Compounds for sight restoration

Abstract

The present invention relates to the discovery of compounds of formula (I), possessing activity as modulators of metabotropic glutamate 6 receptor (mGlu6) whose modulatory activity on the receptor may be controlled by irradiation with suitable light resulting in the optical control of receptor biological function, to pharmaceutical compositions comprising said compounds of formula (I), and to the use of said compounds and pharmaceutical compositions as a medicament, in particular for the treatment and / or prevention of retinitis pigmentosa, and / or for the enhancement of visual performance in healthy subjects.

Description

[0001] COMPOUNDS FOR SIGHT RESTORATION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to compounds which are photoswitchable modulators of the mGlu6 receptor, pharmaceutical compositions comprising said compounds, and to the use of said compounds and pharmaceutical compositions as a medicament, in particular for the treatment and / or prevention of retinitis pigmentosa, and / or for the enhancement of visual performance in healthy subjects.

[0004] BACKGROUND OF THE INVENTION

[0005] Blindness greatly affects human life and personal destinies. In adults, visual impairment causes loss of personal independence and the ability to work in many cases, often leading to the need for disability pensions, social services, and nursing. Around 3 million Europeans suffer from low vision and almost 700,000 have been legally recognized as blind. There are several inherited and acquired forms of blindness, including retinal degenerative diseases such as retinitis pigmentosa, in which photoreceptor cells are progressively lost, leading to visual impairment with very limited treatment options. More common degenerative diseases are age-related macular degeneration (AMD), Leber’s congenital amaurosis, Usher syndrome, X-linked juvenile retinoschisis, and Stargardt disease.

[0006] The retina is a complex tissue made up of different types of cells that receive and process visual information. Starting from the back of the eye, the retina is composed of layers of neurons and support cells that acquire, process, and transmit visual information through the retina and onward to the brain. The retina is separated from the vascular tissue in the back of the eye by the retinal pigment epithelium, a layer of pigmented cells that nourish retinal neurons and provide them with the ability to sense light by replenishing their supply of the chromophore retinaldehyde. In the neural retina, two types of photoreceptor cells, the rods and cones, interdigitate with the retinal pigment epithelium. Three different chromatic types of cones respond to bright light of different wavelengths and mediate high-resolution photopic color vision under daylight conditions (luminance level 101— 106cd / m2). Rods respond to dim light and mediate low-resolution scotopic vision under low-light conditions (luminance level 10’6-10’3cd / m2). After photons are absorbed by the retinaldehyde-conjugated opsin in rods and cones, an intracellular signaling cascade leads to a change in the release of the neurotransmitter glutamate from photoreceptor terminals onto cells in the next layer of the retina-bipolar cells. Horizontal cells with processes that project laterally across many photoreceptors help distribute signals and regulate the release of neurotransmitters from photoreceptors to bipolar cells. Bipolar cells respond to the glutamate released from the photoreceptors either by depolarizing their membrane potential (ON bipolar cells) or by hyperpolarizing their membrane potential (OFF bipolar cells). This establishes two parallel pathways for the transmission of visual information. Bipolar cells then transmit visual information by releasing glutamate onto retinal ganglion cells (RGCs), with ON-center RGCs transiently increasing their firing of action potentials when the light is turned on and OFF-center RGCs transiently increasing their firing of action potentials when the light is turned off. Amacrine cells are inhibitory interneurons projecting across many bipolar cells and RGCs. They help shape and process the visual response by releasing GABA and glycine neurotransmitters. Finally, a very small subset of retinal ganglion cells are intrinsically photosensitive (ipRGCs) due to the presence of melanopsin and can also directly sense light. These ipRGCs are important for establishing and entraining the intrinsic circadian clock to light, through the hypothalamus and thalamus, respectively.

[0007] Treatment options for patients with retinal blinding disorders are very limited.

[0008] Some approaches include stem cells and gene therapies. However, protein overexpression can be persistent, immunogenic and oncogenic. Surgically implanted electronic prostheses are the only clinically approved method for vision restoration but some devices (e.g. Second Sight) have been discontinued (https: / / spectrum.ieee.org / bionic-eye-obsolete).

[0009] Recently, the use of light-regulated ligands of ion channels and receptors (Tochitsky and Kramer, Curr Opin Neurobiol., 2015, 34, 74-78; Izquierdo-Serra et al., Nature Communications, 2016, 7, 12221), and optogenetics (Fine et al., Vision Research, 2015, 111 , 115-123; van Wyk et al., PLOS Biology, 2015, 13(5), e1002143; Soto-Sanchez et al., Nanomedicine, 2015, 11(4), 835-843) to photosensitize degenerated retinal tissue and to restore vision in animal models of blindness and in the first patient in a clinical trial (https: / / www.nature.com / articles / s41591-021-01351-4) have been reported. The results require undergoing gene therapy and wearing light-emitting googles, achieving very limited light sensitivity [Samantha R. De Silva and Anthony T. Moore, J. Physiol., 2022, doi: 10.1113 / JP282076],

[0010] In retinal degeneration, light-regulated drugs do not regenerate cells, but they do support a functional recovery of vision that can be rapid, stable and based in small molecules, i.e. amenable to standard drug development procedures (Tochitsky and Kramer, Curr Opin Neurobiol., 2015, 34, 74-78; Drivas and Bennett, Neuron, 2012, 75, 185-187). Thus, these compounds can be regarded as nanoprostheses (Izquierdo-Serra et al., Nature Communications, 2016, 7, 12221) to remotely drive the endogenous receptors that remain in the cells to be treated. The therapeutic opportunities of photoregulating endogenous proteins with photoswitchable compounds do not require microbial protein overexpression or gene therapy and are only subject to conventional drug assessment tests in vitro and in a variety of animal models.

[0011] Chemical photoswitches have been suggested as useful approaches to restore sight (Tochitsky, Chem Rev., 2018, 118(21), 10748-10773). These compounds can repeatedly and reversibly interconvert between photoisomers when irradiated, but there are significant differences in their chemical properties. The photoswitch should absorb visible light (-400-700 nm) since ultraviolet (UV) light is less relevant and even problematic for vision restoration, because UV light is normally filtered out by the human lens and can damage the retina. Another important consideration is the stability of the light-activated isomer, which thermally back-isomerises with a lifetime that depends on the compound structure and electronic properties. If relaxation lifetime is slow, fast changes in light (e.g. between dark and bright features in a scene) cannot be followed. In addition, if too much of the light-activated isomer builds up with repeated light stimulation, light sensitivity will decrease due to saturation or desensitization. Conversely, if the light-activated isomer relaxes too rapidly and acts too transiently on retinal cells, light detection will require extremely bright light in order to maintain a sufficient amount of the light-activated isomer (photostationary state).

[0012] Optogluram (also termed Hit-1 in this text) has been described as a photoswitchable positive allosteric modulator (PAM) of mGlu4 receptor, which was selective for mGlu4 and mGlu6 (Zussy et al., Molecular Psychiatry, 2018, 23(3), 509-520), for use in the treatment of pain. However, the light-activation of this compound occurs at UV-light. Back-isomerisation takes several minutes in the dark and faster interconversion requires two wavelengths of illumination.

[0013] There remains a need for new treatment options to restore sight which overcome the drawbacks of the prior art.

[0014] SUMMARY OF THE INVENTION

[0015] The inventors have surprisingly found a group of light-regulated drugs that activate and deactivate mGlu6 receptors, which are exclusively expressed in retinal bipolar cells and achieve functional restoration of the physiological signaling process that occurs with light in the retina. These compounds exhibit a very potent activity on mGlu6 (nanomolar range versus micromolar for Optogluram). These compounds also act as agonists and positive allosteric modulators (ago-PAM) on the mGlu6 receptor in the nanomolar range of concentrations and thus do not require co-application of an agonist. One of the main advantages of these compounds is that their regulation is mediated by visible light, rather than UV light, as occurs with Optogluram. Another advantage of the compounds of the invention is that, once activated, they return to the relaxed state in seconds, rather than minutes, which are required in the case of Optogluram.

[0016] Thus, in the first aspect, the present invention relates to a compound of formula (I): wherein

[0017] R1is selected from the group consisting of H, Ci-Ce alkoxy, halogen, and CF3 ;

[0018] R2is selected from the group consisting of H, and Ci-Ce alkyl;

[0019] R3is selected from the group consisting of H, halogen, Ci-Ce alkoxy and CF3;

[0020] R4is selected form the group consisting of H, halogen, Ci-Ce alkoxy and CF3;

[0021] R5is selected from the group consisting of halogen, Ci-Ce alkyl and CF3; m is an integer from 0 to 2;

[0022] R6is a heterocyclyl; and G is a nitrogen containing group selected from the group consisting of N, N+-(Ci-Ce alkyl), C-NO2, C-N(CI-C6alkyl)2, and C-NH(CI-C6alkyl); or a pharmaceutically acceptable salt or stereoisomer thereof.

[0023] The second aspect of the invention relates to a pharmaceutical composition comprising a compound according to the first aspect, and a pharmaceutically acceptable excipient.

[0024] In the third aspect, the invention relates to a compound of formula (I) according to the first aspect or a pharmaceutical composition according to the second aspect, for use in medicine.

[0025] The fourth aspect of the invention relates to a compound of formula (I) according to the first aspect or a pharmaceutical composition according to the second aspect, for use in the treatment and / or prevention of a retinal disease.

[0026] The fifth aspect of the invention relates to the use of a compound of formula (I) according to the first aspect or a pharmaceutical composition according to the second aspect, in the enhancement of retinal function and / or visual performance in healthy subjects.

[0027] DESCRIPTION OF THE FIGURES

[0028] Figure 1 shows the chemical / photochromic properties of the compounds of formula (I) of the examples and Optogluram / hit-1 : mass, aqueous solubility, UV-vis absorption spectra, and thermal relaxation.

[0029] Figure 2 shows the activity screening assay of example compounds in HEK cells overexpressing wild-type mouse mGlu6 receptor.

[0030] Figure 3 shows the activity assay of example compounds in HEK cells overexpressing mutant T148A mGlu6 receptor.

[0031] Figure 4 shows dose-response relationship of example compounds of the invention in wild type (wt) mouse mGlu6. Figure 5(A) shows the reversible photoswitching of mGlu6 receptor activity without glutamate in vitro with the example compounds. (B): Histogram showing the differences in photoresponses (F / F0)trans- (F / F0)c / s, express in %.

[0032] Figure 6 shows the ago-PAM responses of example compounds in mutant T148A mGlu6 receptor.

[0033] Figure 7 shows potency values of Ago-PAM responses of example compounds in mutant T148A mGlu6 receptor. Results are consistently superior to Hit-1 (Optogluram).

[0034] Figures 8-10 show the results of behavioral assays in Opn4' / _mice with intravitreal unilateral administration of hit-1 (optogluram) (Figure 8), intravitreal unilateral administration of compound 1492 (Figure 9), and topical bilateral administration of compound 1492 (Figure 10).

[0035] Figures 11-12 show the results of behavioral assays in Opn4' / _mice with topical bilateral administration of compound 1492 (Figure 11) and topical bilateral administration of compound 1495 (Figure 12).

[0036] Figure 13 shows the visual acuity, quantified as saccades / min, before and after addition of a droplet of compound 1492 in healthy non blinded animals, white bar: prior to addition of 1492 compound; black bar: after adding 1492 compound.

