EA049235B1 - KV3 MODULATORS - Google Patents

Abstract

A compound of formula (I) and aspects related thereto are provided.

Description

Field of technology

The present invention relates to new compounds, pharmaceutical compositions containing them and their use in therapy, in particular in the prevention or treatment of hearing disorders, including hearing loss and tinnitus, as well as schizophrenia, substance abuse, pain and fragile X syndrome.

Prior art

The Kv3 family of voltage-gated potassium channels includes four members: Kv3.1, Kv3.2, Kv3.3, and Kv3.4. Kv3 channels are activated by depolarization of the plasma membrane to voltages greater than -20 mV; in addition, these channels are rapidly deactivated by membrane repolarization. These biophysical properties ensure that the channels open toward the peak of the depolarizing phase of the neuronal action potential to initiate repolarization. The rapid termination of the Kv3-mediated action potential allows the neuron to recover more rapidly to reach subthreshold membrane potentials from which further action potentials can be evoked. As a result, the presence of Kv3 channels in certain neurons contributes to their ability to fire at high frequencies (Rudy et al., 2001). Kv3.1-3 channel subtypes predominate in the CNS, whereas Kv3.4 channels are also found in skeletal muscle neurons and sympathetic neurons (Weiser et al., 1994). Kv3.1-3 channel subtypes are differentially expressed by interneuron subclasses in cortical brain regions and the hippocampus (e.g., Chow et al., 1999; Martina et al., 1998; McDonald et al., 2006; Chang et al., 2007), thalamus (e.g., Kasten et al., 2007), cerebellum (e.g., Sacco et al., 2006; Puente et al., 2010), and auditory brainstem nuclei (Li et al., 2001).

Tetraethylammonium (TEA) has been shown to inhibit these channels at low millimolar concentrations (Rudy et al., 2001), and blood depressant toxins (BDS) from the sea anemone Anemonia sulcata (Diochot et al., 1998) selectively inhibit Kv3 channels with high affinity (Yeung et al., 2005).

Kv3 channels are important determinants of cerebellar function, a brain region important for motor control (Joho et al., 2009). Characterization of mice in which one or more Kv3 subtypes have been deleted shows that the absence of Kv3.1 results in increased locomotor activity, altered electroencephalographic activity, and fragmented sleep patterns (Joho et al., 1999). Deletion of Kv3.2 results in a decreased seizure threshold and altered cortical electroencephalographic activity (Lau et al., 2000). Deletion of Kv3.3 is associated with mild ataxia and motor deficits (McMahon et al., 2004). Double deletion of Kv3.1 and Kv3.3 results in a severe phenotype characterized by spontaneous seizures, ataxia, and increased sensitivity to ethanol (Espinosa et al., 2001; Espinosa et al., 2008). Spontaneous mutation in the Kv3.1 gene (KCNC1) causes progressive myoclonic epilepsy (Muona et al., 2014). Mutations in the Kv3.3 gene (KCNC3) in humans are associated with forms of spinocerebellar ataxia (SCA13) (Figueroa et al., 2010).

Bipolar disorder, schizophrenia, anxiety, and epilepsy are severe central nervous system disorders that are associated with decreased inhibitory interneuron function and gamma-aminobutyric acid (GABA) signaling (Reynolds et al., 2004; Benes et al., 2008; Brambilla et al., 2003; Aroniadou-Anderjaska et al., 2007; Ben-Ari, 2006). Parvalbumin-positive basket cells expressing Kv3 channels in the cerebral cortex and hippocampus play a key role in generating feedback inhibition in local circuits (Markram et al., 2004). Given the relative predominance of excitatory over inhibitory synaptic input to glutamatergic pyramidal neurons in these circuits, rapid firing of interneurons providing inhibitory input is necessary to ensure balanced inhibition. Furthermore, precise timing of inhibitory input is necessary to maintain network synchronization, for example in the generation of gamma field potential oscillations that are associated with cognitive function (Fisahn et al., 2005; Engel et al., 2001). Notably, reduced gamma oscillations have been observed in patients with schizophrenia (Spencer et al., 2004), and evidence suggests reduced expression of Kv3.1, but not Kv3.2, in the dorsolateral prefrontal cortex of patients with schizophrenia who were off antipsychotic medication for at least 2 months before death (Yanagi et al., 2014). Accordingly, positive modulators of Kv3 channels would be expected to enhance the excitatory capacity of certain groups of rapidly excitatory neurons in the brain. These effects may be beneficial in disorders associated with abnormal activity of these neuronal groups. In addition, Kv3.2 channels have been shown to be expressed by neurons of the superchiasmatic nucleus (SCN), the main pacemaker controlling circadian rhythms in the CNS (Schulz et al., 2009).

Voltage-gated ion channels of the Kv3 family are expressed at high levels in the auditory brainstem nuclei (Li et al., 2001), where they mediate rapid excitation of neurons that transmit auditory information from the cochlea to higher brain regions. Phosphorylation of Kv3.1 and Kv3.3 channels in auditory brainstem neurons is thought to contribute to rapid physiological

- 1 049235 adaptation to sound levels, which may play a protective role against noise exposure (Desai et al., 2008; Song et al., 2005). Loss of Kv3.1 expression in central auditory neurons has been observed in hearing-impaired mice (von Hehn et al., 2004); furthermore, decreased Kv3.1 expression may be associated with hearing loss in aged mice (Jung et al., 2005), and loss of Kv3 function may also be associated with noise-induced hearing loss (Pilati et al., 2012). Furthermore, abnormal plasticity of brainstem auditory networks likely contributes to the symptoms experienced by many individuals with various types of hearing loss. Recent studies have shown that regulation of Kv3.1 function and expression plays an important role in controlling the excitability of auditory neurons (Kaczmarek et al., 2005; Anderson et al., 2018; Glait et al., 2018; Olsen et al., 2018; Chambers et al., 2017), suggesting that this mechanism may explain some of the plastic changes that lead to tinnitus. Tinnitus may accompany noise-induced hearing loss as a result of adaptive changes in the central auditory pathways from the brainstem to the auditory cortex (Roberts et al., 2010). Kv3.1 and/or Kv3.2 channels are expressed in many of these circuits and contribute to the function of GABAergic inhibitory interneurons, which may control the function of these circuits.

Modulators of Kv3.1 and/or Kv3.2 are known to be useful in the treatment of pain (WO2017/098254). In the broadest sense, pain can be divided into acute and chronic pain. Acute pain is defined as pain that is self-limited and typically requires treatment for no more than a few weeks, such as postoperative pain or acute musculoskeletal pain such as fractures (US Food and Drug Administration, 2014). Chronic pain can be defined as pain that persists for more than 1 month after resolution of the original injury or pain that persists for more than three months. There is often no clear cause for chronic pain, and a variety of other health problems, such as fatigue, depression, insomnia, mood changes, and decreased mobility, often accompany chronic pain.

