Na2S2O4 (3x) and then purified by The product was filtered through a filtration plate. The residue was purified by distillation (0.2 mmHg, 60°C) to obtain 2.10 g (60%) of a yellow oil identified as (Z)-3-iodo-3-methylprop-2-en-1-ol (X). 1H-NMR (400.16 MHz, C6D6): δ 5.49 (s, 1H), 3.90 (s, 2H), 1.55 (s, 3H) ppm. 13C-NMR (101.63 MHz, C6D6): δ 146.6 (s), 74.1 (d), 67.5 (t), 20.9 (q) ppm. HRMS (ESI+): Calcd. for C4H8IO ([M+H]+), 198.9609; Found, 198.9614.

[0419] (Z)-3-iodo-2-methylacrylic acid (XI). To a solution of (Z)-3-iodo-3-methylprop-2-en-1-ol (IIX) (0.21 g, 1.06 mmol) in CH2Cl2 (53.0 mL) were added MnO2 (0.93 g, 10.65 mmol) and Na2CO3 (1.13 g, 10.65 mmol) and the reaction was stirred at 25°C for 1 h. The mixture was then The pad was filtered and washed with CH2Cl2. The solvent was evaporated to afford 0.15 g (73%) of a yellow oil identified as (Z)-3-iodo-2-methylacrylic acid (XI).

[0420] 2-(1E,3Z)-4-iodo-3-methylbut-1,3-dien-1-yl)-1,3,3-trimethylcyclohex-1-ene (XIII). To a cold (-30°C) solution of the phosphonium salt (XII) (150 mg, 0.31 mmol) in THF (2.5 mL) was added nBuLi (2.5 mL, 0.36 mmol, 2.36 M solution in hexane) and the mixture was stirred at 0°C for 45 min. After cooling to -30°C, a solution of (Z)-3-iodo-2-methylacrylic acid (XI) (70 mg, 0.36 mmol) in THF (2.5 mL) was added and the reaction mixture was stirred for 1.5 h. Water (5 mL) was added and the mixture was extracted with hexane (3x). The organic extract was dried (Na2SO4) and the solvent was evaporated. The residue was purified by column chromatography (C18-silica gel, acetonitrile) to obtain 65 mg (64%) of a yellow oil identified as 2-(1E,3Z)-4-iodo-3-methylbut-1,3-dien-1-yl)-1,3,3-trimethylcyclohex-1-ene (XIII). 1H-NMR (400 MHz, C6D6): δ 6.80 (d, J = 16.1 Hz, 1H), 6.32 (d, J = 16.1 Hz, 1H), 5.80 (s, 1H), 1.88 (m, 2H), 1.78 (s, 3H), 1.66 (s, 3H), 1.57–1.48 (m, 2H), 1.44–1.39 (m, 2H), 1.07 (s, 6H) ppm. 13C-NMR (101 MHz, C6D6): δ 142.3 (s), 137.5 (s), 135.1 (d), 132.1 (d), 130.8 (s), 78.6 (d), 39.7 (t), 34.0 (s), 33.1 (t), 29.1 (q, 2x), 22.0 (q), 20.9 (q), 19.3 (t) ppm. UV (MeOH): λmax 266 nm. IR (NaCl): ν 2925 (m, CH), 2862 (m, CH), 962 (m), 771 (s) cm-1. HRMS (ESI+): Calcd. for C14H21I ([M+H]+), 316.0688; found, 316.0684.

[0421] Silylether (XV). To a THF solution (0.2 mL) of 2-(1E,3Z)-4-iodo-3-methylbut-1,3-dien-1-yl)-1,3,3-trimethylcyclohex-1-ene XIII (161 mg, 0.510 mmol) was added Pd(PPh3)4 (45 mg, 0.039 mmol). After stirring at 25°C for 5 min, a THF solution (1.0 mL) of (R)-boropentanes XIV (150 mg, 0.392 mmol) and a 10% aqueous solution of TlOH (4.26 mL) were added, and the reaction mixture was stirred for 2 h. The mixture was extracted with Et2O (3x), and the combined organic layers were washed with brine (3x), and the solvent was evaporated. The residue was purified by column chromatography (silica gel, 95:5 hexanes / EtOAc) to afford 120 mg (71%) of a colorless oil identified as (3R,4E,6Z,8E)-{[3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-yl]oxy}triisopropylsilane (XV). 1HNMR (400.16MHz, C6D6): δ6.93 (d, J=16.0Hz, 1H), 6.69 (dd, J=14.9, 11.1Hz, 1H), 6.2 9(d,J=16.0Hz,1H),6.01(d,J=11.1Hz,1H),5.46(dd,J=15.0,8.3Hz,1H),3.35(t,J=6. 6 Hz, 2H), 2.34–2.19 (m, 1H), 1.99–1.89 (m, 2H), 1.93 (s, 3H), 1.79 (s, 3H), 1.62–1.53 (m, 2H), 1.50–1.44 (m, 2H), 1.36 (q, J = 6.7 Hz, 2H), 1.11 (s, 6H), 0.91 (d, J = 6.7 Hz, 3H) ppm. HRMS (ESI+): Calcd. for C29H53OSi ([M+H]+), 445.3848; found, 445.3806.

