TECHNICAL FIELD

The present invention relates to novel melatonin ligands capable of modulating the melatonin receptors, particularly the melatonin receptor subtype MT2. Moreover, these compounds have been shown to be effective in the treatment of diseases and disorders associated with MT2 receptor activity such as sleep disorders, major depression disorder, mood disorders, anxiety disorders, autism spectrum disorder, cognitive disorders, dementia, metabolic diseases included diabetes Type 1 and type 2, circadian rhythms disorders, pain, acute pain, inflammatory pain, chronic pain, neuropathic pain.

BACKGROUND

Melatonin (N-acetyl-5-methoxytryptamine, MLT) is a neurohormone essentially produced by the pineal gland in a circadian rhythmic fashion with the highest levels, in both diurnal and nocturnal mammals, during the night dark phase (V. Simonneaux, C. Ribelayga, Pharmacol. Rev. 2003, 55, 325-395).

MLT is involved in numerous physiological processes including circadian rhythms, mood regulation, anxiety, sleep, appetite, immune responses, cardiac functions and pain (Comai and Gobbi 2014). Most of the physiological effects of MLT result from the activation of two high-affinity (Ki =0.1 nM) G-protein coupled receptors (GPCRs) named MT1 and MT2.

Apart the above cited sleep-inducing and chronobiotic properties, several other functions have been attributed to MLT at physiological levels and even more when considering supraphysiological levels, including modulation of the activity of the immune system, regulation of the cardiovascular functions, control of mood and behavior, glucose homeostasis, hormone secretion and pain perception. Other effects of MLT described in the literature include its neuroprotective, anti-inflammatory, retinal, antioxidant and anticancer properties.

However, the therapeutic use of MLT can be limited by its short half-life (<30 min), its high first-pass metabolism and low brain bioavailability, as well as the binding of multiple receptors that can have opposite effects.

Specifically the agonism of MT1 receptors produces vasoconstriction (Doolen, Krause et al. 1998, European Journal of Pharmacology, 345(1): 67-69), increase in REM sleep, decrease in NREM sleep (Comai, Ochoa-Sanchez et al. 2013, Behavioural Brain Research 243(1): 231- 238), anti-depressant-like effects (Comai, Ochoa-Sanchez et al. 2015, Int J Neuropsychopharmacol 18(3)), increases the body temperature (Lopez-Canul, Min et al. Int J Mol Sci 20(10), 2019). On the other hand, the MT2 agonism generates vasodilation (Doolen, Krause et al. 1998, European Journal of Pharmacology, 345(1): 67-69), promotes NREM and decreases the latency to sleep (Ochoa-Sanchez, Comai et al. J Neurosci, 31(50), 2011), has anxiolytic-like effects (Ochoa-Sanchez, Rainer et al. 2012, Prog Neuropsychopharmacol Biol Psychiatry 39(2): S18-S25), analgesic effects in acute model of pain (Lopez-Canul, Comai et al. 2015, EurJ Pharmacol 764: 424-432), as well as in chronic neuropathic pain (Lopez-Canul, Palazzo et al. 2015, Pain 156(2): 305-317). Moreover, each receptor is localized in very specific areas (Lacoste et al., 2015, Journal of pineal research, 58(4), 397-417).

These recent discoveries have incentivized the development of selective MT1 and MT2 compounds targeting specific physiological functions and/or specific pathological disorders.

Until now, a few melatonin analogs have been synthetized and commercialized and all of them are non-selective MT1/MT2 receptors agonists.

Among these, one the most interesting and versatile class was that of the m- methoxyanilinoethylamides, allowing to obtain high affinity nonselective full agonists, MT2 selective partial agonists or very selective MT2 antagonists, depending on the size of the substituent on the aniline nitrogen. Two representative ligands of this class are the MT2- selective partial agonist UCM765 (N-{2-[(3-methoxyphenyl)phenylamino]ethyl}acetamide), and its metabolic more stable congener UCM924 (N-{2-[(3-bromophenyl)-(4- fluorophenyl)amino]ethyl}acetamide)) that evidenced interesting hypnotic and antinociceptive properties in rodents.

However, the physicochemical properties of these compounds greatly limited their use in research and functional application, as they suffer from low aqueous solubility and modest microsomal stability.

Therefore, there is still a need to find melatonin derivatives capable of overcoming the above-mentioned drawbacks.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to novel melatonin ligands that overcome these limitations, and can modulates the melatonin receptors, particularly the melatonin receptor subtype MT2. Moreover, these compounds have been shown to be effective in the treatment of diseases and disorders associated with MT2 activity such as sleep disorders, major depression disorder, mood disorders, anxiety disorders, autism spectrum disorder, cognitive disorders, dementia, metabolic diseases included diabetes Type 1 and type 2, circadian rhythms disorders, and pain disorders.

The present invention also relates to therapeutic compositions comprising such novel melatonin ligands or pharmaceutically acceptable salts thereof for the alleviation or treatment of diseases and disorders associated with MT2 activity such as sleep disorders major depression disorder, mood disorders, anxiety disorders, autism spectrum disorder, cognitive disorders, dementia, metabolic diseases included diabetes Type 1 and type 2, circadian rhythms disorders, and pain disorders.

In an aspect the present disclosure includes a compound of Formula (l.a) or a pharmaceutically acceptable salt thereof

Formula (l.a) wherein:

R is selected from the group consisting of hydrogen, Ci-Cs alkyl, C ₃ -C ₆ cyclo-alkyl, and hydroxy-substituted Ci-Cs alkyl,

Ri is selected from the group consisting of hydrogen, Ci-Cs alkyl, and C ₃ -C ₆ cyclo-alkyl,

R ₂ is selected from the group consisting of heteroaryl, benzyl, C ₁ -C ₆ alkyl, C ₃ -C ₆ cyclo-alkyl, hydroxymethyl phenyl, and C ₁ -C ₆ heterocyclic group, and

R ₃ is selected from the group consisting of hydrogen, halogen, phenyl, Ci-Cs alkyl, hydroxy- substituted Ci-Cs alkyl, -CF ₃ , and -CHF ₂ ,

R _{4 IS} selected from the group consisting of hydrogen, Ci-Cs alkyl, and C ₃ -C ₆ cyclo-alkyl,

R ₅ is selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkyloxy, OCF ₃ , CF ₃ , hydroxyl, hydroxy-substituted Ci-C ₄ and halogen.

In an aspect the present disclosure includes a compound having Formula (l.b) or a pharmaceutically acceptable salt thereof

Formula (l.b) wherein:

R6 is selected from the group consisting of C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyloxy, hydroxy-substituted Ci- C6 alkyl, and -CHF ₂ , R7 is selected from the group consisting of heteroaryl, benzyl, C1-C6 alkyl, C3-C6 cyclo-alkyl,

~N pR _s hydroxymethyl phenyl, and C1-C6 heterocyclic group, and

Rs is selected from the group consisting of hydrogen, halogen, phenyl, C1-C6 alkyl, and hydroxy-substituted C1-C6 alkyl,

Rg is selected from the group consisting of C ₁ -C ₄ alkyl, C ₁ -C ₄ alkyloxy, OCF ₃ , CF ₃ , hydroxyl, hydroxy-substituted C ₁ -C ₄ and halogen.

In an aspect the present disclosure includes a use of compound of Formula (la) or of compound of Formula (lb) as defined herewith for preparing a prodrug.

In an aspect the present disclosure includes a compound of Formula (la) or a compound of Formula (lb) as defined herewith for preparing a prodrug.

In an aspect the present disclosure includes a prodrug of compound of Formula (la) or of compound of Formula (lb) as defined herewith.

In an aspect the present disclosure includes a composition comprising compound of Formula (l.a) or of Formula (l.b) and a pharmaceutically acceptable excipient.

In an aspect the present disclosure includes a use of compound of Formula (l.a) or compound (l.b) or of a composition comprising compound of Formula (l.a) or compound (l.b) for treating/alleviating sleep disorders, major depression disorder, mood disorders, anxiety disorders, cognitive disorders, dementia, metabolic diseases included diabetes Type 1 and type 2, circadian rhythms disorders and pain disorders.

In an aspect the present disclosure includes therapeutically effective compositions for treating a condition mediated by the MT1 and/or MT2 receptor, the compositions comprising one or more pharmaceutically acceptable excipients and a compound of Formula (l.a) or (l.b) or a pharmaceutically acceptable salt thereof.

In an aspect the present disclosure includes a method of interacting with at least one of MT1 and MT2 receptor subtypes for treating/alleviating sleep disorders, anxiety, depression, or chronobiological disorders, and pain disorders comprising administering to a subject in need thereof compound of Formula (l.a) or compound (l.b) or a composition comprising compound of Formula (la) or compound (l.b), and one or more pharmaceutically acceptable excipients.

In an aspect the present disclosure includes a method of treating/alleviating sleep disorders, anxiety, depression, or chronobiological disorders and pain disorders comprising administering to a subject in need thereof compound of Formula (l.a) or compound (l.b) or the composition comprising these compounds thereof and one or more pharmaceutically acceptable excipients. Other objects and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating illustrative embodiments are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Plasma concentration profiles of compounds 19 (A) and 14 (B) in Sprague-Dawley rats after single intravenous (5 mg/kg) or oral (40 mg/kg) administration.

DETAILED DESCRIPTION

In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" may mean at least a second or more.

The term "Ci-Cs alkyl", as used herein, is understood as being straight chain or branched chain alkyl groups non-limiting examples of which include methyl, ethyl, propyl, isopropyl, n- butyl, isobutyl, f-butyl, amyl, hexyl, heptyl and octyl.

The term "Ci-Cs alkyloxy", as used herein, is understood as being straight chain or branched chain alkyloxy groups, non-limiting examples of which include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy and t-butoxy.

The term "C3-C6 cycloalkyl", as used herein, is understood as being a carbon-based ring system, non-limiting examples of which include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term "hydroxy-substituted Ci-Cs alkyl", as used herein, is understood as being a Ci-Cs alkyl alcohol, i.e. an aliphatic alcohol in which the aliphatic alkane chain is substituted by a hydroxy group at unspecified position.

The term "heteroaryl", as used herein, is understood as being unsaturated rings of five or six atoms containing one or two O- and/or S-atoms and/or one to four N-atoms, provided that the total number of hetero-atoms in the ring is 4 or less. The heteroaryl ring is attached by way of an available carbon or nitrogen atom. Non-limiting examples of heteroaryl groups include 2-, 3-, or 4-pyridyl, 4-imidazolyl, 4-thiazolyl, 2- and 3-thienyl, and 2- and 3-furyl. The term "heteroaryl", as used herein, is understood as also including bicyclic rings wherein the five or six membered ring containing O, S and N-atoms as defined above is fused to a benzene or pyridyl ring. Non-limiting examples of bicyclic rings include but are not limited to 2- and 3-indolyl as well as 4- and 5-quinolinyl.