[0037] Figure 14 shows the visual acuity, quantified as saccades / min, before and after addition of a droplet of compound 1496, 1497 or 1498 in blinded zebrafish larvae, white box: prior to addition of 1496, 1497 or 1498 compound; grey box: after adding 1496, 1497 or 1498 compound.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] In the first aspect, the present invention relates to compounds of formula (I): wherein

[0040] R1is selected from the group consisting of H, Ci-Ce alkoxy, halogen and CF3;

[0041] R2is selected from the group consisting of H, and Ci-Ce alkyl;

[0042] R3is selected from the group consisting of H, halogen, Ci-Ce alkoxy and CF3;

[0043] R4is selected form the group consisting of H, halogen, Ci-Ce alkoxy and CF3;

[0044] R5is selected from the group consisting of halogen, Ci-Ce alkyl and CF3; m is an integer from 0 to 2;

[0045] R6is a heterocyclyl; and

[0046] G is a nitrogen containing group selected from the group consisting of N, N+-(Ci-Ce alkyl), C-NO2, C-N(CI-C6alkyl)2, and C-NH(CI-C6alkyl); or a pharmaceutically acceptable salt or stereoisomer thereof.

[0047] The term “alkyl” as employed herein alone or as part of another group designates both straight- and branched-chain saturated hydrocarbons containing the number of carbon atoms indicated along the invention and attached to the rest of the molecules through a single bond. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, isobutyl, tert-butyl, sec-butyl, n-butyl, n-pentyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl, 3- pentyl, sec-isopentyl, active pentyl, and n-hexyl.

[0048] The term “alkoxy” as employed herein alone or as part of another group refers to a radical of the formula -O-alkyl, where alkyl has been previously defined, e. g., methoxy, ethoxy, n-propoxy, isopropoxy, isobutoxy, tert-butoxy, sec-butoxy, n-butoxy, n-pentoxy, tert- pentoxy, neopentoxy, isopentoxy, sec-pentoxy, 3-pentoxy, sec-isopentoxy, active pentoxy and n-hexoxy.

[0049] The term “heterocyclyl” as employed herein alone or as part of another group refers to a stable radical of 4 to 8 members that consists of carbon and hydrogen atoms and from one to three heteroatoms selected from the group consisting in N, O and S, preferably a ring of 5 or 6 members with one or more heteroatoms, more preferably a ring of 5 or 6 members with one or more nitrogen atoms, still more preferably with one or two nitrogen atoms. For this invention, the heterocycle can be a system of monocyclic or bicyclic rings, which can include condensed ring systems, and the heterocyclyl radical can be saturated, partially or fully unsaturated or be aromatic and it is linked to the compound by a ring carbon atom. Preferably the heterocycle is aromatic, more preferably a 5- or 6- memberred heteroaromatic ring, even more preferably a 5- or 6-membered heteroaromatic ring having one or two nitrogen atoms, still more preferably a 5- or 6- membered heteroaromatic ring wherein the heteroatom is one N atom. Examples of heterocycles may be, not limited to pyridyl, pyrazyl, thiazolyl, benzimidazolyl, benzothiazolyl, furanyl, isothiazolyl, indolyl, piperidinyl, piperazinyl, thiadiazolyl, tetrahydrofuranyl, coumarine, morpholinyl, pyrrolyl, pyrazolyl, imidazolyle, pyrrolidinyl, tetrahydrofuranyl, etc. The heterocyclyl groups may also bear substituents, preferably those selected from the group consisting of halogens, cyano, nitro, amino, hydroxyl, Cis-alkyl, Ci-3-alkoxy, NH(Ci-3-alkyl) and N(Ci-3-alkyl)2, preferably halogen, more preferably from Cl.

[0050] The term “halogen” as used herein alone or as part of another group refers to chlorine, bromine, fluorine, and iodine.

[0051] As used herein, the term “pharmaceutically acceptable salt” embraces salts with a pharmaceutically acceptable acid or base, which are synthesized from the parent compound which contains an acidic moiety by addition of a pharmaceutically acceptable base, or which are synthesized from the parent compound which contains a basic moiety by addition of a pharmaceutically acceptable acid. Pharmaceutically acceptable acids include both inorganic acids, for example, hydrochloric (HCI), sulfuric (H2SO4), phosphoric (H3PO4), diphosphoric (H4P2O7), hydrobromic (HBr), hydroiodic (HI), and nitric acid (HNO3), and organic acids, for example, citric, fumaric, maleic, malic, mandelic, ascorbic, oxalic, succinic, tartaric, benzoic, acetic (AcOH), methanesulfonic, ethanesulfonic, benzenesulfonic, or p-toluenesulfonic acid. Pharmaceutically acceptable bases include alkali metal (e.g., sodium or potassium) and alkali earth metal (e.g., calcium or magnesium) hydroxides and organic bases, such as alkyl amines, arylalkyl amines, and heterocyclic amines.

[0052] All stereoisomers of the compounds of this invention are contemplated either alone or as mixtures thereof. Stereoisomers refer to compounds having stereogenic centres, e.g. enantiomers, diastereomers, meso and racemic forms, and to compounds having different substituents on the atoms linked to form a multiple bond, such as a double or triple bond (e.g. -HC=CH-, -C=C-, -N=N-, etc.), i.e. cis (Z) and trans (E) isomers. When diastereomeric or enantiomeric products are prepared, they can be separated by conventional methods, for example, chromatographic or functional crystallization. When cis and trans isomers of the compounds of formula (I) according to the present invention are prepared they can be reversibly interconverted from one isomer to the other isomer by irradiation at the appropriate wavelength, i.e., to obtain the trans isomer from the cis isomer, a wavelength between 420 and 900 nm may be used, preferably from 420 to 700 nm, and to obtain the cis isomer from the trans isomer irradiation at a wavelength between 300 and 760 nm may be used, preferably from 300 to 600 nm.

[0053] Preferably, R1is selected from the group consisting of H, Ci-Ce alkoxy and halogen; more preferably, R1is selected from the group consisting of H, C1-C3 alkyl and halogen; more preferably, R1is selected from the group consisting of H, methoxy and halogen; more preferably, R1is selected form the group consisting of H, methoxy and Cl; even more preferably R1is H.

[0054] In one embodiment, R1is H. In another embodiment, R1is Ci-Ce alkoxy, preferably, R1is C1-C3 alkoxy, more preferably methoxy. In another embodiment, R1is halogen, preferably Cl.

[0055] In one embodiment, R2is H. In another embodiment, R2is Ci-Ce alkyl, preferably R2is C1-C3 alkyl. In another embodiment, R2is selected from the group consisting of H and methyl. Preferably, R2is methyl.

[0056] Preferably, R3is selected from the group consisting of H and halogen; more preferably R3is selected from the group consisting of H and Cl. In one embodiment, R3is H. In another embodiment, R3is halogen, preferably Cl. More preferably, R3is H.

[0057] Preferably, R4is selected from the group consisting of H and halogen; more preferably R4is selected from the group consisting of H and Cl. In one embodiment, R4is H. In another embodiment, R4is halogen, preferably Cl. More preferably, R4is Cl.

[0058] In one preferred embodiment, m is 0, i.e. R5is absent. In another embodiment, m is 1 or 2, preferably 1.

[0059] Preferably, R5is selected from the group consisting of halogen, Ci-Ce alkyl and CF3; more preferably R5is selected from the group consisting of halogen, C1-C3 alkyl and CF3; more preferably selected from the group consisting of halogen and C1-C3 alkyl.

[0060] In one embodiment, m is 1 and R5is selected from the group consisting of halogen, Ci- Ce alkyl and CF3, preferably R5is selected from the group consisting of halogen and C1- C3 alkyl, more preferably R5is selected from the group consisting of halogen and C1-C3 alkyl. In another embodiment, m is 1 and R5is halogen, preferably Cl. In another embodiment, m is 1 and R5is Ci-Ce alkyl, preferably C1-C3 alkyl, more preferably methyl. In another embodiment, m is 2 and each R5is independently selected from the group consisting of halogen, Ci-Ce alkyl and CF3, preferably R5is independently selected from the group consisting of halogen, C1-C3 alkyl and CF3, more preferably R5is independently selected from the group consisting of halogen and C1-C3 alkyl. In another embodiment, m is 2 and each R5is independently selected from the group consisting of Cl and methyl.

[0061] In one embodiment R6is a 5- or 6-membered heterocyclyl containing at least one N atom, preferably one or two N atoms, still more preferably one N atom. In a preferred embodiment, R6is a 5- or 6-membered aromatic heterocyclyl containing at least one N atom, preferably one or two N atoms, still more preferably one N atom. In a more preferred embodiment, R6is selected from the group consisting of a pyridyl, pyrazyl and imidazolyl optionally substituted with one, two or three halogen atoms, preferably one, two or three Cl atoms. In a more preferred embodiment, R6is selected from the group consisting of a pyridyl, pyrazyl and imidazolyl optionally substituted with one halogen atoms, preferably one Cl atom. In a more preferred embodiment, R6is selected from the group consisting of a pyridyl, pyridyl substituted with one Cl atom, pyrazyl and imidazolyl. In a more preferred embodiment, R6is a pyridyl. In another embodiment, R6is selected from the group consisting of 2-pyridyl, 3-pyridyl and 4-pyridyl. Preferably, R6is 2-pyridyl.

[0062] In one embodiment, G is a nitrogen containing group selected from the group consisting of N, N+-(CI-C3alkyl), C-NO2, C-N(CI-C3alkyl)2, and C-NH(CI-C3alkyl); preferably G is a nitrogen containing group selected from the group consisting of N, N+-methyl, C- N(methyl)2, and C-NO2. In one embodiment, G is N+-(Ci-Ce alkyl), preferably N+-(Ci-Cs alkyl), more preferably N+-methyl. In another embodiment, G is C-N(Ci-Ce alkyl)2, preferably C-N(CI-C3 alkyl)2, more preferably C-N(methyl)2. In another embodiment, G is C-NO2. In a preferred embodiment, G is N.

[0063] When G is N+-(Ci-Ce alkyl), preferably N+-(CI-C3 alkyl), more preferably N+-methyl, the compound of the invention is in the form of a pharmaceutically acceptable salt with any suitable counterion, such as a halogen, preferably I'.

[0064] In one embodiment, in the compound of formula (I),

[0065] R1is selected from the group consisting of H, C1-C3 alkoxy, halogen and CF3;

[0066] R2is selected from the group consisting of H, and C1-C3 alkyl;

[0067] R3is selected from the group consisting of H, halogen, C1-C3 alkoxy and CF3;

[0068] R4is selected form the group consisting of H, halogen, C1-C3 alkoxy and CF3;

[0069] R5is selected from the group consisting of halogen, C1-C3 alkyl and CF3; m is an integer from 0 to 2;

[0070] R6is a heterocyclyl; and

[0071] G is a nitrogen containing group selected from the group consisting of N, N+-(CI-C3 alkyl), C-NO2, C-N(CI-C3alkyl)2, and C-NH(CI-C3alkyl).

[0072] In one embodiment, in the compound of formula (I),

[0073] R1is selected from the group consisting of H, C1-C3 alkoxy and halogen;

[0074] R2is selected from the group consisting of H and C1-C3 alkyl;

[0075] R3is selected from the group consisting of H and halogen ;

[0076] R4is selected form the group consisting of H and halogen;

[0077] R5is selected from the group consisting of halogen and C1-C3 alkyl; m is an integer from 0 to 2;

[0078] R6is a heterocyclyl; and

[0079] G is a nitrogen containing group selected from the group consisting of N, N+-(CI-C3 alkyl), C-NO2, C-N(CI-C3alkyl)2, and C-NH(CI-C3alkyl).