[0422] (R)-9-cis-13,14-dihydro-retinol XVI. To a cold (0°C) solution of silyl ether (XV) (91.2 mg, 0.212 mmol) in THF (3.5 mL) was added Bu4NF (0.32 mL, 0.32 mmol) and the mixture was stirred at 25°C for 0.5 h. A saturated aqueous solution of NaHCO3 was added and the mixture was extracted with Et2O (3x). The combined organic layers were washed with brine (3x) and dried (Na2SO4), and the solvent was evaporated. The residue was purified by column chromatography (silica gel, gradient from 95:5 to 70:30 hexanes / EtOAc) to obtain 41 mg (67%) of a colorless oil, which was identified as (R)-9-cis-13,14-dihydro-retinol (XVI). 1H NMR (400MHz, C6D6): δ6.93 (d, J=16.0Hz, 1H), 6.69 (dd, J=14.9, 11.1Hz, 1H), 6.29 (d, J=16.0Hz,1H),6.01(d,J=11.1Hz,1H),5.46(dd,J=15.0,8.3Hz,1H),3.35(t,J=6.6Hz ,2H),2.34–2.19(m,1H),1.99–1.89(m,2H),1.93(s,3H),1.79(s,3H),1.62–1.53(m, 2H),1.50–1.44(m,2H),1.36(q,J=6.7Hz,2H),1.11(s,6H),0.91(d,J=6.7Hz,3H)ppm. 13C NMR (101MHz, C6D6): δ140.0(d),138.6(s),132.5(s),130.9(d),129.6(d),129.2(s),128.0(d),124.9(d),60.9( t),40.2(t),39.9(t),34.5(s),34.3(d),33.2(t),29.21(q),29.19(q),22.0(q),20.9(q),20.7(q),19.7(t)ppm. IR(NaCl): ν3326(br,OH),2966(m,CH),2865(m,CH),965(s)cm-1. HRMS (ESI+): calcd. for C20H33O ([M+H]+), 289.2584; found, 289.2525.

[0423] (R)-9-cis-13,14-dihydro-β,β-carotene (XIX). To a solution of alcohol (XVI) (25 mg, 0.087 mmol) in CHCl (3.7 mL) at 0°C was added Dess-Martin periodinane (73.5 mg, 0.173 mmol) and pyridine (0.014 mL, 0.173 mmol), and the reaction was stirred at 0°C for 30 min and at 25°C for 2 h. EtO (5 mL), a saturated aqueous solution of NaHCO (3 mL), and a saturated aqueous solution of NaSO (3 mL) were then added. The layers were separated, and the aqueous layer was extracted with EtO (3x). The combined organic layers were washed with a saturated aqueous solution of NaHCO (2x) and brine (2x), dried (NaSO), and the solvent evaporated. The residue was purified by column chromatography (silica gel, gradient from 95:5 to 70:30 hexanes / EtOAc) to afford 14 mg (57%) of a colorless oil identified as (R)-9-cis-13,14-dihydro-retinal (XVII). It was observed that the product was very unstable and readily isomerized. IR (NaCl): ν 2927 (m, CH), 2864 (m, CH), 1461 (s), 1105. HRMS (ESI+): Calcd. for C20H31O ([M+H]+), 287.2362; Found, 287.2369. To a solution of phosphonium salt VII (24.9 mg, 0.044 mmol) in THF (0.2 mL) was added nBuLi (0.027 mL, 0.044 mmol, 1.6 M in hexane) at -78°C, and the reaction was stirred for 30 min. A solution of (R)-9-cis-13,14-dihydro-retinal (XVII) (9.0 mg, 0.031 mmol) in THF (0.13 mL) was then added, and the mixture was stirred at -78°C for 1 h and at 25°C for 30 min. Water was added and the mixture was extracted with Et2O (3x). The combined organic layers were washed with a saturated aqueous solution of NaCl (2x) and dried (Na2SO4). The solvent was evaporated and the residue was purified by column chromatography (C18-silica gel, acetonitrile) to obtain 11.3 mg (67%) of a red foam identified as (R)-9-cis-13,14-dihydro-β,β-carotene (XIX).1H-NMR (400 MHz, C6D6) δ 6.80-6.63 (m, 2H), 6.57-5.90 (m, 7H), 5.80-5.35 (m, 4H), 2.32-2.10 (m, 3H), 1.94 (s, 3H), 1.91 (s, 3H), 1.89-1.82 (m, 4H), 1.78 (s, 9H), 1.67-1.43 (m, 8H), 1.33 (s, 6H), 1.10 (s, 12H), 0.96 (m, 3H) ppm. HRMS (ESI+): Calcd. for C40H59 ([M+H]+), 539.2528; found, 539.4611.

[0424] References used in the synthetic procedures incorporated herein:

[0425] Okitsu, T. Chem. Comm. 2008, 47, 6330-6332.

[0426] Englert,G.Helv.Chim.Acta 1975,58(8),2367-2390.

[0427] Zanoun,AJMol.Struct.:THEOCHEM 2006,777(1-3),113-120.

[0428] Koyama, Y.; Hosomi, M.; Hashimoto, H. Shimamura, TJMol.Struct.1989,193,185-201.

[0429] Koukal, P.; Ulc, J.; Necas, D.; Kotora, M. Eur. J. Org. Chem. 2016, 2110-2114.

[0430] (a)Isler,0.;Gutmann,H.;Lindlar,H.;Montaron,M.;Riiegg,R.;Ryser,G.;Zeller,P.Helv.Chim.Acta 1956, 39, 463. (b) Broek, AD; Muradin-Szweykowska, M.; Courtin, JML; Lugtenburg, J. Red. Trav. Chim. Pays-Bas 1983, 102, 46.

[0431]

[0432] Example 3: Animal Experiment

[0433] animal:

[0434] Male mice (Charles River, France) were housed in individually ventilated cages (Techniplast, Italy) in groups of 3 mice per cage with a 7am-7pm light / dark cycle. Food (standard diet, derived from D04 in SAFE, France) and water were available ad libitum. All experiments were conducted in accordance with the European Community Council Directive (86 / 609 / EEC) of November 24, 1986, and in accordance with the guidelines of the French National Center for Scientific Research (CNRS) and the French Ministry of Agriculture and Forestry (Decree No. 87848).