The term "halogen", as used herein, is understood as including fluorine, chlorine, bromine and iodine.

The term "phenyl", as used herein, is understood as being an aromatic substituent which is a single C ₆ ring which may optionally be substituted.

The term "benzyl", as used herein, is understood as being an aromatic substituent having the structure C6H5CH2 which may optionally be substituted.

The term "C1-C6 heterocyclic group", is understood as being a carbon-based ring system, where at least one carbon atom is substituted with a "heteroatom", i.e. oxygen, sulfur or nitrogen.

The term "patient" or "subject", as used herein, is understood as being any individual treated with the melatonin ligands of the present disclosure for the alleviation or treatment of pain. Patients include humans, as well as other animals such as companion animals, livestock and rodents such as for example rats.

Administration and uses of solvates of the melatonin ligands of the present disclosure are also contemplated herein. Solvates of the compounds of Formula I are preferably hydrates. The term "derivative" as used herein, is understood as being a substance which comprises the same basic carbon skeleton and carbon functionality in its structure as a given compound but can also bear one or more substituents or rings.

The term "analogue" as used herein, is understood as being a substance similar in structure to another compound but differing in some slight structural detail.

The term "antagonist" as used herein, is understood as being any molecule that blocks, inhibits, or neutralizes a biological activity of the high affinity MLT receptors subtypes MT2 and/or T ^L In a similar manner, the term "agonist" as used herein, is understood as being any molecule that mimics a biological activity of native MLT. The term "partial agonist" as used herein, is understood as being any molecule that mimics the activity of endogenous MLT but is unable to achieve the maximal pharmacological stimulation of MLT.

The term "salt(s)" as used herein, is understood as being acidic and/or basic salts formed with inorganic and/or organic acids or bases. Zwitterions (internal or inner salts) are understood as being included within the term "salt(s)" as used herein, as are quaternary ammonium salts such as alkylammonium salts. Nontoxic, pharmaceutically acceptable salts are preferred, although other salts may be useful, as for example in isolation or purification steps.

Examples of acid addition salts include but are not limited to acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, phosphoric, 2-hydroxyethanesulfonate, lactate, maleate, mandelate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.

Examples of base addition salts include but are not limited to alkali metal salts and alkaline earth metal salts. Non limiting examples of alkali metal salts include lithium, sodium and potassium salts. Non-limiting examples of alkaline earth metal salts include magnesium and calcium salts.

The term "mood disorders" as used herein, is understood as a mental health problem that primarily affects a person's mood. It is a disorder in which a person experiences long periods of extreme happiness (i.e. mania, hypomania), extreme sadness (depression), or both. It includes major depression, bipolar disorder type I and II, persistent depressive disorders, etc.(DSM-5; American Psychiatric Association, 2013).

The term "major depression" as used herein, is understood as a persistent mood disorder marked by varying degrees of sadness, despair, and loneliness that is typically accompanied by inactivity, guilt, loss of concentration, social withdrawal, sleep disturbances, somatic symptoms and sometimes suicidal tendencies.

The term "anxiety disorders" as used herein, is understood as an abnormal and overwhelming sense of apprehension and fear often marked by physical signs (such as tension, sweating, and increased pulse rate), by doubt concerning the reality and nature of the threat, and by self-doubt about one's capacity to cope with it. It includes Generalized anxiety disorders, social phobia, agoraphobia, etc. (DSM-5; American Psychiatric Association, 2013).

Th term "Circadian rhythm disorders" as used herein is understood as a problem that occur when your sleep-wake cycle is not properly aligned with your environment and interferes with your daily activities. Circadian rhythm sleep disorders involve either difficulty falling asleep, waking up during the sleep cycle or waking up too early and being unable to fall back to sleep (DSM-5; American Psychiatric Association, 2013).

"The term "sleep disorders" as used herein refers to conditions that affect sleep quality, timing, or duration and impact a person's ability to properly function while they are awake. These disorders can contribute to other medical problems, and some may also be symptoms for underlying mental health issues (DSM-5; American Psychiatric Association, 2013).

The term "metabolic disorders" as used herein refers to a disorder that negatively alters the body's processing and distribution of macronutrients such as proteins, fats, and carbohydrates. Metabolic disorders can happen when abnormal chemical reactions in the body alter the normal metabolic process. The term "Type 1 diabetes" (or juvenile) as use herein, is understood as a chronic condition in which the pancreas produces little or no insulin. Insulin is a hormone needed to allow sugar (glucose) to enter cells to produce energy.

The term "Type 2 diabetes" (adult-onset) as use herein, is understood as an impairment in the way the body regulates and uses sugar (glucose) as a fuel. This long-term (chronic) condition results in too much sugar circulating in the bloodstream. Eventually, high blood sugar levels can lead to disorders of the circulatory, nervous and immune systems. The pancreas does not produce enough insulin and cells are resistant to insulin.

The term "dementia" as use herein, is understood as the loss of cognitive functioning, thinking, remembering, and reasoning, and behavioural abilities to such an extent that it interferes with a person's daily life and activities. It includes, as example, Alzheimer's dementia, vascular dementia, Alzheimer's Disease, dementia with Lewy Bodies (DLB), Parkinson's Disease Dementia, Mixed Dementia, Frontotemporal Dementia (FTD), Huntington's Disease, Creutzfeldt-Jakob Disease.

The term "pain disorders" as use herein is understood as an uncomfortable sensation in the body, sharp stab or dull ache, also be described as throbbing, pinching, stinging, burning, or sore. It includes these examples: inflammatory pain, acute pain, diabetic neuropathic pain, trigeminal neuralgia, peripheral nerve injury, painful polyneuropathy, postherpetic neuralgia, painful radiculopathy and fibromyalgia. Chronic pain including: (1) chronic primary pain, (2) chronic cancer pain, (3) chronic posttraumatic and postsurgical pain, (4) chronic neuropathic pain, (5) chronic headache and orofacial pain, (6) chronic visceral pain, and (7) chronic musculoskeletal pain.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods and uses of the disclosure.

Within the present invention is provided a compound of Formula (l.a) or a pharmaceutically acceptable salt thereof selected from the group comprising or, alternatively, consisting of:

Formula (l.a) wherein:

R is selected from the group consisting of hydrogen, Ci-Cs alkyl, C3-C6 cyclo-alkyl, and hydroxy-substituted Ci-Cs alkyl, Ri is selected from the group consisting of hydrogen, Ci-Cs alkyl, and C ₃ -C ₆ cyclo-alkyl,

R ₂ is selected from the group consisting of heteroaryl, benzyl, C ₁ -C ₆ alkyl, C ₃ -C ₆ cyclo-alkyl, hydroxymethyl phenyl, and C ₁ -C ₆ heterocyclic group, and

R ₃ is selected from the group consisting of hydrogen, halogen, phenyl, Ci-Cs alkyl, hydroxy- substituted Ci-Cs alkyl, -CF ₃ , and -CHF ₂ ,

R _{4 IS} selected from the group consisting of hydrogen, Ci-Cs alkyl, and C ₃ -C ₆ cyclo-alkyl,

R ₅ is selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkyloxy, OCF ₃ , CF ₃ , hydroxyl, hydroxy-substituted Ci-C ₄ and halogen.

In one embodiment of the present invention, the compounds of Formula (l.a) are those selected from the group comprising or, alternatively, consisting of: wherein R is selected from the group consisting of C ₁ -C ₃ alkyl, and C ₃ -C ₆ cyclo-alkyl, preferably R is selected from the group consisting of C ₁ -C ₃ alkyl, wherein Ri is selected from the group consisting of hydrogen, and C ₁ -C ₃ alkyl, wherein R ₂ is selected from the group consisting of heteroaryl, benzyl, C ₁ -C ₃ alkyl, C ₃ -C ₆ cyclo-alkyl, hydroxymethylphenyl, and C ₁ -C ₆ heterocyclic group, and wherein R ₃ is selected from the group consisting of hydrogen, phenyl, C ₁ -C ₃ alkyl, Br, and F, wherein R _{4 IS} hydrogen, and wherein R ₅ is selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkyloxy, and halogen.

In one embodiment of the present invention, the group R is selected from the group consisting of C ₁ -C ₃ alkyl,

Ri is selected from the group consisting of hydrogen and C ₁ -C ₃ alkyl _,

R ₂ is phenyl,

R _{3 IS} hydrogen _, and

FUis hydrogen. In a further embodiment of the present invention, the compounds of Formula (la) are those selected from the group comprising or, alternatively, consisting of: l-{2-[(3-Methoxyphenyl)phenylamino]ethyl}urea (compound 6a UCM 1091), l-Ethyl-3-{2-[(3-methoxyphenyl)phenylamino]ethyl}urea (compound 6b UCM 1092), or l-{2-[(3-Methoxyphenyl)phenylamino]ethyl}-3-propylurea (compound 6c UCM 1093).

The present invention also relates to a compound having Formula (l.b) or a pharmaceutically acceptable salt thereof selected from the group comprising or, alternatively, consisting of:

Formula (lb) wherein:

R ₆ is selected from the group consisting of C1-C6 alkyl, C1-C6 alkyloxy, hydroxy-substituted Ci- C ₆ alkyl, and -CHF2,

R7 is selected from the group consisting of heteroaryl, benzyl, C1-C6 alkyl, C3-C6 cyclo-alkyl, hydroxymethyl phenyl, and C1-C6 heterocyclic group,

Rs is selected from the group consisting of hydrogen, halogen, phenyl, C1-C6 alkyl, and hydroxy-substituted C1-C6 alkyl,

Rg is selected from the group consisting of C ₁ -C ₄ alkyl, C ₁ -C ₄ alkyloxy, OCF ₃ , CF ₃ , hydroxyl, hydroxy-substituted C ₁ -C ₄ alkyl and halogen.

In one embodiment of the present invention, the compounds of Formula (l.b) are those selected from the group comprising or, alternatively, consisting of:

R ₆ is selected from the group consisting of C ₁ -C ₃ alkyl, C ₁ -C ₃ alkyloxy, hydroxy-substituted Ci- C ₃ alkyl, and -CHF ₂ ,

R7 is selected from the group consisting of heteroaryl, benzyl, C ₁ -C ₃ alkyl, C ₃ -C ₆ cyclo-alkyl, hydroxymethyl phenyl, and C1-C6 heterocyclic group, and Rs is selected from the group consisting of hydrogen, halogen, phenyl, C1-C6 alkyl, and hydroxy-substituted C1-C6 alkyl,

Rg is selected from the group consisting of C ₁ -C ₃ alkyl, C ₁ -C ₄ alkyloxy, hydroxyl, hydroxy- substituted Ci- C ₄ alkyl, F, and Br,

In another embodiment of the present invention, the compounds of Formula (l.b) are those selelcted from the group comprising or, alternatively, consisting of:

R6 is selected from the group consisting of C ₁ -C ₃ alkyloxy, and -CHF ₂ ,

R7 is selected from the group consisting of benzyl, C ₁ -C ₃ alkyl, thiofenyl, pyridine, fluorophenyl, hydroxymethylphenyl, and phenol, and

Rs is selected from the group consisting of hydrogen, halogen, and phenyl.

