FIELD OF THE INVENTION

The present invention belongs to the technical field of pharmacy, pharmaceutical chemistry and pharmacology. In particular, the present invention relates to a class of novel compounds of general formula I, their use as medicaments and pharmaceutical compositions comprising them. Specifically, the compounds of the invention are useful as voltage-gated potassium ion channels modulators.

BACKGROUND OF THE INVENTION

Potassium channels represent the most widely diffused and heterogeneous class of ionic channels characterized so far. Potassium channels are responsible for the stabilization of the membrane potential in neurons: they shift the membrane potential toward the K ⁺ equilibrium potential, away from the firing threshold. Potassium channels are classified both structurally and functionally in four main families: inward rectifier potassium channels (Kir); two pores potassium channels (K2p); Calcium-activated potassium channels (KCa) and voltage-gated potassium channels (Kv). (Aldrich R. et al. Potassium channels. IUPHAR/BPS Guide to PHARMACOLOGY) Among the 13 classes of Kv channels, the family identified as Kv7 (also known as KNCQ) comprises 5 different members (Kv7.1-Kv7.5), characterized by different tissue distribution and physiopathological role. The Kv7.1 channel subunit is mainly expressed in the heart, representing the molecular basis for the I _ks ventricular repolarization current; Kv7.2 and Kv7.3 subunits are mainly expressed in the central nervous system (CNS) where they generate the M-current, a repolarizing current limiting the neuronal spike discharge rate; Kv7.4 is mainly expressed in the inner ear and in the visceral, vascular and pulmonary smooth muscles, being involved in the regulation of smooth muscle tone; finally, Kv7.5 is mainly expressed in the CNS, in vascular smooth and skeletal muscles (Soldovieri MV et al., Physiology 2011, 26(5), 365-376).

Given the prominent role played by Kv7 channels in controlling neuronal excitability and considering that neuronal hyperexcitability is a common feature of neuropsychiatric disorders such as epilepsy, neuropathic pain, ischemic stroke, amyotrophic lateral sclerosis, and several other neurodegenerative diseases, pharmacological modulation of Kv7 channels appears as a rational pharmacological approach to treat these conditions. Specific members of the Kv7 subfamily of voltage-gated potassium channels expressed in the vascular smooth muscle produce membrane hyperpolarization sufficient to reduce the likelihood of Ca ²⁺ channel opening and hence attenuate vasoconstrictor response; thus their pharmacological modulation reduces the frequency of spontaneous contractions and results in dose-dependent hypotension, with potentially beneficial consequences in vascular hypertension. Similar myolytic responses in the respiratory and genitourinary systems may result in Kv7 activators being potentially useful to treat asthma and bladder hyperreactivity, respectively. Further, given the described role of Kv7 channels in controlling skeletal muscle differentiation, proliferation, and function, it has been suggested that Kv7 channel activators may be useful pharmacological agents against myotonia, myokymia and other skeletal muscle hyperexcitability diseases.

In particular, the pivotal role of Kv7.2 and Kv7.3 in neuronal excitability is evidenced by the occurrence of convulsive disorders with high phenotypic variability in humans, caused by specific mutations of their corresponding genes. For instance, these mutations can be responsible of a benign neonatal epilepsy with autosomal dominant transmission, also known as“Benign familiar neonatal seizures”, or of a severe sporadic neonatal epileptic encephalopathy, leading to neurocognitive impairment, anti-epileptic drugs resistance and typical neuroradiological findings (Miceli et al, GeneReviews 2016).

Current therapeutic options to treat the above-cited diseases could be improved in terms of efficacy and tolerability. Although none of the drugs currently approved for each of the mentioned indications targets Kv7 channels directly, available knowledge suggests that defects in the structure or function of at least one voltage-gated potassium channel of the Kv7 subfamily plays a relevant pathophysiological role in each of the cited conditions. The current relevance of potassium channels (possibly different from Kv7) as pharmacological targets is also highlighted by them being the therapeutic targets class III of antiarrhythmics (Amiodarone, Sotalol, Ibutilide, Dofetilide), oral hypoglycemic agents for treatment of type 2 diabetes (Sulfonylureas, Glinides), antihypertensive vasodilators (minoxidil, diazoxide and hydralazine), and for the treatment of multiple sclerosis (3,4-diaminopyridines).

Focusing on epilepsy, it is a neurologic disorder usually ascribed to impaired and paroxysmal neurons firing, responsible for an impairment in the sensitive, cognitive, mental, vegetative and motor activities. Epilepsy can be both a chronic or acute disease with an incidence rate of about 1% in the world population. Epilepsy is also associated to numerous psychiatric (such as anxiety and depression) or cognitive comorbidities, and to an increased risk of death (Sudden Death in Epilepsy or SUDEP). Seizures represent a typical clinical symptom of the epilepsy; they are involuntary and spontaneous motor manifestations caused by neuronal hyperexcitability. The hyperexcitability can be evidenced recording the brain electrical activity by specific electrodes positioned on the scalp, using a technique known as electroencephalogram (EEG). Two main electrophysiological properties characterize seizures: hyperexcitability (that is the tendency of the cortical or subcortical neurons to overreact to a normal stimulus) and hypersincronicity (that is the tendency to simultaneous activation). Physiological neuron excitability is guaranteed by a proper balance between the main excitatory (glutamaergic) and inhibitory (GABAergic) neural transmissions.

Moreover, the mechanisms regulating seizure threshold, duration and spread, mediated by voltage gated sodium, potassium and calcium channels, play a pivotal role.

The most important anti -epileptic drugs used nowadays in therapy can be classified on the basis of their mechanism of action, corresponding to: sodium voltage-gated channel blockade, increased GABAergic neurotransmission and voltage-gated Calcium channels blockade. In addition to these, additional molecular mechanisms appear to be exploited by newer anti-epileptic drugs. Although anticonvulsants represent important and often critical tools for epilepsy treatment, their risk/benefit ratio is suboptimal, often because of their poor target selectivity and of their wide range of side effects. Among the latest anti-epileptic drugs, retigabine (trade name in Europe Trobalt®; trade name in the US Potiga®), the prototypical Kv7 channel modulator, acts by increasing the activity of Kv7.2-5 channels and has been recommended as an adjunctive treatment for drug-resistant partial epilepsies in adults since 2011-2012 (Porter RJ et al., Neurology, 2007, 68(15), 1197-1204). Unfortunately, over the last five years, the use of this drug has been limited because of its important side effects; due to the unfavourable risk/benefit ratio, the manufacturing Company (GSK) has decided to discontinue the commercialization of retigabine, which is no longer available since June 2017 (http://www.ilae.org/Visitors/News/documents/GSK_Retigabine_ market_withdrawal.pdf).

Retigabine has a peculiar pharmacokinetic profile, with a fast absorption after oral administration (1.5-2. Oh after administration) but also a fast metabolization, mainly leading to the formation of glucuronic derivatives at N-2 and N-4 (Borlak et al., Metabolism: Clinical and Experimental 2006, 55(6):711-21). Moreover, retigabine is poorly stable under oxidative conditions both during synthesis and in its final form. One of the most characteristic side effects revealed during chronic (> 4 years) therapies is the occurrence of side effects represented by blue coloration of skin, lips, nails and, particularly, retina, due to the accumulation of blue-colored precipitates. These precipitates may represent degradation products of the aniline ring of the retigabine molecular structure catalyzed by oxidation triggered by visible light (Kumar et al., Mol. Pharmacol. 2016, 89(6):667-77). Another side effect is represented by urinary retention, possibly due to the interference of retigabine with Kv7 subunits located in the visceral smooth muscle and regulating bladder contraction (Kv7.4).

Different agonists of the Kv7 channels have been described so far, bearing the chemical features summarised below.

U.S. patent No 5,384,330 describes compounds with the following chemical structure, characterized by the presence of two vicinal amino group, as anticonvulsivants, antipyretics, muscle-relaxants and pheripheral analgesics:

WO 2005/087754 relates to compounds with the following chemical structure, in which two amino groups are placed in para-position on the aromatic ring, one of the two groups is part of an aliphatic cycle, while the two ortho-positions to the other amino group are occupied by R1 and R2 substituents:

Such derivatives act as openers of the Kv family of potassium ion channels.

WO 2008/024398 refers to the following structures, characterized by a bicyclic-fused structure between aromatic and aliphatic rings, and their effect on modulation of voltage-gated potassium channels:

US 2014/0336252 describes compounds having the following general formula as Kv potassium channel agonists for treating nervous system diseases Therefore, there is still a need for the development of new Kv7 modulators characterized by increased pharmacodynamic (increased potency and selectivity), and pharmacokinetic properties (increased distribution in the CNS and stability to oxidation), as well as by reduced side effects and good tolerability compared to current therapeutic options for Kv7-mediated disorders. These novel molecules may be used for the pharmacological treatment of central nervous system diseases (such as epilepsy, neuropathic pain, ischemic stroke and neurodegenerative disorders), peripheral nervous system diseases (such as pain), respiratory system diseases (such as asthma), genitourinary system diseases (such as bladder hyper-reactivity) and cardiovascular diseases (such as hypertension).

SUMMARY OF THE INVENTION

The present invention provides a novel class of compounds of formula I as defined below, as well as tautomers, salts, solvates, stereoisomers or isotopes thereof

Compounds of the present invention were tested by patch-clamp measurements, Thallium -Based Fluorescence Assay and stability assays as described in the Examples. They were found to act as voltage-gated potassium channels modulators, with EC50 values for Kv7.2/7.3 in the low micromolar range, and to possess desirable stability and photostability properties.

Notably, compounds of the present invention may act as agonists or antagonists of voltage-gated potassium channels. As a matter of fact, the substituent in position Re is able to modulate the activity of the compounds of general formula I in terms of modulation (agonism/antagonism), potency, efficacy and selectivity.

Furthermore, the NHQ functionality includes sulfonamide, urea, thiourea and guanidine bonds. The inventors found an exceptional activity towards potassium channels in comparison with retigabine, herein used as reference compound, which is known to increase the activity of voltage gated potassium channels Kv7.2-7.5 and has been used in therapy as anticonvulsant. In particular, when compared to retigabine, the compounds object of the present invention have improved pharmacological properties, both pharmacodynamic and pharmacokinetic. Moreover, they can be synthesized using easier and safer chemical procedures and show increased chemical and photochemical stability than retigabine. Therefore, compounds with general formula I are able to overcome the main problems limiting the clinical use of retigabine, in particular since they are more stable (chemically and biologically) and less prone to photoinduced oxidation.

The inventors have further observed that compounds of general formula I, in particular IA and IE, possess a plethora of desiderable properties, including increased potency and stability compared to retigabine.

Another important observation made by the inventors is that the use of a sterically encumbered aliphatic and/or aromatic group in position ¾ is useful to switch the activity of the compounds from agonism to antagonism and confers selectivity over the different isoforms of Kv7 channels. Thus, these specific substitutions represent an important feature in some embodiments of this invention to modulate the activity of some compounds, with some substitutions resulting in a blockade of the ionic currents carried by the channel (with the resulting molecules thereby acting as antagonists or blockers of the channels), whereas others enhance the ionic currents carried by the channels (with the resulting molecules thereby acting as agonists or openers). The functional electrophysiologial experiments carried out and whose results are detailed below allow a direct estimate of the pharmacological effects exerted by each drug, which could therefore be classified as a blocker or as an opener with respect to a specific channel target. Moreover, these specific substitutions represent an important feature in some embodiments of this invention to modulate the selectivity of some compounds, with some substitutions resulting in a selectivity for specific Kv7 subtypes.

Therefore, it is an object of the present invention a compound of general formula I:

I

or a tautomer, salt, solvate, stereoisomer or isotope thereof,

wherein:

Y is selected from: C1-C10 alkylamino, C1-C10 alkyl, C1-C10 alkoxy, C3-C8 cycloalkyl, Cl- C10 alkylcarbonyl, Cl -CIO alkylaminocarbonyl, Cl -CIO alkylthio, aryl, heterocycle, Cl -CIO carbonyl, C1-C10 amide and C1-C10 carboxyl and it is optionally mono-substituted or bi- substituted with a first substituent independently selected from: hydroxyl, amino, thiol, carboxylic acid, amide, carbonyl, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkylcarbonyl, C1-C6 alkoxy carbonyl, C1-C6 alkylamino, C1-C6 alkylaminocarbonyl, C1-C6 alkyloxy, C1-C6 alkylthio, C2-C8 alkenyl, C5-C7 cycloalkenyl, C2-C8 alkynyl and C5-C10 aryl, any of said first substituents being optionally substituted by one or more second substituents independently selected from: hydroxyl, amino, thiol, carboxylic acid, amide, carbonyl, halogen, C1-C6 alkyl, C3- C6 cycloalkyl, C1-C6 alkylcarbonyl, C1-C6 alkoxy carbonyl, C1-C6 alkylamino, C1-C6 alkylaminocarbonyl, C1-C6 alkyloxy, C1-C6 alkylthio, C2-C8 alkenyl, C5-C7 cycloalkenyl, C2- C8 alkynyl and C5-C10 aryl;

b is an integer comprised between 1 and 3;

or Y is NH or O and b is 1;

R-2 is selected from: an aromatic ring, a cycloalkyl, a heterocycle, a linear or branched saturated alkyl and a linear or branched unsaturated alkyl, any of which optionally bearing in one or more positions at least one substituent independently selected from the group consisting of: OH, NO2, CN, halogen, N¾, C1-C3 haloalkyl, C1-C3 haloalkoxy, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkylcarbonyl, C1-C6 alkoxy carbonyl, C1-C6 alkylamino, C1-C6 dialkylamino, C1-C6 alkylaminocarbonyl, acetamidyl, C1-C6 alkyloxy, C1-C6 alkylthio, C2-C8 alkenyl, C5-C7 cycloalkenyl, C2-C8 alkynyl, C5-C10 aryl, thiol and thiol ether;

Ri, R ₃ , R ₄ and R ₅ are each independently selected from the group consisting of: H, NH2, OH, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkylcarbonyl, C1-C6 alkoxy carbonyl, C1-C6 alkylamino, C2-C6 dialkylamino, C1-C6 alkylaminocarbonyl, C1-C6 alkyloxy, C1-C6 alkylthio, C2-C8 alkenyl, C5-C7 cycloalkenyl, heterocycle, C2-C8 alkynyl and C5-C10 aryl, any of which being optionally substituted by one or more substituents independently selected from: hydroxyl, amine, thiol, carboxyl, carboxylic acid, and halogen;

Q is selected from the group consisting of: C=0, SO2, SO, SO2NH, CO H, CS H, C=NH, CNHNH, and OS;

when Q is C=0 or C=S, 5 is selected from: linear or branched C3-C30 alkyl, linear or branched C5-C30 arylalkyl, C3-C8 cycloalkyl, linear or branched C2-C4 alkyl substituted by C3-C8 cycloalkyl, linear or branched C3-C10 cycloalkylamino, linear or branched Cl -CIO alkylamino, linear or branched C2-C10 alkenylamino, linear or branched C2-C10 alkynylamino, C5-C10 aryl, linear or branched C7-C15 arylalkylamino, linear or branched C7-C15 arylalkenylamino, linear or branched C7-C15 arylalkynylamino, C2-C30 alkenyl, C2-C30 alkynyl and C1-C30 alkoxyl, any of which being optionally substituted with one or more substituents independently selected from: linear or branched alkyl group, halogen, hydroxyl group, amine group, alkylamine, dialkylamine, thiol, thioether and cycloalkyl; when Q is SO2, SO, SO2NH, COM3, CSNH, C=NH, or CNHNH, Re is selected from: linear or branched C1-C30 alkyl, C3-C8 cycloalkyl, linear or branched C1-C30 alkylcarbonyl, linear or branched C1-C30 alkoxycarbonyl, linear or branched C1-C30 alkylamino, linear or branched Cl- C30 alkylaminocarbonyl, linear or branched C1-C30 alkyloxy, linear or branched C1-C30 alkylthio, linear or branched C2-C30 alkenyl, C5-C8 cycloalkenyl, linear or branched C2-C30 alkynyl, heterocycle and aryl, any of which being optionally substituted with one or more substituents independently selected from: halogen, C1-C6 alkyl, oxo and C3-C7 cycloalkyl.

In a preferred embodiment, the present invention provides a compound of formula G :

G

or a tautomer, salt, solvate, stereoisomer or isotope thereof,

wherein:

Y is selected from: C1-C10 alkyl, C3-C8 cycloalkyl, C1-C10 alkylcarbonyl, C1-C10 alkylamino, Cl -CIO alkylaminocarbonyl, Cl -CIO alkoxy, Cl -CIO alkylthio, aryl, heterocycle, Cl -CIO carbonyl, C1-C10 amide and C1-C10 carboxyl and it is optionally mono-substituted or bi- substituted with a first substituent independently selected from: hydroxyl, amino, thiol, carboxylic acid, amide, carbonyl, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkylcarbonyl, C1-C6 alkoxy carbonyl, C1-C6 alkylamino, C1-C6 alkylaminocarbonyl, C1-C6 alkyloxy, C1-C6 alkylthio, C2-C8 alkenyl, C5-C7 cycloalkenyl, C2-C8 alkynyl and C5-C10 aryl, any of said first substituents being optionally substituted by one or more second substituents independently selected from: hydroxyl, amino, thiol, carboxylic acid, amide, carbonyl, halogen, C1-C6 alkyl, C3- C6 cycloalkyl, C1-C6 alkylcarbonyl, C1-C6 alkoxy carbonyl, C1-C6 alkylamino, C1-C6 alkylaminocarbonyl, C1-C6 alkyloxy, C1-C6 alkylthio, C2-C8 alkenyl, C5-C7 cycloalkenyl, C2- C8 alkynyl and C5-C10 aryl;

b is an integer comprised between 0 and 3;

Hy is selected from: an aromatic ring, a cycloalkyl, a heterocycle, a linear or branched saturated alkyl and a linear or branched unsaturated alkyl, any of which optionally bearing in one or more positions at least one substituent independently selected from the group consisting of: OH, NO2, CN, halogen, N¾, C1-C3 haloalkyl, C1-C3 haloalkoxy, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkylcarbonyl, C1-C6 alkoxy carbonyl, C1-C6 alkylamino, C1-C6 dialkylamino, C1-C6 alkylaminocarbonyl, acetamidyl, C1-C6 alkyloxy, C1-C6 alkylthio, C2-C8 alkenyl, C5-C7 cycloalkenyl, C2-C8 alkynyl, C5-C10 aryl, thiol and thiol ether;

Ri and R2 are each independently selected from the group consisting of: H, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkylcarbonyl, C1-C6 alkoxy carbonyl, C1-C6 alkylamino, C1-C6 alkylaminocarbonyl, C1-C6 alkyloxy, C1-C6 alkylthio, C2-C8 alkenyl, C5-C7 cycloalkenyl, C2- C8 alkynyl and C5-C10 aryl, any of which being independently optionally substituted by one or more substituents independently selected from: hydroxyl, amino, thiol, carboxylic acid, carboxyl and halogen;

R3, R ₄ and R5 are each independently selected from the group consisting of: H, NH2, OH, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C2-C6 alkylcarbonyl, C2-C6 alkoxycarbonyl, C1-C6 alkylamino, C2-C6 dialkylamino, C2-C6 alkylaminocarbonyl, C1-C6 alkyloxy, C1-C6 alkylthio, C2-C8 alkenyl, C5-C7 cycloalkenyl, C2-C8 alkynyl and C5-C10 aryl, any of which being optionally substituted by one or more substituents independently selected from: hydroxyl, amine, thiol, carboxyl and halogen;

Q is selected from the group consisting of: C=0, SO2, SO, SO2NH, CONH, CSNH, C=NH, CNHNH and C=S;

when Q is C=0 or C=S, 5 is selected from: linear or branched C1-C30 alkyl, linear or branched C5-C30 arylalkyl, C3-C8 cycloalkyl, linear or branched C3-C10 cycloalkylamino, linear or branched Cl -CIO alkylamino, linear or branched C2-C10 alkenylamino, linear or branched C2- C10 alkynylamino, C5-C10 aryl, linear or branched C7-C15 arylalkylamino, linear or branched C7-C15 arylalkenylamino, linear or branched C7-C15 arylalkynylamino, C2-C30 alkenyl, C2- C30 alkynyl and C1-C30 alkoxyl, any of which being optionally substituted with one or more substituents independently selected from: linear or branched alkyl group, halogen, hydroxyl group, amine group, alkylamine, dialkylamine, thiol, thioether and cycloalkyl;

when Q is SO2, SO, SO2NH, CONH, CSNH, C=NH, or CNHNH, Re is selected from: linear or branched C1-C30 alkyl, C3-C8 cycloalkyl, linear or branched C1-C30 alkylcarbonyl, linear or branched C1-C30 alkoxycarbonyl, linear or branched C1-C30 alkylamino, linear or branched Cl- C30 alkylaminocarbonyl, linear or branched C1-C30 alkyloxy, linear or branched C1-C30 alkylthio, linear or branched C2-C30 alkenyl, C5-C8 cycloalkenyl, linear or branched C2-C30 alkynyl, heterocycle and aryl, any of which being optionally substituted with one or more substituents independently selected from: halogen, C1-C6 alkyl, oxo and C3-C7 cycloalkyl.

The following preferred embodiments equally refer to compounds of formula I or G and may be combined among each other in any way that would give rise to a stable compound.