Chronic pain can be divided into the following groups: neuropathic pain, chronic musculoskeletal pain, and mixed chronic pain. Neuropathic pain usually accompanies tissue injury and is caused by or is triggered by damage to the nervous system (peripheral nervous system and/or central nervous system), such as amputation, stroke, diabetes, or multiple sclerosis. Chronic musculoskeletal pain can be a symptom of conditions such as osteoarthritis and chronic low back pain, and can occur after muscle tissue damage or trauma to the area, such as fractures, strains, and dislocations. Mixed chronic pain encompasses all other types of long-term pain and includes non-neuropathic pain conditions such as cancer pain and fibromyalgia, as well as headaches and tendonitis.

Chronic pain is a highly heterogeneous condition that remains one of the most distressing and difficult to treat clinically (McCarberg et al., 2008; Woolf, 2010; Finnerup et al., 2015). Despite years of drug research and development, little progress has been made in identifying treatments that can match opioids in efficacy without significant side effects and the risk of addiction. Voltage-gated ion channels represent important targets for the treatment of specific pain symptoms, particularly neuropathic pain conditions. Furthermore, genetic mutations in certain ion channels have been associated with several chronic pain disorders (Bennett et al., 2014). Examples of voltage-gated ion channels being investigated as pharmaceutical targets include: sodium channels (particularly NaV1.7) - Sun et al., 2014; Dib-Hajj et al., 2013; N-muna calcium channels - Zamponi et al., 2015; Kv7 potassium channels - Devulder, 2010; Wickenden et al., 2009; and SLACK - Lu et al., 2015.

The hypothesis underlying these approaches is that chronic pain states are associated with increased excitability and/or aberrant firing of peripheral sensory neurons, in particular neurons involved in the transmission of painful sensory stimuli, such as C-fibers of the dorsal root ganglia and specific circuits within the spinal cord (Baranauskas et al., 1998; Cervero, 2009; Woolf et al., 2011; Baron et al., 2013). Animal models of neuropathic and inflammatory chronic pain provide the main support for this hypothesis, although evidence of a causal relationship is still lacking (Cervero, 2009).

Drugs that target hyperexcitability, such as sodium channel blockers (e.g. CNV1014802, lamotrigine, carbamazepine, and local anesthetics), Kv7 positive modulators (e.g. flupertine and retigabine), and N-type calcium channel modulators (e.g. gabapentin, which interacts with the α2δ subunit of the N-type calcium channel, and ziconitide, derived from cone snail toxin), have shown efficacy in models of inflammatory and/or neuropathic pain. However, there is conflicting evidence of clinical efficacy among these drugs, such as countervailing efficacy and increased severity of central nervous system side effects. The discrepancy between efficacy in animal models and efficacy in humans is likely due to a number of factors, but in particular, the achievable drug concentration in humans (due to poor

- 2 049235 tolerance) and heterogeneity of human pain states. For pain indications, it is also necessary to identify targets by which pain relief can be achieved with decreased tolerance or tachyphylaxis and reduced susceptibility to abuse and/or risk of dependence.

Thus, improvements in pharmacological treatment of pain focus on mechanisms that can provide good efficacy with reduced severity of side effects, decreased tolerance or tachyphylaxis, and reduced abuse liability and/or risk of dependence.

Recently, Kv3.4 channels have emerged as a target of interest for the treatment of chronic pain. Kv3.4 channels are expressed on dorsal root ganglia neurons (Ritter et al, 2012; Chien et al, 2007), where they are predominantly expressed on sensory C-fibers (Chien et al, 2007). Kv3 channels are also expressed by specific subsets of spinal cord neurons. In particular, Kv3.1b (Deuchars et al, 2001; Brooke et al, 2002), Kv3.3 (Brooke et al, 2006), and Kv3.4 (Brooke et al, 2004) subunits have been identified in the rodent spinal cord, although not always associated with circuits involved in sensory processing. It is likely that Kv3 channels shape the excitatory properties of spinal cord neurons, including motor neurons.

Furthermore, recent studies have shown that Kv3.4 channels expressed in dorsal root ganglia (DRG) nociceptors exert a significant influence on glutamatergic synaptic transmission (Muqeem et al., 2018). Animal models support negative feedback regulation of Kv3.4 surface expression in DRG neurons following spinal cord injury associated with hypersensitivity to pain stimuli (Ritter et al., 2015; Zemel et al., 2017; Zemel et al., 2018). Similarly, negative feedback regulation of Kv3.4 expression has been observed in rodent DRG following spinal cord ligation (Chien et al., 2007). This latter study also showed that intrathecal administration of an antisense oligonucleotide to suppress Kv3.4 expression in rats resulted in hypersensitivity to mechanical stimuli. It has been shown that Kv3.4 channel inactivation can be influenced by protein kinase C-dependent phosphorylation of the channels, and that this physiological mechanism may allow DRG neurons to alter their firing characteristics in response to painful stimuli (Ritter et al., 2012). These studies support a causal relationship between the occurrence of mechanical allodynia and reduced Kv3.4 channel expression or function. None of these studies assessed the expression of Kv3.1, Kv3.2, or Kv3.3 in SC or DRG neurons, and expression of these two subtypes has not been clearly demonstrated in DRG neurons (although, as mentioned above, they are distributed in certain regions of the spinal cord). The in vivo studies described above provide a rationale for modulating Kv3.4 as a novel approach to treating certain neuropathic pain conditions.

Dementia with Lewy bodies (DLB) and Parkinson's disease (PD) are severe neurodegenerative disorders that are associated with the accumulation of the protein alpha-synuclein in Lewy bodies, leading to loss of connectivity and death of neuronal cells. Symptoms of DLB include progressive cognitive deficits, particularly difficulty with planning and attention. Visual hallucinations are also common, occurring in approximately 60% of patients. PD is associated with early motor deficits, primarily due to the loss of dopamine neurons. Although there are currently no studies directly linking Kv3 channels to DLB or PD, the location and role of Kv3 channels, particularly Kv3.1, in cortical and basal ganglia circuits suggest that modulators of these channels may improve symptoms of DLB or PD, either alone or in combination with current treatments such as acetylcholinesterase inhibitors for DLB or L-DOPA for PD.

International applications WO2011/069951, WO2012/076877, WO2012/168710, WO2013/175215, WO2013/083994, WO2013/182850, WO2017/103604, WO2018/020263 and WO2018/109484 disclose compounds that are modulators of Kv3.1 and Kv3.2. In addition, the usefulness of such compounds has been demonstrated in animal models of seizures, hyperactivity, sleep disorders, psychosis, hearing impairment and bipolar disorders.

International application publication WO2013/182851 discloses modulation of Kv3.3 channels by certain compounds.