[0080] In a particular embodiment, in the compound of formula (I),

[0081] R1is selected from the group consisting of H, C1-C3 alkoxy and halogen;

[0082] R2is C1-C3 alkyl;

[0083] R3is selected from the group consisting of H and halogen;

[0084] R4is selected form the group consisting of H and halogen; R5is selected from the group consisting of halogen and C1-C3 alkyl; m is an integer from 0 to 1 ;

[0085] R6is a pyridyl; and

[0086] G is selected from the group consisting of N, N+-(CI-C3 alkyl), C-NO2, and C-N(CI-C3 alkyl)2.

[0087] In another particular embodiment, in the compound of formula (I),

[0088] R1is selected from the group consisting of H, methoxy and Cl;

[0089] R2is methyl;

[0090] R3is selected from the group consisting of H and Cl;

[0091] R4is selected form the group consisting of H and Cl;

[0092] R5is selected from the group consisting of halogen and methyl; m is an integer from 0 to 1 ;

[0093] R6is a pyridyl; and

[0094] G is selected from the group consisting of N, N+-(methyl), C-NO2, and C-N(methyl)2.

[0095] The compounds of the invention have a double bond between the two aromatic rings (the phenyl ring bearing the R1and OR2substituents and the aromatic ring bearing the R3, R4and optionally R5substituents). Therefore, the compounds of the invention can be in the form of the E-isomer (trans) or in the form of the Z-isomer (cis). In a preferred embodiment, the compounds of the invention are in the form of the E-isomer. In another embodiment, the compounds of the invention are in the form of the Z-isomer.

[0096] In a particular embodiment, the compound of formula (I) according to the invention is selected from the group consisting of:

[0097] (E)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide, (Z)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide, (E)- / V-(3-Methoxy-4-((3-methyl-4-nitrophenyl)diazenyl)phenyl)picolinamide, (Z)- / V-(3-Methoxy-4-((3-methyl-4-nitrophenyl)diazenyl)phenyl)picolinamide, (E)- / V-(4-((4-(Dimethylamino)phenyl)diazenyl)-3-methoxyphenyl)picolinamide, (Z)- / V-(4-((4-(Dimethylamino)phenyl)diazenyl)-3-methoxyphenyl)picolinamide, (E)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3,5-dimethoxyphenyl)picolinamide, (Z)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3,5-dimethoxyphenyl)picolinamide, (E)- / V-(3,5-Dimethoxy-4-((3-methyl-4nitrophenyl)diazenyl)phenyl)picolinamide, (Z)- / V-(3,5-Dimethoxy-4-((3-methyl-4nitrophenyl)diazenyl)phenyl)picolinamide, (E)- / V-(4-((4-(Dimethylamino)phenyl)diazenyl)-3,5-dimethoxyphenyl)picolinamide,

[0098] (Z)- / V-(4-((4-(Dimethylamino)phenyl)diazenyl)-3,5-dimethoxyphenyl)picolinamide,

[0099] (E)- / V-(4-((3-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide,

[0100] (Z)- / V-(4-((3-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide,

[0101] (E)- / V-(3-Chloro-4-((3,5-dichloropyridin-4-yl)diazenyl)-5-methoxyphenyl)picolinamide,

[0102] (Z)- / V-(3-Chloro-4-((3,5-dichloropyridin-4-yl)diazenyl)-5-methoxyphenyl)picolinamide,

[0103] (E)- / V-(3-Methoxy-4-(pyridin-4-yldiazenyl)phenyl)picolinamide,

[0104] (Z)- / V-(3-Methoxy-4-(pyridin-4-yldiazenyl)phenyl)picolinamide,

[0105] (E)-4-((2-Methoxy-4-(picolinamido)phenyl)diazenyl)-1-methylpyridin-1-ium iodide,

[0106] (Z)-4-((2-Methoxy-4-(picolinamido)phenyl)diazenyl)-1-methylpyridin-1-ium iodide,

[0107] (E)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-1 / 7-imidazole-2- carboxamide,

[0108] (Z)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-1 / 7-imidazole-2- carboxamide,

[0109] (E)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)pyrazine-2-carboxamide,

[0110] (Z)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)pyrazine-2-carboxamide, (E)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-5-fluoropicolinamide, and (Z)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-5-fluoropicolinamide.

[0111] In a more preferred embodiment, the compound of formula (I) according to the invention is selected from the group consisting of:

[0112] (E)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide,

[0113] (E)- / V-(3-Methoxy-4-((3-methyl-4-nitrophenyl)diazenyl)phenyl)picolinamide,

[0114] (E)- / V-(4-((4-(Dimethylamino)phenyl)diazenyl)-3-methoxyphenyl)picolinamide,

[0115] (E)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3,5-dimethoxyphenyl)picolinamide,

[0116] (E)- / V-(3,5-Dimethoxy-4-((3-methyl-4nitrophenyl)diazenyl)phenyl)picolinamide,

[0117] (E)- / V-(4-((4-(Dimethylamino)phenyl)diazenyl)-3,5-dimethoxyphenyl)picolinamide,

[0118] (E)- / V-(4-((3-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide,

[0119] (E)- / V-(3-Chloro-4-((3,5-dichloropyridin-4-yl)diazenyl)-5-methoxyphenyl)picolinamide,

[0120] (E)- / V-(3-Methoxy-4-(pyridin-4-yldiazenyl)phenyl)picolinamide,

[0121] (E)-4-((2-Methoxy-4-(picolinamido)phenyl)diazenyl)-1-methylpyridin-1-ium iodide,

[0122] (E)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-1 H-imidazole-2- carboxamide, (E)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)pyrazine-2-carboxamide, and

[0123] (E)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-5-fluoropicolinamide.

[0124] Compounds of formula I may be synthesized by using some of the methods described below, as well as other processes known in the field of the organic chemistry. Preferred methods include, but are not limited to, the general procedures shown in the Schemes 1 and 2.

[0125] In general, compounds formula (I) may be obtained by amidation of intermediates of formula II using compound 3 (scheme 1) in the presence of coupling reagents such as HATLI or EDC, among others, together with an organic base (DIPEA, TEA, etc.). At the same time, intermediates of formula II can be obtained by diazotization with NaNCh in acid media (HCI, H2SO4, HNO3, H3PO4, etc.) and reaction in situ with aminoanisole 2 in basic media (NaOH, AcONa, Na2COs, NaHCCh, etc.).

[0126] Scheme 1

[0127] In addition to Scheme 1 , when G contains a group N+-(Ci-Ce alkyl), compounds of formula (I) can be obtained by following the route described in Scheme 2. In this way, Compound of formula I can be obtained via an alkylation of appropriate pyridine by using as alkylating agent of formula 4. The "leaving group" or LG, can be, without limitation, chloride, bromide, Nosyl (Ns), Tosyl (Ts), Mesyl (Ms) and other suitable groups in each case.

[0128] Scheme 2

[0129] Any starting material (compounds 1 to 4) may be commercially available or can be obtained following reactions well known in the field of organic chemistry. Preferred methods include, but are not limited to, the procedures described in the experimental section.

[0130] Pharmaceutical compositions comprising the compound of formula (I)

[0131] In a second aspect, the invention is directed to a pharmaceutical composition comprising a compound of formula (I) as defined above, and a pharmaceutically acceptable excipient.

[0132] The term "pharmaceutically acceptable excipient" refers to a vehicle, diluent, or adjuvant that is administered with the active ingredient. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and similar, surfactant, bile acid, chelating agents, preservatives, cyclodextrins, among others. Water or saline aqueous solutions and aqueous dextrose and glycerol solutions, particularly for injectable solutions, are preferably used as vehicles. Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 21st Edition, 2005. Other suitable excipients are cyclodextrins and polyethylene glycols (e.g. liquid PEGs such as PEG400). Surfactants, bile acids, chelating agents, preservatives, and cyclodextrins increase chemical stability and bioavailability and decrease local irritation [Brian G. Short, Toxicologic Pathology, 2008, 36(1), 49-62],

[0133] Compounds formula (I) according to the present invention may be administered by the topical, intraocular (such as intravitreal injections or subretinal injections or by means of an ocular drug delivery device -such as (non)biodegradable reservoirs and / or matrix, orocular implants like subconjunctival or intravitreal implants-, or drug delivery systems - e.g., nanoparticles and microparticles- ), periocular injections (e.g., subconjunctival, retrobulbar, peribulbar, and posterior sub-Tenon injections), iontophoresis,), oral, sublingual, parenteral, subcutaneous, intramuscular, intravenous, transdermal, intranasal, and / or rectal routes; preferably topical administration, more preferably topical administration to the eye. The compounds may be administered alone or in combination with one or more other compounds of the invention or one or more other drugs. In general, the compounds should be administered as a formulation in association with one or more pharmaceutically acceptable excipients. Any administration method commonly used for drugs, such as solutions (including eye drops), cream, ointment, powder, tablets, coated tablets, capsules, syrup, powder, and suppository, may be used. The pharmaceutical composition can be formulated employing conventional liquid or solid vehicles or diluents and pharmaceutical additives, according to the desired mode of administration. The mentioned formulations will be prepared using standard methods, such as those described or referred to in the Spanish, E.U., and U.S. Pharmacopoeias and similar reference texts.

[0134] The compounds of formula (I) may be administered in a therapeutically effective amount. “Effective” amount or a “therapeutically effective amount” of a compound is meant a nontoxic but sufficient amount of the drug or agent to provide the desired effect. The amount that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, the particular active agent or agents, and the like. Thus, it is not always possible to specify an exact “effective amount”. However, an appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. The compounds will typically be administered once or more times a day in the case of topical route, for example 1 , 2, 3 or 4 times daily, with typical concentrations around 30 pM and enough volume to cover most of the cornea (in mice, 5-10 pl; in humans, 100-500 pl). In the case of intraocular route, typical injected concentrations are around 1-30 pM and a volume that takes into consideration the relevant regulations (in mice, 1 pl; in humans, 50-100 pl) every 3 days or more.

[0135] Medical uses of the compound of formula (I) The mGlu6 receptor is considered a potential drug target for treatment of retinal diseases. As shown in the examples, the present compound are potent agonists and allosteric modulators of the mGlu6 receptor, in particular, the compounds show a photoswitching activity on said receptor that mimics the physiological photoresponses in non-degenerated retina. As also shown in the examples the compounds are active in an in vivo model of retinal degeneration.

[0136] Thus, the compounds of formula (I) according to the present invention may be used for treating or preventing a condition in a mammal, including a human, the treatment or prevention of which is affected or facilitated by the modulatory effect of mGlu6 ligands controlled by light.

[0137] Therefore, in a third aspect, the present invention provides a compound of formula (I) as defined above, or a pharmaceutical composition comprising a compound of formula (I) as defined above, for use as a medicament.

[0138] The invention also relates to the use of a compound of formula (I) as defined above, or a pharmaceutical composition comprising a compound of formula (I) as defined above, for the manufacture of a medicament.