In a further embodiment of the present invention, the compounds of Formula (lb) are those selected from the group comprising or, alternatively, consisting of:

N-{2-[(4-Methoxy-[l,l'-biphenyl]-2-yl)(methylamino)]ethyl }acetamide (compound 18, UCM 1094),

N-{2-[(2-Bromo-5-methoxyphenyl)methylamino]ethyl}acetamid e (compound 19, UCM 1095), N-{2-[(3-Methoxyphenyl)(thiophen-3-yl)amino]ethyl}acetamide (compound 11, UCM 1096), N-(2-{[3-(Difluoromethyl)phenyl](4-fluorophenyl)amino}ethyl) acetamide (compound 10, UCM 1097),

N-{2-[(3-Methoxyphenyl)(phenethyl)amino]ethyl}acetamide (compound 12, UCM1098), N-{2-[(3-Methoxyphenyl)(pyridine-4-yl)amino]ethyl}acetamide (compound 13, UCM 1099), N-{2-[(3-Hydroxymethylphenyl)(3-methoxyphenyl)amino]ethyl} acetamide (compound 14, UCM 1118), or

N-{2-[(3-Hydroxyphenyl)(3-methoxyphenyl)amino]ethyl} acetamide (compound 15, UCM 1119).

Preferred compounds of formula (l.a) are those selected from Table a:

Table a

Preferred compounds of formula (l.b) are selected from those of Table b:

Table b

In another aspect, there is provided a pharmaceutical composition comprising the compounds or pharmaceutically acceptable salts thereof according to Formula (l.a) and/or Formula (l.b), and one or more optional pharmaceutically acceptable carriers or excipients.

Such compositions may be prepared in a manner well known in the pharmaceutical art by mixing the compounds or pharmaceutically acceptable salts thereof having a suitable degree of purity with one or more optional pharmaceutically acceptable carriers or excipients. The carrier/excipient can be suitable for administration of the compounds or pharmaceutically acceptable salts thereof by any conventional administration route, for example, for oral, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration. In an embodiment, the carrier/excipient is adapted for administration of the compounds or pharmaceutically acceptable salts thereof by the oral route. In an embodiment, the pharmaceutical composition comprises from 0.1 % to 99% by weight of the compound/s of Formula (la) or (lb) disclosed herein or a pharmaceutically acceptable salt thereof, and from 99.9% to 1% by weight of one or more optional pharmaceutically acceptable carriers or excipients.

In another embodiment, the pharmaceutical composition comprises from 10% to 60% by weight of the compound/s of Formula (la) or (lb) disclosed herein or a pharmaceutically acceptable salt thereof, and from 90% to 40% by weight of one or more optional pharmaceutically acceptable carriers or excipients.

In an embodiment, the pharmaceutical composition comprises from 20% to 50% by weight of the compound of Formula (la) or (lb) disclosed herein or a pharmaceutically acceptable salt thereof, 30% to 40% by weight of the compound of Formula I disclosed herein or a pharmaceutically acceptable salt thereof, 43% to 47% by weight of the compound of Formula (la) or (lb) disclosed herein or a pharmaceutically acceptable salt thereof.

The compounds according to the invention may be administered orally in the form of solutions which may contain colouring and/or flavouring agents. The compounds according to the invention may also be administered sublingually in the form of tracheas or lozenges in which the active ingredient(s) is/are mixed with sugar or corn syrups, flavouring agents and dyes, and then dehydrated sufficiently to make the mixture suitable for pressing into solid form.

The compositions described herein may be in the form of tablets, coated tablets, capsules, caplets, powders, granules, lozenges, suppositories, reconstitutable powders, syrups, liquid preparations such as oral or sterile parenteral solutions or suspensions, as well as injectable formulations and transdermal formulations.

The solid oral compositions may be prepared by conventional methods of blending, granulation, compression, coating, filling, tabletting, or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are, of course, conventional in the art. The tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.

Oral liquid preparations may be in the form of emulsions, suspensions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may or may not contain conventional additives. Non limiting examples of conventional additives include suspending agents such as sorbitol, syrup, natural gums, agar, methyl cellulose, gelatin, pectin, sodium alginate, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fats; emulsifying agents such as sorbitan monooleate or acaci; non-aqueous vehicles (which may include edible oils) such as almond oil, fractionated coconut oil, oily esters selected from the group consisting of glycerine, propylene glycol, ethylene glycol, and ethyl alcohol; preservatives such as for instance methyl para-hydroxybenzoate, ethyl para hydroxybenzoate, n-propyl parahydroxybenzoate, n-butyl parahydroxybenzoate or sorbic acid; and, if desired conventional flavoring such as saccharose, glycerol, mannitol, sorbitol, or coloring agents. For parenteral administration, fluid unit dosage forms may be prepared by utilizing the melatonin ligands or pharmaceutically acceptable salts thereof and a sterile vehicle (i.e. sterile water), and, depending on the concentration employed, the melatonin ligands or pharmaceutically acceptable salts thereof may be either suspended or dissolved in the vehicle. Other suitable vehicles may include olive oil, ethyl oleate, and glycols. If needed, a suitable quantity of lidocaine hydrochloride may also be included. Once in solution, the melatonin ligands or pharmaceutically acceptable salts thereof may be injected and filter sterilized before filling a suitable vial or ampoule followed by subsequently sealing the carrier or storage package. Adjuvants, such as a local anesthetic, a preservative or a buffering agent, may be dissolved in the vehicle prior to use. Stability of the pharmaceutical composition may be enhanced by freezing the composition after filling the vial and removing the water under vacuum, (e.g., freeze drying).

The melatonin ligands or pharmaceutically acceptable salts thereof may be administered in the form of suppositories. Suppositories may contain pharmaceutically acceptable vehicles such as cocoa butter, polyethylene glycol, sorbitan, esters of fatty acids, lecithin and the like.

The present disclosure relates to the use of the compounds or pharmaceutically acceptable salts thereof described herein (or pharmaceutical compositions comprising same) for managing or treating a disease or disorder associated with melatonin MT2 receptor activity such as sleep disorders, major depression disorder, mood disorders, anxiety disorders, cognitive disorders, attention deficit and hyperactivity disorder, dementia, metabolic diseases included diabetes Type 1 and type 2, circadian rhythms disorders.

Thus, in another aspect, the present disclosure relates to a method for managing or treating a disease or disorder associated with melatonin MT2 receptor activity in a subject in need thereof comprising administering to the subject an effective amount of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the same, described herein. The present disclosure also relates to the compounds of Formula (l.a) or (l.b), pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, for use in the treatment of a disease or disorder associated with melatonin MT2 receptor activity in a subject. The present disclosure also relates to the use of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein for the manufacture of a medicament for managing or treating a disease or disorder associated with melatonin MT2 receptor activity in a subject. The present disclosure also relates to the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein, for use in managing or treating a disease or disorder associated with melatonin MT2 receptor activity in a subject.

In another aspect, the present disclosure relates to a method for treating a sleep, chronobiological and/or circadian rhythm disorder in a subject comprising administering to the subject an effective amount of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the same, described herein. The present disclosure also relates to the use of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein for treating a sleep, chronobiological and/or circadian rhythm disorder in a subject. The present disclosure also relates to the use of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein for the manufacture of a medicament for treating a sleep, chronobiological and/or circadian rhythm disorder in a subject. The present disclosure also relates to the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein for use in treating a sleep, chronobiological and/or circadian rhythm disorder in a subject. In an embodiment, the sleep disorder is insomnia or sleep-wake disorder. In an embodiment, the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, improve the quality of sleep, sleep latency and/or daytime function.

In another aspect, the present disclosure relates to a method for treating a metabolic disorder in a subject comprising administering to the subject an effective amount of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same described herein. The present disclosure also relates to the use of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same described herein for treating a metabolic disorder in a subject. The present disclosure also relates to the use of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same described herein for the manufacture of a medicament for treating a metabolic disorder in a subject. The present disclosure also relates to the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same described herein for use in treating a metabolic disorder in a subject. Examples of metabolic disorders include impaired glucose tolerance, insulin resistance and diabetes. In an embodiment, the metabolic disorder is diabetes, such as type 2 diabetes.

In another aspect, the present disclosure relates to a method for treating a neuropsychiatric disorder in a subject comprising administering to the subject an effective amount of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein. The present disclosure also relates to the use of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein for treating a neuropsychiatric disorder in a subject. The present disclosure also relates to the use of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein for the manufacture of a medicament for treating a neuropsychiatric disorder in a subject. The present disclosure also relates to the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein for use in treating a neuropsychiatric disorder in a subject. Neuropsychiatric disorders include dementia, attention deficit disorders, cognitive deficit disorders, autism spectrum disorder, migraine headaches, addictions, eating disorders as well as mood disorders such as depression and anxiety. In an embodiment, the neuropsychiatric disorder is a mood disorder. In a further embodiment, the mood disorder is depression, for example major depressive disorder or seasonal affective disorder (SAD). In a further embodiment, the mood disorder is anxiety.

In another aspect, the present disclosure relates to a method for alleviating pain in a subject comprising administering to the subject an effective amount of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein. The present disclosure also relates to the use of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein for alleviating pain in a subject. The present disclosure also relates to the use of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein for the manufacture of a medicament for alleviating pain in a subject. The present disclosure also relates to the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising same, described herein for use in alleviating pain in a subject. In an embodiment, the pain to be alleviated or treated is chronic pain. In an embodiment, the pain to be alleviated or treated is acute pain. The acute pain may be, for example, acute tonic pain, pain relating to surgery (e.g. post-surgical pain, surgical pain), and/or pain relating to trauma. In an embodiment, the pain is hyperalgesia pain or allodynic pain. In another embodiment, the pain is myalgic pain and/or inflammatory pain (e.g., pain associated with an inflammatory disease or condition). In another embodiment the pain is neuropathic pain and/or nociceptive pain. In a further embodiment, the nociceptive pain is visceral pain or somatic pain, for example musculo-skeletal pain or post-traumatic pain. In another embodiment, the neuropathic pain is peripheral neuropathic pain or central neuropathic pain. In a further embodiment, the pain is back pain or joint pain. In yet another embodiment, the pain is head pain (e.g., headache). In another embodiment, the pain is pain associated with a disorder or condition. In an embodiment, the disorder or condition is chosen from fibromyalgia, irritable bowel syndrome, arthritis, ulcer, diabetic neuropathy, sciatica and migraine. In another embodiment, the ulcer is a gastric ulcer. In another embodiment the pain associated to vulvodynia. The skilled person would understand that one or more types of pain can for example be treated and/or alleviated at the same time.