International application publication WO2013/175211 discloses that modulation of Kv3.1, Kv3.2 and/or Kv3.3 channels has been found to be useful in preventing or limiting the onset of permanent hearing loss due to exposure to loud noise. The benefits of such prevention can be observed even after discontinuation of administration of the Kv3.1, Kv3.2 and/or Kv3.3 modulator.

International application publication WO2017/098254 discloses that modulation of Kv3.1, Kv3.2 and/or Kv3.3 channels has been found to be useful in the prevention or treatment of pain, in particular neuropathic or inflammatory pain.

International application publication WO2019/222816 discloses meta-linked pyridinyl compounds of the general formula:

which are said to be modulators of Kv3.1 and/or Kv3.2 channels.

International application publication WO2020/000065 discloses meta-linked diazine and triazine compounds of the general formula:

which are said to be modulators of Kv3.1 and/or Kv3.2 channels.

There remains a need to identify alternative modulators of Kv3.1, Kv3.2 and/or Kv3.3, in particular modulators of Kv3.1 and/or Kv3.2. Such modulators may exhibit high in vivo potency, channel selectivity, an improved safety profile, or desirable pharmacokinetic parameters, such as high brain availability and/or a low clearance rate that reduces the dose required for therapeutic effect in vivo. Alternative modulators may provide the advantage of having metabolites that are distinct from known modulators. Compounds that have balanced modulatory properties with respect to Kv3.1, Kv3.2 and/or Kv3.3 may be desirable, such as compounds that modulate Kv3.1 and Kv3.2 to the same or similar extent. For certain therapeutic indications, there is also a need to identify compounds with differential modulatory effects on Kv3.1, Kv3.2 and/or Kv3.3 channels, such as compounds that alter the kinetics of channel opening or channel inactivation and that may behave in vivo as negative channel modulators.

Brief summary of the invention

According to the present invention, there is provided a compound of formula (I):

Where:

R1 is H or methyl;

R2 and _R3 are both methyl, or R2 and _R3 , together with the carbon atom to which they are attached, form a spirocyclopropyl ring;

R ₄ represents methyl or ethyl;

R ₅ is H or methyl;

or R4 and R5, together with the carbon atom to which they are attached, form _C3 - _C4 spirocarbocyclyl.

The compound of formula (I) may be provided in the form of a salt and/or solvate thereof. Suitably, the compound of formula (I) may be provided in the form of a pharmaceutically acceptable salt and/or solvate and/or derivative thereof. In one embodiment of the invention, the compound of formula (I) is provided in the form of a pharmaceutically acceptable salt.

The compounds of formula (I) can be used as medicinal products, in particular for use in the prevention or treatment of hearing disorders, including hearing loss and tinnitus, as well as schizophrenia, substance abuse, pain or fragile X syndrome.

In addition, a method for preventing or treating hearing disorders, including hearing loss and tinnitus, as well as hearing disorders, including hearing loss and tinnitus, as well as schizophrenia, substance abuse, pain, or fragile X syndrome is provided.

The compounds of formula (I) can be used in the manufacture of a medicinal product for the prevention or treatment of hearing disorders, including hearing loss and tinnitus, as well as schizophrenia, substance abuse, pain or fragile X syndrome.

- 4 049235

Also provided are pharmaceutical compositions comprising a compound of formula (I) and a pharmaceutically acceptable carrier or excipient.

Methods for preparing compounds of formula (I) and new intermediate compounds used in preparing compounds of formula (I) are also proposed.

Additionally, prodrug derivatives of compounds of formula (I) are proposed.

Detailed description of the invention

According to the present invention, compounds of formula (I) are provided:

Where:

R1 is H or methyl;

R2 and _R3 are both methyl, or R2 and _R3 , together with the carbon atom to which they are attached, form a spirocyclopropyl ring;

R4 represents methyl or ethyl;

R5 is H or methyl;

or R ₄ and R5, together with the carbon atom to which they are attached, form C ₃ -C ₄ spirocarbocyclyl;

or a pharmaceutically acceptable salt and/or solvate and/or derivative thereof.

The embodiments set out below, concerning the relative stereochemistry and the nature of the groups including R1, R2, _R3 , R4, R5, are considered to be independently fully combinable with one another, where appropriate under the circumstances (i.e. where chemically appropriate), to yield further embodiments of the invention. Such embodiments apply equally to intermediates that can be used in the synthesis of a compound of formula (I), for example compounds of formulae (II), (IV), (VI), (VII) and (XVI).

Compounds of formula (I) may optionally be provided in the form of a pharmaceutically acceptable salt and/or solvate. In one embodiment of the invention, a compound of formula (I) is provided in the form of a pharmaceutically acceptable salt. In a second embodiment of the invention, a compound of formula (I) is provided in the form of a pharmaceutically acceptable solvate. In a third embodiment of the invention, a compound of formula (I) is not in the form of a salt or solvate.

In one embodiment, R ₁ is H. In a second embodiment, R ₁ is methyl.

In one embodiment, R ₂ is methyl and R ₃ is methyl. In another embodiment, R 2 and R ₃ are spirocyclopropyl to form the following moiety:

In one embodiment, R4 is methyl. In a second embodiment, R4 is ethyl.

In one embodiment, R5 is hydrogen. In a second embodiment, R5 is methyl.

In one embodiment, R4 and R5 are the same (i.e., are methyl).

In embodiments where R4 and R5 are different, they may have the following stereochemical configuration:

In this embodiment, for example, R ₄ is methyl and R ₅ is H, R ₄ is ethyl and R ₅ is H, or R ₄ is ethyl and R ₅ is methyl.

In embodiments where R ₄ and R ₅ are different, they may alternatively have the following stereochemical configuration:

- 5 049235

In this embodiment, for example, R4 is methyl and R5 is H, _R4 is ethyl and R5 is H, or R4 is ethyl and R5 is methyl.

In one embodiment, R4 and R5, together with the carbon atom to which they are attached, form spirocyclopropyl.

In another embodiment, R4 and R5, together with the carbon atom to which they are attached, form spirocyclobutyl.