Preferably, b is 1. Preferably, Q is C=0 or SO2.

Preferably, Y is C1-C10 alkylamino, C1-C10 alkyl or C1-C10 alkoxy. More preferably, Y is selected from the group consisting of: -N(H)CH2-; -N(H)CH2CH2-; -N(H)CH2CH2CH2-;

-N(CH )CH ₂ -; -N(CH ₂ CH ₃ )CH ₂ -; -N(CH ₂ CH ₂ CH ₃ )CH ₂ -; -N(H)CH(CH ₃ )-; -OCH2-; -CH ₂ CH ₂ - and - CH2CH2CH2-. In particular, it is to be understood that when Y is C1-C10 alkylamino, it is bound to the phenyl via the nitrogen atom and to R2 via the alkyl moiety. In particular, it is to be understood that when Y is C1-C10 alkoxy, it is bound to the phenyl via the oxygen atom and to Pi via the alkyl moiety.

Preferably, R2 is an aromatic ring optionally substituted with one or more substituents independently selected from the group consisting of: OH, NO2, halogen, N¾, C1-C3 haloalkyl, acetamidyl, C1-C6 alkyloxy, C1-C3 haloalkoxy, and C1-C6 alkylcarbonyl.

More preferably, said aromatic ring in position R2 is phenyl, pyridine or pyrrole.

Still preferably, R2 is phenyl optionally substituted with one substituent in para- position, said substituent being selected as defined above.

Yet preferably, R2 is selected from: a p-halogenphenyl, a dihalogenphenyl, a p- (trihalogenomethyl)phenyl and a p-(trihalogenomethoxy)phenyl.

Preferably, Ri, R ₃ , R4 and R5 are each independently: H, C1-C6 alkyl, C3-C6 cycloalkyl, N¾, NH(C1-C6 alkyl), N(C1-C6 alkyl)2 , azepane, pirrolidine, piperidine, aziridine or halogen.

Preferably, Ri is H. Preferably, R ₃ is H or C1-C6 alkyl, R ₄ is H or C1-C6 alkyl and R ₅ is H or halogen; or R ₃ , R ₄ and R ₅ are all H; or R ₃ and R ₄ are both H and R ₅ is halogen; or R ₃ is H, R ₄ is NH2 orNHalkyl or N(alkyl)2 or azepane or pirrolidine or piperidine or aziridine and R5 is H or halogen.

Preferably, when Q is C=0 or C=S, R45 is selected from: linear or branched C3-C30 alkyl, linear or branched C5-C30 arylalkyl, C3-C8 cycloalkyl, linear or branched C2-C4 alkyl substituted by C3-C8 cycloalkyl, linear or branched C1-C10 alkylamino, linear or branched C7-C15 arylalkylamino, C2-C30 alkenyl, C2-C30 alkynyl and C1-C30 alkoxyl, any of which being optionally substituted with one or more substituents independently selected from: halogen and cycloalkyl. More preferably, when Q is C=0 or C=S, said linear or branched C3-C30 alkyl is a branched C3-C30 alkyl or a linear C6-C30 alkyl. Preferably, said linear or branched C2-C4 alkyl substituted by C3-C8 cycloalkyl is CH2CH2Cyclohexyl or CH2CH2CH2 cyclohexyl.

Preferably, when Q is S0 ₂ , SO, SO2NH, CONH, CSNH, C-NH, or CNHNH, Re is C3-C8 cycloalkyl or aryl and is optionally substituted with one or more C1-C6 alkyl.

In a particularly preferred embodiment, the present invention provides a compound of formula I as defined above or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein: Y is Cl -CIO alkylamino, Cl -CIO alkyl or Cl -CIO alkoxy;

b is 1;

R.2 is an aromatic ring optionally substituted with one or more substituents independently selected from the group consisting of: OH, NO2, halogen, NH2, C1-C3 haloalkyl, acetamidyl, C1-C6 alkyl oxy, C1-C3 haloalkoxy, and C1-C6 alkylcarbonyl;

Ri, R.3, R4 and R5 are each independently: H, C1-C6 alkyl, C3-C6 cycloalkyl, M¾, NH(C1-C6 alkyl), N(C1-C6 alkyl)2 , azepane, pirrolidine, piperidine, aziridine or halogen;

Q is: C=0 or SO2;

when Q is C=0, R ₆ is selected from: linear or branched C3-C30 alkyl, linear or branched C5-C30 arylalkyl, C3-C8 cycloalkyl, linear or branched C2-C4 alkyl substituted by C3-C8 cycloalkyl, linear or branched C1-C10 alkylamino, linear or branched C7-C15 aryl alkyl amino, C2-C30 alkenyl, C2-C30 alkynyl and C1-C30 alkoxyl, any of which being optionally substituted with one or more substituents independently selected from: halogen and cycloalkyl;

when Q is SO2, Ro is C3-C8 cycloalkyl or aryl and is optionally substituted with one or more Cl- C6 alkyl.

Preferably, the invention provides a compound of formula I wherein, when Q is C=0 or C=S, Rr, is selected from: linear or branched C5-C30 alkyl, linear or branched C5-C30 arylalkyl, C3-C8 cycloalkyl, linear or branched C3-C10 cycloalkylamino, linear or branched C7-C10 alkylamino, linear or branched C2-C10 alkenylamino, linear or branched C2-C10 alkynylamino, linear or branched C5-C15 arylalkylamino, linear or branched C5-C15 arylalkenylamino, linear or branched C5-C15 arylalkynylamino, C5-C30 alkenyl, C5-C30 alkynyl and C5-C30 alkoxyl, any of which being optionally substituted with one or more substituents independently selected from: a linear or branched alkyl group, a halogen, a hydroxyl group, an amine group, an alkylamine, a dialkylamine, a thiol, a thioether and a cycloalkyl, or a tautomer, salt, solvate, stereoisomer or isotope thereof

Preferably, the invention provides a compound of formula I wherein, when Q is C=0 or C=S, R ₆ is selected from: linear or branched C7-C30 alkyl, linear or branched C6-C30 arylalkyl, C3-C8 cycloalkyl, linear or branched C3-C10 cycloalkylamino, linear or branched C7-C10 alkylamino, linear or branched C2-C10 alkenylamino, linear or branched C2-C10 alkynylamino, linear or branched C7-C15 arylalkylamino, linear or branched C7-C15 arylalkenylamino, linear or branched C7-C15 arylalkynylamino, C7-C30 alkenyl, C7-C30 alkynyl and C7-C30 alkoxyl, any of which being optionally substituted with one or more substituents independently selected from: a linear or branched alkyl group, a halogen, a hydroxyl group, an amine group, an alkylamine, a dialkylamine, a thiol, a thioether and a cycloalkyl, or a tautomer, salt, solvate, stereoisomer or isotope thereof.

Preferably, the invention provides a compound of formula I wherein Q is C=0 or C=S and Re i s a C5-C10 aryl optionally substituted by one or more substituents independently selected from: a linear or branched alkyl group, a halogen, a hydroxyl group, an amine group, an alkylamine, a dialkylamine, a thiol and a thioether, preferably said C5-C10 aryl being selected from the group consisting of: pyridine, pyrazine, naphthalene and tetrahydronaphtalene, or a tautomer, salt, solvate, stereoisomer or isotope thereof.

Preferably, the invention provides a compound of formula I, wherein Y is Cfh, b is an integer between 1 and 3, and R2 is an aromatic ring optionally substituted by one or more substituents independently selected from: a linear or branched C1-C6 alkyl group, a C1-C3 haloalkyl, a halogen, a hydroxy group, a C1-C6 alkoxy, a C1-C3 haloalkoxy, an amine, a C1-C6 monoalkylamine, a C1-C6 dialkylamine, a thiol and a thioether, or a tautomer, salt, solvate, stereoisomer or isotope thereof.

Preferably, the invention provides a compound of formula I, wherein Y is CH2CO, COCH2 or CH=CH, b is 1 and R2 is an aromatic ring optionally substituted by one or more substituents independently selected from: a linear or branched C1-C6 alkyl group, a C1-C3 haloalkyl, a halogen, a hydroxy group, a C1-C6 alkoxy, a C1-C3 haloalkoxy, an amine, a C1-C6 monoalkylamine, a C1-C6 dialkylamine, a thiol and a thioether, or a tautomer, salt, solvate, stereoisomer or isotope thereof.

Preferably, the invention provides a compound of formula I wherein Y is NH or O, b is 1 and R2 is an aromatic ring optionally substituted by one or more substituents independently selected from: a linear or branched C1-C6 alkyl group, a C1-C3 haloalkyl, a halogen, a hydroxy group, a C1-C6 alkoxy, a C1-C3 haloalkoxy, an amine, a C1-C6 monoalkylamine, a C1-C6 dialkylamine, a thiol and a thioether, or a tautomer, salt, solvate, stereoisomer or isotope thereof.

A preferred object of the present invention is a compound or a tautomer, salt, solvate, stereoisomer or isotope thereof as defined above having formula IA, IB, IC, ID or IE: wherein Ri, R2, Y, b, R3, R4, Rs and R6 are as defined above.

In a preferred embodiment, this invention contemplates molecules with general structure I, reported above, wherein the group NH-Q-R6 is selected from: H-SO2-R6, H-SO2- H-R6, NH- CO-Re, NH-CO-NH-Re, NH-CS- H-Re and NH-CNH-NH-Re.

Thus, in one of its embodiments the present invention contemplates a compound with general formula I in which NH-Q-Rs is represented by NH-SO2-R6.

In yet another embodiment, the present invention contemplates a compound with general formula I in which NH-Q-R ₆ is represented by NH-SO2-NH-R6.

In another specific embodiment, the present invention contemplates a compound with general formula I in which NH-Q-Ibs is represented by NH-CO-R6.

In another specific embodiment, this invention provides a compound with general formula I in which NH-Q-R ₆ is represented by NH-CS-NH-Re. In another specific embodiment, this invention provides a compound with general formula I in which NH-Q-Rf, is represented by NH-CNH-NH-Rr,.

In a preferred embodiment, the present invention contemplates compounds with formula IA, IB, IC, ID and IE wherein Ri, R3, R4 and R5 are independently selected from: H, MB, OH, alkyl (preferably methyl), alkoxy (preferably methoxyl or ethoxyl), cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), halogen (preferably fluoro, chloro or bromo), Mlalkyl, N(alkyl)2, azepane, pirrolidine, piperidine, aziridine.

In another preferred embodiment, the present invention contemplates compounds with formula IA, IB, IC, ID or IE, wherein R3 and R4 are both alkyl groups and Rs is H or F.

In another preferred embodiment, the present invention contemplates compounds with formula IA, IB, IC, ID and IE, wherein R5 is halogen while Ri, R3 and R4 are independently selected from hydrogen, H, MB, OH, alkyl (preferably methyl), cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), alkoxy (preferably methoxyl or ethoxyl), halogen (preferably fluoro, chloro or bromo), Mlalkyl, N(alkyl)2, azepane, pirrolidine, piperidine, aziridine.

In another preferred embodiment, the present invention contemplates compounds with formula IA, IB, IC, ID and IE, wherein R2 is an aryl group optionally bearing in one or more positions a substituent independently selected from: alkyloxy, nitrile, amino, hydroxyl, nitro, halogen, haloalkyl (preferably trifluoroalkyl, trichloroalkyl, difluoroalkyl, dichloroalkyl), alkylcarbonyl, haloalkoxy (preferably trifluorom ethoxy), alkyl.

In another preferred embodiment, the present invention contemplates compounds with formula IA, IB, IC, ID and IE, wherein R2 is selected from: pyridine, piperidine, pyrazine, piperazine, thiophene, tetrahydrothiophene, furan and tetrahydrofuran, any of which optionally bears in one or more positions a substituent independently selected from: alkyloxy, nitrile, amino, hydroxyl, nitro, halogen, haloalkyl (preferably trifluoroalkyl, trichloroalkyl, difluoroalkyl, dichloroalkyl), alkylcarbonyl, haloalkoxy (preferably trifluoromethoxy), alkyl.

Preferably, the present invention provides a compound of formula I, wherein when Q is C=0, IB is selected from: linear or branched C5-C30 alkyl (preferably C6-C30 alkyl, more preferably C7- C30 alkyl), linear or branched C6-C30 arylalkyl, C3-C8 cycloalkyl, linear or branched C3-C10 cycloalkylamino, linear or branched C1-C10 alkylamino (preferably C5-C30 alkylamino, still preferably C6-C30 alkylamino, more preferably C7-C30 alkylamino), linear or branched C2-C10 alkenylamino, linear or branched C2-C10 alkynylamino, C5-C10 aryl, linear or branched C7-C15 arylalkylamino, linear or branched C7-C15 arylalkenylamino, linear or branched C7-C15 arylalkynylamino, C2-C30 alkenyl (preferably C5-C30 alkenyl, still preferably C6-C30 alkenyl, more preferably C7-C30 alkenyl), C2-C30 alkynyl (preferably C5-C30 alkynyl, still preferably C6-C30 alkynyl, more preferably C7-C30 alkynyl) and C1-C30 alkoxyl (preferably C5-C30 alkoxy, still preferably C6-C30 alkoxy, more preferably C7-C30 alkoxy), any of which being optionally substituted with one or more substituents independently selected from: linear or branched alkyl group, halogen, hydroxyl group, amine group, alkylamine, dialkylamine, thiol, thioether and cycloalkyl.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein Rs is halogen. More preferably, R5 is fluorine. Still more preferably, R3 is H, R4 is NH2 or NHalkyl or N(alkyl)2 or azepane or pirrolidine or piperidine or aziridine and R5 is halogen. Also preferably, R ₃ and R ₄ are both H and R ₅ is halogen. Preferably, R ₃ , R ₄ and Rs are all H. Preferably, R ₃ and R ₅ are both H and R4 is NH2 or NHalkyl or N(alkyl)2 or azepane or pirrolidine or piperidine or aziridine. Preferably, R3 is linear or branched C1-C6 alkyl, R4 and Rs are both H. Preferably, R4 is linear or branched C1-C6 alkyl, cycloalkyl R3 and R5 are both H. Preferably, R3 and R4 are both methyl and R ₅ is H. Preferably, R ₃ and R ₄ are both H and R ₅ is F. Preferably, R ₃ is H, R ₄ is N¾ and Rs is F. Preferably, R3 is methyl, R ₄ and R5 are both H.

In a preferred embodiment, the present invention provides a compound of formula I as defined above, wherein R ₃ is H. In a preferred embodiment, the present invention provides a compound of formula I as defined above, wherein R4 is H.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein R45 is“Cl- 30 alkyl” refers to C2-30 alkyl, C3-30 alkyl, C4-30 alkyl, C5-30 alkyl, C6-30 alkyl, C7-30 alkyl, Cl 0-30 alkyl, C12-30 alkyl, or C15-30 alkyl.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein R ₆ is“C3- C8 cycloalkyl” refers to C4-30 cycloalkyl, C5-30 cycloalkyl, C6-30 cycloalkyl. In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein R ₆ is“C3-C8 cycloalkyl” refers to C3-6 cycloalkyl

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein R45 is“Cl- C30 alkylcarbonyl” refers to C2-30 alkylcarbonyl, C3-30 alkylcarbonyl, C4-30 alkylcarbonyl, C5- 30 alkylcarbonyl, C6-30 alkylcarbonyl, C7-30 alkylcarbonyl, CIO-30 alkylcarbonyl, C12-30 alkylcarbonyl, or C15-30 alkylcarbonyl. In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“Cl- C30 alkoxycarbonyl” refers to C2-30 alkoxycarbonyl, C3-30 alkoxycarbonyl, C4-30 alkoxycarbonyl, C5-30 alkoxycarbonyl, C6-30 alkoxycarbonyl, C7-30 alkoxycarbonyl, CIO-30 alkoxycarbonyl, C12-30 alkoxycarbonyl, or C15-30 alkoxycarbonyl.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“Cl- 30 alkylamino” refers to C2-30 alkylamino, C3-30 alkylamino, C4-30 alkylamino, C5-30 alkylamino, C6-30 alkylamino, C7-30 alkylamino, CIO-30 alkylamino, C12-30 alkylamino, or Cl 5-30 alkylamino.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“Cl- C30 alkylaminocarbonyl” refers to C2-30 alkylaminocarbonyl, C3-30 alkylaminocarbonyl, C4-30 alkylaminocarbonyl, C5-30 alkylaminocarbonyl, C6-30 alkylaminocarbonyl, C7-30 alkylaminocarbonyl, CIO-30 alkylaminocarbonyl, C12-30 alkylaminocarbonyl, or C15-30 alkylaminocarbonyl .

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“Cl- 30 alkoxyl” refers to C2-30 alkoxyl, C3-30 alkoxyl, C4-30 alkoxyl, C5-30 alkoxyl, C6-30 alkoxyl, C7-30 alkoxyl, CIO-30 alkoxyl, C12-30 alkoxyl, or C15-30 alkoxyl.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“Cl- C30 alkylthio” refers to C2-30 alkylthio, C3-30 alkylthio, C4-30 alkylthio, C5-30 alkylthio, C6- 30 alkylthio, C7-30 alkylthio, CIO-30 alkylthio, C12-30 alkylthio, or C15-30 alkylthio.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“C5- C8 cycloalkenyl” refers to C6-C8 cycloalkenyl.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“C2- 30 alkenyl” refers to C2-30 alkenyl, C3-30 alkenyl, C4-30 alkenyl, C5-30 alkenyl, C6-30 alkenyl, C7-30 alkenyl, CIO-30 alkenyl, C12-30 alkenyl, or C15-30 alkenyl.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“C2- 30 alkynyl” refers to C2-30 alkynyl, C3-30 alkynyl, C4-30 alkynyl, C5-30 alkynyl, C6-30 alkynyl, C7-30 alkynyl, CIO-30 alkynyl, C12-30 alkynyl, or C15-30 alkynyl.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“C5- C30 arylalkyl” refers to C6-C30 arylalkyl.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“C3- C10 cycloalkylamino” refers to C4-C10 cycloalkylamino, C5-C10 cycloalkylamino or C6-C10 cycloalkylamino.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“Cl- C10 alkylamino” refers to C2-C10 alkylamino, C3-C10 alkylamino, C4-C10 alkylamino, C5-C10 alkylamino, C6-C10 alkylamino or C7-C10 alkylamino.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“C2- C10 alkenylamino” refers to C3-C10 alkenylamino, C4-C10 alkenylamino, C5-C10 alkenylamino, C6-C10 alkenylamino or C7-C10 alkenylamino.

In a preferred embodiment, the present invention provides a compound of the above-defined general formula I or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein ¾ is“C2- C10 alkynylamino” refers to C3-C10 alkynylamino, C4-C10 alkynylamino, C5-C10 alkynylamino, C6-C10 alkynylamino or C7-C10 alkynylamino.