The present invention also discloses a use of compound of Formula (la) or compound (lb) for preparing a prodrug.

These prodrugs are poorly active or inactive compound containing a parental drug that undergoes some in vivo biotransformation through chemical or enzymatic cleavage, enabling the delivery of said parental drug at efficacious levels (Jornada, dos Santos Fernandes et al. Molecules, 2015 Dec 29;21(1):42. 2015).

The present invention also discloses a compound of Formula (la) or a compound of Formula (lb) as defined herewith for preparing a prodrug.

The present disclosure also discloses a prodrug of compound of Formula (la) or of compound of Formula (lb) as defined herewith.

Prodrugs of formula (la) or compound (lb) may include but are not limited to aminoacid prodrugs, ester prodrugs and carbamate prodrugs.

Preferably, the prodrugs of compound formula (la) or compound (lb) are selected from the group consisting of aminoacid prodrugs, ester prodrugs and carbamate prodrugs.

Advantageously, prodrugs may optimize the physicochemical and pharmacological properties of Formula (la) or compound (lb) to improve their solubility and pharmacokinetics. The prodrugs as described here can also be comprised in the compositions as described and claimed herewith, and can be used for the medical uses described in the present contest.

EXPERIMENTAL PART

General procedures

The compounds of Formula (l.a) can be prepared according to the method reported below in scheme 1. Other procedures, as well as variations thereof, could also be employed for preparing the compounds of Formula (l.a), these should be all considered part of the present invention.

Synthesis Examples

Compounds 6a-c were prepared as described in the scheme 1. The monosubstituted urea (6a) was synthesized by reaction of the crude N ¹ -3-(methoxyphenyl)-N ¹ -phenylethane-l,2- diamine with potassium cyanate in acidic aqueous medium; dissymmetric disubstituted analogues (6b-c) were obtained by treatment of the same ethane-1, 2-diamine with ethyl- or n-propyl isocyanate.

The crude N ¹ -3-(methoxyphenyl)-N ¹ -phenylethane-l, 2-diamine was prepared following a previously reported procedure involving the N-cyanoalkylation of 3-methoxy-N- phenylaniline (4a) with bromoacetonitrile in the presence of sodium hydride, and subsequent hydrogenation of the intermediate nitrile 5 with Raney Nickel, in the presence of 1M NHs-EtOH.

Scheme 1: Reagents and conditions: a) BrCFhCN, NaH, DMF, 100 °C, 24 h; b) H2, Raney-Ni, NHs-EtOH, THF, 60 °C, 6h; c) KOCN, AcOH/THF/H ₂ 0, r.t., 4 h (for 6a); d) RNCO, DCM, r.t, 0.5 h (for 6b-c).

The compounds of Formula (l.b) can be prepared according to the method reported below in schemes 2 and 3. Other procedures, as well as variations thereof, could also be employed for preparing these compounds, and are as well part of the present invention.

Scheme 2. Synthesis of N-[2-(diarylamino)ethyl]amides 10-11, 13-15 and N-[2-(N- phenethylanilino)ethyl]acetamide 12. The (anilinoethyl)acetamido derivatives (7-12) were obtained by reductive /V-alkylation of the suitable aniline 4b-g with /V-(2,2-dimethoxyethyl)acetamide in the presence of triethylsilane/trifluoroacetic acid (scheme 2). The N- pyridine aniline derivative 13 was synthesized by palladium-catalyzed amination [Pd2(dba)3/XantpPhos/t-BuONa] of 7 with 4- bromopyridine hydrochloride (scheme 2). To prepare the 3-(hydroxymethyl)phenyl derivative 14, the corresponding methyl ester 8 was reduced with lithium aluminum hydride, while the 3-hydroxyphenyl analog 15 was obtained by O-debenzylation of compound 9, using Ph/Pd-C (scheme 2). Compounds 7, 8 and 9 are intermediate of reaction and can be used to prepare respectively compounds 13, 14, and 15. These intermediates are also disclosed and claimed herewith.

The /V-methyl-2-substituted-aniline derivatives 18-19 were prepared by reductive N- alkylation of the corresponding 2-substituted anilines 4h-i with N-( 2,2- dimethoxyethyl)acetamide in the presence of triethylsilane/trifluoroacetic acid, followed by /V-methylation with Mel (18) or reductive amination with HCHO/NaBHsCN (19), as depicted in Scheme 3.

Scheme 3. Synthesis of N-[2-(2-substituted-5-methoxyanilino)ethyl]acetamides 18-19 Reagents and conditions: a) H2, 10% Pd-C, EtOAc/EtOH, r.t., 6h; b) TES, TFA, DCM, r.t., 2 h; c) Mel, NaHCOs, MeOH, 50 °C, 24 h (for 18); d) HCHO (37% in aqueous solution), NaBH ₃ CN, MeOH/AcOH, r.t., 1 h (for 19).

The key starting anilines are commercially available (4a-b, 4i) or have been synthesized using known procedures for similar compounds as outlined in Scheme 4. Briefly, the 6-phenyl-m- anisidine 4h was prepared by hydrogenation (Eh/Pd-C) of 4-methoxy-2-nitro-l,l'-biphenyl (Scheme 3).

The /V-arylanilines 4d-f were prepared by palladium-catalyzed amination of the suitable aniline with l-(benzyloxy)-3-bromobenzene, 3-bromothiophene or 3- (difluoromethyl)bromobenzene (Scheme 4). The /V-phenethylaniline 4g was prepared by N- acylation of 3-methoxyaniline with phenylacetyl chloride and subsequent amide reduction by treatment with lithium aluminum hydride (Scheme 4).

Compounds 4a-4f and 4i are starting materials disclosed and claimed herewith.

Scheme 4. Synthesis of N,N-diarylamines (4d-f) and /V-phenethyl-3-methoxyaniline (4g).

Scheme 4: Synthesis of N,N-diarylamines (4d-f) and N-phenethyl-3-methoxyaniline (4g). Reagents and conditions: a) t-BuONa, Pd2(dba)3, XantPhos for 4d, RuPhos for 4f toluene, 100 °C, 18 h; b) TEA , DCM, r.t. 0.5 h; c) UAIH4, THE, reflux, 1 h; d) BINAP, Pd(0Ac)2, t-BuOK, toluene, 100 °C, 3 h.

Materials and methods

Melting points were determined on a Buchi B-540 capillary melting point apparatus and are uncorrected. ¹ H NMR and ¹³ C NMR spectra were recorded on a Bruker AVANCE 200 or 400, using CDCU as solvent unless stated otherwise. Chemical shifts (d scale) are reported in parts per million (ppm) relative to the central peak of the solvent; coupling constants (J) are given in hertz (Hz). ESI MS spectra were taken on a Waters Micromass ZQ instrument; molecular ions [M+H] ⁺ are given. High-resolution mass spectroscopy was performed on a Micromass Q- ToF Micro mass spectrometer (Micromass, Manchester, UK) using an ESI source. The purity of tested compounds, determined by high performance liquid chromatography (HPLC), was greater than 95%. These analyses were performed on a Waters HPLC/DAD/MS system (separation module Alliance HT2795, Photo Diode Array Detector 2996, mass detector Micromass ZQ; software: MassLynx 4.1). Column chromatography purifications were performed under "flash" conditions using Merck 230-400 mesh silica gel. Analytical thin- layer chromatography (TLC) was carried out on Merck silica gel 60 F254 plates.

3-Methoxy-/V-phenylaniline (4a), 3-methoxyaniline (4b), l-(benzyloxy)-3-bromobenzene, 3- (difluoromethyl)bromobenzene, 3-bromothiophene, 2-bromo-5-methoxyaniline (4i) and 4- bromopyridine HCI were purchased from commercial suppliers and used without further purification.

Synthesis Example 1: (l-{2-[(3-Methoxyphenyl)phenylamino]ethyl}urea) (Compound 6a, UCM 1091)

A solution of the nitrile 5 (see scheme 1) (262 mg, 1.1 mmol) in THF (7 mL) and 1M NH3 in EtOH (1.5 mL) was hydrogenated over Raney nickel at 4 atm of H2 for 6 h at 60 °C. The catalyst was filtered on a celite pad, the filtrate was concentrated in vacuo, to give a crude oily amine which was used in the next step without any further purification. A solution of KOCN (150 mg, 1.85 mmol) in H ₂ O (0.85 mL) was added dropwise to a solution of the above crude amine in THF-H ₂ O-ACOH (1-1-0.5 mL) and the resulting mixture was stirred at room temperature for 4 h. The reaction mixture was neutralized with a saturated aqueous solution of NaHC0 ₃ , and then extracted with EtOAc. The combined organic phases were washed with brine, dried (Na2S04) and concentrated under reduced pressure to give a crude residue that was purified by flash chromatography (silica gel; EtOAc as eluent). White solid, mp 78-9°C (diethyl ether-petroleum ether), 45% yield. ^X H NMR (400 MHz, CDCI ₃ ) d: 3.34 (bt, 2H, J = 6.5 Hz), 3.73 (s, 3H), 3.80 (t, 2H, J = 6.5 Hz), 4.57 (brs, 2H), 5.39 (brs, 1H), 6.47 (dd, 1H, J = 2.5 and 8.0 Hz), 6.54-6.57 (m, 2H), 6.96-7.00 (m, 1H), 7.03-7.06 (m, 2H), 7.13 (dd, 1H, h = J ₂ = 8.0 Hz), 7.24-7.28 (m, 2H). ¹³ C NMR (100 MHz, CDCI ₃ ) d: 38.2, 51.8, 55.2, 106.1, 106.3, 112.7, 122.2, 122.4, 129.5, 130.0, 147.6, 149.3, 159.2, 160.6. ESI MS ( m/z ): 286 [M+H] ⁺ . HRMS (ESI): m/z calculated for C ₁₆ H ₂₀ N ₃ O ₂ , [M+H] ⁺ 286.1556. Found: 286.1555. Synthesis Example 2: l-Ethyl-3-{2-[(3-methoxyphenyl)phenylamino]ethyl}urea (6b, UCM

1092)

A solution of the nitrile 5 (see scheme 1) (262 mg, 1.1 mmol) in THF (7 mL) and 1M NH ₃ in EtOH (1.5 mL) was hydrogenated over Raney nickel at 4 atm of H ₂ for 6 h at 60 °C. The catalyst was filtered on a celite pad, the filtrate was concentrated in vacuo, to give the crude oily /V^-S-imethoxyphenylJ-A/^-phenylethane-l, 2-diamine which was used in the next step without any further purification. Ethyl isocyanate (1.2 mmol) was added to a solution of the above crude amine in DCM (4 mL) and the resulting mixture was stirred at room temperature for 30 min. The solvent was removed by distillation under reduced pressure to give a crude residue that was purified by flash chromatography (silica gel, EtOAc/cyclohexane 1 : 1 as eluent). White solid, mp 96-7°C (diethyl ether); 87% yield. ¹ H NMR (400 MHz, CDCI ₃ ) d: 1.06 (t, 3H, J = 7.0 Hz), 3.11 (q, 2H, J = 7.0 Hz), 3.38 (t, 2H, J = 6.5 Hz), 3.73 (s, 3H), 3.82 (t, 2H, J = 6.5 Hz), 4.69 (brs, 1H), 4.90 (brs, 1H), 6.48 (dd, 1H, J = 2.5 and 8.0 Hz), 6.53-6.55 (m, 1H), 6.59 (dd, 1H, J = 2.5 and 8.0 Hz), 6.96-7.00 (m, 1H), 7.05-7.07 (m, 2H), 7.15 (dd, 1H, Ji = J ₂ = 8.0 Hz), 7.24-7.28 (m, 2H). ¹³ C NMR (100 MHz, CDCI ₃ ) d: 15.4, 35.3,

38.3, 52.0, 55.2, 106.1, 106.2, 112.7, 122.0, 122.2, 129.5, 130.0, 147.6, 149.3, 158.4, 160.6. ESI MS (m/z): 314 [M+H] ⁺ . HRMS (ESI): m/z calculated for C ₁₈ H ₂₄ N ₃ O ₂ , [M+H] ⁺ 314.1869. Found: 314.1853.