In one embodiment, the compound of formula (I) is selected from the group consisting of:

5,5-dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4yl)oxypyrazin-2-yl]imidazolidin-2,4-dione;

3-[5-[(3,3-dimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5,5-dimethylimidazolidine-2,4-dione;

(5R)-5-ethyl-5-methyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4yloxypyrazin-2-yl)azolidin-2,4-dione imide;

5,5-dimethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2yl)imide azolide in-2,4-dione;

(5R)-5-ethyl-5-methyl-3-[5-(7-methylpyro[2H-benzofuran-3,Γ-cyclopropane]-4yl)oxypyrazin-2-yl]imidazolidine-2,4-dione;

(5R)-3 -[5 - [(3,3-d imethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5 -ethyl-5 methyl-imidazolidine-2,4-dione;

5,5-dimethyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2yl]imide in-2,4-dione azolide;

(5R)-5-ethyl-5-methyl-3-[5-[(3,3,7-trimethyl-2H-benzopyran-4-yl)oxy]pyrazin-2-yl]imide azolide in-2,4-dione;

(5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4yl)oxypyrazin-2-yl]imidazolidin-2,4-dione;

(5R)-5-ethyl-3-(5-cypo[2H-benzopyran-3,Γ-cyclopropane]-4-yloxypyrazin-2yl)imide azolide in-2,4-dione;

(5R)-3 -[5 - [(3,3-d imethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5-ethylimidazolidine-2,4-dione;

(5R)-5-ethyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2yl]imide azolide ine-2,4-dione;

7-[5-(7-methylspiro[2H-benzofuran-3,N-cyclopropan]-4-yl)oxypyrazin-2-yl]5,7-diazaspiro[3.4]octane-6,8-dione;

or a pharmaceutically acceptable salt and/or solvate and/or derivative thereof.

In one embodiment, the compound of formula (I) is:

6-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]-4,6diazaspiro[2.4]heptane-5,7-dione;

or a pharmaceutically acceptable salt and/or solvate and/or derivative thereof.

In one embodiment, the compound of formula (I) is:

(5 S)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2yl]imidazolidine-2,4-dione;

or a pharmaceutically acceptable salt and/or solvate and/or derivative thereof.

When a compound contains a C _1-3 alkyl group, whether used alone or as part of a larger group, the alkyl group may be straight, branched, or cyclic. Examples of C _1-3 alkyl are methyl, ethyl, n-propyl, isopropyl, and cyclopropyl. Reference to propyl includes n-propyl, isopropyl, and cyclopropyl.

The term halogen, as used here, refers to an atom of fluorine, chlorine, bromine, or iodine. Specific examples of halogen are fluorine, chlorine, and bromine, such as chlorine or bromine.

- 6 049235

The term C _3-4 spirocarbocyclyl as used herein means a cyclic ring system containing 3 or 4 carbon atoms, namely a cyclopropyl or cyclobutyl group, wherein the cyclic ring system is attached to a secondary carbon through a spiro center such that the secondary carbon is one of the 3-4 carbon atoms in the cyclic ring, as shown below:

s

Szspirocarbocyclyl Sapirocarbocyclyl

It will be understood that for use in medicine, the salts of the compounds of formula (I) must be pharmaceutically acceptable. Suitable pharmaceutically acceptable salts will be apparent to those skilled in the art. Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse J. Pharm. Sci. (1977) 66, pp. 1-19. Such pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric or phosphoric acid and organic acids such as succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid. Salts which are not pharmaceutically acceptable may be used, for example, in the isolation of the compounds of formula (I) and are included within the scope of the present invention.

Certain compounds of formula (I) may form acid addition salts with one or more equivalents of acid. The present invention includes within its scope all possible stoichiometric and non-stoichiometric forms.

The compounds of formula (I) may be obtained in crystalline or non-crystalline form and, in the case of crystalline forms, they may optionally be solvated, for example as a hydrate. The present invention includes within its scope stoichiometric solvates (e.g. hydrates) as well as compounds containing variable amounts of solvent (e.g. water).

It is to be understood that the invention includes pharmaceutically acceptable derivatives of the compounds of formula (I) and that they are included within the scope of this invention.

As used herein, the term pharmaceutically acceptable derivative includes any pharmaceutically acceptable ester or salt of such ester of a compound of formula (I) which, when administered to a recipient, is capable of providing (directly or indirectly) a compound of formula (I) or an active metabolite or residue thereof.

A pharmaceutically acceptable prodrug can be formed by functionalizing a secondary nitrogen of the hydantoin, for example, with a group L, as shown below (where R ₄ and R ₅ are as described above):

In one embodiment of the invention, the compound of formula (I) is functionalized via the secondary nitrogen of the hydantoin with a group L selected from:

a) -PO(OH)O--M ⁺ , where M ⁺ is a pharmaceutically acceptable monovalent counterion,

b) -PO(O-)2-2M ⁺ ,

c) -PO(O ^- ) ₂ -D ²⁺ , where D2 ⁺ is a pharmaceutically acceptable divalent counterion,

d) -CH(R ^X )-PO(OH)O--M ⁺ , where R ^X is hydrogen or C _1-3 alkyl,

e) -CH(R ^x )-PO(O ^- )2-2M ⁺ ,

e) -CH(R ^x )-PO(O ^- )2-D ²⁺ ,

g) -SOf-M,

c) -CH(R ^X )-SO3--M ⁺ , and

i) -co-cn ₂ cn ₂ -co ₂ ⁺ .

It is to be understood that the present invention embraces all isomers of formula (I) and their pharmaceutically acceptable derivatives, including all geometric, tautomeric and optical forms and mixtures thereof (e.g. racemic mixtures). When additional chiral centers are present in the compounds of formula (I), the present invention includes within its scope all possible diastereoisomers, including mixtures thereof. The various isomeric forms may be separated or separated from each other by conventional methods, or any given isomer may be obtained by conventional synthetic methods or by stereospecific or asymmetric synthesis.

- 7 049235

The present invention includes all isotopic forms of the compounds of the invention provided herein, whether they are in the form (1), wherein all atoms of a given atomic number have a mass number (or set of mass numbers) that predominates in nature (referred to herein as a natural isotopic form), or in the form (2), wherein one or more atoms are replaced by an atom having the same atomic number but a mass number different from the mass number of atoms that predominates in nature (referred to herein as a non-natural isotopic form variant). It should be understood that an atom may naturally exist as a set of mass numbers. The term non-natural variant of an isotopic form also includes embodiments in which the proportion of an atom of a given atomic number having a mass number less common in nature (referred to herein as an uncommon isotope) has been increased compared to that which occurs in nature, such as to a level of greater than 20%, greater than 50%, greater than 75%, greater than 90%, greater than 95%, or greater than 99% by number of atoms of that atomic number (the latter embodiment being referred to as an isotopically enriched variant form). The term non-natural variant of an isotopic form also includes embodiments in which the proportion of the uncommon isotope has been reduced compared to that which occurs in nature. Isotopic forms may include radioactive forms (i.e., they include radioisotopes) and non-radioactive forms. Radioactive forms are typically isotopically enriched variant forms.

Thus, an unnatural variant of an isotopic form of a compound may contain one or more artificial or uncommon isotopes, such as deuterium ( ^2H or D), carbon-11 ( ^12C ), carbon-13 ( ^13C ), carbon-14 ( ^14C ), nitrogen-13 ( ^13N ), nitrogen-15 (15N), oxygen-15 ( ^15O ), oxygen-17 ( ^17O ), oxygen-18 ( ^18O ), phosphorus-32 ( ^32P ), sulfur-35 ( ^35S ), chlorine-36 ( ^36Cl ), chlorine-37 ( ^37Cl ), fluorine-18 ( ^18F ), iodine-123 ( ^123I ), iodine-125 ( ^125I ), in one or more atoms or may contain an increased proportion of said isotopes compared to the proportion that predominates in nature in one or more atom.