In a preferred embodiment, the present invention refers to a compound of formula I as defined above or a tautomer, salt, solvate, stereoisomer or isotope thereof, wherein:

Ri isH or C1-C6 alkyl, preferably Ri is H; and/or

Y is NH, O, C1-C10 alkyl, C1-C10 alkylamino, C1-C10 alkoxy, C1-C10 alkyl carbonyl, C1-C10 carbonyl, preferably Y is NH, O, NHCH2, OCH2, C¾, CH2CH2, COCH2 or C=0; and/or b is 1, 2 or 3, preferably b is 1; and/or

R2 is an aryl ring, preferably phenyl, pyridine or pyrrole, optionally substituted with one or more substituents independently selected from: OH, NO2, halogen (preferably fluorine, chlorine or bromine), NH2, C1-C3 haloalkyl (preferably trifluorom ethyl), C1-C6 alkyloxy (preferably m ethoxy), C1-C3 haloalkoxy (preferably trifluoromethoxy), and C1-C6 alkylcarbonyl (preferably acetamide), preferably R2 IS selected from: fluorophenyl, trifluoromethylphenyl, methoxy phenyl, trifluoromethoxy phenyl, hydroxyphenyl, nitrophenyl, aminophenyl, pyridine, pyrrole, acetamidophenyl, difluorophenyl, difluorophenyl, chlorophenyl, bis(trifluoromethyl)phenyl, bromophenyl, nitrophenyl and bromo-nitro-phenyl, more preferably R2 is selected from: 4- fluorophenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl, 4-trifluoromethoxyphenyl, 4- hydroxyphenyl, 4-nitrophenyl, 4-aminophenyl, pyridine, pyrrole, 4-acetamidophenyl, 2,4- difluorophenyl, 3,4-difluorophenyl, 4-chlorophenyl, 2,4-bis(trifluoromethyl)phenyl, 4- bromophenyl, 2-nitrophenyl and 4-bromo-2-nitro-phenyl; and/or

R3, R4 and R5 are each independently: H, C1-C6 alkyl (preferably methyl), cycloalkyl, NH2, NHalkyl or N(alkyl)2 or azepane or pirrolidine or piperidine or aziridine or halogen (preferably fluorine), cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), NH2, NHalkyl or N(alkyl)2 or azepane or pirrolidine or piperidine or aziridine preferably R3 is H or Cl- C6 alkyl (preferably methyl), R4 is H, C1-C6 alkyl (preferably methyl) or NH2 and R5 is H or halogen (preferably fluorine), also preferably R ₃ , R _t and R ₅ are all H, also preferably R ₃ and R ₄ are both C1-C6 alkyl (preferably methyl) and R5 is H, also preferably R3 and R ₄ are both H and R5 is halogen (preferably fluorine), also preferably R ₃ is H, R is NH2 C1-C6 alkyl (preferably methyl), cycloalkyl, N¾, NHalkyl or N(alkyl)2 or azepane or pirrolidine or piperidine or aziridine and R5 is halogen (preferably fluorine), also preferably R3 and R5 are both H and R4 is MH;

Q is: S0 ₂ , CONH, CSNH, CNHNH or C=0; and/or

when Q is SO2, CONH, CSNH or CNHNH, R ₆ is selected from: linear or branched C1-C30 alkyl (preferably butyl, hexyl, CH2C(CH3)3 or CH2CH(CH3)2), C3-C8 cycloalkyl (preferably cyclohexyl), aryl (preferably phenyl) and heterocycle (preferably piperazine, azepane or piperidine), and is optionally substituted with one or more substituents independently selected from: halogen (preferably fluorine), C1-C6 alkyl (preferably methyl) and oxo, preferably when Q is SO2, CONH, CSNH or CNHNH, R ₆ is: butyl, cyclohexyl, 4-fluorocyclohexyl, CH2C(CH3)3, phenyl, CH2CH(CH3)2, hexyl, piperazine, methylpiperazine, azepane, dimethyl-2- oxobicyclo[2.2.1]heptanyl, dimethylpiperidine or QHQHcyclohexyl; and/or

when Q is C=0, Re is linear or branched C5-C30 alkyl (preferably CH ₂ C(CH3) ₃ , hexyl, heptyl, octyl, nonyl, decyl or undecyl), linear or branched C3-C7 cycloalkyl (preferably cyclohexyl), linear or branched C6-C30 arylalkyl (preferably CFbl _n CrHi where n is a number from 0 to 10), linear or branched C1-C30 alkenyl (preferably nonenyl), linear or branched C1-C30 alkynyl (preferably butenyl) or C1-C30 alkoxy and is optionally substituted with one or more substituents independently selected from: C1-C6 alkyl (preferably methyl or isopropyl), halogen (preferably fluorine), C3-C7 cycloalkyl (preferably cyclopropyl or cyclohexyl) and N¾, preferably when Q is C=0 or 0=CO, R ₆ is selected from: hexyl, CH2C(CH3)3, cyclohexyl, isopropyl-methyl- cyclohexyl, difluorohexyl (including all possible isomers and stereoisomers), heptyl, difluoroheptyl (including all possible isomers and stereoisomers), octyl, difluorooctyl (including all possible isomers and stereoisomers), nonyl, difluorononyl (including all possible isomers and stereoisomers), decyl, difluorodecyl (including all possible isomers and stereoisomers), undecyl, difluoroundecyl (including all possible isomers and stereoisomers), CH2Cyclohexyl, CPpCfbcyclohexyl, (Cbh/eCeHs, (CFh^fluorophenyl, aminohexyl (including all possible isomers and stereoisomers), aminoheptyl (including all possible isomers and stereoisomers), aminooctyl (including all possible isomers and stereoisomers), aminononyl (including all possible isomers and stereoisomers), aminodecyl (including all possible isomers and stereoisomers), aminoundecyl (including all possible isomers and stereoisomers), (CH2)6aniline, CH2CH2CHCH, Cl 12 C I ICI Icyclopropyl, nonenyl (including all possible isomers and geometrical isomers), CH ₂ 0(CH ₂ ) ₂ 0CH ₃ or (CH ₂ ) ₂ 0(CH ₂ ) ₂ 0CH ₃ .

Preferably, for compounds of formula I as defined above, wherein Q = CO, R„ is not C5 alkyl. Preferably, for compounds of formula I as defined above, ¾ is not C5 alkyl.

In an embodiment, the present invention discloses each one of the following compounds:

- Retigabine;

- 2-Amino-4-(4-fluorobenzylamino)-l-ethoxycarbonylaminobenzene ;

2-Amino-4-(4-trifluoromethylbenzilamino)-l ethoxy carbonylamino-benzene;

2-Amino-4-benzylamino-l-ethoxycarbonylamino-benzene;

2-Amino-4-(3,5-dichlorobenzylamino)-l-ethoxycarbonylamino benzene;

2-Amino-4-(3,5-dichlorobenzylamino)-l-propyloxycarbonylamino benzene;

2-Amino-4-(2-chlorobenzylamino)-l-(diethylcarbamoylamino) benzene;

2-Amino-4-(2,4-dichlorobenzylamino)-l-(dimethylcarbamoylamin o) benzene;

K15 K19

K29 K33

In a preferred embodiment, the compound of the invention is selected from:

In a preferred embodiment, the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is a voltage-gated potassium channel modulator. Preferably the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is a modulator of at least one member of the Kv7 subfamily of voltage-gated potassium channels. More preferably the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is a Kv7.1, Kv7.2, Kv7.3, Kv7.4 and/or Kv7.5 modulator. Still more preferably, the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is a Kv7.2 and Kv7.3 modulator.

In another preferred embodiment, the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is for use as a medicament.

Preferably, the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is for use in the treatment and/or prevention of a condition characterised by a defect in the structure and/or function of at least one voltage-gated potassium channel. Preferably, the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is for use in the treatment and/or prevention of a condition characterised by a defect in the structure and/or function of at least one member of the Kv7 subfamily of voltage-gated potassium channels. Also preferably, the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is for use in the treatment and/or prevention of a condition characterised by a defect in the structure and/or function of at least one of Kv7.1, Kv7.2, Kv7.3, Kv7.4 and/or Kv7.5, more preferably of both Kv7.2 and Kv7.3.

In another preferred embodiment, the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is for use as an anticonvulsant, analgesic, anti-ischemic, anti -arrhythmic, neuroprotective, anti-tinnitus, antiasthmatic, gastrointestinal motility modulator, antihypertensive, antimyotonic and/or anti-skeletal muscle dystrophia medicament.

It is another object of the present invention a pharmaceutical composition comprising the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above and a pharmaceutically acceptable carrier. Preferably, said pharmaceutical composition comprises a further therapeutic agent selected from: an anticonvulsivant, an analgesic and a nonsteroidal anti-inflammatory drug (NSAID). Preferably, said anticonvulsant is selected from the group consisting of: Acetazolamide, Brivaracetam, Carbamazepine, Clobazam, Clonazepam, Diazepam, Eslicarbazepine, Ethosuximide, Fosphenytoin, Gabapentin, Lacosamide, Lamotrigine, Levetiracetam, Lorazepam, Methosuximide, Nitrazepam, Oxcarbazepine, Perampanel, Phenobarbital, Phenytoin, Pregabalin, Primidone, Rufmamide, Stiripentol, Topiramate, Valproic Acid, Vigabatrin, Felbamate, Tiagabine and Zonisamide. Preferably, said analgesic is acetaminophen or an opioid. Preferably, said nonsteroidal anti-inflammatory drug (NSAID) is selected from the group consisting of: aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine, oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetin and zomepirac. Preferably, said opioid is morphine.

In a preferred embodiment, the pharmaceutical composition as defined above is for use in the treatment and/or prevention of a condition characterised by a defect in the structure and/or function of at least one voltage-gated potassium channel. Preferably, the pharmaceutical composition as defined above is for use in the treatment and/or prevention of a condition characterised by a defect in the structure and/or function of at least one member of the Kv7 subfamily of voltage-gated potassium channels. Also preferably, said pharmaceutical composition is for use in the treatment and/or prevention of a condition characterised by a defect in the structure and/or function of at least one of Kv7.1, Kv7.2, Kv7.3, Kv7.4 and/or Kv7.5, more preferably of both Kv7.2 and Kv7.3. In another preferred embodiment, the pharmaceutical composition as defined above is for use as an anticonvulsant, analgesic, anti -ischemic, anti-arrhythmic, neuroprotective, anti-tinnitus, antiasthmatic, gastrointestinal motility modulator, antihypertensive, antimyotonic and/or anti- skeletal muscle dystrophia medicament.

Preferably, said condition characterised by a defect in the structure and/or function of at least one voltage-gated potassium channel is selected from the group consisting of: a central nervous system disease, a peripheral nervous system disease, a sensory sytem disease, a respiratory system disease, a genitourinary system disease, a gastrointestinal system disease, a cardiovascular disease, a skeletal muscle disease and a channelopathy.

Also preferably, said condition characterised by a defect in the structure and/or function of at least one voltage-gated potassium channel is selected from the group consisting of: epilepsy, a convulsive disorder, a seizure, a seizure disorder, a disorder characterised by hyperexcitability of the nervous system, a neonatal epilepsy, spontaneous contractions, an early-onset epileptic disorder, Ohtahara syndrome, early infantile epileptic encelopathy with suppression-burst, benign familiar neonatal seizures, epileptic encelopathy, severe sporadic neonatal epileptic encelopathy, an intellectual disability, a psychiatric or cognitive comorbidity, a cognitive defect, anxiety, depression, schizophrenia, sudden death in epilepsy, drug-resistant epilepsy, neuropathic pain, an ischemic stroke, angina, migraine, a neurodegenerative disorder, a neurologic disorder, amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, tremors, paroxysmal choreoathetosis, tinnitus, pain, asthma, bladder hyper-reactivity, urinary incontinence, constipation, irritable bowel syndrome, diarrhea, Krohn’s disease, hypertension, vascular hypertension, muscular dystrophy, a skeletal muscle hyperexcitability disease, myokymia, myotonia, a genetic channelopathy, a transcriptional channelopathy, aKv7.2- and/or Kv7.3 -related disorder, a Kv 7.2 encelopathy, autosomal dominant type 2 deafness (DFNA2), ataxia, episodic ataxia type 1, episodic ataxia with myokymia syndrome, neuromyotonia, hypokalaemic periodic paralysis, an arrhythmia, a congenital arrhythmia, a long QT syndrome, Romano-Ward syndrome, Jervell and Lange-Nielsen syndrome, Bartter syndrome, hyperinsulinemic hypoglycaemia of infancy (HHI), attenuate vasoconstrictor response, type 2 diabetes and multiple sclerosis.

It is another object of the present invention a method of treatment of a condition characterised by a defect in the structure and/or function of at least one voltage-gated potassium channel as defined above, by administering a therapeutic dose of the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above.

In yet another preferred embodiment, the compound for use as defined above or the pharmaceutical composition for use as defined above is administered to an animal. Preferably, said animal is a companion animal or livestock.

The present invention also provides the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above for use in a method of modulating a voltage-gated potassium channel. Preferably, the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is for use in a method of modulating at least one member of the Kv7 subfamily of voltage-gated potassium channels. Also preferably, the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is for use in a method of modulating Kv7.1, Kv7.2, Kv7.3, Kv7.4 and/or Kv7.5. More preferably, the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above is for use in a method of modulating Kv7.2 and Kv7.3.

It is also an object of the present invention an in-vitro method of modulating a voltage-gated potassium channel by using the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above. Preferably, said in-vitro method is an in-vitro method of modulating at least one member of the Kv7 subfamily of voltage-gated potassium channels by using the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above. Also preferably, said in-vitro method is an in-vitro method of modulating Kv7.1, Kv7.2, Kv7.3, Kv7.4 and/or Kv7.5 by using the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above. More preferably, said in-vitro method is an in-vitro method of modulating Kv7.2 and Kv7.3 by using the compound or tautomer, salt, solvate, stereoisomer or isotope thereof as defined above.

It is to be understood that any embodiment of the present invention may be combined with any other embodiment of the present invention to give rise to yet another embodiment of the present invention.

As used herein, the term“alkylcarbonyl” preferably refers to groups such as CH3(CH2) _n CO where n is a number comprised between 0 and 10 and to branched isomers thereof. Examples of “alkylcarbonyl” include acetamide, CH ₃ (CH ₂ ) ₆ CO, (CH ₃ ) ₃ CCH ₂ CO, CH ₃ (CH ₂ ) ₂ CH(CH ₃ )CO, (CH ₃ ) ₃ C(CH ₂ ) ₆ CO, CH ₃ (CH ₂ ) ₅ C(CH ₃ ) ₃ CO, CH ₃ (CH ₂ ) ₄ C(CH ₃ ) ₃ CH ₂ C0.

As used herein, the term“alkoxycarbonyl” preferably refers to groups such as CH ₃ (CH ₂ )nOCO where n is a number comprised between 0 and 10 and to branched isomers thereof. Examples of “alkoxycarbonyl” include CH ₃ (CH ₂ ) ₆ OCO, (CH ₃ ) ₃ CCH ₂ OCO, CH ₃ (CH ₂ ) ₂ CH(CH ₃ )OCO, (CH ₃ ) ₃ C(CH ₂ ) ₆ OCO, CH ₃ (CH ₂ ) ₅ C(CH ₃ ) ₃ OCO, CH ₃ (CH ₂ ) ₄ C(CH ₃ ) ₃ CH ₂ OCO.

As used herein, the term“alkylamino”, preferably refers to linear or branched alkyl groups bearing a NH or H ₂ substituted, preferably a terminal NH or NH ₂ substituted. Preferred alkylamino groups include groups such as CH ₃ (CH ₂ ) _n NH where n is a number comprised between 0 and 10 and their branched isomers, as well as groups such as H ₂ (CH ₂ ) _n CH ₂ where n is a number comprised between 0 and 10 and their branched isomers. “Alkylamino” as used herein also preferably refers to Cl -CIO linear or branched alkyl chains bearing a primary, secondary or tertiary amine as side chain Examples of“alkylamino” include NHMe, NHEt, NHiPr, N(Me) ₂ , N(Et) ₂ , N(iPr) ₂ , CH ₃ (CH ₂ ) ₆ NH, (CH ₃ ) ₃ CCH ₂ NH, CH ₃ (CH ₂ ) ₂ CH(CH ₃ )NH, (CH ₃ ) ₃ C(CH ₂ ) ₆ NH, CH ₃ (CH ₂ ) ₅ C(CH ₃ ) ₃ NH, CH ₃ (CH ₂ ) ₄ C(CH ₃ ) ₃ CH ₂ NH NH ₂ CH ₂ (CH ₂ ) ₅ CH ₂ , [NH ₂ (CH ₃ ) ₂ ]CCH ₂ NH, NH ₂ CH ₂ (CH ₂ ) ₂ CH(CH ₃ ), H ₂ (CH ₃ ) ₂ C(CH ₂ ) ₆ , NH ₂ CH ₂ (CH ₂ ) ₅ C(CH ₃ ) ₂ ,

NH ₂ CH ₂ (CH ₂ ) ₄ C(CH ₃ ) ₃ CH _2, CH ₃ CHNH ₂ (CH ₂ ) ₅ , CH ₃ (CH ₂ ) ₂ C H ₂ (CH ₂ ) ₂ ,

CH ₃ (CH ₂ ) ₂ CH ₂ [N(CH ₃ ) ₂ ], CH ₃ (CH ₂ ) ₂ C[N(CH ₃ ) ₂ ](CH ₂ ) _2, CH ₃ (CH ₂ ) ₂ C[NH(CH ₃ )](CH ₂ ) _2.

The term“alkenylamino” as used herein refers to a linear or branched alkylamino group as defined above comprising at least one carbon-carbon double bond.

The term“alkynylamino” as used herein refers to a linear or branched alkylamino group as defined above comprising at least one carbon-carbon triple bond. As used herein, the term “alkylaminocarbonyl” preferably refers to groups such as NH ₂ (CH ₂ ) _n CH ₂ CO where n is a number comprised between 0 and 10 and to branched isomers thereof.“Alkylaminocarbonyl” as used herein also preferably refers to C3-C10 linear or branched CEt ₃ X(CH ₂ ) _n CO or CH ₃ (CH ₂ ) _n X(CH ₂ ) _n CO where X is a primary, secondary or tertiary amine and n is comprised between 1 and 10. Examples of “alkylaminocarbonyl” include NH ₂ CH ₂ (CH ₂ ) ₅ CH ₂ CO, NH ₂ C(CH ₃ ) ₂ CH ₂ CO, NH ₂ CH ₂ (CH ₂ ) ₂ CH(CH ₃ )CO,

NH ₂ (CH ₃ ) ₂ C(CH ₂ ) ₆ CO, NH ₂ CH ₂ (CH ₂ ) ₅ C(CH ₃ ) ₃ CO, NH ₂ CH ₂ (CH ₂ ) ₄ C(CH ₃ ) ₃ CH ₂ CO _,

CH ₃ CH(NH ₂ )(CH ₂ ) ₅ CO, CH ₃ (CH ₂ ) ₂ C(NH ₂ )(CH ₂ ) ₂ CO, CH ₃ (CH ₂ ) ₂ CH ₂ [N(CH ₃ ) ₂ ]CO,

CH ₃ (CH ₂ ) ₂ C[N(CH ₃ ) ₂ ](CH ₂ ) ₂ C0 _, CH ₃ (CH ₂ ) ₂ CH[NH(CH ₃ )](CH ₂ ) ₂ C0.

As used herein, the term“alkyloxy” or“alkoxy” refers to groups bearing at least one oxygen atom linked to an alkyl chain. Preferred alkyloxy groups include groups such as: CH ₃ (CH ₂ ) _n O where n is a number comprised between 0 and 10 and their branched isomers, HO(CH ₂ )nCH ₂ where n is a number comprised between 0 and 10 and their branched isomers, as well as groups such as CH ₃ X(CH ₂ ) _n CH ₂ , CH ₃ (CH ₂ ) _n XCH ₂ and CH ₃ (CH ₂ ) _n X(CH ₂ ) _n where X is an oxygen atom and n is comprised between 1 and 10. Examples of“alkyloxy” or“alkoxy” include methoxy, ethoxy, HOCH ₂ (CH ₂ ) ₅ CH ₂ , HO(CH ₃ ) ₂ CCH ₂ , HOCH ₂ (CH ₂ ) ₂ CH(CH ₃ ), H0(CH ₃ ) ₂ C(CH ₂ ) ₆ , HOCH ₂ (CH ₂ ) ₅ C(CH ₃ ) ₃ , HOCH ₂ (CH ₂ ) ₄ C(CH ₃ ) ₃ CH _2, H0CH ₂ CH ₂ 0(CH ₂ ) ₅ ,

CH ₃ (CH ₂ )2C(0H)(CH ₂ ) ₂ , CH ₃ (CH ₂ ) ₂ CH ₂ OCH ₃ , CH ₃ (CH ₂ ) ₂ C(0CH ₃ )(CH2)2.

CH ₃ (CH ₂ ) ₂ C(OCH ₃ )(CH ₂ ) _2, CH ₃ (CH ₂ ) ₆ 0, (CH ₃ ) ₃ CCH ₂ 0, CH ₃ (CH ₂ ) ₂ CH(CH ₃ )0,

(CH ₃ ) ₃ C(CH ₂ ) ₆ 0, CH ₃ (CH ₂ ) ₅ C(CH ₃ ) ₃ 0, CH ₃ (CH ₂ ) ₄ C(CH ₃ ) ₃ CH ₂ 0, CH ₃ CH ₂ OCH ₂ CH ₂ OH,

CH ₃ CH ₂ OCH ₂ CH ₂ O, CH ₂ CH ₂ OCH ₂ CH ₂ O

As used herein, the term“alkylthio” refers to groups bearing at least one sulfur atom linked to an alkyl chain. Preferred alkylthio groups include groups such as: SMe, SEt, CH ₃ (CH ₂ ) _n S where n is a number comprised between 0 and 10 and their branched isomers, HS(CH ₂ ) _n CH2 where n is a number comprised between 0 and 10 and their branched isomers, as well as groups such as CH ₃ X(CH ₂ ) _n CH _2, CH ₃ (CH ₂ )„XCH ₂ or CH ₃ (CH ₂ )„X(CH ₂ )„ where X is a sulfur atom and n is comprised between 1 and 10. Examples of “alkylthio” include HSCH ₂ (CH ₂ )5CH ₂ , HS(CH ₃ ) ₂ CCH ₂ CO, HSCH ₂ (CH ₂ ) ₂ CH(CH ₃ ), HS(CH ₃ ) ₂ C(CH ₂ ) ₆ , HSCH ₂ (CH ₂ ) _S C(CH ₃ ) ₃ , HSCH ₂ (CH ₂ ) ₄ C(CH ₃ ) ₃ CH _2. HSCH ₂ CH ₂ S(CH ₂ ) ₅ , CH ₃ (CH ₂ ) ₂ CSH(CH ₂ ) ₂ , CH ₃ (CH ₂ ) ₂ CH ₂ SCH ₂ , CH ₃ (CH ₂ ) ₂ C(SCH ₃ )(CH ₂ ) ₂ CH ₃ (CH ₂ ) ₂ C(SCH ₃ )(CH ₂ ) ₂ CH ₃ (CH ₂ ) ₆ S, (CH ₃ )3CCH ₂ S,

CH ₃ (CH ₂ ) ₂ CH(CH ₃ )S, (CH ₃ ) ₃ C(CH ₂ ) ₆ S, CH ₃ (CH ₂ ) ₅ C(CH ₃ ) ₃ S, CH ₃ (CH ₂ ) C(CH ₃ ) ₃ CH ₂ S,

CH ₃ CH ₂ S(CH ₂ ) _5. CH ₃ CH ₂ SCH ₂ CH ₂ SH, CH ₃ CH ₂ SCH ₂ CH ₂ SCH ₂ CH ₂ SCH ₂ CH ₂ S.