[0139] In a fourth aspect, the present invention provides a compound of formula (I) as defined above, ora pharmaceutical composition comprising a compound of formula (I) as defined above, for use in the treatment and / or prevention of a retinal disease.

[0140] The present invention also relates to the use of a compound of formula (I) as defined above, ora pharmaceutical composition comprising a compound of formula (I) as defined above, for manufacturing a medicament for the treatment and / or prevention of a retinal disease.

[0141] The invention also relates to a method of treatment and / or prevention of a subject in need thereof of a retinal disease, which comprises the administration of a compound of formula (I) as defined above, or a pharmaceutical composition comprising a compound of formula (I) as defined above. The terms “treat”, “treatment”, or “treatment of” as used herein refer to reducing the potential for a certain disease or disorder, reducing the occurrence of a certain disease or disorder, and / or a reduction in the severity of a certain disease or disorder, preferably, to an extent that the subject no longer suffers discomfort and / or altered function due to it. It also refers to mitigating or decreasing at least one clinical symptom and / or inhibition or delay in the progression of the condition and / or prevention or delay of the onset of a disease or illness.

[0142] The terms “prevention”, “preventing” or “prevent” as used herein refer to avoiding the appearance of a certain disease or disorder. The prevention can be complete (e.g. the total absence of a disease). The prevention can also be partial, such that for example the occurrence of a disease in a subject is less than that which would have occurred without the administration of the combination or composition of the present invention. Prevention also refers to reduced susceptibility to a clinical condition. The prevention also includes reducing the risk of suffering the disease.

[0143] Preferably, the retinal disease is a retinal degenerative disease, such as retinitis pigmentosa, age-related macular degeneration, Leber’s congenital amaurosis, Usher syndrome, X-linked juvenile retinoschisis, and Stargardt disease. More preferably, the reginal degenerative disease is retinitis pigmentosa.

[0144] The subject to which the compounds and pharmaceutical compositions described herein are administered is a human or animal; preferably subjects are mammals, more preferably humans.

[0145] The invention also relates to the use of the compounds of formula (I) as defined in the first aspect for the enhancement of retinal function and / or visual performance in a healthy subject.

[0146] The term “healthy subject” refers to a subject which does not suffer or is not in risk of suffering any of the diseases or disorders which may be treated and / or prevented with the compounds of the invention, in diseases or conditions affected or facilitated by the modulatory effect of mGlu6 ligands controlled by light, such as retinal diseases, in particular retinitis pigmentosa, age-related macular degeneration, Leber’s congenital amaurosis, Usher syndrome, X-linked juvenile retinoschisis, and Stargardt disease. The healthy subject to which the compounds and pharmaceutical compositions described herein are administered is a human or animal; preferably subjects are mammals, more preferably humans.

[0147] The enhancement in the retinal function and / or visual performance is to be understood as having a retinal function and / or visual performance which is greater after the administration of the compounds of the invention when compared to the retinal functions and visual performance before said administration.

[0148] In particular, the retinal function (physiological transduction of light reflected or emitted by objects into neural activity in the optic nerve) and / or visual performance (ability to perform visually-guided tasks) refers to photosensitivity (minimum light fluence of any wavelength that can be detected by the retina), visual acuity (size and speed of features that can be detected in a visual scene), spectral sensitivity (wavelength range that produces retinal responses).

[0149] The following examples represent specific embodiments of the present invention. They do not intend to limit in any way the scope of the invention defined in the present description.

[0150] EXAMPLES

[0151] Abbreviations

[0152] ACN, Acetonitrile; AcOEt, Ethyl acetate; DCM, Dichloromethane; DMF, Dimethylformamide; ESI, Electrospray Ionization; FIA, Flux Injected Analysis; HATU, (1- [Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HPLC, High Performance Liquid Chromatography; MeOH, Methanol; MS, Mass Spectrometry; m / z, mass / charge ratio; ND, Not determined; RT, retention time.

[0153] Analytical and spectroscopic information

[0154] NMR spectra were recorded on a Bruker-400 (1 H at 400.10 MHz). The following abbreviations were used to designate multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, p=quintuplet, br=broad, dd=double-doublet, ddd=double-double- doublet, dt=double-triplet, td=triplet-doublet. In 1 H-NMR chemical shifts were expressed in ppm relative to TMS and coupling constant (J) in Hz.

[0155] High-performance Liquid Chromatography (HPLC) 2795 Alliance Waters Aquity coupled to Detector DAD Agilent 1100 and Detector MS Waters ESI triple cuadrupolo Quattro micro, 10 pL of sample in MeOH was injected. Mass spectroscopy (MS) analyzed by FIA (flux injected analysis) with coupled to LCT Premier Orthogonal Accelerated Time of Flight Mass Spectrometer, acquiring data by electrospray ionization (ESI) in positive mode. Spectra have been scanned between 50 and 1500 Da with values every 0.2 seconds and peaks are given m / z (% of basis peak).

[0156] - Stationary phase: ZORBAX Extend-C18 3.5 pm 2.1x50mm (Ta35°C)

[0157] - Mobile Phase:

[0158] _ Mobile Phase A _ Mobile Phase B _

[0159] 1 water + 0.05% formic acid Acetonitrile + 0.05% formic acid

[0160] - Gradient:

[0161] Synthesis of Intermediates II

[0162] Intermediates II were synthesized following one of the following general diazotization methods:

[0163] Diazotization Procedure A

[0164] To a stirred solution of Amines 1 (1 eq.) (see Scheme 1) in water or methanol (0.5 M) at -10-0°C, HCIconc (3 eq.) was added slowly keeping the temperature between 0-5°C. The solution was stirred at 0-5°C for 5 min. Then, a solution of sodium nitrite (1 eq.) in water (2 M) was added dropwise to generate the azonium intermediate (temp 0-5°C), and the reaction mixture was stirred for 45 min. On the other hand, a solution of Aminoanisoles 2 (1 .2 eq.) (see Scheme 1) and sodium acetate (3 eq.) in water or methanol (0.6 M) was prepared in a separate reaction vessel and stirred in an ice bath. Finally, with stirring, the diazonium solution was added in dropwise while keeping the temperature of the reaction at 0-5°C. It was left 1 h from 0-5°C to room temperature. The mixture was treated with AcOEt / NaHCOssat. / water, the organic layer was dried over anhydrous MgSO4, filtered and concentrated in vacuum. The residue was purified by flash chromatography (silica gel) to afford pure compounds.

[0165] Diazotization Procedure B

[0166] To a stirred solution of sodium nitrite (1 eq.) in water (0.5 M) at 0°C, an ice-cold solution of Amines 1 (1 eq.) (see Scheme 1) in phosphoriCconc / nitricConc acid 2:1 (0.1 M) was added slowly keeping the temperature between 0-5°C. The solution was stirred at 0-5°C for 45 min. Then, it was added dropwise into a solution of Aminoanisoles 2 (1.2 eq.) (see Scheme 1) in water / phosphoric acidC0nc 2:1 (0.1M) keeping temperature of the reaction at 0-5°C. It was left 1h from 0-5°C to room temperature. The mixture was treated with AcOEt / Na2CO3sat. / water, the organic layer was dried over anhydrous MgSCU, filtered and concentrated in vacuum. The residue was purified by flash chromatography (silica gel) to afford pure compounds.

[0167] (E)-4-((2-Chloropyridin-4-yl)diazenyl)-3-methoxyaniline (11-1 )

[0168] According to General Diazotization Procedure A, from 713 mg of 2-chloropyridin-4-amine and 0.75 mL of 3-methoxyaniline using water as solvent, 224 mg of (11-1) were obtained (15% yield).1H NMR (400 MHz, Chloroform-d) 5 8.45 (dd, J = 5.4, 0.7 Hz, 1 H), 7.75 (d, J = 9.4 Hz, 1 H), 7.63 (d, J = 1 .5 Hz, 1 H), 7.56 (dd, J = 5.3, 1 .7 Hz, 1 H), 6.35 - 6.21 (m, 2H), 4.33 (s, 2H), 4.00 (s, 3H). HPLC-PDA-MS: RT: 2.87 min, m / z (ESI+) 263 (MH+, 100%). HRMS (ESI+): found 263.0689, C12H12N4OCI (MH+) requires 263.0700.

[0169] (E)-3-Methoxy-4-((3-methyl-4-nitrophenyl)diazenyl)aniline (II-2)

[0170] According to General Diazotization Procedure A, from 500 mg of 3-methyl-4-nitroaniline and 0.44 mL of 3-methoxyaniline using methanol as solvent, 132 mg of (II-2) were obtained (14% yield).1H NMR (400 MHz, Chloroform-d) 5 8.09 (d, J = 9.4 Hz, 1 H), 7.78 - 7.69 (m, 3H), 6.30 - 6.27 (m, 2H), 4.29 (s, 2H), 3.99 (s, 3H), 2.68 (s, 3H). HPLC-PDA- MS: RT: 3.38 min, m / z (ESI+) 287 (MIT, 100%). HRMS (ESI+): found 287.1142, C14H15N4O3 (MIT) requires 287.1144.

[0171] (E)-4-((4-Amino-2-methoxyphenyl)diazenyl)- / V, / V-dimethylaniline (II-3)

[0172] According to General Diazotization Procedure A, from 490 mg of / V1 , / V1- dimethylbenzene-1 ,4-diamine and 0.49 mL of 3-methoxyaniline using methanol as solvent, 31.2 mg of (II-3) were obtained (3% yield).1H NMR (400 MHz, Chloroform-d) 5 7.79 (d, J = 9.1 Hz, 2H), 7.65 (d, J = 8.6 Hz, 1 H), 6.74 (d, J = 8.9 Hz, 2H), 6.42 - 6.29 (m, 2H), 3.98 (s, 3H), 3.05 (s, 6H). HPLC-PDA-MS: RT: 3.10 min, m / z (ESI+) 271 (MH+, 100%). HRMS (ESI+): found 271.1557, C15H19N4O (MH+) requires 271.1559.

[0173] (E)-4-((2-Chloropyridin-4-yl)diazenyl)-3,5-dimethoxyaniline (II-4)

[0174] According to General Diazotization Procedure A, from 200 mg of 2-chloropyridin-4-amine and 286 mg of 3,5-dimethoxyaniline using water as solvent, 149 mg of (II-4) were obtained (33% yield).1H NMR (400 MHz, DMSO-d6) 5 8.39 (d, J = 5.4 Hz, 1 H), 7.45 (dd, J = 5.3, 1.7 Hz, 1 H), 7.40 (d, J = 1.5 Hz, 1 H), 6.76 (s, 2H), 5.97 (s, 2H), 3.78 (s, 6H). HPLC-PDA-MS: RT: 2.78 min, m / z (ESI+) 293 (MH+, 100%). HRMS (ESI+): found 293.0797, C13H14N4O2CI (MH+) requires 293.0805.

[0175] (E)-3,5-Dimethoxy-4-((3-methyl-4-nitrophenyl)diazenyl)aniline (II-5)

[0176] According to General Diazotization Procedure A, from 210 mg of 3-methyl-4-nitroaniline and 254 mg of 3,5-dimethoxyaniline using water as solvent, 100 mg of (II-5) were obtained (23% yield).1H NMR (400 MHz, DMSO-d6) 5 8.09 (d, J = 8.8 Hz, 1 H), 7.60 - 7.55 (m, 1 H), 7.52 (d, J = 8.7 Hz, 1 H), 6.53 (s, 2H), 5.97 (s, 2H), 3.77 (s, 6H), 2.60 (s, 3H). HPLC-PDA-MS: RT: 3.02 min, m / z (ESI+) 317 (MH+, 100%). HRMS (ESI+): found 317.1250, C15H17N4O4 (MH+) requires 317.1250.