Unnatural variant isotopic forms containing radioisotopes may, for example, be used for tissue distribution studies of the drug and/or substrate. The radioactive isotopes tritium, i.e., ³ H, and carbon-14, i.e., ¹⁴ C, are particularly useful for this purpose due to their ease of incorporation and ready means of detection. Unnatural variant isotopic forms that include deuterium, i.e., ² H or D, may offer certain therapeutic advantages due to greater metabolic stability, such as increased half-life in vivo or reduced dosage requirements, and may therefore be preferable in some circumstances. In addition, unnatural variant isotopic forms that include positron-emitting isotopes such as ⁿ C, ¹⁸ F, ¹⁵ O, and ¹³ N may be prepared and may be useful in positron emission tomography (PET) studies to study receptor occupancy by substrate.

In one embodiment, the compounds of the invention are provided in natural isotopic form.

In one embodiment, the compounds of the invention are provided in a non-natural variant of an isotopic form. In a particular embodiment, the non-natural variant of an isotopic form is a form in which deuterium (i.e. ² H or D) is included, wherein hydrogen is indicated in the chemical structure in one or more atoms of a compound of the present invention. In one embodiment, the atoms of the compounds of the invention are in an isotopic form that is not radioactive. In one embodiment, one or more atoms of the compounds of the invention are in an isotopic form that is radioactive. Suitable radioactive isotopes are stable isotopes. Suitably, the non-natural variant of an isotopic form is a pharmaceutically acceptable form.

In one embodiment, a compound of the invention is provided, wherein one atom of the compound exists in a non-natural isotopic form. In another embodiment, a compound of the invention is provided, wherein two or more atoms exist in a non-natural isotopic form.

Unnatural isotopic variant forms can generally be prepared by conventional methods known to those skilled in the art or by methods described herein, for example methods similar to those described in the accompanying Examples for the Preparation of Natural Isotopic Forms. Thus, unnatural isotopic variant forms can be prepared using suitable isotopic variant (or labeled) reagents instead of the conventional reagents used in the Examples. Since the compounds of formula (I) are intended for use in pharmaceutical compositions, it will be readily understood that each of them is preferably provided in a substantially pure form, for example at least 60% pure, more preferably at least 75% pure, and preferably at least 85% pure, especially at least 98% pure (% by weight). Contaminated preparations of the compounds can be used to prepare purer forms used in pharmaceutical compositions.

Since the compounds of formula (I) are intended for use in pharmaceutical compositions, it will be readily understood that each of them is preferably provided in a substantially pure form,

- 8 049235 example, with a purity of at least 60%, more preferably with a purity of at least 75% and preferably at least 85%, especially with a purity of at least 98% (% is indicated based on weight). Contaminated preparations of the compounds can be used to obtain purer forms used in pharmaceutical compositions.

In general, compounds of formula (I) can be prepared according to methods of organic synthesis known to those skilled in the art, as well as by means of the illustrative methods set forth herein below, the methods set forth in the Examples, and modifications thereof.

In the publications of international applications WO2011/069951, WO2012/076877, WO2012/168710, WO2013/175215, WO2013/083994, WO2013/182850, WO2017/103604,

WO2018/020263 and WO2018/109484 provide methods for synthesizing intermediates that can be used in the preparation of the compounds of the present invention.

General synthesis schemes

The following schemes detail the routes for synthesizing the compounds of the invention and the intermediates in the synthesis of such compounds. In the following schemes, reactive groups may be protected with protecting groups and deprotected according to established procedures well known to those skilled in the art.

The compounds of formula (I) and their salts and solvates can be prepared by the general methods described below. In the following description, the groups R1, _R2 , _R3 , R4 and R5 have the meanings defined above for the compounds of formula (I), unless otherwise indicated.

Scheme 1a.

(II) (III) (I) step (1): Compounds of formula (I) can be prepared by carrying out metal-catalyzed cross-coupling reactions. In this reaction, a halopyrazine derivative of formula (II), wherein X is typically Br, and a hydantoin of formula (III) are reacted in the presence of a metal catalyst such as copper(I) oxide in a suitable solvent, for example N,N-dimethylacetamide, under normal heating or microwave heating.

Scheme 1b.

Compounds of formula (I) wherein R4 and R5 are not H can be prepared by nucleophilic aromatic substitution. In this reaction, a halopyrazine derivative of formula (IV), wherein Y is typically Cl, and a phenol of formula (V) are reacted in the presence of a suitable base such as potassium carbonate, in a suitable solvent such as N,N-dimethylformamide or acetonitrile, with normal heating or microwave heating.

Scheme 1c.

step (2): Compounds of formula (I) can be prepared by cyclization of compounds of formula (VI) in a suitable solvent, such as dichloromethane, with a carbonylating agent, such as triphosgene, preferably previously diluted in the same solvent and added a second time at 0°C, in the presence of a suitable base, such as triethylamine. Alternatively, compounds of formula (I) can be prepared by cyclization of compounds of formula (VI) using a carbonylating agent, such as carbonyldiimidazole, in a suitable solvent, such as

- 9 049235 as ethyl acetate, in the presence of a base such as triethylamine or DIPEA (diisopropylethylamine).

Step (1): Compounds of formula (VI) can be prepared by deprotecting compounds of formula (VII) where PG is a protecting group, a suitable protecting group being BOC (tert-butoxycarbonyl), BOC can be removed under acidic conditions, such as TFA (trifluoroacetic acid) in a suitable solvent, such as dichloromethane, at a temperature from about 0°C to room temperature.

Scheme 1d.

(IX) (XVI) (XVII) (I) Step (2): Compounds of formula (I) can be prepared by reacting a urea of formula (XVII) and a suitable base such as sodium methoxide in a suitable solvent such as methanol at a temperature in the range from 0°C to room temperature.

Step (1): Ureas of formula (XVII) can be prepared by reacting anilines of formula (XVI) and amino esters (e.g. in the form of a hydrochloride) of formula (IX) in a suitable solvent, e.g. dichloromethane or ethyl acetate, with a carbonylating agent, e.g. triphosgene, preferably previously diluted in the same solvent, in the presence of a suitable base, e.g. triethylamine or diisopropylethylamine, at a temperature in the range from 0°C to room temperature.

Scheme 2a.

(IX) (X) (VIII) (IV) step (2): Compounds of formula (IV) can be prepared by reacting a urea of formula (VIII) and a suitable base such as sodium methoxide in a suitable solvent such as methanol at a temperature in the range from 0°C to room temperature.