[0435] For the social defeat stress protocol, we used 6-7 week old C57BL / 6N mice (Charles River, France). All mice were housed in individually ventilated cages (Techniplast, Italy) with a 7am-7pm light / dark cycle in groups of 4-5 mice per cage until they were adapted to the stress protocol in the same cage as the social defeat stress mice (see below). CD1 male mice purchased from Charles River (France) were used as aggressors in this test (see below).

[0436] All experiments were performed in accordance with the European Communities Council Directive of 24 November 1986 (86 / 609 / EEC) and in compliance with the guidelines of the French National Center for Scientific Research (CNRS) and the French Ministry of Agriculture and Forestry (Decree No. 87848) and with authorization from the French Research Institute.

[0437] Behavioral protocol and tissue sample collection:

[0438] Behaviorally naive groups of mice were tested in the DNMTP of the T-maze test according to a previously developed protocol (Wietrzych et al., 2005) and modified to facilitate pharmacological testing (Wietrzych-Schindler et al., 2011). Specifically, in this protocol, animals were first trained for 10 consecutive days with a minimum intertrial interval (ITI) of approximately 15 seconds, which was necessary to achieve a criterion of 90% or higher correct choices for four consecutive days (Days 9-10). Following this period, the ITI was semi-randomly increased in 6-minute blocks for five consecutive days (Days 11-15 shown) to determine each animal's "test ITI," defined as the shortest ITI that a mouse performed at chance level. Starting on Day 16, mice were tested using the corresponding "test ITI" delay after injection of vehicle on Day 16 (to test the effect of vehicle alone) and after injection of 9CDHROL (40 mg / kg) on ​​Day 17. Three mice were excluded from the test because their latency to choose an arm during the retention phase exceeded 3 minutes per trial / day for three consecutive days (exclusion criteria). On day 18, mice were injected with 9CDHROL (40 mg / kg) again and sacrificed 9 hours later for chemical analysis. Mice that received vehicle injections for three consecutive days served as a vehicle-treated control group for analysis of 9CDHROL metabolism. Another group of three naive mice were also injected with 9CDHROL (40 mg / kg) to test 9CDHROL metabolism after acute treatment.

[0439] Forced swimming test: The forced swimming paradigm was performed between 1 p.m. and 4 p.m. in a 3-liter glass beaker half-filled with water (water depth 17 cm) at 22-23°C. All mice were tested only once in this task. To this end, each mouse was gently placed in the water and the immobility time was scored during a 6-minute test period. A mouse was judged to be immobile when it floated upright and made only a small amount of movement to keep its head above the water. After 6 minutes, the mouse was removed from the water, dried under a red light and returned to its home cage. The immobility score of each animal was used as an indicator of despair behavior.

[0440] Sucrose preference experiment: This task designed to test the hedonic behavior of mice is based on the palatable properties of sucrose observed in many mouse species. On the first day of the test, mice that were not treated with sucrose were placed in individual cages at 11 am and left there with water and food for an adaptation period. At 5 pm, one water bottle was replaced with two bottles: one containing water and the other containing a 0.8% sucrose solution. Three hours later (8 pm), the bottles were weighed to measure liquid consumption and replaced in the cage until morning. Overnight consumption was then measured on a separate day to assess sucrose preference. In order to measure spontaneous sucrose preference and exclude any potential emotional confounds caused by water deprivation stress, mice were not deprived of water at any time. Sucrose preference is expressed as the percentage of sucrose solution consumed relative to total liquid consumption.

[0441] Social defeat stress: The social defeat stress protocol was a modification of a previously described protocol (Berton, McClung et al. 2006). C57BL / 6N mice were continuously defeated for 10 consecutive days. Each day, they were physically contacted with an unfamiliar CD1 aggressor in their home cage, with a maximum interaction time of 5 minutes. After each physical stress, C57BL / 6N and CD1 mice were separated by a perforated wall and maintained in perceptual contact for 24 hours. After the last stress, mice were transferred to new cages and housed separately throughout the behavioral testing period. Similar to the experimental animals, C57BL / 6N control mice were housed two per cage in cages separated by perforated metal walls. Each day, they were physically contacted for 5 minutes.

[0442] Animal treatment

[0443] For behavioral analysis, 9CDHROL and 9CDHBC were dissolved in ethanol and then mixed with sunflower oil so that the final solution contained 3% ethanol. Vehicle treatment consisted of a 3% ethanol solution in sunflower oil. Treatments were administered via intraperitoneal injection at a volume / weight ratio of 3 ml / kg between 7:00 AM and 7 hours before the start of testing. For each substance, treatment was administered orally at 10 mg / kg and immediately after the stress period starting on the first day of the stress protocol, at a volume / weight ratio of 3 ml / kg, between 5:00 PM and 6:00 PM on both days of the stress protocol.

[0444] For metabolic analysis, ATROL, 9CDHROL, 9CDHBC, and control solutions were prepared as described above. A single oral dose of 40 mg / kg of each substance was used to treat mice in the evening. 11 hours later, samples were collected under light-protected conditions, weighed, frozen in liquid nitrogen, and stored at -80°C until analysis. Serum was prepared and immediately stored in brown vials at -80°C until further analysis.

[0445] Statistical analysis

[0446] Comparison of behavioral performance during the learning phase was performed using a one-way ANOVA for repeated measures, with time as the dependent parameter and the percentage of correct choices as the independent parameter. Pharmacological data were analyzed by comparing the performance of animals treated with vehicle and 9CDHROL using a one-way ANOVA for repeated measures. Post-hoc statistical analysis was performed using a group t-test to compare animal performance with the 50% chance level. Significant differences are shown in the corresponding figures.