[0177] (E)-4-((4-Amino-2,6-dimethoxyphenyl)diazenyl)- / V, / V-dimethylaniline (II-6)

[0178] According to General Diazotization Procedure A, from 200 mg of / V1 , / V1- dimethylbenzene-1 ,4-diamine and 270 mg of 3,5-dimethoxyaniline, 196 mg of (ll-6) were obtained (44% yield).1H NMR (400 MHz, DMSO-d6) 5 7.53 (d, J = 9.1 Hz, 2H), 6.77 (d, J = 9.1 Hz, 2H), 5.95 (s, 2H), 5.70 (s, 2H), 3.69 (s, 6H), 2.99 (s, 6H). HPLC-PDA-MS: RT: 2.94 min, m / z (ESI+) 301 (MH+, 100%). HRMS (ESI+): found 301.1659, C16H21N4O2 (MH+) requires 301.1665.

[0179] (E)-4-((3-Chloropyridin-4-yl)diazenyl)-3-methoxyaniline (II-7)

[0180] According to General Diazotization Procedure B, from 200 mg of 3-chloropyridin-4-amine and 0.21 mL of 3-methoxyaniline, 222 mg of (II-7) were obtained (54% yield).1H NMR (400 MHz, Chloroform-d) 5 8.68 (s, 1 H), 8.49 (d, J = 5.3 Hz, 1 H), 7.83 (d, J = 8.6 Hz, 1 H), 7.44 (d, J = 5.3 Hz, 1 H), 6.33 - 6.25 (m, 2H), 4.34 (s, 2H), 3.99 (s, 3H). HPLC-PDA- MS: RT: 2.67 min, m / z (ESI+) 263 (MH+, 100%). HRMS (ESI+): found 263.0687, C12H12N4OCI (MH+) requires 263.0700.

[0181] (E)-3-Chloro-4-((3,5-dichloropyridin-4-yl)diazenyl)-5-methoxyaniline (II-8)

[0182] According to General Diazotization Procedure B, from 200 mg of 3,5-dichloropyridin-4- amine and 232 mg of 3-chloro-5-methoxyaniline, 93.0 mg of (II-8) were obtained (23% yield).1H NMR (400 MHz, Chloroform-d) 5 8.53 (s, 2H), 6.51 (d, J = 2.3 Hz, 1 H), 6.18 (d, J = 2.3 Hz, 1 H), 4.32 (s, 2H), 3.88 (s, 3H). HPLC-PDA-MS: RT: 3.59 min, m / z (ESI+) 331 (MH+, 100%). HRMS (ESI+): found 330.9930, C12H10N4OCI3 (MH+) requires 330.9920.

[0183] (E)-3-Methoxy-4-(pyridin-4-yldiazenyl)aniline (II-9)

[0184] According to General Diazotization Procedure B, from 200 mg of pyridin-4-amine and 0.29 mL of 3-methoxyaniline, 100 mg of (II-9) were obtained (21% yield).1H NMR (400 MHz, Chloroform-d) 5 8.68 (d, J = 6.2 Hz, 2H), 7.74 (d, J = 9.4 Hz, 1 H), 7.63 - 7.56 (m, 2H), 6.34 - 6.22 (m, 2H), 4.34 (s, 2H), 3.97 (s, 3H). HPLC-PDA-MS: RT: 2.76 min, m / z (ESI+) 229 (MH+, 100%). HRMS (ESI+): found 229.1083, C12H13N4O (MH+) requires 229.1089.

[0185] Synthesis of Compounds of formula I

[0186] Compound of formula I were synthesized following one of the following general methods: Amidation Procedure C

[0187] To a stirred solution of Intermediate II (1 eq.), carboxylic acid 3 (1 .5 eq.) (see Scheme 1) and HATLI (3 eq.) in DMF (0.05 M) at 40°C, triethylamine (6 eq.) was added. The solution was stirred at 40°C overnight. Then, the mixture was treated with AcOEt / Na2CO3sat. / water, the organic layer was dried over anhydrous MgSCU, filtered and concentrated in vacuum. The residue was purified by flash chromatography (silica gel) to afford pure compounds.

[0188] Alkylation Procedure D

[0189] To a stirred solution of compound I (1 eq.) in anhydrous DCM (0.05 M) at room temperature, alkylating agent (4.5 eq.) (see Scheme 2) was added. The solution was stirred at room temperature overnight. The precipitated solid was filtered and then washed with cold DCM to afford pure compounds.

[0190] (E)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide (1-1, compound 0794)

[0191] According to General Amidation Procedure C, from 27.0 mg of 11-1 and 19.0 mg of 2- picolinic acid, 39.0 mg of (1-1) were obtained (>99% yield).1H NMR (400 MHz, Chloroform-d) 5 10.33 (s, 1H), 8.65 (ddd, J = 4.8, 1.7, 0.9 Hz, 1 H), 8.51 (dd, J = 5.3, 0.7 Hz, 1 H), 8.31 (dt, J = 7.8, 1.1 Hz, 1 H), 8.10 (d, J = 2.1 Hz, 1 H), 7.96 (td, J= 7.7, 1.7 Hz, 1 H), 7.81 (d, J = 8.8 Hz, 1 H), 7.70 (dd, J = 1.7, 0.6 Hz, 1 H), 7.63 (dd, J = 5.3, 1.6 Hz, 1 H), 7.54 (ddd, J = 7.6, 4.8, 1.2 Hz, 1 H), 7.13 (dd, J = 8.9, 2.1 Hz, 1 H), 4.12 (s, 3H). HPLC-PDA-MS: RT: 4.23 min, m / z (ESI+) 368 (MH+, 100%). HRMS (ESI+): found 368.0925, C18H15N5O2CI (MH+) requires 368.0914.

[0192] (E)- / V-(3-Methoxy-4-((3-methyl-4-nitrophenyl)diazenyl)phenyl)picolinamide (I-2, compound 1331)

[0193] According to General Amidation Procedure C, from 30.1 mg of II-2 and 19.4 mg of 2- picolinic acid, 41.0 mg of (I-2) were obtained (>99% yield).1H NMR (400 MHz, Chloroform-d) 5 10.29 (s, 1 H), 8.63 (ddd, J = 4.7, 1.7, 0.9 Hz, 1 H), 8.30 (dt, J = 7.8, 1.1 Hz, 1 H), 8.12 - 8.05 (m, 2H), 7.94 (td, J = 7.7, 1.7 Hz, 1 H), 7.82 - 7.77 (m, 3H), 7.52 (ddd, J = 7.6, 4.8, 1.3 Hz, 1 H), 7.13 (dd, J = 8.8, 2.2 Hz, 1 H), 4.11 (s, 3H), 2.69 (s, 3H). HPLC-PDA-MS: RT: 4.37 min, m / z (ESI+) 392 (MIT, 100%). HRMS (ESI+): found 392.1358, C20H18N5O4 (MT) requires 392.1359.

[0194] (E)- / V-(4-((4-(Dimethylamino)phenyl)diazenyl)-3-methoxyphenyl)picolinamide (I-3, compound 1335)

[0195] According to General Amidation Procedure C, from 15.0 mg of II-3 and 10.3 mg of 2- picolinic acid, 8.8 mg of (I-3) were obtained (42% yield).1H NMR (400 MHz, Chloroformdi 10.21 (s, 1 H), 8.64 (d, J = 4.5 Hz, 1 H), 8.31 (d, J = 7.8 Hz, 1 H), 8.01 (d, J = 2.1 Hz, 1 H), 7.97 - 7.87 (m, 3H), 7.76 (d, J = 8.7 Hz, 1 H), 7.51 (dd, J = 7.8, 4.7 Hz, 1 H), 7.13 (dd, J = 8.7, 2.1 Hz, 1 H), 6.89 (s, 2H), 4.09 (s, 3H), 3.09 (s, 6H). HPLC-PDA-MS: RT:

[0196] 4.31 min, m / z (ESI+) 376 (MH+, 100%). HRMS (ESI+): found 376.1761 , C21H22N5O2 (MH+) requires 376.1774.

[0197] (E)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3,5-dimethoxyphenyl)picolinamide (I-4, compound 1463)

[0198] According to General Amidation Procedure C, from 15.1 mg of II-4 and 9.5 mg of 2- picolinic acid, 8.3 mg of (I-4) were obtained (41 % yield).1H NMR (400 MHz, Chloroform- d) 5 10.28 (s, 1 H), 8.66 (ddd, J = 4.7, 1.7, 0.9 Hz, 1 H), 8.50 (dd, J = 5.3, 0.7 Hz, 1 H),

[0199] 8.31 (dt, J = 7.9, 1.1 Hz, 1 H), 7.97 (td, J = 7.7, 1.7 Hz, 1 H), 7.65 (dd, J = 1.7, 0.6 Hz, 1 H), 7.61 (dd, J = 5.3, 1.7 Hz, 1 H), 7.55 (ddd, J = 7.6, 4.8, 1.2 Hz, 1 H), 7.27 (s, 2H), 3.99 (s, 6H). HPLC-PDA-MS: RT: 3.92 min, m / z (ESI+) 398 (MH+, 100%). HRMS (ESI+): found 398.1002, C19H17N5O3CI (MH+) requires 398.1020.

[0200] (E)- / V-(3,5-Dimethoxy-4-((3-methyl-4nitrophenyl)diazenyl)phenyl)picolinamide (I-5, compound 1464)

[0201] According to General Amidation Procedure C, from 27.1 mg of II-5 and 15.8 mg of 2- picolinic acid, 20.7 mg of (I-5) were obtained (58% yield).1H NMR (400 MHz, Chloroform- d) 5 10.24 (s, 1 H), 8.65 (ddd, J = 4.8, 1.7, 0.9 Hz, 1 H), 8.30 (dt, J = 7.9, 1.1 Hz, 1 H), 8.10 (d, J = 9.3 Hz, 1 H), 7.95 (td, J = 7.7, 1.7 Hz, 1 H), 7.80 - 7.73 (m, 2H), 7.53 (ddd, J = 7.6, 4.8, 1.2 Hz, 1 H), 7.26 (s, 2H), 3.96 (s, 6H), 2.69 (s, 3H). HPLC-PDA-MS: RT: 4.25 min, m / z (ESI+) 422 (MH+, 100%). HRMS (ESI+): found 422.1463, C21 H20N5O5 (MH+) requires 422.1464. (E)-A / -(4-((4-(Dimethylamino)phenyl)diazenyl)-3,5-dimethoxyphenyl)picolinamide

[0202] (I-6, compound 1465)

[0203] According to General Amidation Procedure C, from 36.0 mg of II-6 and 22.1 mg of 2- picolinicacid, 20.3 mg of (I-6) were obtained (42% yield).1H N MR (400 MHz, Chloroform- d) 510.12 (s, 1H), 8.64 (ddd, J= 4.8, 1.7, 1.0 Hz, 1H), 8.30 (dt, J= 7.9, 1.1 Hz, 1H), 7.94 (td, J = 7.7, 1.7 Hz, 1 H), 7.87 (d, J = 9.1 Hz, 2H), 7.51 (ddd, J = 7.6, 4.8, 1.2 Hz, 1 H), 7.23 (s, 2H), 6.80 - 6.73 (m, 2H), 3.89 (s, 6H), 3.07 (s, 6H). HPLC-PDA-MS: RT: 3.87 min, m / z (ESI+) 406 (MH+, 100%). HRMS (ESI+): found 406.1864, C22H24N5O3 (MH+) requires 406.1879.