As used herein, the term“carbonyl” preferably refers to Cl -CIO aliphatic aldheydhes or ketones. Examples of “carbonyl” include HOCCH ₂ (CH ₂ ) ₅ CH ₂ , HOC(CH ₃ ) ₂ CCH ₂ CO, HOCCH ₂ (CH ₂ ) ₂ CH(CH ₃ ), H0C(CH ₃ ) ₂ C(CH ₂ ) ₆ , HOCCH ₂ (CH ₂ ) ₄ C(CH ₃ ) ₃ ,

H0CCH ₂ (CH ₂ ) ₃ C(CH ₃ ) ₃ CH _2, H0CCH ₂ CH ₂ 0(CH ₂ ) ₅ , and CH ₃ (CH ₂ ) _n CO(CH ₂ ) _m groups where n is a number comprised between 0 and 10 and m is a number comprised between 1 and 10 and branched isomers thereof.

As used herein, the term “amide” preferably refers to groups such as CH ₃ CONH, CH ₃ (CH ₂ )nCONH, H ₂ NCO(CH ₂ ) _n where n is a number comprised between 0 and 10 and to branched isomers thereof. In the present invention, amide functionalities are preferably substituted at the nitrogen position.

As used herein, the term“carboxyl”, preferably refers to groups such as XOOC(CH ₂ ) _n where n is a number comprised between 0 and 10, X is selected between H and alkyl chain and to branched isomers thereof. “Carboxyl” also preferably refers to groups such as CH ₃ (CH ₂ ) _n CH ₂ (COOX) where X is selected between H and alkyl chain, and to branched isomers.

As used herein, the term“amine” preferably refers to groups such as NH ₂ , NHX, NXIX ₂ where Xi and X ₂ represents any of the groups described above, in particular linear or branched alkyl chains. As used herein, the terms“aryl” or“aromatic ring” are used interchangeably and refer to a monocyclic, bicyclic or polycyclic aromatic ring system comprising carbon atoms and hydrogen atoms, preferably comprising from 5 to 10 carbon atoms. In the present invention, aromatic rings optionally comprise as members of the ring one or more heteroatoms, preferably 1 to 3 heteroatoms, preferably selected from N, O and S, in which case they are also referred to as “heteroaryl” or“heteroaromatic rings”. Aryl groups of the present invention only need to have some degree of aromatic character. Preferred aryl groups are formed by a phenyl ring and/or phenyl rings optionally substituted in one position or in more than one position, or in any possible position by any kind of possible substituents. Examples of“aryl” include benzene, naphthalene, anthracene, pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine, pyrimidine, quinazoline, pyridazine, cinnoline, phthalazine, 1,2,3-triazine, 1,2,4-triazine, 1,3,5 triazine, furan, benzofuran, isobenzofuran, pyrrole, indole, isoindole, thiophene, benzothiophene, benzo[c]thiophene, imidazole, benzoimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, benzoisoxazole, thiazole, benzothi azole. Preferably, aromatic rings are phenyl, pyridine and pyrrole or Such aryls may optionally be substituted with one or more substituents, preferably selected from alkyloxy, nitrile, amino, hydroxyl, halogen, trifluoroalkyl, trichloroalkyl, difluoroalkyl, dichloroalkyl, alkylcarbonyl, trifluoromethoxy, alkyl alkoxycarbonyl, alkylamino, alkylaminocarbonyl, alkylthio, carbonyl, amide and/or carboxyl.

As used herein, the term“cycloalkyl” refers to a saturated hydrocarbon ring, preferably bearing from 3 to 8 cabon atoms, more preferably bearing from 3 to 6 carbon atoms, that can be unsubstituted or substituted in one, more than one or any of the possible position by any kind of possible substituents. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl, cycloheptyl, cyclooctyl, norbomane, optionally substituted with one or more substituents, preferably selected from: alkyloxy, nitrile, amino, hydroxyl, halogen, trifluoroalkyl, trichloroalkyl, difluoroalkyl, dichloroalkyl, alkylcarbonyl, trifluoromethoxy, alkyl, alkoxycarbonyl, alkylamino, alkylaminocarbonyl, alkylthio, carbonyl, amide and carboxyl.

As used herein, the term“heterocycle”, refers to a saturated or partially saturated aliphatic ring, preferably bearing from 3 to 7 carbon atoms and comprising as ring members one or more than one heteroatoms, preferably selected from N, O and S. Heterocycles can be unsubstituted or substituted in one, more than one or any of the postion possible position by any kind of possible substituents. Examples of “heterocycle” comprise piperidine, quinoline, decahydroquinoline decahydroisoquinoline, pyperazine, decahydroquinoxaline, tetradecahydroacridine, hexahydropyrimidine, decahydroquinazoline, hexahydropyridazine, decahydrocinnoline, decahydrophthalazine, tetrahydrofuran, octahydrobenzofuran, octahydroisobenzofuran, pyrrolidine, octahydro-lH-indole, octahydro-lH-isoindole, tetrahydrothiophene, octahydrobenzothiophene, octahydrobenzo[c]thiophene, imidazoline, inidazolidine, octahydrobenzoimidazole, octahydro-lH-purine, octahydro-lH-indazole, oxazolidine, octahydrobenzoxazole, isoxazolidine, octahydrobenzoisoxazole, thiazolidine, octahydrobenzothiazole optionally substituted with one or more substituents, preferably selected from: alkyloxy, nitrile, amino, hydroxyl, halogen, trifluoroalkyl, trichloroalkyl, difluoroalkyl, dichloroalkyl, alkylcarbonyl, trifluoromethoxy, alkyl, alkoxycarbonyl, alkylamino, alkylaminocarbonyl, alkylthio, carbonyl, amide and carboxyl.

As used herein, the term“alkyl” refers to a linear or branched hydrocarbon chain. Suitable examples of said alkyl include but are not limited to methyl, ethyl, n-propyl, isopropyl, butyl, sec- butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decanyl, hexadecanyl, eicosanyl, CH2C(CH3)3, CH2CH(CH3)2 etc. Long alkyl chains, e.g. C5-30 alkyl, preferably C6- C30 alkyl, more preferably C7-C30 alkyl, still preferably C10-C30 alkyl, are particularly preferred in some embodiments of the invention.

As used herein, the term“alkenyl” refers to a linear or branched alkyl chain as defined above comprising at least one carbon-carbon double bond. Long alkenyl chains, e.g. C5-30 alkenyl, preferably C6-C30 alkenyl, more preferably C7-C30 alkenyl, still preferably C10-C30 alkenyl, are particularly preferred in some embodiments of the invention.

The term“alkynyl” as used herein refers to a linear or branched alkyl group as defined above comprising at least one carbon-carbon triple bond. Long alkynyl chains, e.g. C5-30 alkynyl, preferably C6-C30 alkynyl, more preferably C7-C30 alkynyl, still preferably C10-C30 alkynyl, are particularly preferred in some embodiments of the invention.

As used herein, the term“haloalkyl” refers to a linear or branched alkyl group, wherein at least one hydrogen atom is replaced by a halogen atom, preferably fluorine and/or chlorine. Preferred haloalkyl groups comprise 1-3 carbon atoms. Examples of “haloalkyl” include difluoroalkyl, trifluoroalkyl, trichloroalkyl, difluoroalkyl, dichloroalkyl, 2,2,2 trifluoroethyl, such as CF ₃ , CHF2, CH ₂ F, CCh, CHCh, CH ₂ CI.

As used herein, the term“haloalkoxy” refers to a linear or branched alkoxy group, wherein at least one hydrogen atom is replaced by a halogen atom, preferably fluorine and/or chlorine. Preferred haloalkoxy groups comprise 1-3 carbon atoms. Examples of “haloalkoxy” include trifluoroalkoxyl, trichloroalkoxyl, difluoroalkoxyl, dichloroalkoxyl, such as OCF ₃ , OCHF ₂ , OCH ₂ F, OCCI3, OCHCE, OCHiCl

Preferably, halogen is fluorine, chlorine or bromine.

As used herein, the term“linear or branched C6-C30 arylalkyl” refers to aryl groups, as previously defined, bearing linear or branched alkyl chain substituents in one, more or all possible positions. As used herein, the term“C3-C7 cycloalkylamino” refers to cycloalkyl groups, as previously defined, bearing one or more amines and/or alkylamines and/or dialkylamines as substituents.

As used herein, the term“C7-C15 arylalkylamino” refers to arylalkyl groups, as previously defined, bearing one or more amines and/or alkylamine and/or dialkylamines as substituents.

As used herein, the term“C7-C15 arylalkenylamino” refers to to aryl groups, as previously defined, bearing linear or branched alkenyl chain, as defined above, and further substituted by one or more amines and/or alkylamine and/or dialkylamines.

As used herein, the term“C7-C15 arylalkynylamino” refers to to aryl groups, as previously defined, bearing one or more linear or branched alkynyl chain, as defined above, and further substituted by one or more amines and/or alkylamine and/or dialkylamines.

It is to be understood that in the present invention any aliphatic substituent, such as alkyl, cycloalkyl, alkylcarbonyl, alkoxycarbonyl, alkylamino, alkyl aminocarbonyl, alkyloxy, alkylthio, alkenyl, cycloalkenyl, alkynyl, may be linear or branched.

In a preferred embodiment, compounds of the invention are voltage-gated potassium channel modulators. Hence, they are useful in the treatment of a condition or disorder that is responsive to the modulation of voltage-gated potassium channels, preferably to the blocking or the opening of voltage-gated potassium channels. Compounds of the invention may also be useful in the treatment of a voltage-gated potassium channel-mediated disease or disorder. Such condition, disease or disorder may also be referred to as a condition characterised by a defect in the structure and/or function of at least one potassium channel.

In the present invention, a potassium channel is preferably a voltage-gated potassium channel, more preferably Kv7.1 (KCNQ1; NM_000218.2), Kv7.2 (KCNQ2; M_004518.4), Kv7.3 (KCNQ3; M_004519.2), Kv7.4 (KCNQ4; M_004700.4) or Kv7.5 (KCNQ5; NM_019842.3), still more preferably Kv7.2 (KCNQ2), Kv7.2/3, Kv7.3 (KCNQ3), Kv7.4 or Kv7.5. Still preferably, the potassium channel is Kv7.2 (KCNQ2), Kv7.3 (KCNQ3) or Kv7.2/Kv7.3.

In different human tissues, various classes of voltage-gated potassium channels are expressed, each with peculiar functional and pharmacological properties, reflecting the large genetic heterogeneity of this protein class, with over 70 human genes encoding for voltage-gated potassium channel subunits. Given the overall structural similarity among the different voltage-gated potassium channels, common also to other types of ion channels with different gating and selectivity properties (namely, non voltage-gated potassium channels and sodium and calcium channels), in a preferred embodiment, compounds of the present invention can modulate other ionic channels, such as non voltage-gated potassium channels and sodium and calcium channels.

“Modulation”, as used herein in its various forms, comprises antagonism, agonism, partial antagonism and/or partial agonism of the activity associated with voltage-gated potassium channels. In particular,“modulation”, as used herein in its various forms, includes opening, activating, closing, blocking, partial opening, partially-activating, partial closing or partial booking of voltage-gated potassium channels.

As used herein, the term“voltage-gated potassium channel modulator” includes compounds able to increase or decrease the basal potassium currents, or otherwise able to increase or decrease the channel opening. Moreover, as used herein, the term“voltage-gated potassium channel modulator” refers to a compound able to modulate prevalently but not exclusively voltage-gated potassium channels.

In an embodiment, compounds of the invention are useful as voltage-gated potassium channel blockers. As used herein, the term“antagonist” or“blocker” includes a molecule that blocks the ionic current(s) carried by an ionic channel. In particular, voltage-gated potassium channel antagonists can be compounds that bind to and decrease, close, deactivate, or hamper voltage gated potassium channel activity. Antagonism of voltage-gated potassium channels may include either or both of: (1) decreasing current through a voltage-gated potassium channel; or (2) shifting the half-activation potential of voltage-gated potassium channels to more positive voltages (i.e. a depolarizing shift of the V1/2 for activation). In another embodiment, compounds of the invention are useful as voltage-gated potassium channel openers. As used herein, the term“agonist” or“opener” includes a molecule that enchances the ionic current(s) carried by an ionic channel. In particular, voltage-gated potassium channel agonists can be compounds that bind to and stimulate, increase, open, activate, or facilitate voltage gated potassium channel activity. Activation of voltage-gated potassium channels may include either or both of: (1) increasing current through a voltage-gated potassium channel; or (2) shifting the half-activation potential of voltage-gated potassium channels to less depolarized voltages (i.e. a hyperpolarizing shift of the V1/2 for activation).

Example 8 and 10 provide illustrative procedures to discriminate whether a compound acts as a modulator, in particular as an antagonist or as an agonist, of a voltage-gated potassium channel. In particular, a compound may be classified as a voltage-gated potassium channel antagonist when it possesses a value of I _d mg/E _ti , as defined in Example 8, < 1 and a compound may be classified as a voltage-gated potassium channel agonist when it possesses a value of I _d m _g /I _cti , as defined in Example 8, > 1. In particular, a compound may be classified as a voltage-gated potassium channel antagonist when it possesses a value of F/Fo < 1.3 and a value of Slope < 0.009, as defined in Example 10, and a compound may be classified as a voltage-gated potassium channel agonist when it possesses a value of F/Fo > 1.3 and a value of Slope > 0.009, as defined in Example 10.

Kv7 channels are directly implicated in genetic channelopathies, namely human monogenic diseases caused by specific mutations in the primary sequence of changes Kv7 genes; these include congenital arrhythmias, such as the long QT syndrome, epileptic and non-epileptic diseases known altogether as Kv7.2- and Kv7.3-related disorders, and autosomal dominant type 2 deafness (DFNA2), a progressive form of sensorineural hearing loss. In addition to these rare monogenic diseases, and given their prominent role in controlling neuronal, smooth muscle, and skeletal muscle excitability, changes in the expression of Kv7 channels underlie diverse physiopathologial conditions (so-called transcriptional channelopathies) including, but not limited to: epilepsy, neuropathic pain, ischemic stroke, amyotrophic lateral sclerosis, and several other neurodegenerative diseases, for the neuronal component; vascular hypertension, asthma and bladder hyperreactivity for the smooth muscle component; myotonia, myokymia and other skeletal muscle hyperexcitability diseases. All such conditions may be prevented and/or treated using compounds of the present invention or pharmaceutical compositions comprising them.

As used herein,“defect in the structure and/or function of at least one voltage-gated potassium channel” includes mutations in genes coding for voltage-gated potassium channels and changes in the expression of voltage-gated potassium channels. Preferably, a defect in the structure and/or function of at least one voltage-gated potassium channel is a mutation occurring de novo or inherited by one of the parents which produces gain-of-function or loss-of-function.

Also preferably, a defect in the structure and/or function of at least one voltage-gated potassium channel, which is responsible for changes in the function of specific ion channels, involves accessory subunits or small molecules mostly acting as cytoplasmic regulators, known to interfere with the K ⁺ channel by direct or indirect mechanisms. For example, for Kv7.2 and Kv7.3, such accessory subunits or small molecules include but are not limited to: PIP2 (Zhang H et al., Neuron 2003, 37: 963-975,), the Calmodulin (CaM) (Yus-Najera E et al., JBC 2002, 277: 28545-28553; Wen H et al., J Neurosci 2002, 22: 7991-8001), the protein kinase A anchoring (AECAP) (Hoshi N et al., Nat Neurosci 2003, 6: 564-571), the Ankyrin G (Ank-G), which regulates the subcellular distribution in level of the axonal compartment (Pan Z et al., J Neurosci 2006, 26: 2599-2613) and the ligase ubiquitin Nedd4-2 (Ekberg J et al., JBC 2007, 282: 12135-12142).

As used herein,“pain” includes disorders or conditions related to pain, such as neuropathic pain (including diabetic polyneuropathy), nociceptive pain, persistent pain, osteoarthritic pain, cancer pain, inflammatory pain, postoperative pain, fibromyalgia, chronic widespread pain, musculoskeletal pain, myofascial pain, Temporomandibular joint pain (TMJ pain).

Compounds of the invention may be used to modulate the M-current, for instance to increase or to decrease the M-current. Therefore, the present invention provides compounds of formula I as defined above for use in modulating the M-current, especially in a patient characterized by aberrant M-current.

Compounds of the invention may also be used in a method to open a potassium channel.

Compounds of the invention may also be used in a method to block a potassium channel.

This invention contemplates any tautomer, salt, stereoisomer of the compounds with general formula I. Moreover, it must be considered as part of the present invention any molecule having general formula I in which one or more atoms are replaced by isotopes.

In the context of the invention the salts of the compounds of the present invention are also included. On account of their potential use in medicine, the salts of the compounds of formula I are preferably pharmaceutically acceptable. Pharmaceutically acceptable salts comprise the conventional non-toxic salts obtained by salification of a compound of formula I with inorganic acids (e g. hydrochloric, hydrobromic, sulphuric or phosphoric acid), or with organic acids (e g. acetic, propionic, succinic, benzoic, sulfanilic, 2-acetoxy-benzoic, cinnamic, mandelic, salicylic, glycolic, lactic, oxalic, malic, maleic, malonic, fumaric, tartaric, citric, p-toluenesulfonic, methanesulfonic, ethanesulfonic, or naphthalenesulfonic acid). For a review of suitable pharmaceutical salts see SM Berge, et al., J. Pharm. Sci. 1977, 66, 1-19; PL Gould, Int. J Pharm. 1986, 33, 201-217; LD Bighley, et al., Encyclopedia of Pharmaceutical Technology, Marcel Dekker Inc, New York 1996, Volume 13, page 453-497. Other salts which are not pharmaceutically acceptable, such as trifluoroacetate salt, can be useful in the preparation of compounds of the present invention and these form a further aspect of the invention. The invention includes within its scope all possible stoichiometric and non-stoichiometric forms of the salts of the compounds of formula I.

Furthermore, the compounds of formula I can exist in non-solvated forms as well as in forms solvated with pharmaceutically acceptable solvents such as water, EtOH and the like.

Some compounds of formula I can exist in stereoisomeric forms (e.g. they may contain one or more asymmetric carbon atoms). The individual stereoisomers (enantiomers and diastereoisomers) and any mixture, in particular racemic mixture, comprising these are included in the scope of the present invention. The present invention also covers the individual isomers of the compounds represented by formula I as well as mixtures with isomers in which one or more chiral centres are inverted.

Similarly, it is understood that the compounds of formula I can exist in tautomeric forms other than those shown in the formula and these are included in the scope of the present invention. Though structural representations can show only one of the possible tautomeric or stereoisomeric forms, it is to be understood that the invention encompasses any tautomeric or stereoisomeric form, and mixtures thereof, and is not to be limited merely to any one tautomeric or stereoisomeric form utilized within drawings or the naming of the compounds.

The invention also includes all the suitable isotopic variations of a compound of the invention. An isotopic variation of a compound of the invention is defined as one in which at least one atom is replaced by an atom having the same atomic number but a different atomic mass from the atomic mass usually found in nature. In particular, the term“isotope” as used herein refers to any molecule having the general formula I in which one or more atoms are replaced by isotopes, preferably radioactive isotopes. Examples of isotopes which may be incorporated in the compounds of the invention include isotopes such as ¾, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ 0, ¹⁸ 0, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ C1, respectively. Certain isotopic variations of the invention, for example those in which a radioactive isotope such as ³ H or ¹⁴ C is incorporated, are useful in studies on tissue distribution of the medicament and/or substrate Furthermore, substitution with isotopes such as deuterium ¾, may provide certain therapeutic advantages resulting from a greater metabolic stability. The isotopic variations of the compounds of the invention can generally be prepared by conventional procedures as well as with the methods illustrated or by the preparations described in the examples below using appropriate isotopic variations of suitable reagents.

The invention further comprises pharmaceutical compositions containing at least one compound of the present invention or a pharmaceutically acceptable tautomer, salt, solvate, stereoisomer or isotope thereof and one or more pharmaceutically acceptable carriers. As used herein a pharmaceutically acceptable ingredient comprises any excipient, diluent, binder, lubricant, tablettig agent, disintegrant, preservative, buffering agent and any other ingredient tipically used in the art of formulation of pharmaceuticals and veterinary. An exemplary but not exhaustive list of examples of pharmaceutically acceptable carriers is reported in Kibbe, Handbook of Pharmaceutical Excipients, London, Pharmaceutical Press, 2000.

The pharmaceutical compositions can be chosen according to the needs of treatment. Such compositions are prepared by mixing and are suitably adapted to oral or parenteral administration, and as such can be administered in the form of tablets, capsules, oral preparations, powders, granules, pills, or injectable or infusible liquid solutions, suspensions, suppositories, preparation by inhalation.

Tablets and capsules for oral administration are usually presented in unit dosage form and contain conventional excipients such as ligands, fillers (including cellulose, mannitol, lactose), diluents, tablet agents, lubricants (including magnesium stearate), detergents, disintegrants (for example polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate), colorants, flavourings and wetting agents (for example sodium lauryl sulphate).

The solid oral compositions may be prepared by conventional methods of mixing, filling or tabletting. The mixing operation can be repeated to distribute the active substance in all the compositions containing large quantities of fillers. Such operations are conventional.

The oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or with a suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatine, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents such as lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils) such as almond oil, fractionated coconut oil, oily esters such as glycerine esters, propylene glycol, or ethyl alcohol; preservatives such as methyl or propyl p- hydroxybenzoate or sorbic acid, and if desired, conventional flavourings or colourants. Oral formulations also include conventional slow-release formulations such as gastro-resistant coated tablets or granules. The pharmaceutical preparation for administration via inhalation can be provided by an insufflator or by a pressurized nebulizer.

For parenteral administration, the unit dosage of fluid can be prepared comprising the compound and a sterile vehicle. The compound may be suspended or dissolved, depending on the vehicle and concentration. Parenteral solutions are normally prepared by dissolving the compound in a vehicle, sterilizing the latter by filtration, filling suitable vials and sealing. Advantageously, adjuvants such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle. To increase stability, the composition can be frozen after having filled the vials and removed water under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound may be suspended in the vehicle instead of being dissolved and sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or a wetting agent may be included in the composition to facilitate uniform distribution of the compound of the invention.

For oral or sublingual administration the compositions may be tablets, lozenges, or gel.

The compounds may be pharmaceutically formulated as suppositories or retention enemas, e g. suppositories containing conventional bases such as cocoa butter, polyethylene glycol, or other glycerides, for rectal administration.

Another route of administration of the compounds of the invention regards topical treatment. The topical formulations may contain for example ointments, creams, lotions, gels, solutions, pastes and/or can contain liposomes, micelles and/or microspheres. Examples of ointments include oleaginous ointments such as vegetable oils, animal fats, semi-solid hydrocarbons, emulsifiable ointments such as hydroxystearic sulphate, anhydrous lanolin, hydrophilic petrolatum, cetyl alcohol, glycerol monostearate, stearic acid, water soluble ointments containing polyethylene glycols of various molecular weights. The creams, as known to persons skilled in formulation, are viscous liquids or semi-solid emulsions, and contain an oily phase, an emulsifier and an aqueous phase. The oily phase generally contains petrolatum and an alcohol such as cetyl or stearyl alcohol. The formulations suitable for topical ocular administration also include eye drops, in which the active ingredient is dissolved or suspended in a suitable vehicle, in particular in an aqueous solvent for the active ingredient.

A further method of administration of the compounds of the invention regards transdermal vehiculation. Typical transdermal formulations comprise conventional aqueous and non-aqueous vectors, such as creams, oils, lotions or pastes or may be in the form of membranes or medicated patches. A reference for the formulations is Remington's book (Remington“The Science and Practice of Pharmacy”, Lippincott Williams & Wilkins, 2000).

The compounds of the present invention can be used in the treatment and/or prevention of the conditions referred to herein as a single therapy or in combination with other therapeutic agents, either by separate administrations, or by including the two or more active substances in the same pharmaceutical formulation. The compounds can be administered simultaneously or sequentially. The other therapeutic agents may be compounds currently on the market.

Examples of suitable therapeutic agents that can be used in combination with compounds of the invention include anticonvulsants (for example, but not limited to, Acetazolamide, Brivaracetam, Carbamazepine, Clobazam, Clonazepam, Diazepam, Eslicarbazepine, Ethosuximide, Fosphenytoin, Gabapentin, Lacosamide, Lamotrigine, Levetiracetam, Lorazepam, Methosuximide, Nitrazepam, Oxcarbazepine, Perampanel, Phenobarbital, Phenytoin, Pregabalin, Primidone, Rufmamide, Stiripentol, Topiramate, Valproic Acid, Vigabatrin, Felbamate, Tiagabine, Zonisamide), analgesics (for example, acetaminophen or opioids such as, but not limited to, morphine) and nonsteroidal anti-inflammatory drugs (NSAIDs). Examples ofNSAIDs include, but are not limited to, aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine, oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetin and zomepirac.

The combination can be administered as a separate composition (simultaneous, sequential) of the individual components of the treatment or as a single dosage form containing both agents. When the compounds of the present invention are combined with other active substances, the active substances may be formulated separately in the form of single ingredient preparations of one of the forms described above, and then given as combined preparations, administered at the same time or different times, or may be formulated together in preparations with two or more ingredients.

The compounds of general formula I may be administered to a patient in a total daily dose of, for example, 0 001 to 1000 mg/kg of body weight per day. The compositions of the dosage units may contain amounts of sub-multiples thereof to make up the daily dose. The compound can also be administered weekly or every other day. The determination of optimum dosages for a specific patient is well known to those skilled in the art. As is common practice, the compositions are normally accompanied by instructions for use in the treatment in question written or printed. Preferably, compounds with general formula I are useful for systemic administration (oral, parenteral, or rectal), and/or topical with a variable dosing between 50 to 4000 mg die. Consequently, this invention contemplates different compositions suitable for the human or animal administration of compounds with general formula I, and/or their mixtures, and/or their isotopes, and/or any possible isomer, mixed with pharmaceutically acceptable ingredients. Hence, the present invention contemplates solutions or suspensions (sterile or not), syrups, any kind of tablets and capsules (chewable, dispersible etc.), topical ointments, controlled release pharmaceutical or veterinary formulations, nanotechnology -based pharmaceutical or veterinary formulations suitable for the administrations of compounds with general formula I, and/or their mixtures, and/or their isotopes, and/or any possible isomer.

DETAILED DESCRIPTION OF THE INVENTION

SYNTHETIC PROCEDURES

The following schemes are examples of synthetic procedures that may be used to prepare the compounds of the invention. In the following schemes, unless otherwise indicated, Ri, R ₂ , R ₃ , R ₄ , R5 and 5 are as defined herein above for general formula I. The substituent R as used in the following schemes refers to the one or more substituents that are optionally present on the R ₂ group of general formula I.

It will be understood by those skilled in the art that these synthetic procedures may be modified to still obtain compounds of the invention. For instance, by choosing the appropriate reagents, those skilled in the art will obtain compounds of general formula I wherein (Y) _b -R2 is different from CH2PI1, as shown below. In addition, reactive groups can be protected with protecting groups and deprotected according to well established techniques. Further, certain compounds of the invention can be converted into other compounds of the invention according to standard chemical methods.

Example 1 - Synthesis of compounds of general formula 2

The synthetic procedures for compounds with general formula 2 is shown in Scheme 1.

Scheme 1 :

Synthesis of intermediates of formula 2

Briefly, variously substituted 1,4-phenylendiamines (synthesized using typical and well described synthetic methods or, otherwise, commercially available) were dissolved in THF added with DIPEA (1.2 eq) and the proper acyl chloride (1.2 eq). and then stirred at room temperature for 1 hour. The reaction was then washed with a saturated solution of K ₂ CO ₃ and brine. The organic phase was extracted, dried over anhydrous Na ₂ SC> ₄ , filtered and concentrated in vacuo. Crude product was purified using a linear gradient of n-hexane/ethyl acetate. The obtained intermediates of general formula 1 were alkylated using 1.2 eq of the proper benzyl bromide and 1.2 eq of DIPEA in DMF at 180°C. The reaction was then washed with a solution of K ₂ CO ₃ and brine. The organic phase was extracted, dried over anhydrous INteSCri, filtered and concentrated in vacuo. Crude product was purified using a linear gradient of n-exane/ethyl acetate.

Below follows an example of one product synthesized following the above described procedure.

N-(4-((4-(trifluoromethyl)benzyl)aniino)phenyl)heptanaini de

Synthesized starting from 1,4 phenylenediamine, 4-trifluorobenzyl bromide and heptanoyl chloride. ¹ H NMR (CD ₃ OD, 400 MHz) d 0.92 (t, 3H, G ¾ J=6.9 Hz); 1.32-1.40 (m, 6H, 3 CH>): 1.64-1.71 (m, 2H, C Hi ₎ , 2.31 (t, 2H, C H ₂ , J=7.6 Hz); 4.40 (s, 2H, CH ₂ ); 6.58 (d, 2H, aryl, J=8.9 Hz); 7.23 (d, 2H, aryl, J=8.8 Hz); 7.53-7.61 (m, 4H, aryl); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 13.0; 22.2; 25.7; 28.6; 31.3; 36.4;112.6; 122.1; 124.9; 127.3; 128.2; 128.5; 128.8; 145.1; 145.5; 173.0.

¹⁹ F NMR (CDCb, 376.3 MHz) d: -62.44 (3F, CF ₃ ) HR-MS m/r. calcd for C ₂₁ H ₂₅ F ₃ N ₂ O, [(M+H) ⁺ ] : 379.1997; found 379.2001;

Example 2 - Synthesis of compounds of general formula 6 and 8

The general synthetic procedure for compounds of general formula 6 and 8 is shown in Scheme 2. Scheme 2:

Synthesis of final products of formula 6 and 8

To a solution of the properly substituted 4-amino-3-nitrophenol (3, synthesized using typical and well described synthetic methods or, otherwise, commercially available) differently substituted benzyl bromides (1.2 eq) were added, together with K2CO3 (1.2 eq) and a catalytic amount of KI. The mixture was refluxed for 3 hours. Reaction was washed with a saturated solution of Na ₂ S ₂ 0 ₃ , K2CO3 and brine. The organic phase was extracted, dried over anhydrous Na ₂ SC> ₄ , filtered and concentrated in vacuo. Crude products of general formula 4 were purified using a linear gradient of n-exane/ethyl acetate. The obtained intermediates of general formula 4 were dissolved in THF and coupled with R6SO2CI (1.2 eq) using DBU (1.2 eq) as base, to give intermediates of general formula 5, or with ReCOCl (1.2 eq) using DIPEA (1.2 eq) as base, to give intermediates of general formula 7, under magnetic stirring at room temperature for 3-5 hours. The mixtures were then extracted with a saturated solution of K ₂ CO ₃ and brine and the organic phase were extracted, dried over anhydrous NaiSCU, filtered and concentrated in vacuo. Intermediates of general formula 5 and 7 were purified using a linear gradient of n-exane/ethyl acetate. Then, the intermediates were subjected to catalytic hydrogenation by solubilisation in Methanol/THF (1 :9, v:v) and reaction with ammonium formate (10 eq) and Pd/C (cat) under reflux. Upon cooling to room temperature, the reactions were filtered over a Celite pad, dried in vacuo, reconstituted in DCM and then extracted with NaOH (IN). The organic phase was extracted, dried over anhydrous Na ₂ SC> ₄ , filtered and concentrated in vacuo. Final products of general formula 6 and 8 were obtained by chromatographic purification using linear gradients of n-hexane/ethyl acetate.

The following molecues were synthesized following the procedure described above.

N-(2-amino-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)cyclo hexanesulfonamide

Synthesized starting from 4-amino-3-nitrophenol, 4-trifluorobenzyl bromide and cyclohexyl sulfonyl chloride. ¾ NMR (CD3OD, 400 MHz) d 1.23-1.37 (m, 3H, C//2 and CM): 1.51-1.60 (m, 2H, CH ₂ ); 1.71 (d, 1H, C H, J=10.7 Hz); 1.89 (d, 2H, C Hi, J=12.3 Hz); 2.22 (d, 2H, G ¾, J=12.3 Hz); 3.00 (t, 1H, C H, J=11.9 Hz); 5.13 (s, 2H, G%); 6.35 (d, 1H, aryl, J= 8.7 Hz); 6.48 (s, 1H, aryl); 7.03 (d, 1H, aryl, J=8.6 Hz); 7.62 (d, 2H, aryl, J=8.1 Hz);7.68 (d, 2H, aryl, J=8.1 Hz); ¹³ C NMR (CD3OD, 100 MHz) d: 24.8; 26.3; 59.3; 68.6; 102.3; 104.2; 115.5; 124.9; 127.3; 129.1; 142.1; 146.3; 158.6. ¹⁹ F NMR (CD3OD, 376.3 MHz) d: -62.59 (3F, CF _S ) HR-MS m/z: calcd for C20H25F3NO3S, [(M+H) ⁺ j: 430.1538; found 430.1543;

N-(2-amino-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)hepta namide

Synthesized starting from 1,4 phenylenediamine, 4-trifluorobenzyl bromide and heptanoyl chloride. ¾ NMR (CD ₃ OD, 400 MHz) d 0.82 (t, 3H, CH J=6.6 Hz); 1.26-1.33 (m, 6H, 3 G %); 1.59-1.66 (m, 2H, G %); 2.37 (t, 2H, G %, J=7.6 Hz); 5.15 (s, 2H, G %); 6.96 (s, 1H, aryl); 7.04 (d, 1H, aryl, J=9.8 Hz); 7.13 (d, 1H, aryl, J=8.8 Hz); 7.55 (d, 2H, aryl, J=8.1 Hz); 7.60 (d, 2H, aryl, J=8.1 Hz); ¹³ C NMR (CD OD, 100 MHz) d: 13.0; 22.2; 25.1; 28.7; 31.3; 35.8; 69.3; 110.2; 115.3; 124.6; 125.1; 126.5; 126.7; 127.5; 129.7; 130.0; 141.1; 157.4; 174.6. ¹⁹ F NMR (CD ₃ OD, 376.3 MHz) d: -62.66 (3F, C ); HR-MS m/r. calcd for C ₂₁ H ₂₆ F ₃ N ₂ O ₂ , [(M+H) ⁺ ]: 395.1946; found 395.1948;

Example 3 - Synthesis of compounds of general formula 12

The general procedure for the synthesis of compounds with general formula 12 is depicted in Scheme 3.

Scheme 3 :

Synthesis of final products of formula 12

The properly substituted 2-nitro-l,4-phenylendiamines (synthesized using typical and well described synthetic methods or, otherwise, commercially available) were dissolved in DMF, then 1.2 eq of the proper benzyl halide and 1.2 eq of DIPEA were added. The reaction mixture was refluxed for 3 hours under magnetic stirring. The crude products were then washed with a solution of K2CO3 and brine. The organic phases were extracted, dried over anhydrous NaiSCri, fdtered and concentrated in vacuo. Crude products were purified using a linear gradient of n-hexane/ethyl acetate to give intermediates of general formula 9. These intermediates were reacted with benzyl chloroformate (1.2 eq) using TEA (1.2 eq), to give the corresponding N-protected intermediates. Afterward, the reaction mixtures were diluted with dichloromethane and the resulting solutions were washed with K2CO3, dried over Na ₂ SC> ₄ , and evaporated to dryness. Chromatography of the residues, using DCM or DCM/MeOIT as eluents, yielded the corresponding N-protected carbamate derivatives of general formula 10. Acylation reactions to afford intermediates of general formula 11 were performed in accordance with the procedure described above for the products with general formula 1. Intermediates of general formula 11 were then subjected to the catalytic hydrogenation protocol described above for 6 and 8 to afford the final products of general formula 12.

The following examples include some of the molecules synthesized according to the above described protocol.

N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)heptan amide

Synthesized starting from 2-nitro-l,4-phenylenediamine, 4-trifluorobenzyl bromide and heptanoyl chloride. ¾ NMR (CDCh, 400 MHz) d 0.81 (t, 3H, G%, J=6.5 Hz); 1.19-1.25 (m, 6H, 3 G %); 1.55-1.62 (m, 2H, C Hi), 2.25 (t, 2H, C Hi, 1=1.6 Hz); 4.27 (s, 2H, C%); 5.95-6.00 (m, 2H, aryl); 6.65 (d, 1H, aryl, J=8.4 Hz); 7.42 (d, 2H, aryl, J=8.1 Hz); 7.48 (d, 2H, aryl, J=8.1 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 13.0; 22.2; 25.7; 28.7; 31.3; 35.8; 100.9; 104.2; 114.1; 124.8; 126.9; 127.3; 128.4; 128.8; 142.9; 145.2; 148.0; 174.1. ¹⁹ F NMR (CDCh, 376.3 MHz) d: -62.43 (3F, CF ₃ ) HR- MS m/z: calcd for C ₂₁ H ₂₇ F ₃ N ₃ O, [(M+H) ⁺ ]: 394.2106; found 394.2098;

N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)oct anainide Synthesized starting from 2-nitro-l,4-phenylenediamine, 4-trifluorobenzyl bromide and octanoyl chloride. ¾ NMR (CD ₃ OD, 400 MHz) d 0.93 (t, 3H, CH J=6.9 Hz); 1.34-1.40 (m, 8H, 4 C H ₂ ) 1.67-1.74 (m, 2H, C H ₂ ) 2.37 (t, 2H, CH ₂ , J=7.6 Hz); 4.40 (s, 2H, G %); 6.08-6.12 (m, 2H, aryl); 6.77 (d, 1H, aryl, J=8.4 Hz); 7.55 (d, 2H, aryl, J=8.3 Hz); 7.60 (d, 2H, aryl, J=8.3 Hz); ¹³ C NMR (CD3OD, 100 MHz) d: 13.0; 22.3; 25.8; 28.8; 29.0; 31.5; 35.8;100.7; 114.1; 124.8; 126.79; 126.82; 127.3; 128.4; 128.7; 129.2; 142.8; 145.2; 148.0; 174.1. ¹⁹ F NMR (CD ₃ OD, 376.3 MHz) d: -63.86 (3F, C Fs),· HR-MS m/z calcd for C22H29F3N3O, [(M+H) ⁺ ]: 408.2263; found 408.2271;

N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)dec anamide

Synthesized starting from 2-nitro-l,4-phenylenediamine, 4-trifluorobenzyl bromide and decanoyl chloride. ¾ NMR (CD3OD, 400 MHz) d 0.92 (t, 3H, C H 1=1.0 Hz); 1.23-1.42 (m, 12H, 6 C H ₂ ) 1.67-1.74 (m, 2H, C H ₂ ) 2.37 (t, 2H, C H ₂ , 1=1.6 Hz); 4.40 (s, 2H, C H ₂ ); 6.07-6.12 (m, 2H, aryl); 6.78 (d, 1H, aryl, J=8.4 Hz); 7.55 (d, 2H, aryl, J=8.3 Hz); 7.60 (d, 2H, aryl, J=8.3 Hz); ¹³ C NMR (CD3OD, 100 MHz) d: 13.0; 22.3; 25.8; 29.0; 29.2; 31.6; 35.8; 100.7; 104.2; 114.1; 124.8; 126. 9; 127.3; 142.8; 145.2, 148.0; 174.1. ¹⁹ F NMR (CDCI3, 376.3 MHz) d: -62.43 (3F, C F ₃ ) HR-MS m/z: calcd for C24H33F3N3O, [(M+H) ⁺ ]: 436.2576; found 436.2582;

N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)dod ecanainide

Synthesized starting from 2-nitro-l,4-phenylenediamine, 4-trifluorobenzyl bromide and dodecanoyl chloride.