[0447] Results of Animal Supplementation Studies

[0448] To test the possibility that 9CDHROL could serve as a substrate for the production of 9CDHRA in vivo, we tested working memory in the delayed non-match to place (DNMTP), a behavioral paradigm sensitive to 9CDHRA activity (Ruhl et al., 2015). To this end, we trained a group of C57BL6J male mice in the DNMTP task to achieve a maximal performance criterion (greater than 90%) for more than 4 consecutive days ( Figure 1 a). When trained with a 15-second inter-trial interval (ITI), wild-type C57BL6N male mice acquired the working memory task, as shown by a significant effect of the training day (F[9,39]=8,28; p<0.001, one-way ANOVA with repeated measures). When tested at a longer ITI of 3 min, memory performance decreased. However, when the ITI reached an average of 13 min (mean value for the entire group, shown as gr13 in the figure), the mice showed complete memory loss in the DNMTP task, as their performance did not differ from the 50% chance level ((p<0.05, one-group t-test)). The ITI at which each animal showed complete forgetting was determined as the "test ITI" for each individual animal and used when testing the thyroid-stimulating activity of 9CDHROL ( Figure 1 b). During this test, vehicle application failed to improve memory, as the percentage of correct choices was comparable to the 50% chance level (p = 0.2, not significant; one-group t-test); whereas 9CDHROL treatment significantly enhanced memory performance (F[1,4]16; p < 0.05, one-way ANOVA with repeated measures for treatment effect). 9CDHROL-treated mice performed significantly better than the 50% chance level (p < 0.05; one-group t-test).

[0449] Example 4: Analytical protocol and HPLC and LC-MS analysis

[0450] LC-MS analysis of tissue samples

[0451] A. Retinoid Analysis Only: Analytical Protocol: Analytical analysis was performed using a previously validated protocol (Ruhl, 2006; Ruhl et al., 2015) under dim yellow / amber light using high-performance liquid chromatography-mass spectrometry (Agilent 1260 Infinity LC System; Madrid, Spain)-mass spectrometry (SCIEX Triple Quad 3500 System; Sciex, Madrid, Spain). For detection of 13,14-dihydroretinoic acid, the MS-MS settings were 303->207 m / z; for detection of 13,14-dihydroretinol, the MS-MS settings were 290->69 m / z, using the same dwell times and impact energies as those specified for retinoic acid. Therefore, for sample preparation, 100 mg of material (if the sample is less than 100 mg, water is added until the standard weight used is 100 mg) or 100 μl of serum is diluted with three volumes of isopropanol, the tissue is minced with scissors, vortexed for 10 seconds, placed in an ultrasonic bath for 5 minutes, shaken for 6 minutes and centrifuged at 13000 rpm at +4°C in a Heraeus BIOFUGE Fresco. After centrifugation, the supernatant is dried at 30°C in an Eppendorf concentrator 5301 (Eppendorf, Germany). The dried extract is resuspended with 60 μl of methanol, diluted with 40 μl of a 60 mM aqueous ammonium acetate solution and transferred to an autosampler for subsequent analysis.

[0452] B. Analysis of bound retinoids and carotenoids: Analytical protocol: HPLC-MS / MS (Agilent 1260 Infinity LC system; Madrid, Spain)-MS / MS (SCIEX Triple Quad 3500 system; Absorbance, Madrid, Spain) with an additional online diode array detector (Waters 966DAD, Waters, Santiago de Compostella, ES) was performed under dim yellow / amber light using the previously validated protocol (LIT) with the addition of a third and fourth eluent after a 20 min elution time. The linear gradient was from 20% (isopropanol:methanol:methyl tert-butyl ether (MTBE) / 30:30:40) to 80% (isopropanol:methanol / 50:50) in 20 min, 40% (isopropanol:methanol:methyl tert-butyl ether (MTBE) / 30:30:40) to 60% (isopropanol:methanol / 50:50) in 25 min, 70% (isopropanol:methanol:methyl tert-butyl ether (MTBE) / 30:30:40) to 30% (isopropanol:methanol / 50:50) in 29 min, and 30 min. 0% (isopropanol:methanol:methyl tert-butyl ether (MTBE) / 30:30:40)-100% (isopropanol:methanol / 50:50), 30, 1 min 20% (isopropanol:methanol:methyl tert-butyl ether (MTBE) / 30:30:40)-80% (isopropanol:methanol / 50:50). For detection of 13,14-dihydroretinoic acid, MS-MS settings were 303->207 m / z, for detection of 13,14-dihydroretinol, MS-MS settings were 290->69 m / z, and for detection of 9CDHBC, 405->405 / 405->95 m / z. Therefore, for sample preparation, 100 mg of material (if the sample was less than 100 mg, water was added to the standard weight used: 100 mg) or 100 μl of serum was diluted with three volumes of isopropanol, the tissue was minced with scissors, vortexed for 10 seconds, placed in an ultrasonic bath for 5 minutes, shaken for 6 minutes, and centrifuged at 13,000 rpm at +4°C in a Heraeus BIOFUGE Fresco. After centrifugation, the supernatant was dried at 30°C in a GYROZEN centrifugal vacuum concentrator equipped with an ILMAC MPC 301-Z vacuum pump (CONTROLTECNICA, Madrid, ES). The dried extract was resuspended with 30 μl of methanol-MTBE (50–50) and transferred to an autosampler, and 10 μl was subsequently analyzed.

[0453] Statistical analysis

[0454] Statistical analysis of behavioral analysis and retinoid / carotenoid analysis was performed using t-test for two-group comparisons within each group. Significant differences are shown in the corresponding figures.

[0455] Results of HPLC and LC-MS analysis

[0456] Preliminary determination of endogenous 9CDHROL and 9CDHROL after treatment of mice

[0457] Initially, using our LC-MS assay, we analyzed a mixed standard of 9-cis-13,14-dihydroretinol (9CDHROL) and all-trans-dihydroretinol (ATDHROL) ( Figure 2 ), and later we analyzed liver samples from vehicle-treated mice ( Figure 2 (bottom image). Two peaks from the standards 9CDHROL and ATDHROL coeluted with the peaks identified in the liver sample using the same MS-MS fragmentation channels on our LC-MS system. Based on the coelution and the use of identical LC-MS parameters and the same fragmentation mode channels (290->69 m / z), we claim and confirm that 9CDHROL and ATDHROL are endogenous retinoids in mammalian organisms. Furthermore, these coeluting peaks were also observed at lower levels in the serum and brain of mice, and 9CDHROL levels were strongly increased after 9CDHROL treatment ( Figure 3 ).