[0204] (E)- / V-(4-((3-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide (I-7, compound 1492)

[0205] According to General Amidation Procedure C, from 47.5 mg of II-7 and 33.4 mg of 2- picolinicacid, 40.2 mg of (I-7) were obtained (60% yield).1H NMR(400MHz, Chloroform- d) 510.33 (s, 1H), 8.75 (s, 1H), 8.65 (ddd, J= 4.8, 1.7, 0.9 Hz, 1H), 8.55 (d, J= 5.2 Hz, 1H), 8.31 (dt, J= 7.8, 1.1 Hz, 1H), 8.14 (d, J= 2.2 Hz, 1H), 7.96 (td, J=7.7, 1.7 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.54 (ddd, J= 7.7, 4.8, 1.2 Hz, 1H), 7.45 (d, J= 5.2 Hz, 1H), 7.10 (dd, J= 8.9, 2.1 Hz, 1H), 4.13 (s, 3H). HPLC-PDA-MS: RT: 4.05 min, m / z (ESI+) 368 (MH+, 100%). HRMS (ESI+): found 368.0920, C18H15N5O2CI (MH+) requires 368.0914.

[0206] (E)- / V-(3-Chloro-4-((3,5-dichloropyridin-4-yl)diazenyl)-5-methoxyphenyl) picolinamide (I-8, compound 1493)

[0207] According to General Amidation Procedure C, from 37.0 mg of II-8 and 20.6 mg of 2- picolinic acid, 9.4 mg of (I-8) were obtained (19% yield).1H NMR (400 MHz, Chloroform- d) 510.29 (s, 1H), 8.65 (ddd, J= 4.7, 1.7, 0.9 Hz, 1H), 8.58 (s, 2H), 8.30 (dt, J= 8.0, 1.1 Hz, 1H), 7.96 (td, J=7.7, 1.7 Hz, 1H), 7.88 (d, J= 2.1 Hz, 1H), 7.55 (ddd, J= 7.6, 4.8, 1.2 Hz, 1H), 7.47 (d, J= 2.1 Hz, 1H), 3.99 (s, 3H). HPLC-PDA-MS: RT: 4.56 min, m / z (ESI+) 438 (MH+, 100%). HRMS (ESI+): found 436.0131, C18H13N5O2CI3 (MH+) requires 436.0135. (E)-A / -(3-Methoxy-4-(pyridin-4-yldiazenyl)phenyl)picolinamide (I-9, compound 1495)

[0208] According to General Amidation Procedure C, from 20.1 mg of II-9 and 16.2 mg of 2- picolinic acid, 23.7 mg of (I-9) were obtained (81% yield).1H N MR (400 MHz, Chloroformdi 10.28 (s, 1 H), 8.74 - 8.72 (m, 2H), 8.61 (ddd, J = 4.8, 1.7, 0.9 Hz, 1 H), 8.27 (dt, J = 7.7, 1.1 Hz, 1 H), 8.05 (d, J = 2.1 Hz, 1 H), 7.92 (td, J = 7.7, 1.7 Hz, 1 H), 7.78 (d, J = 8.8 Hz, 1 H), 7.68 - 7.60 (m, 2H), 7.50 (ddd, J = 7.6, 4.8, 1.2 Hz, 1 H), 7.12 (dd, J = 8.8, 2.2 Hz, 1 H), 4.08 (s, 3H). HPLC-PDA-MS: RT: 3.10 min, m / z (ESI+) 334 (MH+, 100%). HRMS (ESI+): found 334.1318, C18H16N5O2 (MH+) requires 334.1304.

[0209] (E)-4-((2-Methoxy-4-(picolinamido)phenyl)diazenyl)-1 -methylpyridin-1 -ium iodide (1-10, compound 1494)

[0210] According to General Alkylation Procedure D, from 17.5 mg of I-9 and 15 pL of methyl iodide, 9.4 mg of (1-10) were obtained (38% yield).1H NMR (400 MHz, Chloroform-d) 5

[0211] 11.22 (s, 1 H), 9.09 (d, J = 6.4 Hz, 2H), 8.81 (d, J = 4.8 Hz, 1 H), 8.27 (d, J = 6.4 Hz, 2H),

[0212] 8.22 (d, J = 7.8 Hz, 1 H), 8.14 (d, J = 1.9 Hz, 2H), 7.85 (s, 2H), 7.78 - 7.74 (m, 1 H), 4.39 (s, 3H), 4.06 (s, 3H). HPLC-PDA-MS: RT: 2.70 min, m / z (ESI+) 348 (MH+, 100%). HRMS (ESI+): found 348.1461 , C19H18N5O2 (MH+) requires 348.1461.

[0213] (E)-A / -(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-1H-imidazole-2- carboxamide (1-11, compound 1496)

[0214] According to General Amidation Procedure C, from 1.6 g of II-7 and 820.0 mg of 1 / 7- imidazole-2-carboxylic acid, 114.2 mg of (1-11) were obtained (13% yield).1H NMR (400 MHz, DMSO-d6) 5 13.37 (s, 1 H), 10.84 (s, 1 H), 8.86 (s, 1 H), 8.63 (d, J = 5.1 Hz, 1 H), 8.01 (d, J = 2.0 Hz, 1 H), 7.77 - 7.66 (m, 2H), 7.59 - 7.01 (m, 3H), 3.99 (s, 3H). HPLC- PDA: RT: 2.83 min. MS (ESI+): found 357.05, Ci6Hi4CIN6O2 (MH+) requires 357.09.

[0215] (E)-A / -(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)pyrazine-2- carboxamide (1-12, compound 1497)

[0216] According to General Amidation Procedure C, from 2.0 g of II-7 and 491.2 mg of pyrazine-2-carboxylic acid, 112.3 mg of (1-12) were obtained (10% yield).1H NMR (400 MHz, Chloroform-d) 5 9.96 (s, 1 H), 9.57 (s, 1 H), 8.90 (d, J = 2.4 Hz, 1 H), 8.79 (s, 1 H), 8.66 (s, 1 H), 8.59 (s, 1 H), 8.14 (d, J = 2.2 Hz, 1 H), 7.91 (d, J = 8.8 Hz, 1 H), 7.50 (d, J = 5.3 Hz, 1 H), 7.11 (dd, J = 9.0, 2.1 Hz, 1 H), 4.16 (s, 3H). HPLC-PDA: RT: 5.16 min. MS (ESI+): found 369.10, C17H14CIN6O2 (MH+) requires 369.09.

[0217] (E)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-5-fluoropicolinamide (1-13, compound 1498)

[0218] According to General Amidation Procedure C, from 2.0 g of II-7 and 558.6 mg of 5- fluoropicolinic acid, 102.3 mg of (1-13) were obtained (9% yield).1H NMR (400 MHz, Chloroform-d) 5 10.13 (s, 1 H), 8.78 (s, 1 H), 8.58 (d, J = 5.3 Hz, 1 H), 8.52 (d, J = 2.7 Hz, 1 H), 8.38 (dd, J = 8.7, 4.5 Hz, 1 H), 8.13 (d, J = 2.1 Hz, 1 H), 7.91 (d, J = 8.8 Hz, 1 H), 7.73-7.63 (m, 1 H), 7.52 (d, J = 5.2 Hz, 1 H), 7.11 (dd, J = 8.8, 2.1 Hz, 1 H), 4.15 (s, 3H). HPLC-PDA: RT: 5.66 min. MS (ESI+): found 386.10, C18H14CIFN5O2 (MH+) requires 386.08.

[0219] Photochemical characterization and solubility

[0220] UV-vis absorption spectra and aqueous solubility

[0221] A collection of samples were prepared with 25 pM of compounds (50 pM in the case of compound 1464) in different solvent configurations ranging from 100% DMSO to 1% DMSO (by volume) in PBS at 25 °C. UV-Vis absorbance spectra of the samples was measured between 300 nm and 600 nm with 2 nm fixed intervals in 96-well transparent plates (200 pL of compound solution / well) using the Spark microplate reader. Illumination at the different wavelengths (380, 455, 470, 550 nm) was achieved using the LED array plate (LEDA Teleopto) during 3 min. For compounds 1496, 1497 and 1498, UV-Vis absorbance spectra of the samples was measured between 300 nm and 600 nm with 1 nm fixed intervals in 1 cm optical path cuvettes (2 mL of compound solution / cuvette) using 8453 Agilent spectrophotometer. Illumination at 395 nm was achieved using a LED (Chanzon, 380 nm - 415 nm). The aqueous solubility was obtained by comparison of the UV-Vis absorbance spectra in the dark at different DMSO percentages to that of pure DMSO. The approximated aquous solubility was selected by choosing the DMSO percentage that rendered a spectra similar in band intensity to that of the DMSO spectra. Kinetics of thermal relaxation from cis to trans

[0222] Thermal relaxation was studied at 25 °C by prolonged absorbance measuring (in dark conditions) after samples (25-50 pM in DMSO) had been subjected to 3 min illuminations at their corresponding optimal trans to cis wavelength. Samples were illuminated using the LED array plate (LEDA Teleopto) system and measurements were collected using the Spark microplate reader. Compounds 1496, 1497 and 1498 were illuminated using LED (Chanzon, 380 nm - 415 nm) and absorbance was measured before, during illumination until reaching photostationary state (in situ photoexcitation), and after using a 8453 Agilent spectrophotometer. The half-life of the compounds was calculated by plotting absorbance readings at a fixed wavelength versus time and then fitting the obtained curve to an exponential decay function using GraphPad prism version 9.2.0 (San Diego, CA). The measured wavelength corresponded to the point where maximum difference in absorbance was observed between trans and cis isomers.

[0223] Biological activity assays

[0224] Cell culture and transient transfection

[0225] Human Embryonic Kidney Tsa201 (HEK Tsa201 , American Type Culture Collection; ATCC) cells were maintained in Dulbecco’s Modified Eagle’s Medium / Nutrient Mixture F-12 Ham (DMEM / F12 1 :1 , Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), penicillin and streptomycin (1%, Sigma-Aldrich) in a controlled environment (37°C, 98% humidity and 5% CO2). As mGlu6 receptors are coupled to the Gi protein, we co-transfected a chimeric Gq / i-protein (GqTOP) to allow the activation of the phospholipase Cp (PLCP), which hydrolyzes the membrane-bound phosphatidylinositol bisphosphate (PIP2) resulting in diacylglycerol (DAG) and inositol trisphosphate (IP3). The latter will spread throughout the cytoplasm and activates IP3- dependent calcium channels at the membrane of the endoplasmic reticulum, which induces the release of calcium into the cytoplasm. Mouse wild-type (WT) mGlu6 or mutant (T148A), where the mutant receptor is devoid to be activated by orthosteric ligands, were transiently co-transfected with GqTOP and the calcium indicator Gcamp6s (ratio 1 :0.25:0.6) through X-tremeGENE 9 DNA Transfection Reagent (Roche Applied Science) following the manufacturer’s instructions. The mouse mGlu6 plasmid was a kind gift from Prof. Takahisa Furukawa (Institute for Protein Research, Osaka University, Osaka, Japan). After 24 h, the cells were harvested with accutase (Sigma-Aldrich) and 80,000 cells / well were seeded onto a 96-well sterile polystyrene plate black with transparent bottom (dD, Biolab), which were pretreated with poly-L-Lysine (Sigma- Aldrich). Finally, the seeded cells were used for the experiments after 24 h.