^ NMR CDsOD, 400 MHz) d 0.92 (t, 3H, G ft, J=7.0 Hz); 1.23-1.38 (m, 16H, 8 C H ₂ ) 1.67-1.74 (m, 2H, C Hi)· 2.37 (t, 2H, G%, J=7.6 Hz); 4.39 (s, 2H, C Hi), 6.07-6.12 (m, 2H, aryl); 6.78 (d, 1H, aryl, J=8.4 Hz); 7.55 (d, 2H, aryl, J=8.3 Hz); 7.60 (d, 2H, aryl, J=8.3 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 13.0; 22.3; 25.8; 29.0; 29.3; 31.7; 35.8; 100.7; 104.2; 114.1; 124.8; 126. 9; 127.3; 142.9; 145.2; 148.0; 174.1. ¹⁹ F NMR (CDCb, 376.3 MHz) d: -62.43 (s, 3F, C F ₃ ) HR-MS m/r calcd for C26H37F3N3O, [(M+H) ⁺ ]: 464.2889; found 464.2885;

N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)-3,3-d imethylbutanamide

Synthesized starting from 2-nitro-l,4-phenylenediamine, 4-trifluorobenzyl bromide and 3, 3 dimethyl butyryl chloride. ¾ NMR (CD3OD, 400 MHz) d 1.11 (s, 9H, 3 G %); 2.25 (s, 2H, C H ₂ ); 4.40 (s, 2H, C H ₂ ) 6.07-6.12 (m, 2H, aryl); 6.77 (d, 1H, aryl, J=8.4 Hz); 7.55 (d, 2H, aryl, J=8.3 Hz); 7.60 (d, 2H, aryl, J=8.3 Hz); ¹³ C NMR (CD3OD, 100 MHz) d: 28.9; 30.5; 49.2; 100.7; 104.2; 114.2; 124.79; 124.83; 126. 9; 127.3; 142.9; 145.2; 148.0; 172.4. ¹⁹ F NMR (CDCb, 376.3 MHz) d: -62.43 (3F, CFs ); HR-MS m/ calcd for C20H25F3N3O, [(M+H) ⁺ ]: 380.1950; found

380.1958;

N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)cyclop ropanecarboxamide

Synthesized starting from 2-nitro-l,4-phenylenediamine, 4-trifluorobenzyl bromide and cyclopropane carbonyl bromide. 'H NMR (CD3OD, 400 MHz) d 0.84 (d, 2H, C//2, J=6.9 Hz); 0.93 (s, 2H, CHi) 1.77 (t, 1H, C H, J=4.0 Hz); 4.39 (s, 2H, C H ₂ ) 6.08-6.12 (m, 2H, aryl); 6.81 (d, 1H, aryl, J=8.0 Hz); 7.55 (d, 2H, aryl, J=7.6 Hz); 7.60 (d, 2H, aryl, J=7.6 Hz); ¹³ C NMR (CD3OD, 100 MHz) d: 6.3; 13.5; 100.7; 104.2; 114.4; 124.8; 126.8; 127.3; 142.8; 145.3; 148.0; 174.2. ¹⁹ F NMR (CDCb, 376.3 MHz) d: -62.44 (3F, C F ₃ ); HR-MS m/r calcd for C18H19F3N3O, [(M+H) ⁺ ]: 350.1480; found 350.1469;

N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)-3-cyc lohexylpropanainide

Synthesized starting from 2-nitro-l,4-phenylenediamine, 4-trifluorobenzyl bromide and cyclohexyl propanoyl bromide. ¾ NMR (CD3OD, 400 MHz) d 0.93-1.02 (m, 2H, CHi); 1.16-1.34 (m, 4H, 2 G ¾); 1.57-1.82 (m, 7H, 3 G Hi and C H); 2.38 (t, 2H, C/ , J=8.0 Hz); 4.39 (s, 2H, Cl 12): 6.07-6.12 (m, 2H, aryl); 6.77 (d, 1H, aryl, J=8.4 Hz); 7.55 (d, 2H, aryl, J=8.2 Hz); 7.60 (d, 2H, aryl, J=8.2 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 26.0; 26.3; 32.85;33.36; 37.41; 100.7; 104.2; 114.1; 124.8; 126.9; 127.3; 142.9; 145.2; 148.0; 174.4. ¹⁹ F NMR (CDCI3, 376.3 MHz) d: -62.42 (3F, G F ₃ ); HR-MS m/z: calcd for C23H29F3N3O, [(M+H) ⁺ ]: 420.2263; found 420.2271;

N-(2-amino-4-((4-(trifluoromethoxy)benzyl)amino)phenyl)he ptanamide

Synthesized starting from 2-nitro-l,4-phenylenediamine, 1 -(bromomethyl)-4- (trifluoromethoxy)benzene and heptanoyl chloride. ¾ NMR (DMSO-d6, 400 MHz) d 0.87 (t, 3H, G¾ , J=6.5 Hz); 1.27 (bs, 6H, 3 C Hi); 1.53-1.57 (m, 2H, C Hi); 2.22 (t, 2H, CH ₂ , J=7.4 Hz); 4.22 (d, 2H, CHi, J=6.0 HZ); 4.51 (bs, 2H, M2); 5.84 (d, 1H, aryl, J=8.4 Hz); 5.95-5.97 (m, 1H, aryl); 6.70 (d, 1H, aryl, J=8.4 Hz); 7.04 (d, 1H, aryl, J=9.8 Hz); 7.30 (d, 1H, aryl, J=8.2 Hz); 7.45 (d, 2H, aryl, J=8.3 Hz); 7.60 (d, 2H, aryl, J=8.3 Hz); 8.80 (s, 1H, CON H); ¹³ C NMR (DMSO-d6, 100 MHz) d: 14.4; 22.5; 25.8; 28.8; 31.5; 36.1; 46.3; 99.7; 102.2; 114.2; 119.3; 121.9; 127.2; 129.2; 140.8; 143.7; 147.5; 171.6. ¹⁹ F NMR (CDCh, 376.3 MHz) d: -62.66 (3F, GF ); HR-MS m/z: calcd for C21H27F3N3O2, [(M+H) ⁺ ] : 410.2055; found 410.2059;

N-(2-amino-4-(benzylamino)phenyl)heptanamide

Synthesized starting from 2-nitro-l,4-phenylenediamine, benzyl bromide and heptanoyl chloride. ‘H NMR (CD ₃ OD, 400 MHz) d 0.82 (t, 3H, C Hi, J=6.4 Hz); 1.19-1.25 (m, 6H, 3 C Hi); 1.55-1.62 (m, 2H, CHi ); 2.25 (t, 2H, C Hi, J=7.6 Hz); 4.17 (s, 2H, G Hi); 5.99 (d, 1H, aryl, J=8.4 Hz); 6.04 (s, 1H, aryl); 6.65 (d, 1H, aryl, J=8.4 Hz); 7.07-7.11 (m, 1H, aryl); 7.17 (t, 1H, aryl, J=7.2 Hz); 7.24 (d, 2H, aryl, J=7.4 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 13.0; 22.2; 25.7; 28.7; 31.3; 35.8; 100.9; 104.3; 114.0; 126.3; 126.8; 126.9; 128.0; 140.2; 142.7; 148.4; 174.1. HR-MS m/z: calcd for C20H28N3O, [(M+H) ⁺ ] _: 326.2232; found 326.2230;

N-(2-amino-4-((4-fluorophenethyl)amino)phenyl)heptanamide Synthesized starting from 2-nitro-l,4-phenylenediamine, l-(2-bromoethyl)-4-fluorobenzene and heptanoyl chloride. ¾ NMR (CD ₃ OD, 400 MHz) d 0.94 (t, 3H, Cfh, J=6.8 Hz); 1.35-1.43 (m, 6H, 3 C Hi); 1.68-1.76 (m, 2H, C// ₂ ); 2.38 (t, 2H, G %, J=7.6 Hz); 2.86 (t, 2H, G%, J=7.2 Hz); 3.29 (t, 2H, G ¾, J=7.6 Hz); 6.11 (d, 1H, aryl, Ji=6.0 Hz, J ₂ =2.4 Hz); 6.19 (d, 1H, aryl, J=2.4 Hz); 6.82 (d, 1H, aryl, J=8.4 Hz); 7.02 (t, 2H, aryl, J=8.8 Hz); 7.23-7.27 (m. 2H, aryl); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 13.0; 22.2; 25.7; 28.7; 31.3; 34.2; 35.8; 45.3; 100.8; 104.3; 114.2; 114.7; 126.9; 130.1; 135.8; 142.9; 148.2; 160.3; 162.7; 174.1. ¹⁹ F NMR (CDCb, 376.3 MHz) d: -62.41 (IF, C F)· HR-MS m/ calcd for C ₂₁ H ₂₉ FN ₃ O, [(M+H) ⁺ ]: 358.2295; found 358.2296;

N-(2-amino-4-((pyridin-2-ylmethyl)amino)phenyl)heptanamid e

Synthesized starting from 2-nitro-l,4-phenylenediamine, 2-(bromomethyl)pyridine and heptanoyl chloride. ¾ NMR (CD ₃ OD, 400 MHz) d 0.94 (t, 3H, CH J=6.8 Hz); 1.31-1.43 (m, 6H, 3 G %); 1.67-1.74 (m, 2H, G %); 2.37 (t, 2H, C Hi, J=7.7 Hz); 4.41 (s, 2H, C H) 6.07 (d, 1H, aryl, Ji=6.1 Hz, J ₂ =2.3 HZ); 6.12 (d, 1H, aryl, J=2.4 Hz); 6.78 (d, 1H, aryl, J=8.4); 7.28 (t, 1H, aryl, J=6.7 Hz); 7.47 (d. 1H, aryl, J=7.9 Hz); 7.76 (t, 1H, aryl, J=6.1 Hz); 8.48 (d, 1H, aryl, J=4.5 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 13.0; 22.2; 25.7; 28.7; 31.3; 35.8; 48.5; 100.6; 104.0; 114.2; 121.6; 122.1; 127.0; 137.3; 143.0; 147.9; 148.2; 160.0; 162.7; 174.1. HR-MS m/r calcd for C ₁₉ H ₂₇ N ₄ O, [(M+H) ⁺ ] : 327.2185; found 327.2191;

N-(2-amino-4-((pyridin-4-ylmethyl)amino)phenyl)heptanamide

Synthesized starting from 2-nitro-l,4-phenylenediamine, 4-(bromomethyl)pyridine and heptanoyl chloride. ¾ NMR (CD ₃ OD, 400 MHz) d 0.94 (t, 3H, CH J=6.7 Hz); 1.34-1.43 (m, 6H, 3 G %); 1.67-1.74 (m, 2H, C Hi),· 2.37 (t, 2H, G %, J=7.6 Hz); 4.38 (s, 2H, C H) 6.04-6.09 (m, 2H, aryl); 6.78 (t, 1H, aryl, J=3.8 Hz); 6.78 (d, 1H, aryl, J=8.4); 7.28 (t, 1H, aryl, J=6.7 Hz); 7.44 (d. 2H, aryl, J=5.5 Hz); 8.43 (d, 2H, aryl, J=5.5 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 13.0; 22.2; 25.7; 28.7; 31.3; 35.8; 46.0; 100.5; 103.9; 114.2; 122.5; 127.0; 143.0; 147.8; 148.4; 151.8; 174.1. HR- MS in r: calcd for C19H27N4O, [(M+H) ⁺ ]: 327.2185; found 327.2179;

N-(2-amino-4-((2-fluorobenzyl)amino)phenyl)heptanainide

Synthesized starting from 2-nitro-l,4-phenylenediamine, l-(bromomethyl)-2-fluorobenzene and heptanoyl chloride ¾ NMR (CD ₃ OD, 400 MHz) d 0.94 (t, 3H, CH 1-6.6 Hz); 1 31-1.43 (m, 6H, 3 G %); 1.67-1.74 (m, 2H, CH ₂ ); 2.37 (t, 2H, G%, J=7.5 Hz); 4.35 (s, 2H, G¾); 6.11 (d, 1H, aryl, J=8.5 Hz); 6.16 (s, 1H, aryl); 6.78 (d, 1H, aryl, J=8.5 Hz); 7.06-7.11 (m, 2H, aryl); 7.22-7.27 (m, 1H, aryl); 7.40 (t, 1H, aryl, J=7.4 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 13.0; 22.2; 25.7; 28.7; 31.3; 35.8; 40.6; 100.7; 104.1; 114.2; 114.4; 114.6; 123.7; 126.9; 128.1; 129.01; 129.06; 142.9;

148.1; 159.7; 162.1; 174.1. ¹⁹ F NMR (CDCh, 376.3 MHz) d: -119.13 (IF, CF); HR-MS ra/z: calcd for C ₂₀ H ₂₇ FN ₃ O, [(M+H) ⁺ ] : 344.2138; found 344.2141;

N-(3-fluoro-4-((4-fluorobenzyl)amino)phenyl)heptanamide

Synthesized starting from 3-fluoro-l,4-phenylenediamine, 1 -(bromomethyl)-2-fluorobenzene and heptanoyl chloride. ¾ NMR (CD3OD, 400 MHz) d 0.93 (t, 3H, CfF, J=6.7 Hz); 1.31-1.39 (m, 6H, 3 G %); 1.66-1.73 (m, 2H, CH ₂ ); 2.38 (t, 2H, CH ₂ , J=7.6 Hz); 4.52 (s, 2H, C H ₂ ); 7.06-7.15 (m, 4H, aryl); 7.25 (d, 1H, aryl, J=8.7 Hz); 7.42 (dd, 2H, aiyl, Ji=3.1 Hz, J ₂ =5.3 Hz); 7.72 (dd, 1H, aryl, Ji=11.4 Hz, J ₂ =1.8 Hz); ¹³ C NMR (CD3OD, 100 MHz) d: 13.0; 22.2; 25.3; 28.6; 31.3; 36.5; 51.6; 107.4; 107.7; 115.2; 115.4; 115.8; 131.3; 152.9; 155.3; 161.9; 164.4; 173.4. ¹⁹ F NMR

(CDCh, 376.3 MHz) d: -117.01 (IF, CF); -120.97 (IF, CF); HR-MS m/r. calcd for C20H25F2N2O, [(M+H) ⁺ ] : 347.1935; found 347.1939;

N-(2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)aniino)phe nyl)heptanainide

Synthesized starting from 2-nitro-3-fluoro-l,4-phenylenediamine, 1 -(bromomethyl)-4- trifluoromethylbenzene and heptanoyl chloride. 'H NMR (DMSO-d6, 400 MHz) d 0.87 (t, 3H, G ¾, J=6.7 Hz); 1.27-1.31 ( 1.57 (m, 2H, C Hi)· 2.24 (t, 2H, C Hi, J=7.4 Hz); 4.39 (d, 2H, C %, J=6.0 Hz); 4.57 (bs, 2H, N %); 5.78, (t, 1H, N//, J=8.8 Hz); 6.02 (t, 1H, aryl, J=5.8 Hz); 6.58 (d, 1H, aryl, J=8.4 Hz); 7.55 (d, 2H, aryl, J=8.0 Hz); 7.66 (d, 2H, aryl, J=8.0 Hz); 8.98 (s, 1H, CONH); ¹³ C NMR (DMSO-d6, 100 MHz) d: 14.3; 22.5; 25.7, 28.8; 31.5; 36.0; 46.2; 100.1; 115.6; 121.5; 125.5; 127.6; 128.0; 131.4; 131.5; 134.5; 139.6; 141.8; 146.2; 171.9. ¹⁹ F NMR (CDCF, 376.3 MHz) d: -60.72 (3F, C F ₃ ); -154.89 (IF, C F) HR-MS m/ _å : calcd for C21H26F4N3O, [(M+H) ⁺ ] : 412.2012; found 412.2018;

N-(2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)aniino)phe nyl)isobutyramide

Synthesized starting from 2-nitro-3-fluoro-l,4-phenylenediamine, 1 -(bromomethyl)-4- trifluoromethylbenzene and 2-methylpropanonyl chloride. ¾ NMR (DMSO-d6, 400 MHz) d 1.06 (d, 3H, CHs, J= 1.9 Hz); 1.08 (d, 3H, CH3, J=1.9 Hz); 2.54-2.5 9 (m, 1H, C H) 4.39 (d, 2H, CH ₂ , J=5.6 Hz); 4.54 (s, 2H, CH ₂ ); 5.79 (t, 1H, aryl, J=8.6 Hz); 6.02 (t, 1H, NT/, J=5.4 Hz); 6.59 (d, 1H, aryl, J=8.6 Hz); 7.55 (d, 2H, aryl, J=7.5 Hz); 7.66 (d, 2H, aryl, J=7.5 Hz); 8.96 (s, 1H, CON H) ¹³ C NMR (DMSO-d6, 100 MHz) d: 20.1; 34.6; 46.2; 100.2; 115.5; 121.5; 125.6; 128.0; 131.5; 134.4; 134.5; 139.6; 141.9; 146.2; 175.8. ¹⁹ F NMR (DMSO-d6, 376.3 MHz) d: -60.74 (3F, CFs); -154.88 (IF, OF); HR-MS m/z: calcd for C18H20F4N3O, [(M+H) ⁺ ]: 37.1543; found 370.1549. N-(2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)amino)phen yl)-3-methylbutanamide

Synthesized starting from 2-nitro-3-fluoro-l,4-phenylenediamine, 1 -(bromomethyl)-4- trifluoromethylbenzene and 3-methylpbutyryl chloride. ¹ H NMR (CD ₃ OD, 400 MHz) d 1.03 (d, 6H, 2 CH ₃ , J=6.6 HZ); 2.13-2.19 (m, 1H, C H) 2.25 (d, 2H, G %, J=7.0 Hz); 4.47 (s, 2H, G %); 5.98 (t, 1H, aryl, J=8.8 Hz); 6.60 (dd, 1H, aryl, Ji=8.6 Hz, J ₂ =1.8 Hz); 7.55 (d, 2H, aryl, J=8.0 Hz); 7.60 (d, 2H, aryl, J=8.0 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 21.3; 26.2; 44.9; 46.3; 101.49; 115.06; 121.23; 124.82; 127.15; 135.0; 135.1; 140.1; 142.4; 145.0; 173.4. ¹⁹ F NMR (CDCh, 376.3 MHz) d: -63.84 (3F, C F ₃ ); -154.79 (IF, C F); HR-MS m/z: calcd for C19H22F4N3O, [(M+H) ⁺ ]: 384.1699; found 384.1691.

N-(2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)aniino)phe nyl)-3,3-dimethylbutanainide

Synthesized starting from 2-nitro-3-fluoro-l,4-phenylenediamine, 1 -(bromomethyl)-4- trifluoromethylbenzene and 3,3-dimethylbutyryl chloride. 'H NMR (CD ₃ OD, 400 MHz) d 1.10 (s, 9H, 3 G H ₃ ); 2.25 (s, 2H, C H ); 4.47 (s, 2H, C%); 5.99 (t, 1H, aryl, J=8.8 Hz); 6.60 (dd, 1H, aryl, Ji=6.7 Hz, J ₂ =l .9 Hz); 7.54 (d, 2H, aryl, J=8.2 Hz); 7.60 (d, 2H, aryl, J=8.2 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 28.9; 30.5; 46.3; 49.1; 101.5; 115.2; 121.2; 124.90; 124.93; 127.2; 130.9; 131.0; 135.0; 0.5.1; 140.2; 142.5; 145.0; 172.5. ¹⁹ F NMR (CD3OD, 376.3 MHz) d: -62.48 (3F,

CFs ); -158.0 (IF, CP); HR-MS m/z : calcd for C20H24F4N3O, [(M+H) ⁺ ]: 398.1856; found 398.1867. N-(2-amino-4-((4-hydroxybenzyl)amino)phenyl)heptanamide

Synthesized starting from 2-nitro-l,4-phenylenediamine, benzyl (4-(iodomethyl)phenyl) carbonate and heptanoyl chloride. 'H NMR (CD3OD, 400 MHz) d 0.94 (t, 3H, CH3, J=6.5 Hz); 1.31-1.44 (m, 6H, 3 C Hi); 1.67-1 74 (m, 2H, CH ), 2.37 (t, 2H, C H ₂ , J-7.6 Hz); 4.17 (s, 2H, C Hi); 6.12 (d, 1H, aryl, J=8.4 Hz); 6.18 (s, 1H, aryl); 6.72-6.79 (m, 3H, aryl); 7.18 (d, 2H, aryl, J=8.2 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 13.0; 22.2; 25.7; 28.7; 31.3; 35.8; 101.2; 104.5; 114.1; 114.7; 126.8; 128.3; 130.7; 142.7; 148.5; 156.0; 174.1. HR-MS z: calcd for C20H28NO2, [(M+H) ⁺ ] : 342.2182; found 342.2171;

N-(2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)amino)phen yl)-3- cyclohexylpropanamide

Synthesized starting from 2-nitro-3-fluoro-l,4-phenylenediamine, 1 -(bromomethyl)-4- trifluoromethylbenzene and 3-cyclohexane propanoyl chloride. 'H NMR (DMSO-d6, 400 MHz) d 0.86-0.92 (m, 2H, C Hi)· 1.12-1.24 (m, 4H, 2 G¾); 1.43-1.49 (m, 2H, G% ); 1.62-1.72 (m, 3H, G¾ and C/7); 2.23-2.27 (m, 2H, CH ₂ ); 4.39 (bs, 2H, N/7 ₂ ); 4.56-4.57 (m, 2H, C/7 ₂ ); 5.78 (t, 1H, N Ή, J=8.3 Hz); 6.00-6.04 (m, 1H, aryl); 6.56-6.58 (m, 1H, aryl); 7.55 (d, 2H, aryl, J=7.6 Hz); 7.67 (d, 2H, aryl, J=7.6 Hz); 8 97 (s, 1H, CON/7); ¹³ C NMR (DMSO-d6, 100 MHz) d: 26.3; 26.6; 33 1; 33.3; 33.6; 37.3; 46.2; 100.1; 115.6; 121.5; 125.5; 127.6; 127.9; 128.0; 131.4; 139.5; 141.8; 146.2; 172.1. ¹⁹ F NMR (CDCI3, 376.3 MHz) d: -62.54 (3F, C F ₃ ); -154.87 (IF, C F); HR-MS m/r. calcd for C23H28F4N3O, [(M+H) ⁺ ] : 438.2169; found 438.2172.

N-(2,6-difluoro-4-((4-(trifluoromethyl)benzyl)amino)pheny l)heptanamide

Synthesized starting from 2,6-difluoro-l,4-phenylenediamine, 1 -(bromomethyl)-4- trifluoromethylbenzene and heptanoyl chloride. 'H NMR (CD3OD, 400 MHz) d 0.84 (t, 3H, C//3. J=6.5 Hz); 1.22-1.26 (m, 6H, 3 C Hi); 1.55-1.60 (m, 2H, C Hi); 2.23 (t, 2H, G Hi, J=7.6 Hz); 4.39 (s, 2H, G Hi); 7.03 (d, 2H, aryl, J=10.5 Hz); 7.42 (d, 2H, aryl, J=7.9 Hz); 7.50 (d, 2H, aryl, J=7.9 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 12.9; 22.2; 25.4; 28.6; 31.3; 36.5; 49.2; 103.3; 103.6; 124.8; 127.6; 130.2; 130.3; 130.5; 145.1; 152.11; 152.21; 154.49; 154.58; 173.12. ¹⁹ F NMR (CDCh, 376.3 MHz) d: -62.49 (3F, C F ₃ ) -126.78 (2F, C F) HR-MS m/z : calcd for C _2i H ₂₄ F ₅ N ₂ 0, [(M+H) ⁺ ]: 415.1809; found 415.1810;

N-(2,6-difluoro-4-((4-(trifluoromethyl)benzyl)amino)phenyl)- 3,3-dimethylbutanamide

Synthesized starting from 2,6-difluoro-l,4-phenylenediamine, 1 -(bromomethyl)-4- trifluoromethylbenzene and 3,3-dimethylbutyryl chloride. ¹ H NMR (CD ₃ OD, 400 MHz) d 0.94 (s, 9H, 3 C/73); 2.07 (s, 2H, C/7 ₂ ); 4.36 (s, 2H, C/7 ₂ ); 5.99 (t, 1H, aryl, J=8.8 Hz); 6.95-7.04 (m, 2H, aryl); 7.38 (d, 2H, aryl, J=8.1 Hz); 7.46 (d, 2H, aryl, J=8.2 Hz), ¹³ C NMR (CD ₃ OD, 100 MHz) d: 28.8; 30.7; 49.2; 49.8; 103.4; 103.7; 121.8; 123.0; 124.8; 125.7; 127.6; 128.6; 129.0; 130.1; 145.1; 154.4; 154.5; 171.6. ¹⁹ F NMR (CD3OD, 376.3 MHz) d: -62.51 (3F, CF ₃ ); -127.51 (2F, CF) HR- MS m/z: calcd for C20H22F5N2O, [(M+H) ⁺ ]: 401.1652; found 401.1655.