[0458] Determination of Endogenous 9CDHROL in Mouse, Human, and Human Food Matrix:

[0459] Mouse brains were prepared according to the above protocol. Briefly, after a single oral administration of 40 mg / kg of 9CDHROL (or other referenced substances) at night, mice were sacrificed 11 hours later, and tissue samples, including the brain, were dissected under light-protected conditions, weighed, frozen in liquid nitrogen, and stored at -80°C until analysis. Serum was prepared and immediately stored in brown vials at -80°C until further analysis.

[0460] Human serum samples were obtained from the blood of healthy volunteers with written informed consent from all subjects.

[0461] Food samples: Beef liver was purchased from a local butcher in Vigo, Spain. Canned peaches (Metades, Pessago em calda, Auchan / Alcampo-home-brand / 420 g can) were purchased from Alcampo, Vigo, Spain.

[0462] Initially, using our LC-MS analysis, we analyzed 9-cis-13,14-dihydroretinol (9CDHROL, Figure 14A We then analyzed control brain samples from control treated mice ( Figure 14A The peak of the 9CDHROL standard co-eluted with the peak identified in the brain sample using the same MS-MS dispersion channel in our LC-MS system.

[0463] In addition, we determined that human serum ( Figure 14B ) and related human food / beef liver 9CDHROL ( Figure 14C Based on the co-elution and the use of identical LC-MS parameters and the use of the same dispersive mode channels (290->69 m / z), we claim and identify 9CDHROL as a novel endogenous retinoid in the mammalian organisms mouse and human and in human food represented by bovine liver, which is also a mammalian organism.

[0464] Determination of 9CDHBC in human food matrix ( Figure 15 ):

[0465] 9CDHBC was determined in canned peaches (canned) using co-elution and comparable UV / VIS spectroscopy.

[0466] Example 5: Determination of Compounds after Treatment of Mice

[0467] Determination of 9CDHRA after 9CDHROL treatment in mice

[0468] We recently discovered that 9CDHRA is an endogenous derivative in mice (Ruhl et al., 2015) and humans (unpublished data). The next step is to identify the precursor of 9CDHRA in in vivo and in vitro models.

[0469] Animal treatment, 9CDHROL administration, and sample analysis were performed as described in Examples 3 and 4.

[0470] In the mouse supplementation experiment, we orally administered 100 mg / kg body weight (mg / kg bw) of 9CDHROL to n=6 mice, and sacrificed the mice 9 h after treatment to obtain blood, brain, and liver. Blood was drawn and centrifuged to obtain serum.

[0471] In serum, brain and liver, low levels of 9CDHRA and ATDHRA were found endogenously. In addition, after 9CDHROL-treatment, a strong increase in 9CDHRA levels was determined in the analyzed samples of serum, brain and liver ( Figure 4 As a comparison, we Figure 5 Increased levels of 9CDHRA in liver samples are shown in FIG.

[0472] Identification of 9CDHROL in mice

[0473] 9CDHROL accumulation in 9CDHROL-treated mice and metabolites of 9CDHBC in in vitro oligodendrocyte cultures: compared with control-treated brains ( Figure 14A The middle chromatogram of the chromatogram), treatment with 9CDHROL in the orally supplemented mice increased 9CDHROL in the mouse brain ( Figure 14A , bottom chromatogram). Here we note that administration of 9CDHBC and 9CDHROL increased 9CDHROL levels in human oligodendrocyte cultures, whereas ATBC did not show any increase. 9CDHBC (and ATROL) showed no or only a weak non-isomer-selective increase in 9CDHROL in oligodendrocytes ( Figure 14D In conclusion, 9CDHBC is an excellent precursor of 9CDHROL in human oligodendrocyte culture in vitro.

[0474] Example 6: Cell culture experiments

[0475] Cell culture experiments

[0476] Normal immortalized oligodendrocyte cell line 158N was cultured as previously reported (Feutz et al., 2001). When cells reached 70% confluency, they were treated with a 10-2 M ethanolic solution of one of the following compounds: 9CDHROL, 9CDHROL-acetate, 9CDHRA-ethyl ester. 9CDHRA and ethanol served as controls, and vehicle was used. After 18 h of treatment, cells were harvested in brown Eppendorf tubes and stored at -80°C until analysis. Treatment and cell harvesting were performed under light-limited conditions to avoid photolysis of retinoids.

[0477] High performance liquid chromatography mass spectrometry (LC-MS) was performed as described in Example 4.

[0478] Conversion of ATROL and 9CROL

[0479] In similar experiments, we used the following retinol species in the same cell culture for comparison: ATROL and 9CROL. As a standard, a normal ROL standard (black line) was used. Species are also indicated by arrows ( Figure 11 ).

[0480] Close attention was also paid to the conversion of retinol species. Figure 12In the figure, the measurement of 13,14-dihydro-retinoic acid (DH-RA) species 9CDHRA and ATDHRA (blue and red lines, respectively) is shown after administration of the same retinol species ATROL and 9CROL. It is clearly shown that at most very small amounts of dihydroretinoic acid are present in the cells or none at all (9CROL).

[0481] In a similar experiment, measurements of the retinoic acid species 13-cis retinoic acid (13CRA) and 9CRA and ATRA are shown (blue and red lines, respectively) following administration of the same retinol species ATROL and 9CROL. It is clearly shown that if any very small amounts of these acid forms are present in the cells, they are barely visible against the background noise. Therefore, the alcohol is converted into the corresponding The acid form is very weak, and the alcohol form is clearly unsuitable as a precursor.