Synthesis Example 3: l-{2-[(3-Methoxyphenyl)phenylamino]ethyl}-3-propylurea (6c, UCM

1093)

Compound 6c was prepared following the above described procedure for the synthesis of 6b, using n-propyl isocyanate instead of ethyl isocyanate. White solid, mp 63-5°C (diethyl ether-petroleum ether); 91% yield. ^X H NMR (400 MHz, CDCI ₃ ) d: 0.79 (t, 3H, J = 7.5 Hz), 1.32- 1.41 (m, 2H), 2.95 (t, 2H, J = 7.0 Hz), 3.31 (t, 2H, J = 6.5 Hz), 3.65 (s, 3H), 3.75 (t, 2H, J = 6.5 Hz), 4.60 (brs, 1H), 4.71 (brs, 1H), 6.40 (dd, 1H, J = 2.5 and 8.0 Hz), 6.45-6.46 (m, 1H), 6.51 (dd, 1H, J = 2.5 and 8.0 Hz), 6.88-6.90 (m, 1H), 6.92-6.99 (m, 2H), 7.06 (dd, 1H, Ji = J ₂ = 8.0 Hz), 7.16-7.20 (m, 2H). ¹³ C NMR (100 MHz, CDCI ₃ ) d: 11.3, 23.3, 38.4, 42.3, 52.1, 55.2, 106.2,

106.3, 112.7, 122.0, 122.2, 129.4, 130.0, 147.6, 149.3, 158.5, 160.6. ESI MS (m/z): 328 [M+H] ⁺ . HRMS (ESI): m/z calculated for C ₁₉ H ₂₆ N ₃ O ₂ , [M+H] ⁺ 328.2025. Found: 328.2013.

Synthesis Example 4: 3-Benzyloxy-/V-(3-methoxyphenyl)aniline (Compound 4d) (starting material for the synthesis of Compounds according to Formula (lb)

A Schlenk flask was charged with 3-methoxyaniline 4b (see scheme 4) (123 mg, 1 mmol) and l-(benzyloxy)-3-bromobenzene (264 mg, 1 mmol). Dry toluene (10 mL) was added, followed by t-BuONa (144 mg, 1.5 mmol), Pd ₂ (dba) ₃ (18 mg, 0.02 mmol) and XantPhos (34 mg, 0.06 mmol). The mixture was evacuated and purged with argon (3 cycles), then heated to 100 °C under argon for 18 h . The mixture was cooled to room temperature, quenched by addition of water and then extracted with EtOAc. After drying over Na ₂ SC> ₄ , the combined organic layers were concentrated under reduced pressure and the resulting crude product was purified by flash chromatography (silica gel, cyclohexane/EtOAc 8 : 2 as eluent). Amorphous solid, 88% yield. ^X H NMR (200 MHz, CDCI ₃ ) d: 3.78 (s, 3H), 5.05 (s, 2H), 5.73 (brs, 1H), 6.49- 6.74 (m, 5H), 7.13-7.43 (m, 8H). ESI MS (m/z): 306 [M+H] ⁺ . Synthesis Example 5: 3-(Difluoromethyl)-/V-(4-fluorophenyl)aniline (Compound 4e) (starting material for the synthesis of Compounds according to Formula (lb)

A Schlenk flask was charged with Pd(OAc)2 (14 mg, 0.06 mmol), (±)-BINAP (41 mg, 0.06 mmol), t-BuOK (190 mg, 1.7 mmol) 3-(difluoromethyl)bromobenzene (250 mg, 1.2 mmol) and 4-fluoroaniline (133 mg, 1.2 mmol) under nitrogen atmosphere. Dry toluene (2 mL) was added via syringe and after the addition was completed the mixture was stirred at 100 °C for 3 h. After cooling to room temperature the reaction mixture was quenched with water and then extracted with DCM. The combined organic phases were dried over Na2SC>4 and evaporated under reduced pressure to yield a crude product which was purified by flash chromatography (silica gel, cyclohexane/EtOAc 9 : 1 as eluent). Oil, 44% yield. ¹ H NMR (200 MHz, CDCIs) d: 5.70 (brs, 1H), 6.58 (t, 1H, J = 56.5 Hz), 7.03-7.14 (m, 7H), 7.28-7.36 (m, 1H). ESI MS (m/z): 238 [M+H] ⁺ .

Synthesis Example 6: /V-(3-Methoxyphenyl)thiophen-3-amine (Compound 4f)

An oven dried Schlenk tube containing a magnetic stirrer bar was evacuated and backfilled with argon. The tube was then charged with Pd2(dba)3 (11 mg, 0.012 mmol), 2- dicyclohexylphosphino-2',6'-diisopropoxybiphenyl (RuPhos, 23 mg, 0.05 mmol), and t-BuONa (82 mg, 0.85 mmol). The tube was evacuated and argon backfilled once and capped with a septa. 3-Bromothiophene (99 mg, 58 pi, 0.61 mmol) and 3-methoxyaniline 4b (91 mg, 83 mI, 0.74 mmol) were added via syringe followed by toluene (1.25 mL). The tube was sealed under a positive pressure of argon with a Teflon screw cap and placed into a pre-hated oil bath (100 °C) for 18 h. The reaction mixture was cooled to room temperature and filtered through a celite pad washing with EtOAc. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (silica gel, cyclohexane/EtOAc 9 : 1 as eluent). Oil, 92% yield. ^X H NMR (200 MHz, CDCI ₃ ) d: 3.79 (s, 3H), 5.71 (brs, 1H), 6.44 (dd, 1H, J = 2.0 and 8.5 Hz), 6.55-6.58 (m, 2H), 6.77-6.79 (m 1H), 6.94 (dd, 1H, J = 1.5 and 5.0 Hz), 7.16 (dd, 1H, Ji = J ₂ = 8.5 Hz), 7.25-7.29 (m,lH). ESI MS (m/z): 206 [M+H] ⁺ .

Synthesis Example 7: 3-Methoxy-/V-phenethylaniline (Compound 4g)

First, /V-(3-Methoxyphenyl)-2-phenylacetamide was prepared. A solution of 2-phenylacetyl chloride (0.4 mL, 2.47 mmol) in DCM (3.5 mL) was added dropwise to an ice-cooled solution of 3-methoxyaniline 4b (300 mg, 2.44 mmol) and TEA (0.51 mL, 3.66 mmol) in DCM (3.5 mL), and the resulting reaction mixture was stirred at room temperature for 30 min. After dilution with DCM the mixture was washed with a saturated aqueous solution of NaHC0 ₃ followed by brine. After drying over Na2SC>4, the solvent was removed by distillation to give a crude residue that was purified by flash chromatography (silica gel; cyclohexane/EtOAc 8 : 2 as eluent). White solid, 84% yield. Physicochemical data are in agreement with those previously reported.

Solid UAIH4 (154 mg, 3.95 mmol) was added portionwise to a stirred ice-cooled solution of /V-(3-methoxyphenyl)-2-phenylacetamide (320 mg, 1.33 mmol) in dry THF (15 mL) under nitrogen atmosphere. Upon completion of the addition, the mixture was refluxed for 1 h. The unreacted UAIH4 was destroyed by careful addition of water at 0 °C, and the resulting mixture was filtered through a celite pad. The filtrate was concentrated in vacuo, and the residue was partitioned between EtOAc and water. The combined organic phases were washed with brine, dried (Na2SC>4), and concentrated by distillation under reduced pressure to give a crude residue which was purified by flash chromatography (silica gel, cyclohexane/EtOAc 8 : 2 as eluent). Oil; 70% yield. Physicochemical data are in agreement with those previously reported.

Synthesis Example 8: 4-Methoxy-[l,l'-biphenyl]-2-amine (Compound 4h)

A solution of 4-methoxy-2-nitro-l,l'-biphenyl (530 mg. 2.31 mmol) in a 1:1 mixture of EtOAc and EtOH (46 mL) was hydrogenated over 10% Pd-C (43 mg) at 4 atm of Eh for 6 h at room temperature. The catalyst was filtered on a celite pad, the filtrate was concentrated in vacuo, to give the corresponding amine, which was used without any further purification. Oil, 99%. Physicochemical data are in agreement with those previously reported.

General procedure for reductive /V-alkylation of anilines. TFA (1 mL) and TES (0.4 mL, 2.5 mmol) were added to a solution of the opportune aniline (1 mmol) and N-( 2,2- dimethoxyethyl)acetamide (206 mg, 1.4 mmol) in DCM (2 mL), and the resulting mixture was stirred at room temperature for 2 h under a nitrogen atmosphere. After cooling to 0 °C, the reaction mixture was carefully neutralized with NaHC0 ₃ aqueous saturated solution of and diluted with DCM. The aqueous phase was extract with DCM and the combined organic phases were washed with brine, and dried over Na2SC>4. The solvent was removed by distillation, and the crude residue was purified by column chromatography to afford the desired compound.

Synthesis Example 9: Methyl 3-[(2-acetamidoethyl)(3-methoxyphenyl)amino) benzoate (Intermediate Compound 8)

Compound 8 was prepared following the above-described general procedure starting from 4c. Flash chromatography: silica gel, EtOAc as eluent. Oil, 81% yield. ¹ H NMR (200 MHz, CDCIs) d: 3.80 (s, 3H), 3.91 (s, 3H), 5.83 (brs, 1H), 6.54 (dd, 1H, J = 2.0 and 8.0 Hz), 6.65-6.71 (m, 2H), 7.21 (dd, 1H, Ji = J ₂ = 8.0 Hz), 7.26-7.38 (m, 2H), 7.58-7.61 (m, 1H), 7.74-7.75 (m, 1H). ESI MS (m/z): 258 [M+H] ⁺ .