Step (1): Ureas of formula (VIII) can be prepared by reacting a commercially available halopyrazine derivative of formula (X), wherein Y is typically Cl, and amino esters (e.g. in the form of a hydrochloride) of formula (IX), in a suitable solvent, e.g. dichloromethane or ethyl acetate, with a carbonylating agent, e.g. triphosgene, preferably previously diluted in the same solvent, in the presence of a suitable base, e.g. triethylamine or diisopropylethylamine, at a temperature in the range from 0°C to room temperature.

Scheme 2b.

(XII) (X) (XI) (IV) step (2): Compounds of formula (IV) can be prepared by cyclizing compounds of formula (XI) in a suitable solvent, such as dichloromethane, with a carbonylating agent, such as triphosgene, preferably previously diluted in the same solvent and added a second time at 0°C, in the presence of a suitable base, such as triethylamine.

Step (1): Compounds of formula (XI) can be prepared from anilines of formula (X), where typically Y is Cl, and amino acids (as free base or hydrochloride) of formula (XII) by amide coupling in the presence of a coupling agent, for example T3P, in a suitable solvent such as ethyl acetate, acetonitrile or a mixture thereof.

- 10 049235

Scheme 3.

(XIV) (XIII) (VII) step (2): Compounds of formula (IV) can be prepared by metal-catalyzed cross-coupling reactions. In this reaction, a halopyrazine derivative of formula (II), wherein typically X is Br, and an amide of formula (XIII) are reacted in the presence of a metal catalyst such as tris(dibenzylideneacetone)dipalladium(0), a suitable ligand such as dicyclohexyl-[2-(2,4,6-triisopropylphenyl)phenyl]phosphane (XPhos), and a suitable base such as cesium carbonate, in a suitable solvent such as 1,4-dioxane, under normal heating or microwave heating. Alternatively, in this reaction, a halopyrazine derivative of formula (II), wherein typically X is Br, and an amide of formula (XIII) are reacted in the presence of a metal catalyst such as copper(I) iodide, a suitable ligand such as N,N'-dimethylethane-1,2-diamine, and a suitable base such as potassium carbonate, in a suitable solvent, for example 1-butanol, under ordinary heating or microwave heating. Another alternative for preparing compounds of formula (IV) is to react a halopyrazine derivative of formula (II), wherein typically X is Br, and an amide of formula (XIII), in the presence of a metal catalyst such as palladium(II) acetate, a suitable ligand such as Xantphos, and a suitable base such as cesium carbonate, in a suitable solvent, for example 1,4-dioxane, under ordinary heating or microwave heating.

Step (1): Compounds of formula (XIII) can be prepared from N-protected (e.g. by BOC) amino acids of formula (XIV) and an amine such as hexamethyldisilazane by amide coupling in the presence of a base such as DIPEA and a coupling agent such as HATU ((O-7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) or TBTU (benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate) in a solvent such as N,N-dimethylformamide.

Scheme 4.

(XV) (V) (II)

Step (1): Compounds of formula (II), wherein X is typically Br, can be prepared by nucleophilic aromatic substitution. In this reaction, a halopyrazine derivative of formula (XV), wherein X and Z are typically both Br, and a phenol of formula (V) are reacted in the presence of a base such as potassium carbonate in a suitable solvent, such as N,N-dimethylformamide, under normal heating or microwave heating.

Scheme 5.

(XVIII) (V) (XVI) step (1): anilines of formula (XVI) can be prepared by carrying out metal-catalyzed cross-coupling reactions. In this reaction, a halopyrazine derivative of formula (XVIII), wherein Z is typically Br, and a phenol of formula (V) are reacted in the presence of a metal catalyst such as copper(I) iodide, a suitable ligand-like picolinic acid in a suitable solvent, for example N,N-dimethylformamide or N,N-dimethylacetamide, under normal heating or microwave heating, optionally a suitable base such as potassium carbonate or cesium carbonate can be used.

- 11 049235

Scheme 6.

In Scheme 6 shown above, PG1 and PG2 are suitable protecting groups. PG1 in steps (1)-(3) may be different from PG1 in steps (4)-(7). Suitable protecting groups include benzyl, tetrahydropyranyl, or methyloxymethyl. Suitably, _PG2 is the same as _PG1 , e.g., both are benzyl.

Description of the circuit where PG1 and PG2 both represent benzyl.

Step (7): Phenols of formula (V) can be prepared from benzylated compounds of formula (XIX) by deprotection, for example using a metal catalyst such as palladium on carbon and a hydrogen source such as an atmosphere of hydrogen or ammonium formate, in a suitable solvent such as ethanol or methanol, at a temperature in the range from room temperature to reflux temperature.

Step (6): Benzylated compounds of formula (XIX) can be prepared from diols of formula (XX) using a base such as potassium tert-butoxide and a suitable solvent such as dimethyl carbonate at a temperature in the range from room temperature to reflux temperature.

Step (5): Diols of formula (XX) can be prepared from lactones of formula (XXI) using a reducing agent such as lithium aluminum hydride in a suitable solvent such as THF at a temperature in the range from 0°C to room temperature.

Step (4): Lactones of formula (XXI) can be prepared from phenols of formula (XXII) using a benzylating agent such as benzyl bromide in the presence of a base such as potassium carbonate in a suitable solvent such as acetonitrile or THF or a mixture thereof at a temperature in the range from room temperature to reflux temperature.

Step (3): Phenols of formula (XXII) can be prepared from dibenzylated esters of formula (XXIII), wherein Rx is a suitable alkyl group such as methyl or ethyl, using a metal catalyst such as palladium on carbon and a hydrogen source such as an atmosphere of hydrogen or ammonium formate, in a suitable solvent such as ethanol or methanol, at a temperature in the range from room temperature to reflux temperature.

Step (2): Dibenzylated esters of formula (XXIII), wherein Rx is a suitable alkyl group such as methyl or ethyl, can be prepared from dibenzylated bromo derivatives of formula (XXIV) using preformed organozinc derivatives of formula (XXVI), wherein Rx is a suitable alkyl group such as methyl or ethyl, in the presence of a metal catalytic complex such as bis(tri-tert-butylphosphine)palladium(0), in a suitable solvent such as THF or DMF or a mixture thereof, at a temperature in the range from room temperature to reflux temperature.

Step (1): Dibenzylated bromo derivatives of formula (XXIV) can be prepared from commercially available derivatives of formula (XXV) using a benzylating agent such as benzyl bromide in the presence of a base such as potassium carbonate in a suitable solvent such as acetonitrile, THF or acetone or a mixture thereof at a temperature in the range from room temperature to reflux temperature.

When PG1 and/or PG2 are protecting groups such as tetrahydropyranyl or methyloxymethyl, the usual protection/deprotection conditions apply:

Conditions for protecting phenols with tetrahydropyranyl include reacting the phenol with dihydro-2Hpyran in the presence of a catalyst such as C:Py-p-MePhSO ₃ H in a suitable solvent such as dichloromethane at a temperature in the range from 0°C to reflux temperature.