[0482] The analytical protocol was performed as described above.

[0483] Determination of 9CDHRA after addition of 9CDHROL, 9CDHRET-acetate, or 9CDHRA-ethyl ester to oligodendrocytes

[0484] We determined that 9CDHRA is a metabolite of 9CDHROL supplementation in mouse serum, brain, and liver. In addition, we treated oligodendrocyte cell lines with 10-5M 9CDHROL, 9CDHRET-acetate, or 9CDHRA-ethyl ester for 18 h. We observed a strong increase in 9CDHRA-levels after administration of 9CDHROL and 9CDHRET-acetate (top). Figure 6 In addition, we found a very strong increase after administration of 9CDHRA-ethyl ester (bottom Figure 6 ).

[0485] Determination of 9CDHROL after administration of 9CDHBC and 9CDHROL

[0486] Administration of 9CDHBC and 9CDHROL increased 9CDHROL levels in human oligodendrocyte cultures, whereas ATBC did not show any increase. 9CDHBC (and ATROL) showed no or only a weak and non-isomer-selective increase in 9CDHROL levels in oligodendrocyte cultures ( Figure 14D ). In conclusion, 9CDHBC is a key target for human oligodendrocyte culture in vitro. 9CDHROL is an excellent precursor substance.

[0487] Determination of 9CDHBC after administration of 9CBC and ATBC

[0488] 9CDHBC was identified after administration of 9CDHBC and 9CBC, but no 9CDHBC was identified after administration of ATBC to oligodendrocyte cultures in vitro ( Figure 16A and 16B ): In oligodendrocyte cultures, when 9CBC was administered, we could detect 9CBC at a detection wavelength of 411 nm with a residence time of 26.1 min in these treated cells ( Figure 16A , 411nm detection), while 9CBC could not be detected in the control treatment and after ATBC or 9CDHBC-treatment. This was also confirmed by UV / Vis spectra taken by a secondary array detector (data not shown here).

[0489] Focus on 9CDHBC (UV detection at 366nm Figure 16B ), we could detect a very small peak of 9CDHBC after control treatment or ATBC treatment, while a slightly higher peak could be detected after 9CBC-treatment, and even a higher peak could be detected after 9CDHBC-treatment (retention time of 25.1 min), which was confirmed by comparable UV spectra captured by a diode array detector (data not shown here).

[0490] Identification of 9CDHROL, 9CDHROL-ester, 9CDHRA, and 9CDHRA-ester after administration to oligodendrocyte cultures

[0491] In use 10 -5 In oligodendrocyte cell lines treated with 9CDHROL, 9CDHROL-acetate, 9CDHRA, or 9CDHRA-ethyl ester at 18 h, we observed a strong increase in 9CDHRA levels after treatment with 9CDHROL and 9CDHROL-acetate ( Figure 17A Furthermore, we found that a very strong increase ( Figure 17A While 9CDHROL and 9CDHROL-ester are excellent precursors to 9CDHRA, we also determined that ATROL, 9CDHBC, 9CBC, and ATBC were not converted to 9CDHRA (data not shown). In summary, 9CDHROL, 9CDHROL-ester and 9CDHRA-ester is an excellent, selective and isomerically specific RXR ligand precursor in human oligodendrocyte cultures in vitro. body.

[0492] Example 7 Identification of 9CDHRA after 9CDHROL and 9CDHBC Supplementation in Mice The experiment was similar to that described in Example 5.

[0493] In addition to the in vitro experiments, we conducted in vivo supplementation experiments in which mice were orally supplemented with 9CDHBC and 9CDHROL ( Figure 17B and17C A modest but significant increase in 9CDHRA was observed after 9CDHBC-supplementation compared to the control ( Figure 17B Low levels of 9CDHRA and its isomer ATDHRA were observed in the serum, liver, and brain of control mice ( Figure 17C chromatograms), especially after 9CDHROL-supplementation, 9CDHRA was strongly increased ( Figure 17C chromatograms).

[0494] Example 8: Experimental Protocol - Testing (R)-9CDHRA in Animal Tests

[0495] Example 8.1: Method

[0496] animal:

[0497] Rbp1- / - and Rxrγ- / - mutants and their wild-type (WT) control mice were generated as described on a mixed genetic background of heterozygous crosses (60% C57BL / 6J and 40% 129SvEms / j) (Ghyselinck NB 1999; Krezel et al., 1996) and tested at the age of 3-6 months. All mice were housed in groups of 4-5 per cage in individually ventilated cages (Techniplast, Italy) with a light / dark cycle of 7am-7pm. Food and water were available ad libitum. All experiments were performed in accordance with the European Communities Council Directive of 24 November 1986 (86 / 609 / EEC) and in compliance with the guidelines of the French National Center for Scientific Research (CNRS) and the French Ministry of Agriculture and Forestry (Decree No. 87848).

[0498] For the social defeat stress protocol, we used 6-week-old C57BL / 6N mice shipped from Taconic (France) and housed in groups of 4 per cage. After a 1-week acclimation period, they were subjected to social defeat stress. CD1 mice purchased from Charles River (France) were used as aggressors in this test.

[0499] Behavioral plan All behavioral tests were performed according to standard protocols at the Institute Clinique de la Souris (http: / / www.ics-mci.fr / ).

[0500] Forced swim test:

[0501] The forced swim paradigm (Dalvi and Lucki, 1999) was performed between 1 and 4 p.m. in a 2-liter glass beaker half-filled with water (water depth 17 cm) at 22-23°C. All mice were tested only once in this task. To this end, each mouse was gently placed in the water and scored for the time spent immobile during a 6-minute test period. A mouse was judged to be immobile when it floated upright and made only a small amount of movement to keep its head above the water. After 6 minutes, the mouse was removed from the water, dried under a red light, and returned to its home cage. The immobility score for each animal was used as an indicator of despair behavior.