N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)-2-(2- methoxyethoxy)acetamide

Synthesized starting from 2-nitro-l,4-phenylenediamine, 1 -(bromomethyl)-4- trifluoromethylbenzene and (2-Methoxyethoxy)acetyl chloride. H NMR (CD3OD, 400 MHz) d 3.41 (s, 3H, G¾); 3.64 (d, 2H, CH ₂ , 3=2.1 Hz); 3.78 (d, 2H, G ¾, J=2.7 Hz); 4.14 (s, 2H, CH ₂ ); 4.40 (s, 2H, G %); 6.09-6.12 (m, 2H, aryl); 6.88 (d, 1H, aryl, J=8.3 Hz); 7.55 (d, 2H, aryl, J=7.8 Hz); 7.61 (d, 2H, aryl, J=7.8 Hz); ¹³ C NMR (CD3OD, 100 MHz) d: 46.7; 57.8; 70.1; 70.6; 71.4; 100.4; 104.0; 112.8; 124.8; 126.9; 127.3; 142.9; 145.2; 148.2; 170.3. ¹⁹ F NMR (CDCI3, 376.3 MHz) d: -62.42 (3F, G Fs); HR-MS m/z: calcd for C19H23F3N3O, [(M+H) ⁺ ]: 398.1692; found 398.1701;

Example 4 - Synthesis of compounds of general formula 13

The general synthetic procedure for compounds with general formula 13 is depicted in Scheme 4. Scheme 4 (wherein R4 = NH2 in compound 12):

Synthesis of intermediates of formula 13

Products of general formula 11 or 12, used as starting compounds, were dissolved in MeOH in presence of different commercially available aldehydes (1.2 eq) and TFA (1 eq). The reaction was refluxed for 3 hours, then 3 equivalents of NaBTLi were added at 0°C. The organic layer was concentrated in vacuo and further extracted with a solution of K ₂ CO ₃ and brine. After drying over Na2SC>4, the organic phase was concentrated in vacuo. Final thus obtained were purified using linear gradients of n-hexane/ethyl acetate as mobile phase.

Below follow some examples of molecules synthesized following the above described procedure.

N-(2-amino-4-(methyl(4-(trifluoromethyl)benzyl)amino)phen yl)heptanamide Synthesized starting from N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)heptan amide and formaldehyde. ¾ NMR (CD3OD, 400 MHz) d 0.82 (t, 3H, G H ₃ , J=6.5 Hz); 1.19-1.31 (m, 6H, 3 G ¾); 1.56-1.63 (m, 2H, CH ₂ ); 2.26 (t, 2H, G%, J=7.4 Hz); 2.89 (s, 3H, C H ); 4.47 (s, 2H, G %); 6.08 (dd, 1H, aryl, Ji=7.1 Hz, J ₂ =1.5 Hz); 6.16 (s, 1H, aryl); 6.75 (d, 1H, aryl, J=8.6 Hz); 7.29 (d,

2H, aryl, J=7.8 Hz); 7.47 (d, 2H, aryl, J=7.8 Hz); ¹³ C NMR (CD3OD, 100 MHz) d: 13.0; 22.2; 25.7; 28.7; 31.3; 35.8; 37.85; 55.75; 100.8; 103.5; 114.2; 124.9; 126.9; 127.1; 142.9; 144.1; 149.2; 174.1. ¹⁹ F NMR (CDCh, 376.3 MHz) d: -62.41 (3F, C F ₃ ); HR-MS m/ _å : calcd for C22H29F3N3O, [(M+H) ⁺ ] : 408.2263; found 408.2257;

N-(2-amino-4-(methyl(4-(trifluoromethyl)benzyl)amino)phen yl)-3,3-dimethylbutanainide

Synthesized starting from N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)-3,3- dimethylbutanamide and formaldehyde. ¹ H MR (CD3OD, 400 MHz) d 1.12 (s, 9H, 3 C//3): 2.26 (s, 2H, C Hi); 3.01 (s, 3H, G¾); 4.60 (s, 2H, Hi); 6.20 (dd, 1H, aryl, Ji=6.1 Hz, J ₂ =2.6 Hz); 6.28 (d, 1H, aryl, J=2.7 Hz); 6.87 (d, 1H, aryl, J=8.6 Hz); 7.41 (d, 2H, aryl, J=7.8 Hz); 7.59 (d, 2H, aryl,

J=7.8 Hz); ¹³ C NMR (CD3OD, 100 MHz) d: 29.0; 30.5; 37.9; 49.2; 55.7; 100.9; 103.5; 114.3; 124.9; 125.8; 127.1; 128.5; 128.8; 142.9; 144.1; 149.1; 172.4. ¹⁹ F NMR (CD3OD, 376.3 MHz) d: -63.76 (3F, CFs) HR-MS m/z: calcd for C21H27F3N3O, [(M+H) ⁺ ]: 394.2106; found 394.2111; N-(2-amino-4-(propyl(4-(trifluoromethyl)benzyl)amino)phenyl) heptanamide

Synthesized starting from N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)heptan amide and propionaldehyde. ^X H NMR (CD3OD, 400 MHz) d 0.82 (t, 3H, G¾ , J=6.5 Hz); 1.19-1.31 (m, 6H, 3 G %); 1.56-1.63 (m, 2H, CH ₂ ); 2.26 (t, 2H, G %, J=7.4 Hz); 2.89 (s, 3H, G%); 4.47 (s, 2H, G¾); 6.08 (dd, 1H, aryl, Ji=7.1 Hz, J ₂ =1.5 Hz); 6.16 (s, 1H, aryl); 6.75 (d, 1H, aryl, J=8.6 Hz); 7.29 (d, 2H, aryl, J=7.8 Hz); 7.47 (d, 2H, aryl, J=7.8 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) d: 13 0; 22.2; 25.7; 28.7; 31.3; 35.8; 37.85; 55.75; 100.8; 103.5; 1 14.2; 124.9; 126.9; 127.1 ; 142.9; 144.1 ; 149.2; 174.1. ¹⁹ F NMR (CDCF, 376.3 MHz) d: -62.41 (3F, C F ₃ ) HR-MS m/z: calcd for C22H29F3N3O, [(M+H) ⁺ ] : 408.2263; found 408.2257.

N-(2-amino-3-fluoro-4-(methyl(4-(trifluoromethyl)benzyl)amin o)phenyl)-3,3- dimethylbutanamide

Synthesized starting from N-(2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)amino)phen yl)-3,3- dimethylbutanamide and formaldehyde.

Example 5 - Synthesis of compounds of general formula 15

The general procedure for the synthesis of compounds with general formula 15 is shown in Scheme 5.

Scheme 5 :

Synthesis of final products of formula 15

The previously described intermediates 10 were subjected to the reaction protocol with different sulfonyl chlorides described above to afford intermediates 14. These intermediates were subjected to the catalytic hydrogenation protocol previously described to give final products of general formula 15.

An exemplary structure obtined with the above described procedure is represented below:

N-(3-fluoro-4-((4-fluorobenzyl)amino)phenyl)cyclohexanesulfo namide Synthesized starting from 2-fluoro-N ¹ -(4-(trifluoromethyl)benzyl)benzene-l, 4-diamine and cyclohexane sulfonyl chloride. 2-fluoro-N ¹ -(4-(trifluoromethyl)benzyl)benzene- 1,4-diamine may be prepared from the procedure described in Example 3 for the synthesis of compounds of formula 10, starting from: 3-fluoro-l,4-phenylenediamine and l-(bromomethyl)-4-fluorobenzene. 'H NMR (CDCh, 400 MHz) d 1.18-1.30 (m, 3H, Ctf ₂ and C H); 1.57-1.63 (m, 3H, C# ₂ and CH); 1.89- 1.92 (m, 2H, C %); 2.16 (d, 2H, C ¾ J=13.1 Hz); 2.90-2.98 (m, 1H, CH); 4.34 (s, 2H, C H ₂ ); 6.11 (s, lH, aryl); 6.59 (t, 1H, aryl, J=9.0 Hz); 6.83-6.85 (m, 1H, aryl); 7.03-7.09 (m, 2H, aryl); 7.35 (dd, 1H, aryl, Ji=3.0 Hz, J ₂ =5.4 Hz); ¹³ C NMR (CDCh, 100 MHz) d: 25.0; 25.1; 47.3; 59.9; 110.8; 111.0; 112.3; 115.5; 115.7; 119.6; 126.0; 129.0; 134.2; 134.7; 134.9; 149.9; 152.3; 163.4. ¹⁹ F NMR (CDCh, 376.3 MHz) d: -118.41 (IF, C F); -135.31 (IF, C F); HR-MS m/z: calcd for C _I9 H ₂₃ F ₂ N ₂ 0 ₂ S, [(M+H) ⁺ ] : 381.1448; found 381.1454;

N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)cycloh exanesulfonamide

Synthesized starting from 2-nitro-N ¹ -(4-(trifluoromethyl)benzyl)benzene- 1,4-diamine and cyclohexane sulfonyl chloride. 2-nitro-N ¹ -(4-(trifluoromethyl)benzyl)benzene-l, 4-diamine may be prepared from the procedure described in Example 3 for the synthesis of compounds of formula 10, starting from: 2-nitro-l,4-phenylenediamine and 4-trifluorobenzyl bromide. 'H NMR (CD3OD, 400 MHz): d: 1.23-1.37 (m, 3H, CH ₂ d CH); 2.21-2.24 (m, 2H, C H ₂ ); 3.00 (t, 1H, CH, J=12.2 Hz); 5.13 (s, 2H, C H ₂ ); 6.35 (d, 1H, aryl, J=8.1 Hz); 6.48 (s, 1H, aryl); 7.03 (d, 1H, aryl, J= 8.3 Hz); 7.62 (d, 2H, aryl J=8.2 Hz); 7.68 (d, 2H, aryl, J=8.3 Hz);. HR-MS m/z calcd for C ₂ aH ₂₆ F ₃ N ₃ 0 ₂ S, [(M+H) ⁺ ] : 429.1692; found 429.1701.

Example 6 - Synthesis of compounds of general formula 20-22

The general procedure for the synthesis of compounds with general formula 17 or 18 is shown in Scheme 6.

Scheme 6: Synthesis of final products of formula 20-22

Intermediates with general formula 9 with R4HNO2 (1 eq), prepared as described in Example 3, were coupled with Boc anhydride (1.2 eq) using TEA (1.2 eq) to give the correspondent protected intermediates of general formula 16. Afterward, the reaction mixture was diluted with dichloromethane and the resulting solution was washed with K2CO3, dried over Na2S04, and evaporated to dryness. Chromatography of the residues, using different eluent systems, yielded the corresponding N-protected derivatives. These intermediates were N-acylated using the acylation procedure described in Example 3 affording intermediates of general formula 17. These intermediates were reduced to the corresponding amines with general formula 18 using the catalytic hydreogenation protocol previously described. The intermediates 18 were subjected to alkylation reaction using different alkyl monobromides and alkyl dibromides (1.5 eq), to afford compounds with general formula 19, 20 and 21 respectively. Reactions were performed in ACN as solvent using K2CO3 (2 eq) and KI (2 eq) and mixtures were refluxed overnight. The organic phases were concentrated in vacuo and diluted with DCM. The organic phases were then washed with a saturated solution of K2CO3, extracted, dried over anhydrous Na ₂ SC> ₄ , filtered and concentrated in vacuo. Crude products were purified using a linear gradient of n-exane/ethyl acetate. Boc protecting group was removed from intermediates 19, 21 and 22 by reaction in DCM/TFA (3 : 1, v:v) in the presence of catalytic amounts of triethylsilane to give compounds 22, 23 and 24, respectively. The resulting mixtures were diluted with DCM, washed with a saturated solution of NaHCCh and brine, dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated in vacuo. Crude products were purified using a linear gradient of n-exane/ethyl acetate to give the final products.

An exemplary compound obtained with the above described procedure is:

3,3-dimethyl-N-(2-(piperidin-l-yl)-4-((4-(trifluoromethyl)be nzyl)amino)phenyl)butanamide

Synthesized starting from S-nitro-Nk^-itrifluoromethy^benzy^benzene-l, 4-diamine, 3,3- dimethylbutyryl chloride and 1,5-dibromopentane. 3-nitro-N ¹ -(4-

(trifluoromethyl)benzyl)benzene- 1,4-diamine may be prepared from the procedure described in Example 3 for the synthesis of compounds of formula 9, starting from: 2-nitro-l,4- phenylenediamine and 4-trifluorobenzyl bromide. 'H NMR (CDCI ₃ , 400 MHz) d 1.12 (s, 9H, 3 G ¾, J=6.5 Hz); 1.59 (d, 2H, C// _2, J=4.01 Hz); 1.70-1.73 (m, 4H, 2 G %); 2.23 (s, 2H, C /¾; 2.74 (t, 4H, 2 G ¾, J=4.7 Hz); 4.10 (bs, 1H. NH); 4.39 (s, 2H, C/ft); 6.39 (d, 1H, aryl, J=8.7 Hz); 6.44 (s, 1H, aryl); 7.49 (d, 1H, aryl, J=7.9 Hz); 7.60 (d, 2H, aryl, J=7.9 Hz); 8.16 (s, 1H, CON//), 8.20 (d, 1H, aryl, J=8.7 Hz); ¹³ C NMR (CDCb„ 100 MHz) d: 24.1; 27.0; 29.9; 48.2; 52.2; 53.7; 105.8;

108.8; 120.6; 122.8; 125.0; 125.50; 125.55; 127.6; 129.3; 129.6; 143.8; 144.1; 169.2. ¹⁹ F NMR (CDCI ₃ , 376.3 MHz) d: -62.42 (3F, G F ₃ ); HR-MS m/z: calcd for C ₂₅ H ₃₃ F ₃ N ₃ O, [(M+H) ⁺ ]: 448.2576; found 448.2577. Example 7 - Synthesis of compounds of general formula 30 or 31

The general procedure for the synthesis of compounds with general formula 30 or 31 is shown in Scheme 7.

Scheme 7:

The differently substitued 4-nitroaniline of formula 25 or the differently substituted l-fluoro-4- nitrobenzene of formula 27 used as starting compounds in Scheme 7 have been synthesized using typical and well described synthetic methods or, otherwise, are commercially available.

Synthesis of intermediates of formula 26

For the synthesis of compounds with general formula 26, intermediates of formula 25 (1.0 eq) are dissolved in 15 mL of dimethylformamide and added with 1.5 equivalents of triethylamine (TEA) or N,N-Diisopropylethylamine (DIPEA), a catalytic amount of KI (2.5% mol) and 1.1 equivalents of the proper halide with general formula R-benzyl-X. The mixture is stirred at 60°C until completion. The resulting solution is diluted with water and extracted 3 times with ethyl acetate. Organic phases are dried over anhydrous Na ₂ SC> ₄ , filtered and concentrated in vacuo. The crude products are purified by flash chromatography using mixtures of n-hexane/ethyl acetate as mobile phase.

Synthesis of intermediates of formula 28

For the synthesis of compounds with general formula 28, intermediates of formula 27 (1.0 eq) are dissolved in 15 mL of dimethylformamide and added with 1.2 equivalents of triethylamine (TEA), a catalytic amount of h (5% mol) and 3.0 equivalents of the proper amine with general formula R1NH2. The mixture is refluxed at 120°C for 2.5-3.5h. The resulting solution is diluted with water and extracted 2 times with dichloromethane. Organic phases are dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated in vacuo. The crude products are purified by flash chromatography using mixtures of n-hexane/ethyl acetate as mobile phase.

Synthesis of intermediates of formula 29

Intermediates 26 or 28 are dissolved in a mixture of tetrahydrofurane/methanol (1 : 1.5 v/v) at a final concentration of 0.1M and subjected to catalytic hydrogenation using a continuous-flow hydrogenation reactor (H-Cube, TalesNano, Budapest, Hungary). The typical process parameters selected are: 30°C of temperature, 10 atm of pressure and 1 ml/min of flow. Prepacked Pd/C 10% cartridges (TalesNano) are used as supported catalyst. The products of formula 29 obtained at the end of the reduction cycle are used directly in the following steps without further purification. Synthesis of final products of formula 30 or 31

Compounds of general formula 30 and 31 are synthesized according to the procedures described in Example 2 for the synthesis of compounds of formula 5 and 7, respectively. When optical isomers are obtained, separation is performed by HPLC using a teicoplanine-based chiral stationary phase previously described by Ismail et al. (J Chromatogr A. 2016, 4;1427:55-68).

The list below depicts examples of some compounds prepared by using the above-described procedure for the synthesis of compounds of general formula 30 or 31: N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)heptan amide

Synthesized starting from 4-nitrobenzene- 1, 3-diamine, reacted with 4-(trifluoromethyl)benzyl bromide following the general procedure for the synthesis of compounds with general formula 26. The obtained intermediate was reduced under continuous flow hydrogenation condition to give the corresponding N ⁴ -(4-(trifluoromethyl)benzyl)benzene- 1,2, 4-triamine that was lately reacted with heptanoyl choride to give the final compound. 'H NMR (CD3OD, 400 MHz): d: 0.81 (t, 3H, CH ₃ , J=4.2 Hz); 1.19-1.31 (m, 6H, 3 C7¾); 1.55-1.62 (m, 2H, Ci¾; 2.25 (t, 2H, G ¾ J=7.8 Hz); 4.27

(s, 2H, CH ₂ ) 5.95-6.00 (m, 2H, aryl); 6.65 (d, 1H, aiyl, J = 12.2 Hz); 7.43 (d, 2H, aryl, J = 8.2

Hz); 7.48 (d, 2H, aryl, J = 8.4 Hz); HR-MS m/r. calcd for C21H27F3N3O, [(M+H) ⁺ ]: 394,2101; found 394.2109.

N-(3-fluoro-4-((4-fluorobenzyl)amino)phenyl)heptanamide

Synthesized starting from 2-fluoro-4-nitroaniline, reacted with 4-fluorobenzyl bromide following the general procedure for the synthesis of compounds with general formula 26. The obtained intermediate was reduced under continuous flow hydrogenation condition to give the corresponding 2-fluoro-N ¹ -(4-fluorobenzyl)benzene- 1,4-diamine, that was lately reacted with heptanoyl choride to give the final compound. 'H NMR (CD3OD, 400 MHz): d: 0.93 (t, 3H, C//3,

J=7.8 Hz); 1.31-1.42 (m, 6H, 3 G%); 1.66-1.73 (m, 2H, CH ₂ ); 2.38 (t, 2H, C H ₂ , J=8.4 Hz); 4.52 (s, 2H, CH ₂ ); 7.06-7.15 (m, 3H, aryl); 7.25 (d, 1H, aryl, J= 8.3 Hz); 7.40-7.43 (m, 2H, aryl); 7.71

(d, 1H aryl, J= 12.5 Hz). HR-MS m/ calcd for C20H25F2N2O, [(M+H) ⁺ ]: 347,1929; found 347,1936.

N-(2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)aniino)phe nyl)heptanainide

Synthesized starting from 2,3-difluoro-6-nitroaniline, reacted with (4- (trifluoromethyl)phenyl)methanamine following the general procedure for the synthesis of compounds with general formula 28. The obtained intermediate was reduced under continuous flow hydrogenation condition to give the corresponding 3-fluoro-N ⁴ -(4- (trifluoromethyl)benzyl)benzene- 1,2, 4-triamine that was lately reacted with heptanoyl choride to give the final compound. ¾ NMR (DMSO-d6, 400 MHz): d: 0.87 (t, 3H, C// ₃ , J=8.3 Hz); 1.24- 1.31 (m, 6H, 3 C Hi); 1.53-1.57 (m, 2H, C H ₂ ) 2.24 (t, 2H, C Hi, J=8.1 Hz); 4.39 (d, 2H, CH ₂ , J = 7.7 Hz); 4.57 (bs, 2H, N ¾); 5.78 (t, 1H, aryl, J = 7.6 Hz); 6.03 (t, 1H, NT/, J = 3.7 Hz); 6.58 (d, 1H, aryl, J = 8.4 Hz); 7.55 (d, 2H, aryl, 7 = 8.3 Hz); 7.66 (d, 2H, aryl , J = 8.4 Hz); 8.90 (s, 1H, CON//); HR-MS m/r. calcd for C21H26F4N3O, [(M+H) ⁺ ]: 412,2007; found 412.2011.