Synthesis Example 10: /V-{2-[(3-Benzyloxyphenyl)(3-methoxyphenyl)amino]ethyl} acetamide (Intermediate Compound 9)

Compound 9 was prepared following the above-described general procedure starting from 4d. Flash chromatography: silica gel, EtOAc as eluent. Oil, 80% yield. ^X H NMR (400 MHz, CDCIs) d: 1.92 (s, 3H), 3.49 (m, 2H), 3.77 (s, 3H), 3.86 (t, 2H, J = 6.5 Hz), 5.02 (s, 2H), 5.62 (brs, 1H), 6.60 (dd, 1H, J = 2.5 and 8.0 Hz), 6.60-6.63 (m, 2H), 6.65-6.66 (m, 2H), 7.17-7.21 (m, 2H), 7.32-7.41 (m, 6H). ESI MS (m/z) 391 [M+H] ⁺ .

Synthesis Example 11: /V-(2-{[3-(Difluoromethyl)phenyl](4- fluorophenyl)amino}ethyl)acetamide (Compound 10, UCM 1097)

Compound 10 was prepared following the above-described general procedure starting from 4e. Flash chromatography: silica gel, EtOAc/cyclohexane 7 : 3 as eluent. White solid, mp 76- 7°C (diethyl ether-petroleum ether); 77% yield. ^X H NMR (400 MHz, CDCI ₃ ) d: 1.95 (s, 3H), 3.48 (m, 2H), 3.85 (t, 2H, J = 6.5 Hz), 5.80 (brs, 1H), 6.54 (t, 1H, J = 56.5 Hz), 6.93-6.97 (m, 3H), 7.04-7.14 (m, 4H), 7.25-7.60 (m, 1H). ¹³ C NMR (100 MHz, CDCI ₃ ) d: 23.1, 37.6, 51.3, 113.4 (t, J = 6.2 Hz), 114.8 (t, J = 237.2 Hz), 116.4 (t, J = 6.2 Hz), 116.7 (d, J = 22.3 Hz), 118.8 (t, J = 1.8 Hz), 126.7 (d, J = 8.2 Hz), 129.7, 135.5 (t, J = 21.8 Hz), 142.8 (d, J = 3.0 Hz), 148.7, 159 7 (d, J = 43.0 Hz), 170.6. ESI MS (m/z): 323 [M+H] ⁺ . HRMS (ESI): m/z calculated for C17H18F3N2O, [M+H] ⁺ 323.1371. Found: 323.1359.

Synthesis Example 12: /V-{2-[(3-Methoxyphenyl)(thiophen-3-yl)amino]ethyl}acetamide (Compound 11, UCM 1096)

Compound 11 was prepared following the above-described general procedure starting from 4f. Flash chromatography: silica gel, EtOAc as eluent. Yellowish solid, mp 76-7 °C (diethyl ether-petroleum ether); 66% yield. ^X H NMR (400 MHz, CDCI3) d: 1.93 (s, 3H), 3.50 (m, 2H), 3.77 (s, 3H), 3.83 (t, 2H, J = 6.5 Hz), 5.71 (brs, 1H), 6.47 (dd, 1H, J = 2.5 and 8.0 Hz), 6.54-6.56 (m, 1H), 6.59 (dd, 1H, J = 2.5 and 8.0 Hz), 6.70-6.78 (m, 1H), 6.88 (d, 1H. J = 5.0 Hz), 7.15 (t, 1H, Ji = J ₂ = 8.0 Hz), 7.25 (d, 1H, J = 5.0 Hz). ¹³ C NMR (100 MHz, CDCI3) d: 23.2, 37.8, 52.0,

55.2, 104.7, 105.8, 110.4, 111.0, 124.1, 125.4, 129.9, 146.4, 149.5, 160.6, 170.4. ESI MS (m/z): 291 [M+H] ⁺ . HRMS (ESI): m/z calculated for C15H19N2O2S, [M+H] ⁺ 291.1167. Found: 291.1167.

Synthesis Example 13: /V-{2-[(3-Methoxyphenyl)(phenethyl)amino]ethyl}acetamide (Compound 12, UCM 1098)

Compound 12 was prepared following the above-described general procedure starting from 4g. Flash chromatography: silica gel, EtOAc as eluent. Oil, 36% yield. ^X H NMR (400 MHz, CDCI3) d: 1.83 (s, 3H), 2.79 (t, 3H, J = 7.5 Hz), 3.23-3.27 (m, 4H), 3.45 (t, 2H, J = 7.5 Hz), 3.74 (s, 3H), 5.40 (brs. 1H), 6.21-6.38 (m, 3H), 7.06-7.25 (m, 6H). ¹³ C NMR (100 MHz, CDCI ₃ ) d:

23.2, 33.3, 37.5, 50.8, 53.1, 55.2, 99.5, 101.8, 105.9, 126.4, 128.6, 128.9, 130.2, 139.5, 149.2, 161.0, 170.3. ESI MS (m/z): 313 [M+H] ⁺ . HRMS (ESI): m/z calculated for Ci ₉ H ₂₄ N ₂ 0 ₂ Na, [M+H] ⁺ 335.1735. Found: 335.1747.

Synthesis Example 14: /V-{2-[(3-Methoxyphenyl)(pyridine-4-yl)amino]ethyl}acetamide (Compound 13, UCM 1099)

A Schlenk flask was charged with 4-bromopyridine HCI (100 mg, 0.48 mmol) and N-{ 2-[(3- methoxyphenyl)amino]ethyl}acetamide 7 (121 mg, 0.62 mmol). Dry toluene (5 mL) was added, followed by t-BuONa (115 mg, 1.2 mmol), Pd2(dba) ₃ (9 mg, 0.01 mmol) and XantPhos (17 mg, 0.03 mmol). The mixture was evacuated and purged with argon (3 cycles), then heated to 100 °C for 16 h under argon. The mixture was cooled to room temperature, quenched by addition of water and then extracted with EtOAc. After drying over Na2S04, the combined organic layers were concentrated by distillation under reduced pressure and the resulting crude product was purified by filtration on a pad of silica gel (DCM/MeOH 9 : 1 as eluent). Oil; 95% yield. ^X H NMR (400 MHz, CDCI ₃ ) d: 1.90 (s, 3H), 3.45 (m, 2H), 3.78 (s, 3H), 3.84 (t, 2H, J = 6.5 Hz), 6.61 (dd, 2H, J = 1.5 and 5.0 Hz), 6.71 (brt, 1H), 6.73-6.75 (m, 1H), 6.78 (ddd, 1H, J = 1.0, 2.5 and 8.0 Hz), 6.84 (ddd, 1H, J = 1.0, 2.5 and 8.0 Hz), 7.33 (t, 1H, Ji = J ₂ = 8.0 Hz). 8.08 (dd, 2H, J = 1.5 and 5.0 Hz). ¹³ C NMR (100 MHz, CDCI3) d: 23.0, 37.3, 50.4, 55.4, 108.6, 112.5, 113.3, 119.5, 131.0, 145.4, 148.9, 153.9, 161.2, 171.0. ESI MS (m/z): 286 [M+H] ⁺ . HRMS (ESI): m/z calculated for C16H20N3O2, [M+H] ⁺ 286.1556. Found: 286.1549. Synthesis Example 15: /V-{2-[(3-Hydroxymethylphenyl)(3-methoxyphenyl)amino]ethyl} acetamide (Compound 14, UCM 1118)

A solution of compound 8 (150 mg, 0.44 mmol) in dry THF (3 mL) was added dropwise to a stirred ice-cooled suspension of LiAII-U (34 mg, 0.88 mmol) in dry THF (3 mL) under a nitrogen atmpsphere, and the resulting mixture was stirred at 0 °C for 1 h. The unreacted UAIH4 was destroyed by careful addition of water at 0 °C, and the resulting mixture was filtered through a celite pad. The filtrate was concentrated in vacuo, and the residue was partitioned between EtOAc and water. The organic phases were combined and washed with brine, dried (Na2SC>4), and concentrated by distillation under reduced pressure to yield a crude residue, which was purified by flash chromatography (silica gel, EtOAc as eluent). Oil, 84% yield. ^X H NMR (400 MHz, CDCI3) d: 1.84 (s, 3H), 3.36 (brs, 1H), 3.40 (m, 2H), 3.72 (s, 3H), 3.80 (t, 1H, J = 6.5 Hz), 4.57 (s, 2H), 6.35 (brt, 1H), 6.46 (ddd, 1H, J = 1.0, 2.5 and 8.0 Hz), 6.52-6.54 (m, 1H), 6.56 (ddd, 1H, J = 1.0, 2.5 and 8.0 Hz), 6.90-6.93 (m, 2H), 7.08-7.09 (m, 1H), 7.12 (t, 1H, Ji = J ₂ = 8.0 Hz), 7.20 (t, 1H, Ji = J ₂ = 8.0 Hz). ¹³ C NMR (100 MHz, CDCI3) d: 22.9, 37.7, 50.9, 55.2, 64.8, 106.3, 106.4, 112.8, 120.0, 120.7, 120.9, 129.4, 130.0, 142.7, 147.6, 149.1, 106.6, 171.1. ESI MS (m/z): 315 [M+H] ⁺ . HRMS (ESI): m/z calculated for Ci ₈ H ₂ 2N ₂ 03Na, [M+H] ⁺ 337.1528. Found: 337.1541.

Synthesis Example 16: /V-{2-[(3-Hydroxyphenyl)(3-methoxyphenyl)amino]ethyl} acetamide (Compound 15, UCM 1119)

A solution of compound 6 (120 mg, 0.4 mmol), in MeOH (10 mL) was hydrogenated over 10% Pd-C (20 mg, 0.02 mmol) at 1 atm of H ₂ for 2.5 h at room temperature. The catalyst was filtered on a celite pad, the filtrate was concentrated in vacuo, and the crude residue was purified by flash chromatography (silica gel, EtOAc as eluent). White solid, mp 110 °C (diethyl ether); 80% yield. ^X H NMR (400 MHz, CDCI ₃ ) d: 1.93 (s, 3H), 3.47 (m, 2H), 3.76 (s, 3H), 3.83 (t, 2H, J = 6.5 Hz), 5.76 (brs, 1H), 6.47 (ddd, 1H, J = 1.0, 2.5 and 8.0 Hz), 6.52 (ddd, 1H, J = 1.0, 2.5 and 8.0 Hz), 6.55 (ddd, 1H, J = 1.0, 2.5 and 8.0 Hz), 6.59 (dd, 1H, Ji =J ₂ = 2.5 Hz), 6.61 (dd, 1H, Ji = J ₂ = 2.5 Hz), 6.65 (ddd, 1H, J = 1.0, 2.5 and 8.0 Hz), 7.09 (dd, 1H, Ji = J ₂ = 8.0 Hz), 7.18 (dd, 1H, Ji = J ₂ = 8.0 Hz). ¹³ C NMR (100 MHz, CDCI3) d: 23.1, 38.0, 51.0, 55.3, 107.3, 107.5, 107.8, 108.7, 112.5, 114.3, 130.1, 130.2, 148.8, 149.0, 157.3, 160.7, 171.0. ESI MS (m/z): 301 [M+H] ⁺ . HRMS (ESI): m/z calculated for C17H21N2O3, [M+H] ⁺ 301.1552. Found: 301.1563.