[0226] Site-direct mutagenesis

[0227] The mutant mGlu6 receptor had a punctiform T148A mutation ( / .e., a threonine to alanine exchange at position 148) in the venus flytrap domain (VFD), resulting in a receptor that is insensitive to its orthosteric ligands. This site-direct mutagenesis was accomplished through a PCR-driven overlap extension by using the KOD Hot Start Master Mix kit (Sigma-Aldrich). The final product (around 800 bp) containing the mutation of interest was loaded on an agarose gel, purified by a gel extraction kit (Qiagen) and inserted in an expression vector (PGEM-T Easy Vector systems, Promega) to transform bacteria (One Shot™ MAX Efficiency™ DH5a-T1R, Thermofisher). Subsequently, the DNA from random colonies that screened positive was sequenced to verify the correctly established mutation. Then the insert and the vector were digested with the EcoRI (New England Biolabs), a restriction enzyme, whereas the digested vector backbone was treated with the Antarctic phosphatase (New England Biolabs) prior to ligation, preventing the two ends of the vector to re-circularize. The cut insert was ligated into the vector, later the complete insert + vector construct was transformed into the DH5a bacteria. The screening for successful transformation was achieved by mini-prepping (NucleoSpin plasmid kit, Macherey-Nagel) and Sanger sequencing of DNA from random colonies.

[0228] Calcium imaging assay

[0229] The fluorescence-based calcium mobilization assay was performed in the HEK Tsa201 cell line, using a calcium indicator ( / .e., Gcamp6s) to determine changes in intracellular calcium concentrations upon receptor activation. In general, the culture medium was aspirated and 100 pl of assay buffer (140 mM NaCI, 5.4 mM KCI, 1 mM MgCh, 10 mM HEPES, 10 mM glucose and 2 mM CaChwith pH 7.4) was added to each well. Although for the ago-PAM assay in HEK Tsa201 cells co-transfected with WT mGlu6 this step was preceded by a pre-incubation with GlutaMAX (Fisher Scientific) for 30 min, ensuring that the observed effect was solely produced by the molecule. Subsequently, the signal was measured by the TECAN infinite 200 microplate reader (Tecan Austria GmbH), which was controlled by i-Control software (Tecan Group Ltd., version 1.6). The microplate reader was set at 485 nm excitation wavelength and 535 nm emission wavelength, while applying empirically optimized parameters (temperature 37°C; integration time 40 ps; number of flashes 18; gain 120; number of cycles 25). Next, the blank or background signal was measured ( / .e., baseline reading). Later, 20 pl were taken from each well and 20 pl of the assay buffer, containing the compound or the control, were transferred to the cell plate and the changes in fluorescence intensity were recorded for 120 s. Positive and negative controls were included in the experiments. On one hand, Triton X-100 (0.2%, Sigma-Aldrich), which lyses the cells and provokes maximum calcium release to obtain the highest fluorescence intensity, as well as Hit-1 (optogluram, 10 pM), allowing to verify successful transfections and used as a reference to normalize our drugs, were included as positive controls. Of note, the L-2-amino-4-phosphonobutyric acid (L-AP4) was used as a positive control for the experiments with mouse WT type mGlu6. On the other hand, a mix of Triton X-100 and EGTA (ethylene glycol-bis(P-aminoethyl ether)- N,N,N',N '-tetraacetic acid), a divalent calcium chelator, resulted in a minimal fluorescence intensity as well as the vehicle control, to ensure that does not interfere with the receptors, were used as negative controls. However, the L-AP4 and glutamate (Sigma-Aldrich) were used as a negative control with the mutant T148A mGlu6. All the experiments were performed under dark conditions to keep the compounds in their trans conformation.

[0230] Drug application and photoswitching

[0231] Test compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and diluted with the assay buffer to a 2% concentration of DMSO in each well. Every compound was tested at different concentrations and experimental conditions. The ago- PAM activity was studied by testing the molecules alone at 4 concentrations (10 pM, 1 pM, 100 nM and 1 nM) in the mouse WT and mutant mGlu6. Finally, the highest ranked compounds in terms of potency and efficacy were selected for the mGlu6 to study their photoswitchable behaviour. As all of them are fast-relaxing molecules, the photoisomerization of the compounds from the trans to the cis conformation was achieved by pre-illuminating them with 460 nm for 3 minutes, and then they were quickly added to the wells. Data analysis

[0232] All data were analysed by using Excel (Microsoft, version 16.62) and GraphPad Prism (GraphPad Software, version 9). First, the raw data obtained by the Tecan were grouped in a single Excel sheet, because for each run the microplate reader produces a different sheet. The Relative Fluorescence (RF) was calculated through the ratio between the peak fluorescence and baseline for each well. Subsequently, the data were normalized with the maximal response to Hit-1 (10 pM). For the dose-response experiments, the entire set of the normalized values was subsequently fitted by a sigmoidal dose-response curve model with variable slope, while the EC50 and Hill slope values were estimated for each compound. The efficacy was determined by referencing maximal responses to Hit-1. Statistical differences were analyzed by the unpaired Mann-Whitney II test, whereas a value of p < 0.05 was considered as significant. The data are expressed as the mean ± SEM for at least three independent experiments.

[0233] Results

[0234] UV-vis absorption spectra, aqueous solubility and kinetics of thermal relaxation from cis to trans

[0235] The results of UV-vis absorption spectra, aqueous solubility and kinetics of thermal relaxation from cis to trans are shown in Figure 1. In agreement to the relatively fast relaxation values observed in DMSO, the lifetimes in aqueous solution were even faster and not measurable using the current experimental setup (i.e. below a few seconds), as it is required for photoswitching mGlu6 activity with cycles of illumination and darkness in assays in vivo. For compounds 1496, 1497 and 1498 a different setup for lifetimes measurement able to detect lifetimes in the order of a few seconds was employed, which allowed to calculate 1496 and 1498 half-lives under aqueous solvent conditions (DMSO: PBS 99:1) which are used in biological assays, while this was not possible for 1497 that presents already a fast relaxation in DMSO and presumably a millisecond order half-life in aqueous solution.

[0236] Aqo-PAM activity screening assay in HEK cells overexpressinq wild-type mouse mGlu6 receptor Ago-PAM activities were evaluated in HEK cells co-transfected with mouse mGlu6 receptor, GqTOP and GCaMP6s. The 30 minutes precedent to the beginning of the assay, cells were pre-incubated with the medium glutaMAX. The ratio of peak fluorescence to baseline in each well was subsequently normalized to the reference molecule Hit-1 (Optogluram, 10 zM). The results are provided in Figure 2. As it can be seen, each of the compound tested yielded a significant rise in intracellular calcium and most of them elicited a response much higher than the one caused by Hit-1 (see determination of potency and efficacy values in Figure 4). L-AP4 was used as a positive control and vehicle as a negative control. Each bar represents the mean ± SEM of activities measured in at least two or three experiments. The calcium assay served to screen several compounds for the agonistic activity in mGlu6.

[0237] Ago-PAM activity assay in HEK cells overexpressinq mutant T148A mGlu6 receptor

[0238] HEK cells were co-transfected with mouse mGlu6 mutant T148A which is not sensitive to orthosteric ligands, GqTOP and GCaMP6s. The calcium assay was performed by using the TECAN infinite 200 microplate reader. The ratio of peak fluorescence to baseline in each well was subsequently normalized to the reference molecule Hit-1 (Optogluram 10 zM). The results are provided in Figure 3. As it can be seen, each of the compound tested yielded a significant rise in intracellular calcium and most of them elicited a response increase much higher than the one caused by Hit-1 (see determination of potency and efficacy values in Figure 6). L-AP4 and vehicle did not produce calcium responses. Each bar represents the mean ± SEM of activities measured in at least two or three experiments The calcium assay served to screen several compounds for the agonistic activity on mGlu6 receptor mutant T148A.

[0239] Dose response relationship in wt mouse mGlu6

[0240] Dose-response curves to determine the EC50 for each compound in its trans isoform (black solid line). The concentration-dependent dose-curve data were plotted as F / Fo and normalized to 10 zM Hit-1 (Optogluram). Curves were calculated from GraphPad using a sigmoidal dose-response curve model with variable slope. Results are shown in Figure 4 as the mean of at least three independent experiments ± SEM. For the cis isoforms of the compounds two or three points are shown at 100 nM, 1 and 10 zM. The EC50 values determined for the trans are 156 nM for compound 1463; 284 nM for compound 1464; 106 nM for the compound 1465; 65 nM for the compound 1492; 149 nM for compound 1493; 129 nM for the compound 1494 and 259 nM for the compound 1495. The efficacy values compared to hit-1 (100%) are 145% for compound 1465; 150% for compound 1492; 92% for compound 1494 and 154% for compound 1495.

[0241] Photoswitchinq mGlu6 receptor activity without glutamate in vitro

[0242] The cis isoforms were obtained by illuminating the compounds for 3 minutes at 460 nm. As shown in Figure 5(A), the compounds 1463 and 1493 seems to be c / s-active, while the other compounds are frans-active. The compounds 1465, 1492 and 1492 show significant photoswitching at 1 zM, with the trans isoform to be the active one. The data were analyzed by the unpaired Mann-Whitney test (p-value (**) < 0.01 , (*) < 0.05; GraphPad Prism 6). Error bars are ± SEM. Figure 5(B) Histogram showing the differences in photoresponses (F / F0)rrans - (F / F0)c / s, expressed in %.

[0243] Ago-PAM responses in mutant T148A mGlu6 receptor

[0244] Dose-response curves were generated to make estimations of agonist potencies and efficacies. The concentration-dependent dose-curve data were plotted as F / FO and normalized to 10 zM Hit-1 (optogluram). Data shown in Figure 6 are representative of a set of at least three independent experiments ± SEM for each concentration. The EC50 values determined were on the nanomolar range for all the compounds tested. The curves were obtained by nonlinear regression analysis using GraphPad Prism. Figure 7 shows an outline of potency values of Ago-PAM responses in mutant T148A mGlu6 receptor. As it can be seen, results obtained with the compounds of the invention are consistently superior to Hit-1 (Optogluram).

[0245] In vivo assays

[0246] Behavioral assays in mice

[0247] Behavioral assays were carried out in Opn4' / _mice, lacking photosensitive melanopsinic cells, but having otherwise functional retinas (transition test, preference for room perceived as dark) before photoreceptor damage. After photoreceptor damage (intraperitoneal sodium iodate 40 or 65 mg / kg, 3 or more weeks), a photorreceptor degeneration was observed. The results are provided in Figures 8-10.