[0502] Sucrose preference experiment: This task (Moreau, 1997), designed to test the hedonic behavior of mice, is based on the palatable properties of sucrose observed in many mouse species. On the first day of the test, mice that had not been treated with sucrose were placed in separate cages at 11 a.m. and left there with water and food for an adaptation period. At 5 p.m., one water bottle was replaced with two bottles: one containing water and the other containing a 0.8% sucrose solution. Three hours later (8 p.m.), the bottles were weighed to measure liquid consumption and replaced in the cage until morning. Overnight consumption was then measured on another day to assess sucrose preference. In order to measure spontaneous sucrose preference and exclude any potential emotional confounds caused by water deprivation stress, mice were not deprived of water at any time. Sucrose preference was expressed as the percentage of sucrose solution consumed relative to total liquid consumption.

[0503] Social defeat stress: The social defeat stress protocol was a modification of a previously described protocol (Burton et al., 2006). C57BL / 6N mice were continuously defeated for 10 consecutive days. Each day, they were physically contacted with an unfamiliar CD1 aggressor in their home cage, with a maximum interaction time of 5 minutes. After each physical stress, C57BL / 6N and CD1 mice were separated by a perforated metal wall and maintained in perceptual contact for 24 hours. After the last stress, the mice were transferred to a new cage and housed separately throughout the behavioral testing period. Similar to the experimental animals, C57BL / 6N control mice were housed two per cage in cages separated by perforated metal walls. They were physically contacted for 5 minutes each day.

[0504] The animals were then tested in the forced swim and sucrose tests.

[0505] Example 7.2: R-9CDHRA supplementation reverses behavioral changes in Rbp1- / - mice

[0506] To address the relevance of 9CDHRA in regulating RXR function in vivo, the inventors tested whether R-9CDHRA could reverse the behavioral deficits of Rbp1- / - mice. Acute treatment with R-9CDHRA reduced immobility in the forced swim test in knockout mice in a dose-dependent manner, reaching a maximal effect at 1 mg / kg, which was comparable to the anti-desperate effect of the same dose of the synthetic RXR agonist UVI2108 or 5 mg / kg ATRA treatment ( Figure 9 The treatment effect was not evident in WT mice, which may be related to the low baseline immobility of this species and due to a floor effect. These activities are mediated by RXRγ, as 2 mg R-9CDHRA treatment did not improve the performance of Rxrγ- / - mice, which remained immobile for 120 ± 25 seconds compared to 119 ± 19 seconds in vehicle-treated Rxrγ- / - animals.

[0507] Similar to the anti-despair effect in the forced swim test, 1 or 2 mg / kg of R-9CDHRA also improved the performance of Rbp1- / - mice in the memory test. These treatments increased the rate of successful choices in Rbp1- / - mice, which was significantly better than the 50% chance level when tested with a 6-min intertrial interval in the DNMTP test ( Figure 8 ). Treatment with 2 mg / kg R-9CDHRA also improved the performance of WT mice to approximately 70% correct choices when tested at a longer inter-trial interval of 12 or 18 minutes, compared to the vehicle-treated WT mice, which performed at chance levels (50% correct choices). Such treatment did not improve the performance of Rxrγ- / - mice, which performed at 57±7% correct choices, providing further evidence that impaired RXRγ function associated with reduced 9CDHRA levels is the source of the deficits observed in Rbp1- / - animals.

[0508] Example 7.3: R-9CDHRA exhibits antidepressant effects in the chronic social defeat stress model.

[0509] The present inventors found that R-9CDHRA supplementation treated depressive behavior in a chronic stress model of depression. Stress is an important environmental factor in the etiology of depression. To test the efficiency of 9cDHRA in treating depressive behavior caused by stress, we used a social frustration stress animal model (Burton et al., 2006, Hollis and Kabbaj, 2014). Ten days of daily brief physical contact with a resident dominant CD1 male and subsequent perceptual contact effectively induced despair in a forced swim task ( Figure 10a). Treatment with 1 or 3 mg / kg of 9cDHRA during the stress regimen effectively normalized immobility time, which was comparable to that of control non-stressed mice. In contrast to the lower dose, treatment with 3 mg / kg of 9cDHRA showed an anti-despair effect, as it was significantly lower than the immobility time observed in stressed and therefore non-treated mice, indicating that 9cDHRA shows a dose effect in controlling this behavioral parameter. The effects of 9cDHRA were comparable to the activity of the synthetic pan-RXR (panRXR) agonist UVI2108, indicating an important role for RXR activation in achieving the anti-despair activity of 9cDHRA. In addition to despair behaviors, chronic stress also induced anhedonia, manifested by a lack of preference for sugary drinks, with consumption levels of the sugary drinks not significantly different from the 50% of responses chosen randomly ( Figure 10b In stressed mice treated with low-dose 9cDHRA, anhedonia was abolished, as indicated by a sucrose preference significantly exceeding the 50% chance level, although this preference was even more pronounced after treatment with a higher dose of 3 mg / kg 9cDHRA. The pan-RXR agonist UVI2108 exhibited similar activity to 9cDHRA, supporting the involvement of RXR activation in antidepressant activity through 9cDHRA.

[0510] In the previous patent, RXRg- / - was used as a negative control, showing that 9cDHRA acts on RXRg to improve memory - therefore, in the absence of RXRg, it cannot improve memory. Rbp1- / - was used because they show the same type of deficiency as RXRg- / -, indicating decreased RXRg signaling. The fact that expressed 9cDHRA can normalize their memory is proof of concept that what these mice lack is the RXR ligand rather than the receptor itself (or receptor function). Even in previous experiments, we showed that 9cDHRA can improve memory in WT mice, indicating that 9cDHRA can work as a memory enhancer in healthy subjects and can also be used as a memory enhancer in AD. However, 9cDHRA may have teratogenic effects, so we tested the precursors that are most likely to derive this teratogenic activity.