Synthesis Example 17: /V-{2-[(4-Methoxy-[l, -biphenyl]-2-yl)amino]ethyl}acetamide (Compound 16)

Compound 16 was prepared following the above-described reductive /V-alkylation general procedure starting from 4h. Flash chromatography: silica gel, EtOAc as eluent. Amorphous solid, 65% yield. ^X H NMR (200 MHz, CDCI ₃ ) d: 1.93 (s.3H), 3.23-3.29 (m, 2H), 3.37-3.46 (m, 2H), 3.84 (s, 3H), 4.20 (brs, 1H), 5.65 (brs, 1H), 6.34-6.36 (m, 1H), 6.36 (dd, 1H, J = 2.5 and 8.0 Hz), 7.03 (d, 1H, J = 8.0 Hz), 7.31-7.49 (m, 5H). ESI MS (m/z): 285 [M+H] ⁺ . Synthesis Example 18: /V-{2-[(2-Bromo-5-methoxyphenyl)amino]ethyl}acetamide (Compound 17)

Compound 17 was prepared following the above-described reductive /V-alkylation general procedure starting from 4i. Flash chromatography: silica gel, EtOAc as eluent. Amorphous solid, 70% yield. ^X H NMR (200 MHz, CDCI ₃ ) d: 2.02 (s, 3H), 3.29-3.35 (m, 2H), 3.48-3.57 (m, 2H), 3.78 (s, 3H), 4.58 (brs, 1H), 5.84 (brs. 1H), 6.18 (dd, 1H, J = 3.0 and 9.0 Hz), 6.26 (d, 1H, J = 3.0 Hz), 7.30 (d, 1H, J = 9.0 Hz). ESI MS (m/z): 287 [M+H] ⁺ .

Synthesis Example 19: /V-{2-[(4-Methoxy-[l, -biphenyl]-2- yl)(methylamino)]ethyl}acetamide (Compound 18, UCM 1094)

A suspension of compound 16 (284 mg, 1 mmol), NaHC0 ₃ (84 mg, 1 mmol) and methyl iodide (0.4 mL, 6.5 mmol) in dry MeOH (11 mL) was heated at 50 °C for 24 h. After removing the solvent by distillation in vacuo, the residue was poured into water, extracted with EtOAc, and the combined organic phases were washed with brine and dried (Na2S04). The solvent was removed by distillation under reduced pressure and the residue was purified by flash chromatography (silica gel, DCM/EtOAc 8 : 2 as eluent). Oil, 74% yield. ^X H NMR (400 MHz, CDCIs) d: 1.65 (s, 3H), 2.54 (s, 3H), 2.71 (t, 2H, J = 6.5 Hz), 3.04 (m, 2H), 3.75 (s, 3H), 5.16 (brs, 1H), 6.61 (dd, 1H, J = 2.5 and 8.5 Hz), 6.70 (d, 1H, J = 2.5 Hz), 7.09 (d, 1H, J = 8.5 Hz), 7.20- 7.25 (m, 1H), 7.30-7.36 (m, 4H). ¹³ C NMR (100 MHz, CDCI ₃ ) d: 23.1, 36.9, 40.8, 55.3, 55.5, 107.6, 108.5, 126.6, 128.3, 129.5, 130.1, 132.0, 141.1, 151.9, 159.9, 170.1. ESI MS (m/z): 299 [M+H] ⁺ . HRMS (ESI): m/z calculated for Ci ₈ H ₂₃ N ₂ 0 ₂ , [M+H] ⁺ 299.1760. Found: 299.1751.

Synthesis Example 20: /V-{2-[(2-Bromo-5-methoxyphenyl)methylamino]ethyl}acetamide (Compound 19, UCM 1095)

Sodium cyanoborohydride (100 mg, 1.20 mmol) and a 37% HCHO aqueous solution (0.5 mL) were added to a solution of 17 (143 mg, 0.5 mmol), in MeOH (5 mL) and AcOH (to pH = 5), and the resulting mixture was stirred at room temperature for 1 h. After removing the solvent by distillation in vacuo, the residue was taken up in water and extracted with EtOAc. The combined organic layers were dried (Na ₂ S04) and concentrated by distillation under reduced pressure to give a crude residue which was purified by flash chromatography (silica gel, cyclohexane/EtOAc 6 : 4 as eluent). White solid, mp 94-6 °C (diethyl ether-petroleum ether); 78% yield. ^X H NMR (400 MHz, CDCI ₃ ) d: 1.97 (s, 3H), 2.70 (s, 3H), 3.09 (t, 2H, J = 6.0 Hz), 3.39 (m, 2H), 3.78 (s, 3H), 6.30 (brs. 1H), 6.56 (dd, 1H, J = 3.0 and 9.0 Hz), 6.72 (d, 1H, J = 3.0 Hz), 7.45 (d, 1H, J = 9.0 Hz). ¹³ C NMR (100 MHz, CDCI ₃ ) d: 23.3, 36.8, 41.7, 54.2, 55.5, 109.8, 110.6, 111.7, 133.8, 151.3, 160.0, 170.2. ESI MS (m/z): 301 [M+H] ⁺ . HRMS (ESI): m/z calculated for Ci ₂ Hi ₈ BrN ₂ 0 ₂ , [M+H] ⁺ 301.0552. Found: 301.0552.

Biological Evaluation

Melatonin Receptor Binding and Intrinsic Activity Evaluation

The in vitro pharmacology experiments were conducted at Cerep (Celle-Levescault, France) following radioligand binding and cellular functional assays known in the art. Binding affinities were determined using 2-[ ¹²⁵ l]iodomelatonin as the labeled ligand in competition experiments on cloned human MTi and MT2 receptors expressed in CHO cells. 2-[ ¹²⁵ l]lodomelatonin (10 pM for MTi and 50 pM for MT2) and different concentrations of tested compounds (10 ^_11 -10 ⁷ M) were incubated with the receptor preparation for 60 min at room temperature (MTi) or 90 min at 37 °C (MT2). Nonspecific binding was assessed in the presence of melatonin 1 mM. After incubation, the % of radioligand binding inhibition was determined via a scintillation counting method. The radioligand binding experiments were performed in triplicate. IC50 values were determined by nonlinear regression analysis of the competition curves using Hill equation curve fitting. The pKi values were calculated from the IC50 values in accordance with the Cheng-Prusoff equation.

To define the functional activity of the new compounds, cellular functional assays were performed on CHO cells stably expressing human MTi or MT2 receptors. Agonist activity at MTi receptors was evaluated using a cell impedance assay measured by cellular dielectric spectroscopy, and at MT2 receptors with an cAMP assay, measured by a fluorometric method. The EC50 values were determined by non-linear regression analysis of the concentration-response curves. Experiments were performed in triplicate.

MTi and MT2 binding affinities of the Compounds according to Formula (la) and (lb) are reported in Table 1, with data for reference UCM765 and UCM924 tested in the same experimental conditions.

Table 1: °Ki values are calculated from IC50 values obtained from competition curves by the method of Cheng and Prusoff (Y. Cheng, W. H. Prusoff, Biochem. Pharmacol. 1973, 22, 3099-3108), and are the mean ± standard deviation of three determinations. Physicochemical characterization

Metabolic stability assays in rat and human liver microsomes

Stock solutions of melatonin receptor ligands were prepared in DMSO immediately before use; cosolvent concentration in final samples was kept constant at 1% v/v. Metabolic stability was assessed by incubating each compound (final concentration: 5 mM) in the presence of rat or human liver microsomes (final concentration: 1 mg protein/mL) and of a NADPH-regenerating system (final concentrations: 2 mM NADP ⁺ , 10 mM glucose-6- phosphate, 0.4 U mL ^-1 glucose-6-phosphate dehydrogenase, 5 mM MgCh) in a 10 mM Phosphate Buffered Saline (PBS) solution, pH 7.4, at 37 °C. Reaction mixtures were preheated (37 °C) for 10 min before adding the parent compound. At stated time points, depending on compound kinetics, aliquots of samples were withdrawn, deproteinized with two volumes of acetonitrile, centrifuged (9000 g, 10 min, 4 °C) and the supernatant was directly analyzed by injection in a HPLC system. Apparent half-lives (ti/2 in min) for the disappearance of compounds were calculated from the pseudo first-order rate constants (k) obtained by linear regression of the log chromatographic peak area versus time plots and are reported as means of three experiments ± standard deviation. Intrinsic clearance (CLi) values of the compounds were calculated using the following equation: CLi (mί x min ¹ x mg protein ^-1 ) = k x V where V (mί x mg protein ^-1 ) = incubation volume/mg protein added.

Pooled rat and human liver microsomes (20 mg/mL) were supplied by BD Biosciences (BD Biosciences, Woburn, MA, USA). Acetonitrile and methanol were HPLC grade and were supplied by Sigma-Aldrich (Sigma-Aldrich srl, Milan, Italy). Water was freshly bidistilled before use. All other reagents were purchased in the highest purity available.

Analytical methods for stability assays

Metabolic stability of melatonin receptor ligands was analyzed by direct injection of incubation samples in a HPLC-UV-VIS system. A Shimadzu High Performance Liquid Chromatography (HPLC) system (Shimadzu Corp., Kyoto, Japan) was employed, consisting of a LC-lOADvp solvent delivery module, a 20 pL Rheodyne sample injector (Rheodyne LLC, Rohnert Park, CA, USA) and a SPD-lOAvp UV-VIS detector. Data were acquired and HPLC peaks were integrated by PeakSimple 2.83 software. HPLC columns used were a RP-C18 Supelco Discovery (Supelco, Bellefonte, PA, USA), 5 pm, 150 x 4.6 mm i.d. for all analytes except compound 13, and a Phenomenex Gemini, 5 pm, 150 x 4.6 mm i.d. for compound 13. Mobile phases consisted of water and acetonitrile at different percentages and at a flow rate of 1 mL min ^-1 ; UV wavelength was set at 254 nm. For compound 13, the mobile phase consisted of a 10 mM ammonium acetate buffer pH 7.0 and methanol and the UV absorption was set at the relative maximum of 281 nm.