[0248] Animals (Opn^) were subjected to the Light / Dark Transition Test prior to intraperitoneal administration of NalCh. With this Test we are able to measure the light sensitivity of an animal. They are introduced in a wooden experimental set consisting of two compartments, connected by a small opening in the wall, and with different light conditions (Light intensities (A.W. Sperry - SLM-110, A.W. Sperry Instruments, U.S.A.): 380 nm, 9.7 lx; 460 nm, 77 lx 500 nm, 22.6 lx; white light, 39 lx). Animals are allowed to explore both compartments, and the time spent in each compartment is being timed. When it was found that the animals did not show any visual alteration, NalCh was administered systemically by intraperitoneal injection of a dose of 65 mg / kg, with a 25 G needle. The animals were returned to their respective cages and allowed to rest for 3 or more weeks. Prior to administration of the Photoswitches, a new Light / Dark Transition Test was performed, checking that the animals did not show rejection of the compartments with higher light intensity. Subsequently, the animals were anaesthetised with isoflurane and the various photosensitive molecules were administered individually, by unilateral intravitreal injection, with a 10 pL syringe (NanofilTm, World Precision Instruments, Florida, USA) with a 34 G needle (NanofilTm, NF35BV-2, World Precision Instruments, Florida, USA), or by topical application of a 10 pL drop on the ocular surface of both eyes. The animals were allowed to rest for 2 hours and 30 minutes and then reintroduced to the Light / Dark Transition Test. All animals were placed in the darkest compartment in all cases. The test was repeated three times for each animal, each time cleaning each compartment to avoid falsification of the results. All experimental procedures were carried out following very similar schedules, during the night phase of the animals, to ensure a higher activity of the animals, as well as a higher reproducibility.

[0249] Single dose, unilateral intravitreal injections of 30 pM hit-1 (optogluram) were applied to mice and the assay was repeated after 2 h and a half hour, and 2 days after, without supplementary doses. Three out of six mice displayed significant preference for the 500 nm lit room, as it is shown in Figure 8, where the 380 nm illuminated room is shown as a white bar. Topical binocular application of a 10 pl drop of 30 pM hit-1 (optogluram), in separate experiments, did not produce significant effects. No signs of pain or distress were observed in any case.

[0250] Single dose, unilateral intravitreal injections of 1 , 10, or 32 pM of compound 1492 compound were applied to mice and the assay was repeated after 2 and a half hours without supplementary doses. Three out of four mice at each concentration displayed significant preference for the dark room, as it is shown in Figure 9 (black bars), rejecting the 460 nm illuminated room (white bars; which favored cis isomerization of the compound).

[0251] Figure 10 shows that topical application of 10 pl drop of 30 pM of compound 1492 in different mice produced similar effects in five out of six mice. No signs of pain or distress were observed neither for intravitreal nor topical application of compound 1492.

[0252] The behavioral assays of Figures 8-10 were repeated after topical application of compounds 1492 and 1495 (10 pl drop of 30 pM; 1.6% DMSO) but using cool white LEDs (emission centered at 460 nm, which favors cis isomerization of both compounds). The results are shown in Figures 11-12. As shown in Figure 11 , compound 1492 restored preference for the dark room (black bars), 2 and a half hours after topical application in six out of six mice. As shown in Figure 12, compound 1495 restored preference for the dark room (black bars), 2 and a half hours after topical application in six out of nine mice. No signs of pain or distress were observed neither for intravitreal nor topical application of either compound. Light intensity (A.W. Sperry - SLM-110, A.W. Sperry Instruments, U.S.A.): 39 lx.

[0253] Visual Acuity assays in zebrafish

[0254] Tupfel Long-fin (TL) train zebrafish larvae aged 6 days post fertilization (dpf) and raised in stove at 28°C without further treatment (not blinded) were placed in a petri dish containing a droplet of methylcellulose 8% to assess their optokinetic response (OKR, Brockerhoff, S.E., Nat P rot oc., 2006, 1 (5), 2448-2451). Saccades / min were registered by triplicate for each animal at 5 rpm (revolutions per minute), before and after addition of a droplet of compound 1492 dissolved in DMSO 1 % at a concentration of 1 pM. Animals were illuminated using white light with a 460 nm intensity of 5 mW. The results are presented in Figure 13 (n=7, *P<0.05) and demonstrate that the visual acuity (quantified as saccades / min for lines rotating at 5 rpm in the OKR measurement device) can be enhanced two-fold by the 1492 compound in healthy non blinded animals (white bar: prior to addition of 1492 compound; black bar: after adding 1492 compound).

[0255] For compounds 1496, 1497 and 1498 assays, Tupfel Long-fin (TL) strain zebrafish larvae aged 6 days post fertilization (dpf) and raised in stove at 28°C were placed in a petri dish in a closed mirrored chamber, and then exposed to 135.000 lux light emitted by a mercury lamp (model Olympus U-LH100HG) for 30 min for blinding. At 7 dpf, OKR assay was performed. Saccades / min were registered by duplicate for each animal at 11 rpm (revolutions per minute), before and after addition of a droplet of each compound dissolved in DMSO 1 % at a concentration of 10 pM. The results are presented in Figure 14 (n=5, n=7 and n=7, *P<0.05, **P<0.01 paired t-test) and demonstrate that the visual acuity (quantified as saccades / min for lines rotating at 11 rpm in the OKR measurement device) can be partially recovered in blinded animals (white box: prior to addition of 1496, 1497 or 1498 compound; grey box: after adding 1496, 1497 or 1498 compound.

Claims

CLAIMS1. Compound of formulawhereinR1is selected from the group consisting of H, Ci-Ce alkoxy, halogen and CF3;R2is selected from the group consisting of H, and Ci-Ce alkyl;R3is selected from the group consisting of H, halogen, Ci-Ce alkoxy and CF3;R4is selected form the group consisting of H, halogen, Ci-Ce alkoxy and CF3;R5is selected from the group consisting of halogen, Ci-Ce alkyl and CF3; m is an integer from 0 to 2;R6is a heterocyclyl; andG is a nitrogen containing group selected from the group consisting of N, N+-(Ci-Ce alkyl), C-NO2, C-N(CI-C6alkyl)2, and C-NH(CI-C6alkyl); or a pharmaceutically acceptable salt or stereoisomer thereof.

2. Compound of formula (I) according to claim 1 , wherein R1is selected from the group consisting of H, methoxy and Cl.

3. Compound of formula (I) according to any one of the preceding claims, wherein R2is methyl.

4. Compound of formula (I) according to any one of the preceding claims, wherein R3is selected from the group consisting of H and Cl.

5. Compound of formula (I) according to any one of the preceding claims, wherein R4is selected from the group consisting of H and Cl.

6. Compound of formula (I) according to any one of the preceding claims, wherein either m is 0, or m is 1 and R5is selected from the group consisting of Cl and methyl; preferably wherein m is 0.

7. Compound of formula (I) according to any one of the preceding claims, wherein R6is selected from the group consisting of pyridyl optionally substituted with one halogen atom, pyrazyl and imidazolyl.

8. Compound of formula (I) according to any one of the preceding claims, wherein R6is a pyridyl, preferably 2-pyridyl.

9. Compound of formula (I) according to any one of the preceding claims, wherein G is selected from the group consisting of N, N+-methyl, C-N(methyl)2, and C-NO2.

10. Compound of formula (I) according to any one of the preceding claims, which is selected from the group consisting of:(E)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide, (Z)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide, (E)- / V-(3-Methoxy-4-((3-methyl-4-nitrophenyl)diazenyl)phenyl)picolinamide, (Z)- / V-(3-Methoxy-4-((3-methyl-4-nitrophenyl)diazenyl)phenyl)picolinamide, (E)- / V-(4-((4-(Dimethylamino)phenyl)diazenyl)-3-methoxyphenyl)picolinamide, (Z)- / V-(4-((4-(Dimethylamino)phenyl)diazenyl)-3-methoxyphenyl)picolinamide, (E)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3,5-dimethoxyphenyl)picolinamide, (Z)- / V-(4-((2-Chloropyridin-4-yl)diazenyl)-3,5-dimethoxyphenyl)picolinamide, (E)- / V-(3,5-Dimethoxy-4-((3-methyl-4nitrophenyl)diazenyl)phenyl)picolinamide, (Z)- / V-(3,5-Dimethoxy-4-((3-methyl-4nitrophenyl)diazenyl)phenyl)picolinamide, (E)- / V-(4-((4-(Dimethylamino)phenyl)diazenyl)-3,5-dimethoxyphenyl)picolinamide, (Z)- / V-(4-((4-(Dimethylamino)phenyl)diazenyl)-3,5-dimethoxyphenyl)picolinamide, (E)- / V-(4-((3-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide, (Z)- / V-(4-((3-Chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)picolinamide, (E)- / V-(3-Chloro-4-((3,5-dichloropyridin-4-yl)diazenyl)-5-methoxyphenyl)picolinamide, (Z)- / V-(3-Chloro-4-((3,5-dichloropyridin-4-yl)diazenyl)-5-methoxyphenyl)picolinamide, (E)- / V-(3-Methoxy-4-(pyridin-4-yldiazenyl)phenyl)picolinamide, (Z)- / V-(3-Methoxy-4-(pyridin-4-yldiazenyl)phenyl)picolinamide, (E)-4-((2-Methoxy-4-(picolinamido)phenyl)diazenyl)-1-methylpyridin-1-ium iodide,(Z)-4-((2-Methoxy-4-(picolinamido)phenyl)diazenyl)-1-methylpyridin-1-ium iodide, (E)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-1 / 7-imidazole-2- carboxamide,(Z)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-1 / 7-imidazole-2- carboxamide,(E)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)pyrazine-2-carboxamide,(Z)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)pyrazine-2-carboxamide, (E)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-5-fluoropicolinamide, and (Z)- / V-(4-((3-chloropyridin-4-yl)diazenyl)-3-methoxyphenyl)-5-fluoropicolinamide.11 . Compound of formula (I) according to any one of the preceding claims, which is the E-isomer.

12. Pharmaceutical composition comprising a compound according to any one of claims 1 to 11 , and a pharmaceutically acceptable excipient.

13. Compound of formula (I) according to any one of claims 1 to 11 or pharmaceutical composition according to claim 12, for use in medicine.

14. Compound of formula (I) according to any one of claims 1 to 11 or pharmaceutical composition according to claim 12, for use in the treatment and / or prevention of a retinal disease.

15. Compound of formula (I) or pharmaceutical composition for use according to claim 14, wherein the retinal disease is a retinal degenerative disease, preferably retinitis pigmentosa.

16. Use of a compound of formula (I) according to any one of claims 1 to 11 or pharmaceutical composition according to claim 12, in the enhancement of retinal function and / or visual performance in healthy subjects.

17. Use of a compound of formula (I) according to any one of claims 1 to 11 or pharmaceutical composition according to claim 12, in the manufacture of a medicament.

18. Use of a compound of formula (I) according to any one of claims 1 to 11 or pharmaceutical composition according to claim 12, in the manufacture of a medicament for the treatment and / or prevention of a retinal disease.

19. Use according to claim 18, wherein the retinal disease is a retinal degenerative disease, preferably retinitis pigmentosa.

20. Method of treatment and / or prevention of a subject in need thereof of a retinal disease, which comprises the administration of a compound of formula (I) according to any one of claims 1 to 11 or pharmaceutical composition according to claim 12.

21. Method according to claim 20, wherein the retinal disease is a retinal degenerative disease, preferably retinitis pigmentosa.

22. Method for enhancing the retinal function and / or visual performance in healthy subjects, which comprises the administration of a compound of formula (I) according to any one of claims 1 to 11 or pharmaceutical composition according to claim 12.

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