N-(2-amino-4-((4-(trifluoromethoxy)benzyl)amino)phenyl)he ptanamide

Synthesized starting from N^^-methoxybenzy^-d-nitrobenzene-l, 4-diamine, reacted with 4- (trifluoromethyl)benzyl bromide following general procedure for the synthesis of compounds with general formula 26. After reduction of the nitro group, the corresponding amine was coupled with heptanoyl chloride. ¾ NMR (DMSO-d6, 400 MHz): d: 0.87 (t, 3H, C// ₃ , J=6.7 Hz); 1.20-1.31 (m, 6H, 3 G %); 1.53-1.57 (m, 2H, C Hi)\ 2.22 (t, 2H, CH ₂ , J=8.1 Hz); 4.22 (d, 2H, G %, J=7.8 Hz); 4.51 (bs, 2H N ¾); 5.84 (d, 1H, aryl, J= 8.4 Hz); 5.95-5.97 (m, 2H, aryl and N//); 6.70 (d, 1H, aryl, J= 8.2Hz); 7.30 (d, 2H, aryl, J= 11.6 Hz); 7.45 (d, 2H, aryl, J = 11.5 Hz); 8.80 (s, 1H, aryl, CON//); HR-MS m/z\ calcd for C21H27F3N3O2, [(M+H) ⁺ ]: 410,2050; found 410,2048.

N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)cyc lohexanesulfonamide

Synthesized starting from 4-nitrobenzene- 1, 3-diamine, reacted with 4-(trifluoromethyl)benzyl bromide following the general procedure for the synthesis of compounds with general formula 26. The obtained intermediate was reduced under continuous flow hydrogenation condition to give the corresponding N ⁴ -(4-(trifluoromethyl)benzyl)benzene- 1,2, 4-triamine that was lately reacted with hexanesulfonyl chloride to give the final compound. 'H NMR (CD ₃ OD, 400 MHz): d: 1.23-1.37 (m, 3H, CH ₂ and C H), 2.21-2.24 (m, 2H, G%); 3.00 (t, 1H, C H, J=12.2 Hz); 5.13 (s, 2H, G %); 6.35 (d, 1H, aryl, J=8.1 Hz); 6.48 (s, 1H, aryl); 7.03 (d, 1H, aryl, J= 8.3 Hz); 7.62 (d, 2H, aryl J=8.2 Hz); 7.68 (d, 2H, aryl, J=8.3 Hz);. HR-MS m z calcd for C20H26F3N3O2S, [(M+H) ⁺ ]: 429.1692; found 429.1701.

IN VITRO PHARMACOLOGICAL ASSAYS

Example 8 - Electrophysiological assays on mammalian cells transiently expressing Kv7 channel subunits

Cell Culture and Transient Transfection

Channel subunits were expressed in Chinese Hamster Ovary (CHO) cells (ATCC, Catalogue Number CRL-9618) by transient transfection, using plasmids containing cDNAs encoding human Kv7.1, Kv7.2 and Kv7.3 (Soldovieri et al. J Biol Chem. 2006;281(l):418-28; Iannotti et al. J Pharmacol Exp Ther. 2010;332(3):811-20; Landoulsi et al., Mol Pharmacol. 2013;84(5):763-73), all cloned in pcDNA3.1(+) vector (ThermoFisher Scientific, Catalog number: V79020). According to the experimental protocol, these plasmids were expressed individually or in combination (in particular of Kv7.2 and Kv7.3), together with a plasmid-expressing enhanced green fluorescent protein (Clontech, Palo Alto, CA) used as a transfection marker. Total cDNA in the transfection mixture was kept at 4 pg. CHO cells were grown in 100-mm plastic Petri dishes in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, nonessential amino acids (0.1 mM), penicillin (50 U/ml), and streptomycin (50 mg/ml) in a humidified atmosphere at 37°C with 5% C02. The cells were plated on glass coverslips coated with poly-L-lysine, and transfected one day after plating with the appropriate cDNA using Lipofectamine 2000 (Life Technologies), according to the manufacturer’s protocol.

Whole-cell electrophysiology

Currents from CHO cells were recorded at room temperature (20-22°C) 24 hours after trasfection using the whole-cell configuration of the patch-clamp technique, with glass micropipettes of 3-5 MW resistance. During the recording, constant perfusion of extracellular solution was maintained. The extracellular solution contained (in mM): 138 NaCl, 2 CaCh, 5.4 KC1, 1 MgCh, 10 glucose, and 10 HEPES, at pH 7.4, adjusted with NaOH. The pipette (intracellular) solution contained (in mM): 140 KC1, 2 MgCh, 10 EGTA, 10 HEPES, 5 Mg ²⁺ -ATP, at pH 7.3-7 4, adjusted with KOH. Currents were recorded using an Axopatch-200A amplifier, filtered at 5 kHz, and digitized using a DigiData 1440A (Molecular Devices). The pCLAMP software (version 10.2, Molecular Devices) was used for data acquisition and analysis. To evaluate the activity of exemplary compounds on Kv7.2 (see Table 1), as well as on Kv7.1 and Kv7.2/7.3 channels for a selected compound (see Table 2), the cells were clamped at -80 mV and currents were elicited by 3-s voltage ramps from -80 mV to +20 mV in the presence and absence of each compound. To calculate the EC50, cells were perfused with increased concentration of the compund (0.01, 0.1, 1, 10 mM) until saturation was achieved. The effects of drugs were expressed as shift in the ramp- voltage (AV) calculated at 20% of the maximal current (Miceli et al, Proc Natl Acad Sci U S A. 2013, 110(11):4386-91). Curves were fitted using the following equation: y=max/(l+x/EC50)n, where x is the drug concentration, n is the Hill coefficient and max is the maximal value achieved, as previously described (Ambrosino et al, Br J Pharmacol. 2013, 168(6): 1430-1444). Data analysis and fit were performed using Sigmaplot (version 12.3).

Results

Ramp-evoked Kv7.2 currents were analyzed by measuring the currents at -40 mV (a membrane potential value close to the activation threshold) and at 0 mV (a membrane potential value at which Kv7.2 conductance is largely saturated). In particular, -40 mV represents the threshold potential for spike initiation and 0 mV represents instead the membrane potential value at which maximal conductance of the channel is achieved. Using the measurements at -40 and 0 mV, the inventors have approximated drug effects at membrane potential values achieved by the neurons during low and high frequency firing activity, respectively. Results are reported as average values ± standard error from the mean (N±SEM).

In Table 1, the effect of exemplary compounds of the invention is expressed as the ratio between current amplitude in the presence (Umg) and absence (I _c ti) of the drug at these two membrane voltages. Idrug/Icti>l represents activating activity; Idmg/IctU l represents inhibiting activity; Idmg/Icti= l indicates no effect of the drug on this parameter.

Table 1. Effect of exemplary compounds on Kv7.2 in comparison with retigabine

As evidenced in Table 1, the activity the derivatives is modulated in particular by the substituents present in position N4 of the aryldiamino core. It was also found that the activity of the tested compounds is influenced by the substitution in position N1 of the aryldiamine core, as well by the nature of the bond between these substituents and the nitrogen itself (i.e. substituent

Q). Sulfonamide and amide derivatives act, preferentially, as agonists of Kv7.2 channel, depending also on the nature of the side chain. Flexible and highly lipophilic side chains (as for N-(2-amino-4-((4-(trifluoromethoxy)benzyl)amino)phenyl)hepta namide and N-(2-amino-4-((4- (trifluoromethyl)benzyl)amino)phenyl)heptanamide) tend to enhance the agonistic activity.

Contrariwise, the use of rigid substituents is able to reduce currents, leading some of the tested molecules to behave as antagonist. The use of more rigid Q substituents such as urea and guanidine tends to switch the activity to antagonism, as well.

The EC50 of retigabine and of a compound of the invention (N-(2-amino-4-((4-

(trifluoromethyl)benzyl)amino)phenyl)heptanamide) for Kv.7.1 and Kv7.2/3 currents was calculated (Table 2).

Table 2. EC ₅₀ values of retigabine and N-(2-amino-4-((4-

(trifluoromethyl)benzyl)amino)phenyl)heptanamide on different subtype of Kv7 channels

The data shown in Table 2 suggest that the novel compound N-(2-amino-4-((4- (trifluoromethyl)benzyl)amino)phenyl)heptanamide is eight times more potent than retigabine with respect to the ability to increase Kv7.2/3 currents. Due to their higher potency on Kv7.2/3 current, N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)heptan amide is expected to exert its desired pharmacodynamic effects at lower concentrations than retigabine.

Additionally, similar to retigabine, N-(2-amino-4-((4-

(trifluoromethyl)benzyl)amino)phenyl)heptanamide does not potentiate the currents carried by cardiac Kv7.1 channels, thus avoiding detrimental cardiovascular side effects. As a matter of fact, retigabine is selectively active on specific Kv7 channel subtypes, due to its ability to interact with specific channel residues; in particular, a major component of the retigabine binding site seems to be provided by a tryptophane residue at position 236 (W236) along the primary sequence of Kv7.2 subunit. The substitution of this residue with a leucine (the naturally-occurring residue in retigabine-insensitive Kv7.1 channels; W236L substitution) largely hampers the ability of retigabine to act as a Kv7.2 channel modulator (Wuttke et al., Mol. Pharmacol., 2005, 67(4), 1009- 1017).

W236L substitution could remove the activity of N-(2-amino-4-((4- (trifluoromethyl)benzyl)amino)phenyl)heptanamide, one of the most effective compounds herein described as Kv7.2 modulators.

In particular, application of 10 m\1 retigabine in cells expressing Kv7.2 W236L mutant channels (a Kv7.2 mutant channel insensitive to retigabine, Soldovieri et al. J Biol Chem. 2006,281(1):418- 28; Iannotti et al. J Pharmacol Exp Ther. 2010;332(3):811-20; Landoulsi et al., Mol Pharmacol. 2013;84(5):763-73), failed to affect current amplitude. The ratio between current amplitude in the presence and absence of retigabine at -40 mV was 1.2±0.1. Compound of formula I are likely to display a similar mode of action to retigabine.

Example 9 - Stability

Materials and methods

In order to study the solution stability and photostability of the potassium channel modulators object of the present invention, a stock solution of the proper analyte (1 mg/mL) in dimethylsulfoxide (DMSO) has been prepared. This solution has been then diluted to a concentration of 100 ppm using extracellular solution containing (in mM): 138 NaCl, 2 CaCh, 5.4 KC1, 1 MgCh, 10 glucose, and 10 HEPES, at pH 7.4, adjusted with NaOH. To measure solution stability, the diluted solutions have been maintained at ambient temperature for 6 h. To measure photostability, the diluted solutions have been irradiated for 6 h using a Oriel Sol3A Solar Simulator (Newport corporation, Irvine, CA, USA) with a 12x12 inches output beam. Aliquots of solution have been withdrawn after 6h and analyzed by HPLC using a Kinetex C18, 150 mm x 4.6 mm x 2.6 pm (Phenomenex, Bologna, Italy) as stationary phase and a mobile phase constituted by A: H2O/CH3CN (80:20) and B: CH3CN. Gradient elution has been performed from 20% to 90% of B in 7.50 minutes and from 90% to 20% of B in 1 minute. The pump flux has been set to 1.5 mL/min. Column has been thermostatted at 40°C during analysis. Results are espressed in Table 3 as percentage of degradation of the product calculated as ratio between the peak area of the analyte at time 0 (Pao) and after 6h (Pa ₆ ) using the following formula:

% degradation = (Pao-Pa ₆ )/Pao * 100.

Results

Table 3: Stability in solution and photostability of exemplary compounds object of the present invention in comparison with retigabine

A: degradation lower than 20%; B: 20%< degradation<40%; C: degradation > 40%

The data in Table 3 show that retigabine is chemically unstable when exposed to visible light; indeed, after 6 hours of exposure to light, more that 40% of the drug is degraded. By contrast, most of derivatives were degraded by less than 20% after light exposure for the same exposure time showing that in addition to being more potent than retigabine on Kv7.2/3 currents, are also chemically more stable than retigabine. The improved chemical stability shown by the compounds of the invention with respect to retigabine may translate, in vivo, in a lower tendency of these molecules to accumulate as blue-colored precipitates in the skin, mucasae, and in the retina of subjects taking these drugs.

Example 10 - Thallium flux assay and electrophysiological assay on mammalian cells stably expressing Kv7.2+Kv7.3 channel subunits

Generation of a stable cell line expressing Kv7.2 and Kv7.3 subunits To generate the cell line stably expressing Kv7.2 and Kv7.3 subunits the PiggyBac Transposon (PB) System (System Biosciences), a biological technique that uses a transposase to efficiently intergrate a specific vector into chromosomes via a "cut and paste" mechanism, was used. In particular, during transposition, the PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and efficiently integrates them into TTAA chromosomal sites. Two plasmids containing the cDNA for KCNQ2 or KCNQ3 genes inserted into the PiggyBac vector (System Biosciences, Cat. No. PB514B) were used. These vectors also contain a puromicyn resistance gene as selection marker and a red fluorescent protein (RFP) gene. Stable cell lines were established by cotransfection of CHO cells with the plasmids containing the cDNA for KCNQ2 and KCNQ3 genes and a Super PiggyBac Transposase plasmid (System Biosciences, Cat. No. PB210PA-1) by using Lipofectamine 2000 transfection reagent (Invitrogen, Cat. No. 11668019). At 48 hours post transfection, cells were subjected to puromycin (Invitrogen, Cat. No. A1113802) selection (10 pg/mL) for 7 days. The transposed cells were puromycin resistant and RFP positive. The level of Kv7.2/3 expression was evaluated by electrophysiological techniques. The selected clone was grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, nonessential amino acids (0.1 mM), penicillin (50 U/ml), and streptomycin (50 mg/ml) in a humidified atmosphere at 37°C with 5% C02.

Thallium flux assay

The screening assay was performed on stable cell lines co-expressing Kv7.2 plus Kv7.3 channel subunits using a commercially available fluorescence-based assay (FluxOR Green Potassium Ion Channel Assay; ThermoFisher Scientific, Cat. No. F20016). This fluorescent assay uses thallium ions as surrogates of potassium ions and a fluorescent Thallium-sensitive dye. When thallium is added to the extracellular solution with a stimulus to open channels, thallium flows down its concentration gradient into the cells, and channel activity is detected with a proprietary indicator dye that increases in cytosolic fluorescence.

Below is a detailed description of the assay, performed accordingly to the manufacturer’s protocol. Chinese hamster ovary (CHO) cells (ATCC, Catalogue Number CRL-9618) stably expressing Kv7.2 plus Kv7.3 subunits generated using the previously-described technique (see the “Generation of a stable cell line expressing Kv7.2 and Kv7.3 channels” paragraph) were routinely cultured in DMEM growth medium containing 10% FBS, 50 U/ml Penicillin, 50 pg/ml Streptomycin and 4 pg/ml Puromycin. Kv7.2+Kv7.3 cells were seeded into a 96-well white clear bottom plate at a concentration of 16000 cells/well by a multidrop dispenser, and the plate was incubated overnight at 37 °C in a 5% CO2 incubator. The next day, after replacement of the extracellular medium, 80 pL/well dye-loading buffer ( 1000/ FluxOR reagent stock, IOO ^c PowerLoad, l x Hanks’ balanced salt solution, 20 mmol/L HEPES, and 2.5 mmol/L Probenecid, pH 7.40) was added into the cell plate, and the plate was then incubated in the dark at room temperature for 60 min. Once the dye-loading buffer was removed, 70 pL/well assay buffer (l xHank’s balanced salt solution, 20 mmol/L HEPES, and 2.5 mmol L Probenecid, pH 7.40) was added to the cell plates. Then, 10 pL/well of buffer containing the compound of interest at the desired concentration was added into the plate. The final DMSO concentration was 0.1%. The cell plates were then loaded onto a microplate reader (FLUOstar Optima, BMG LABTECH). Baseline fluorescence was recorded for 5 s, then 20 pL/well of stimulus buffer was inj ected and fluorescence was recorded every second for 50 s. The stimulus buffer contained 25 mmol L K ⁺ and 10 mmol/L Tl ⁺ ; the final concentration of K ⁺ was 5.8 mmol/L and the Tl ⁺ was 2 mmol/L. The effects of exemplary compounds of the invention, at a single concentration of 10 mM on Kv7.2+Kv7.3 channels were expressed as the ratio between maximal fluorescence calculated after 50 s of stimulus buffer addition and the baseline fluorescence (F/Fo), and as the slope of fluorescence current traces in the first 5 seconds after stimulus buffer addition. The F Fo and the slope of fluorescence are reported as average values ± standard error from the mean (N±SEM). * indicates values significantly different (p<0.05) from retigabine.

To calculate the EC50, the initial slope of fluorescence signal was plotted towards each concentration of the compound (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 100 mM). These values were then fitted using the following equation: y=max/(l+x/EC5o)n, where x is the drug concentration, n is the Hill coefficient and max is the maximal value achieved, as previously described (Ostacolo et al., Synthesis and Pharmacological Characterization of Conformationally Restricted Retigabine Analogues as Novel Neuronal Kv7 Channel Activators, J. Med. Chem., 2020 January 9;63(1): 163- 185. doi: 10.1021/acs.jmedchem.9b00796). Data analysis and fit were performed using Sigmaplot (version 12.3).

Statistically significant differences in fluorescence data were evaluated with Student t-test.

Whole-cell electrophysiology

Currents from CHO cells that stably expressed Kv7.2 plus Kv7.3 subunits were recorded at room temperature (20-22°C) using the whole-cell configuration of the patch-clamp technique, with glass micropipettes of 3-5 MW resistance. During the recording, constant perfusion of extracellular solution was maintained. The extracellular solution contained (in mM): 138 NaCl, 2 CaCb, 5.4 KC1, 1 MgCh, 10 glucose, and 10 HEPES, at pH 7.4, adjusted with NaOH. The pipette (intracellular) solution contained (in mM): 140 KC1, 2 MgCh, 10 EGTA, 10 HEPES, 5 Mg ²⁺ -ATP, at pH 7.3-7.4, adjusted with KOH. Currents were recorded using an Axopatch-200A amplifier, filtered at 5 kHz, and digitized using a DigiData 1440A (Molecular Devices). The pCLAMP software (version 10.2, Molecular Devices) was used for data acquisition and analysis. To evaluate the activity of exemplary compounds on Kv7.2/7.3 channels, the cells were clamped at -80 mV and currents were elicited by 1.5-second depolarization potentials in 10 mV increments, from -100 to +20 mV, followed by a return step to 0 mV. Currents elicited by each voltage step were measured and used to generate the conductance-voltage (G-V) curves. The Boltzmann function was used to fit the G-V curves and to determine the half-maximal activation voltage (V1 _/ 2) of Kv7.2/7.3 currents.

To calculate the EC50, the shift in V1/2 was plotted against each concentration of the investigated compound (0.01, 0.1, 1, 3, 10, 100 mM). These values were then fitted using the following equation: y=max/(l+x/EC5o)n, where x is the drug concentration, n is the Hill coefficient and max is the maximal value achieved, as previously described (Ostacolo et al., Synthesis and Pharmacological Characterization of Conformationally Restricted Retigabine Analogues as Novel Neuronal Kv7 Channel Activators, J. Med. Chem., 2020 January 9;63(1): 163-185. doi: 10.1021/acs.jmedchem.9b00796). Data analysis and fit were performed using Sigmaplot (version

12.3).

Results

In Table 4, the effect of the compounds is expressed as the ratio between maximal fluorescence calculated and the baseline fluorescence (F/Fo), and as the slope of fluorescence current traces. Table 4. Effect of exemplary compounds on Kv7.2+Kv7.3 currents in comparison to retigabine at a single dose of 10 mM.

The results obtained revealed that most of the tested compounds show agonist activity on channels composed of Kv7.2+Kv7.3 subunits, and one compound is an antagonist (N-(3-fluoro-4-((4- fluorobenzyl)amino)phenyl)cyclohexanesulfonamide). Among the active compounds at least twelve are more active than retigabine.

The EC50 value for retigabine, N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)-3,3- dimethylbutanamide (Cmp No. 63) and N-(2-amino-4-((4- (trifluoromethyl)benzyl)amino)phenyl)-3-cyclohexylpropanamid e (Cmp No. 251), calculated by plotting the initial slope of fluorescence signal, was 4.6±0.7 mM, 0,17±0,02 mM and 2,22±0,17 pM, respectively; suggesting that these two compounds are more potent than retigabine to potentiate Kv7.2/Kv7.3 currents.

The higher potency of the two compounds N-(2-amino-4-((4- (trifluoromethyl)benzyl)amino)phenyl)-3,3-dimethylbutanamide (Cmp No. 63) and N-(2-amino- 4-((4-(trifluoromethyl)benzyl)amino)phenyl)-3-cyclohexylprop anamide (Cmp No. 251) when compared to retigabine was confirmed by electrophysiological experiments; indeed, an EC50 calculated by plotting the shift in V1/2 was 4.8±1.8 pM, 0.19±0.03 pM, and 0.28±0.04 pM for retigabine, N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)-3,3-d imethylbutanamide (Cmp No. 63) and N-(2-amino-4-((4-(trifluoromethyl)benzyl)amino)phenyl)-3- cyclohexylpropanamide (Cmp No. 251), respectively.