The conditions for the cleavage of the tetrahydropyranyl protecting group from phenols include interactions

- 12 049235 reaction of a THP-protected phenol in the presence of an acid such as sulfuric acid or p-MePhSO ₃ H or HCl, in a suitable solvent such as methanol or ethanol, at a temperature in the range from 0°C to reflux temperature.

Conditions for protecting phenols with methyloxymethyl include reacting the phenol with chloromethyl methyl ether in the presence of a base such as potassium carbonate in a suitable solvent such as tetrahydrofuran or acetonitrile at a temperature in the range from 0°C to reflux temperature.

Conditions for the cleavage of the methyloxymethyl protecting group from phenols include reacting the MOM-protected phenol in the presence of an acid such as sulfuric acid or p-MePhSO ₃ H or HCl, in a suitable solvent such as methanol or ethanol, at a temperature in the range from 0°C to reflux temperature.

Scheme 7.

R2 R3

At o ____,

Vg Rx ---------- ⁴

O stage (1) (XXVII)

R2 R3

Ζη Rx

O (XXVI)

Step (1): Organozinc derivatives of formula (XXVI), wherein Rx is a suitable alkyl group such as methyl or ethyl, can be prepared by adding commercially available bromoesters of formula (XXVII) to a refluxing suspension of zinc(0) in the presence of 1,2-dibromoethane and chlorotrimethylsilane in a suitable solvent such as THF.

Methods according to the invention.

According to further aspects of the present invention, methods for preparing compounds of formula (I) and their derivatives, as well as methods for preparing intermediates in the synthesis of compounds of formula (I) are provided.

The methods of the invention are described above and include any individual step of a multi-step scheme.

Intermediate connections.

The present invention also relates to novel intermediates in the synthesis of compounds of formula (I). Such novel intermediates include compounds of formulas (II), (IV), (VI), (VII), (VIII), (XI), (XVI) and (XVII). Also of interest are intermediates of formulas (XIX) to (XXIV). The present invention also provides salts, such as pharmaceutically acceptable salts, of such intermediates.

Thus, the intermediate compounds of the invention include:

compounds of formula (II):

where R1, R2 and R3 are as defined above, X is a halogen such as Br; compounds of formula (IV):

where R1, R2 and R3 are as defined above, Y is a halogen such as Cl; compounds of formula (VI):

where R1, R2, _R3 , R4 and R5 are as defined above;

- 13 049235 compounds of formula (VII):

(VII) where R1, R2, _R3 , R4 and R5 are as defined above, PG is a suitable protecting group such as BOC;

compounds of formula (XVI):

where R1, R2 and R3 are as defined above.

Kv3.1, Kv3.2 and/or Kv3.3 modulation

The compounds of formula (I) of the present invention are modulators of Kv3.1. The compounds of formula (I) may also be modulators of Kv3.2 and/or Kv3.3. The compounds of the invention may be tested in the assay of Biological Example 1 to determine their modulatory properties on Kv3.1 and/or Kv3.2 and/or Kv3.3 channels.

The term modulator, as used herein, refers to a compound that is capable of causing at least a 10% potentiation, and suitably at least a 20% potentiation, of whole-cell currents mediated by human Kv3.1 and/or human Kv3.2 and/or human Kv3.3 channels recombinantly expressed in mammalian cells.

The term Kv3.1, Kv3.2 and/or Kv3.3 shall mean the same as Kv3.1 and/or Kv3.2 and/or Kv3.3 and may also be referred to as Kv3.1/Kv3.2/Kv3.3.

In one embodiment, the modulator is capable of producing at least a 10% potentiation, and suitably at least a 20% potentiation, of whole-cell currents mediated by human Kv3.1 channels recombinantly expressed in mammalian cells. Suitably, the pEC ₅₀ of the modulator is in the range of 4-7 (e.g. 5-6.5).

In one embodiment, the modulator is capable of producing at least a 10% potentiation, and suitably at least a 20% potentiation, of whole-cell currents mediated by human Kv3.2 channels recombinantly expressed in mammalian cells. Suitably, the pEC ₅₀ of the modulator is in the range of 4-7 (e.g. 5-6.5).

In one embodiment, the modulator is capable of producing at least a 10% potentiation, and suitably at least a 20% potentiation, of whole-cell currents mediated by human Kv3.3 channels recombinantly expressed in mammalian cells. Suitably, the pEC ₅₀ of the modulator is in the range of 4-7 (e.g. 5-6.5).

In another embodiment, the modulator is capable of producing at least a 10% potentiation, and suitably at least a 20% potentiation, of whole-cell currents mediated by human Kv3.1 and Kv3.2 channels recombinantly expressed in mammalian cells.

In another embodiment, the modulator is capable of producing at least a 10% potentiation, and suitably at least a 20% potentiation, of whole-cell currents mediated by human Kv3.1 and Kv3.3 channels recombinantly expressed in mammalian cells.

In another embodiment, the modulator is capable of producing at least a 10% potentiation, and suitably at least a 20% potentiation, of whole-cell currents mediated by human Kv3.2 and Kv3.3 channels recombinantly expressed in mammalian cells.

In another embodiment, the modulator is capable of producing at least a 10% potentiation, and suitably at least a 20% potentiation, of whole-cell currents mediated by human Kv3.1, Kv3.2 and Kv3.3 channels recombinantly expressed in mammalian cells.

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates and/or derivatives can be used for the treatment or prevention of a disease or disorder in which

- 14 049235 a modulator of Kv3.1 or Kv3.2 or Kv3.1 and Kv3.2 channels is required. As used herein, a modulator of Kv3.1 or Kv3.2 or Kv3.1 and Kv3.2 is a compound that alters the properties of these channels either positively or negatively. In a particular aspect of the invention, the compound of formula (I) is a positive modulator. Compounds of the invention can be tested in the assay of Biological Example 1 to determine their modulating properties.

In one embodiment of the invention, the compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates and/or derivatives thereof are selective for the modulation of Kv3.1 channels over the modulation of Kv3.2 channels. By selective is meant that the compounds exhibit, for example, at least a 2-fold, 5-fold or 10-fold activity on Kv3.1 channels over Kv3.2 channels. The activity of a compound is suitably quantified by its potency, as indicated by the Ec50 value.

In another embodiment of the invention, the compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates and/or derivatives thereof are selective for the modulation of Kv3.2 channels over the modulation of Kv3.1 channels. Again, by selective is meant that the compounds exhibit, for example, at least 2-fold, 5-fold or 10-fold activity on Kv3.2 channels over Kv3.1 channels.

In a particular embodiment of the present invention, the compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates and/or derivatives thereof exhibit comparable activity between modulation of Kv3.1 and Kv3.2 channels, such as activity on one channel less than 2-fold compared to the other channel, such as less than 1.5-fold higher or less than 1.2-fold higher.