Experimental lipophilicity

Distribution coefficients for melatonin receptor ligands (log D _oc t, 7.4) were determined employing the reference shake-flask method in the biphasic n-octa no I/water partition system, at room temperature (25±1°C) and at physiological pH 7.4. Chosen buffer was 0.05 M MOPS (3-morpholinopropanesulfonic acid), pH 7.4 to avoid ionic couple partitioning, adjusted to 0.15 M ionic strength with KCI. Compounds, after partitioning overnight, and dilution of both phases with CH3OH, were dosed in the RP-HPLC/UV system, as detailed above. Reported log D _oct, 7.4 values are the mean of three measurements ± standard deviation.

Solubility measurements

Thermodynamic solubility values were measured in two different buffers (pH 1.0; pH 7.4) by the shake flask method. 1 mg of each compound was placed in 1 mL of appropriate buffer and shaken at room temperature overnight. The resulting suspension was centrifuged (10 min, 10000 g, 20 °C) and the supernatant analyzed by RP-HPLC/UV. Solubility data were interpolated from appropriate calibration curves prepared from concentrated DMSO stock solutions. Reported solubility values are the mean of three measurements ± standard deviation.

The solubility, metabolic stability and lipophilicity of the new compounds were experimentally evaluated and are reported in Table 2. Thermodynamic solubility was measured at physiological pH and in acidic conditions (pH = 1). In general, diarylamino derivatives had similar solubility at the two pH values, usually slightly higher at pH = 1. The most soluble compounds were the primary urea 6a, the hydrophilic and basic 4-pyridine derivative 13 and the m-hydroxymethyl-substituted compound 14. Compound 14 had solubility greater than 1 mg/mL at both pH values. A significantly higher solubility at acidic pH than at neutral values was observed for phenylalkylamides 12, 18 and 19, with solubility > 1 mg/mL at pH = 1. Compared to UCM765, compound 19 was significantly more soluble also at pH = 7. Metabolic stability was evaluated assessing rat and human microsomal stabilities, expressed as pseudo half-life and as intrinsic clearance in Table 2. All the compounds were susceptible, albeit at different degrees, to oxidative metabolism, with the exception of the 4-pyridine derivative 13 which remained unaltered after 60 min of incubation with microsomes of both species. The primary urea 6a was significantly more stable than UCM765 and the substituted ureas 6b and 6c, with half-lives recalling those of the metabolically protected UCM924. Alkyl substitution of the urea group contributed to reduce the metabolic stability in microsomes of both species, which appeared to be inversely related to the lipophilicity (LogD _oct, 7.4) of the compounds. The meta-substituted derivatives 14 and 15 showed a slight increase (approximately 1.5 fold) of both rat and human microsomal stability, compared to their unsubstituted precursor UCM765. Besides the primary urea 6a, the most stable derivatives were compounds 10 and 19. Compound 10 is a structural analogue of the metabolically protected UCM924, having similar half-life. Compound 19 is significantly less lipophilic and lacks the metabolically labile phenyl ring, both contributing to its improved microsomal stability.

Table 2. Lipophilicity, Rat and Human Microsomal Stability and Solubility of Melatonergic Ligands.

°LM: Liver Microsomes. ^fa CLi = Intrinsic Clearance (mί min ¹ mg prot ¹ ). Percentage of compound left after 60 min, 37 °C. ^Weighted compound completely dissolved in the chosen buffer at 1000 m /ihί. N.D.: not determined. Experiments were performed in triplicate and values are reported as the mean ± standard deviation.

Taking into consideration binding affinity, solubility and metabolic stability, compounds 14, and 19 were selected for further characterization. The m-hydroxymethyl derivative 14 maintained an MT2-selective behavior like that observed for UCM765 and UCM924, with high receptor binding affinity. Its limited metabolic stability could be improved preparing suitable prodrugs, temporarily masking the hydroxyl group, likely responsible for their short half-life. Additionally, compound 14 is highly soluble at both acidic and neutral pH values. The p-Br-phenylalkylamide 19 is a potent IVHVselective ligand, endowed with high solubility and metabolic stability. The agonist behavior of these compounds was evaluated at MTi and MT2 receptors (Table 3). Compound 19 showed a full agonist profile, with high potency at the MT2 receptor, close to that of melatonin (0.17 nM). Compound 14 behaved as an MT2- selective partial agonist, recalling the behavior observed for UCM765 and UCM924.

Table 3: “ECso values obtained from a cell impedance assay. ^fa The Emax values are referred to the % of melatonin response. ^c ECso values obtained from a cAMP assay. Experiments were performed in triplicate and values are reported as the mean ± standard deviation.

Pharmacokinetic evaluation

The in vivo pharmacokinetic experiments were conducted at Nikem Research (Baranzate - Milan - Italy). PK studies were performed using Sprague-Dawley CD (albino) male rats (7-9 weeks-old, weight 160-200 grams, Charles River Lab. Italia, Calco). Animals were housed under standard conditions and had free access to water and standard laboratory rodent diet. Care and husbandry of animals were in conformity with the institutional guidelines, in compliance with national and international laws and policies (EEC Council Directive 86/609, OJL 358m, 1, Dec. 122, 1987; NIH Guide for the care and Use of Laboratory Animals, NIH Publication No. 86-23, 1985). The compounds were dissolved in water containing 3% DMSO and 20% Encapsin (and a stechiometric amount of HCI for 14) at a concentration of 2.5 mg mL-1 for the IV (bolus) dose, or in water containing 5% DMSO and 10% encapsin (and a stechiometric amount of HCI for 14) at a concentration of 4 mg mL-1 for PO (gavage) dose. Rats were randomly assigned to four treatment groups (n = 3) and received a single IV bolus dose (5 mg kg-1) or a single oral administration (40 mg kg-1) through oral gavage of compound 14 or 19. Serial blood samples (200 mL) were collected from caudal vein at 5, 30, 60, 120, 480 and 1440 min after IV injection, or at 15, 30, 60, 120, 480 and 1440 min after PO administration. Blood samples were collected in heparinized Eppendorfs (Heparin Vister 5000 U.I/ml_Marvecs Pharma), gently mixed and placed on ice; then blood was centrifuged (3500xg, at 4 °C for 15 min), the plasma was collected and immediately frozen at -80 °C until submission to UPLC/MS/MS analysis. For the sample preparation, 100 pL of plasma spiked with 5 pL of internal standard (I.S.; 19 (UCM1095) for compound 14 (UCM118) and 14 (UCM118) for compound 19 UCM1095; 2.5 pg mL-1;) were added to a SW96 deep well plate (Waters) containing 300 pL of acetonitrile. The plate was shaken for 10 min and then centrifuged at 3000 rpm for 15 min. UPLC/MS/MS analyses were performed on an Acquity UPLC, coupled with a sample organizer and interfaced with a triple quadrupole Premiere XE (Waters, Milford, USA). LC runs (inj. vol. 5 pL) were carried out at 50 °C on Acquity BEH C18 columns (1.7 pm, 2.1 x 50 mm) at a flow rate of 0.45 mL min-1. Mobile phases consisted of a phase A (water) and a phase B (0.1% formic acid in acetonitrile). The column was conditioned with 2% phase B for 0.2 min, then brought to 100% phase B within 0.6 min and maintained at these conditions for 1.1 min. Analyses were carried out using a positive electrospray ionization [ESI(+)] interface in multiple reaction monitoring (MRM) mode. Capillary 3.5 Kv; extractor 3V; source T 140 °C, desolvation T 450 °C. Transitions: for compound 14 Q1/Q3 315.2/86, CV 14, CE16 + 315.2/238 CV 14, CE19; for I.S. (compound 19): 301.1/86 CV 14 CE16; LLOQ: 1 ng ml-1. Transitions: for compound 19 Q1/Q3: 301.1/86 CV 14 CE16; for I.S. (compound 14) 315.2/238 CV 14, CE19; LLOQ: 1 ng mL-1. Pharmacokinetic analysis was performed by non-compartmental analysis (NCA) using the WinNonlin 5.1 software (Pharsight, Mountain View, CA, USA. Absolute oral bioavailability (F) was calculated by linear trapezoidal rule using the relationship: F = [doselV x AUCoral o- /doseoral x AUCIV o- ] x 100.

To evaluate the in vivo behavior of the two melatonergic ligands 14 and 19, a preliminary pharmacokinetic (PK) study was carried out in male Sprague-Dawley rats. The compounds were administrated as intravenous bolus (IV) or by oral gavage (PO) at doses of 5 or 40 mg kg-1, respectively. Quantitative analysis was carried out by UPLC/MS/MS and the plasma concentration profiles are shown in Figure 1, while the main PK parameters are listed in Table 4.

Table 4. Main Pharmacokinetic Parameters in Rat Plasma Obtained for Compounds 14 and 19.

IV bolus (5 mg kg ¹ ) PO gavage (40 mg kg ¹ )

Parameter ¹³¹ 14 19 14 19

AUC iv o- _' [ng 66508.2 66017.0 2626.5 19129.8 min mL ¹ + + ± 1037.9 ± 4802.3

9656.1 7842.4

C [ng mL ¹ ] 2757.5 2973.0 75.6 455.7

± 478.6 ± 383.9 ± 39.7 ± 178.5 tin [min] 11.5 13.5 19.8 26.2 ± 1.0 ± 0.3 ± 1.9 ± 0.9

CL [mL min 76.2 76.4 ^g ¹ ] ± 10.3 ± 8.8 [L kg ¹ 0.9 0.7

± 0.2 ± 0.1

F [%] 0.5 3.6 [a]AUC: area under the plasma concentration-time curve of the drug; ti/ ₂ : half-life; CL: volume of plasma cleared of the drug per unit time; V _ss : volume of distribution at the steady state defined by the amount of drug in the body over the concentration of the drug in the plasma at the steady state; F: percentage of the dose reaching blood circulation after oral administration.

Upon single IV administration they displayed limited plasma half-life (compound 14, 11.5 min; compound 19, 13.5 min), and a moderately-high plasma clearance (14, 76.2 mL min- lkg-1; 19, 76.4 mL min-lkg-1). For 14 the Cmax was 2757.5 ng mL-1 while for 19 it was 2973.0 ng mL-1. The steady-state volume of distribution (Vss) was moderate (14, 0.9 L kg-1; 19, 0.7 L kg-1) indicating a moderate propensity to distribute out from the plasma compartment. After PO administration their plasma half-life was longer (14, 19.8 min; 19, 26.2 min) with corresponding Cmax of 75.6 mg mL-1 (14), and 455.7 ng mL-1 (19). The oral bioavailability was very low for 14 and moderate for 19. The PK profile of compound 19, albeit far from optimal, was more favorable than that of compound 14, with greater oral absorption and longer plasma exposure, likely related to its higher in vitro liver microsomal stability. The results are shown in Figure 1.