[0511] Example 7.4: 9CDHROL or 9CDHBC prevents depressive behavior in a chronic stress model of depression ( Figure 18 ):

[0512] Stress is an important environmental factor in the etiology of depression. To test the efficacy of 9cDHROL and 9cDHBC in treating stress-induced depressive behaviors, we used a social defeat stress animal model (Burton et al., 2006, Hollis and Kabaja, 2014). Daily brief physical contact and subsequent perceptual contact with a dominant CD1 male for ten days effectively reduced despair in a forced swim task ( Figure 18A). During the stress protocol, treatment with 10 mg / kg of 9CDHROL or 10 mg / kg of 9CDHBC effectively normalized immobility time, which was comparable to that of control non-stressed mice. In addition to despair behavior, chronic stress also induced anhedonia, as evidenced by a lack of preference for the sugary drink, with consumption levels not significantly different from the 50% response to random selection ( Figure 18 B) Anhedonia was abolished in stressed mice treated with 10 mg / kg 9CDHROL or 10 mg / kg 9CDHBC during the stress protocol as indicated by sucrose preference significantly exceeding the 50% chance level.

[0513] Summary of the invention and industrial applicability

[0514] Vitamin A is a family of derivatives that can be converted in vivo into visual pigments and nuclear hormone receptor ligands. While vitamin A1 (retinol) is a well-known precursor of the RAR ligand all-trans retinoic acid (ATRA), the precursor of the recently discovered endogenous ligand for the retinoid X receptor (RXR), 9-cis-13,14-dihydroretinoic acid (9CDHRA), is unknown. In this study, we show that the nutritional precursors of 9cDHRA are 9-cis-13,14-dihydroretinol (9CDHROL) and 9-cis-13,14-dihydro-β-carotene (9CDHBC), respectively, which are novel retinoids and carotenoids that have not been described to date but are present at high levels in food matrices and endogenously in mammals, including humans. Using in vitro and in vivo experiments, we demonstrate that these precursors can be metabolized directly or indirectly into 9CDHRA. In contrast, known endogenous retinoids / carotenoids such as all-trans-retinol, 9-cis-retinol, and all-trans-β-carotene are only weak and non-selective precursors of 9CDHRA. We also demonstrated that the known endogenous carotenoid 9-cis-β-carotene (9CBC) is only weakly converted to 9CDHBC via dehydrogenation. However, 9CDHBC is readily converted to 9cDHRA, thus suggesting that 9CBC may serve as an indirect precursor of the RXR ligand 9cDHRA. Through our metabolic screening, we established for the first time that 9CDHRA-esters, 9CDHROLs, 9CDHROL-esters, and 9CDHBCs are excellent nutritionally and physiologically relevant selective precursors of the endogenous RXR ligand 9CDHRA for inducing RXR-mediated signaling. We further describe this new class of substances as a novel, independent, and selective novel vitamin A, hereinafter referring to 9cDHROL as vitamin A5 and 9CDHBC as provitamin A5. Proof of concept for the preventive / pharmaceutical use of these compounds is shown in the treatment of depressive-like behavior in a chronic stress animal model of depression. Similar uses of these compounds can be envisioned for various diseases in which RXR-mediated signaling is affected or serves as a therapeutic target. These diseases may include neurodegenerative and metabolic diseases, skin and immune dysfunction (including inflammation), and cardiovascular disease. Lifestyle applications such as memory enhancement effects are also contemplated.

[0515] References

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

Claims

1. Use of 9-cis-β-carotene 9CBC in the preparation of a functional food, the functional food containing an added amount of 9-cis-β-carotene 9CBC, the functional food being used for preventing vitamin A5 deficiency for non-therapeutic purposes and for general vitamin A5 supplementation to prevent retinoid X receptor (RXR)-dependent dysfunction, the 9-cis-β-carotene 9CBC being used for administration to a mammalian subject at an effective dose.

2. Use of 9-cis-β-carotene 9CBC as a food supplement for increasing the level of 9-cis-13,14-dihydroretinoic acid 9CDHRA in mammals with non-therapeutic administration, said use being used for preventing vitamin A5 deficiency for non-therapeutic purposes and for general vitamin A5 supplementation to prevent retinoid X receptor RXR-dependent dysfunction, said 9-cis-β-carotene 9CBC being administered to mammalian subjects at an effective dose as part of a daily administered diet.

3. The use according to claim 2, wherein the mammal is a human.

4. The use according to claim 2, wherein the 9-cis-β-carotene 9CBC is used as a food supplement to increase the level of 9-cis-13,14-dihydro-β-carotene 9CDHBC and thereby increase the level of the 9-cis-13,14-dihydroretinoic acid 9CDHRA.

5. The use according to claim 2 or 3, wherein The 9-cis-β-carotene 9CBC is present in the daily administered diet at a daily dose of 1.0 mg to 30 mg.

6. The use according to claim 2 or 3, wherein The 9-cis-β-carotene 9CBC is present in the daily administered diet at a daily dosage of 0.1 mg to 100 mg.

7. Use of 9-cis-β-carotene 9CBC as a precursor of a retinoid X receptor RXR ligand for the preparation of a nutritional composition for preventing vitamin A5 deficiency for non-therapeutic purposes, wherein the retinoid X receptor RXR ligand acts as a precursor of provitamin A5.

8. Use of 9-cis-β-carotene 9CBC as a food ingredient in the preparation of a nutritional composition for the prevention of vitamin A5 deficiency for non-therapeutic purposes and for general vitamin A5 supplementation.

9. Use of 9-cis-β-carotene 9CBC as a food ingredient as part of a diet in the preparation of a composition for non-therapeutic purposes for preventing vitamin A5 deficiency and general vitamin A5 supplementation.

Images