In some disorders, it may be useful to use a modulator of Kv3.3 or Kv3.1, or of Kv3.3 and Kv3.1, that exhibits a particular selectivity profile between these two channels. For example, the compound may be selective for modulation of Kv3.3 channels over modulation of Kv3.1 channels, exhibiting, for example, at least 2-fold, 5-fold, or 10-fold potency on Kv3.3 channels over Kv3.1 channels.

In another embodiment of the invention, the compounds of formula (I) or pharmaceutically acceptable salts and/or solvates and/or derivatives thereof are selective for modulation of Kv3.1 channels over modulation of Kv3.3 channels. Again, by selective is meant that the compounds exhibit, for example, at least 2-fold, 5-fold or 10-fold activity on Kv3.1 channels over Kv3.3 channels.

In a particular embodiment of the present invention, the compound may exhibit comparable activity between modulating Kv3.3 and Kv3.1 channels, such as activity on each channel less than 2-fold compared to the other channel, such as less than 1.5-fold higher or less than 1.2-fold higher.

In certain disorders, it may be useful to use a modulator of Kv3.3 or Kv3.2, or Kv3.3 and Kv3.2, that exhibits a defined selectivity profile between these two channels. The compound may be selective for modulation of Kv3.3 channels over modulation of Kv3.2 channels, exhibiting, for example, at least 2-fold, 5-fold, or 10-fold activity on Kv3.3 channels over Kv3.2 channels.

In another embodiment of the invention, the compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates and/or derivatives thereof are selective for the modulation of Kv3.2 channels over the modulation of Kv3.3 channels. Again, by selective is meant that the compounds exhibit, for example, at least 2-fold, 5-fold or 10-fold activity on Kv3.2 channels over Kv3.3 channels.

In another specific embodiment, the compound may exhibit comparable activity between modulation of Kv3.3 and Kv3.2 channels, such as activity for each channel less than 2-fold compared to the other channel, such as less than 1.5-fold higher or less than 1.2-fold higher.

In another specific embodiment of the invention, the compound may exhibit comparable activity between modulation of Kv3.3, Kv3.2 and Kv3.1 channels, such as activity for each channel less than 2-fold compared to any other channel, such as less than 1.5-fold higher or less than 1.2-fold higher. The activity of the compound is suitably quantified by its potency, as indicated by the EC50 value.

Therapeutic methods.

The present invention also provides a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate (eg a salt) and/or derivative thereof for use in the treatment or prevention of a disease or disorder in which a modulator of Kv3.1, Kv3.2 and/or Kv3.3 is required, such as the diseases and disorders mentioned herein below.

According to the present invention, there is provided a method for the treatment or prevention of a disease or disorder requiring a modulator of Kv3.1, Kv3.2 and/or Kv3.3, such as the diseases and disorders mentioned herein below, comprising administering to a subject in need thereof an effective

- 15,049,235 of the compound of formula (I) or its pharmaceutically acceptable salt and/or solvate (e.g. salt) and/or derivative.

The present invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate (e.g. salt) and/or derivative thereof in the manufacture of a medicament for the treatment or prevention of a disease or disorder in which a modulator of Kv3.1, Kv3.2 and/or Kv3.3 is required, such as the diseases and disorders mentioned herein below.

In one embodiment, there is provided a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate and/or derivative thereof for use as a medicinal product.

As used herein, the term treatment or cure includes control, alleviation, reduction or modulation of a disease state or its symptoms.

The term prophylaxis is used herein to mean the prevention of symptoms of a disease or disorder in a subject or the prevention of the recurrence of symptoms of a disease or disorder in a subject suffering from a disease, and is not limited to the complete prevention of a disease.

The subject suitably represents a human being.

Diseases or disorders that may be mediated by modulation of Kv3.1 and/or Kv3.2 channels can be selected from the list below. The numbers in parentheses after the diseases listed below refer to the classification code in the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) published by the American Psychiatric Association and/or the International Classification of Diseases, 10th edition (ICD-10).

In one embodiment of the invention, the compounds of formula (I) or pharmaceutically acceptable salts and/or solvates and/or derivatives thereof can be used for the treatment or prevention of a disease or disorder selected from the group consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse, anxiety disorders, sleep disorders, hyperacusis and auditory perception disorders, Meniere's disease, balance disorders and inner ear disorders, impulse control disorders, personality disorders, attention deficit hyperactivity disorder, autism spectrum disorders, eating disorders, cognitive impairment, ataxia, pain such as neuropathic pain, inflammatory pain and mixed pain, dementia with Lewy bodies and Parkinson's disease.

In one embodiment of the present invention, the compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates and/or derivatives thereof can be used for the treatment or prevention of a disease or disorder selected from the group consisting of hearing disorders, including hearing loss and tinnitus, schizophrenia, substance abuse, pain such as neuropathic pain, inflammatory pain and mixed pain, dementia with Lewy bodies and Parkinson's disease.

In one embodiment of the present invention, the compounds of formula (I) or pharmaceutically acceptable salts and/or solvates and/or derivatives thereof can be used for the treatment or prevention of a disease or disorder selected from the group consisting of fragile X syndrome, Rett disease and Alzheimer's disease.

According to the present invention, there is provided a method for the prevention or treatment of a disease or disorder selected from the group consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse, anxiety disorders, sleep disorders, hyperacusis and auditory perception disorders, Meniere's disease, balance disorders and inner ear diseases, impulse control disorders, personality disorders, attention deficit hyperactivity disorder, autism spectrum disorders, eating disorders, cognitive impairment, ataxia, pain such as neuropathic pain, inflammatory pain and mixed pain, dementia with Lewy bodies and Parkinson's disease, comprising administering to a subject in need thereof an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate (e.g. a salt) and/or derivative thereof.

The present invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate (e.g. salt) and/or derivative thereof in the manufacture of a medicament for the treatment or prevention of a disease or disorder selected from the group consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse, anxiety disorders, sleep disorders, hyperacusis and auditory perception disorders, Meniere's disease, balance disorders and inner ear disorders, impulse control disorders, personality disorders, attention deficit hyperactivity disorder, autism spectrum disorders, eating disorders, cognitive impairment, ataxia, pain such as neuropathic pain, inflammatory pain and mixed pain, dementia with Lewy bodies and Parkinson's disease.

- 16 049235

In a specific embodiment of the present invention, there is provided a compound of formula (I) or pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof for use in the treatment or prevention of hearing disorders. Hearing disorders include auditory neuropathy, auditory processing disorder, hearing loss, which includes sudden hearing loss, noise-induced hearing loss, substance-induced hearing loss, and hearing loss in adults over 60, over 65, over 70, or over 75 years of age (senile hearing loss), and tinnitus.

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prevention of Meniere's disease, balance disorders and inner ear diseases.

In a specific embodiment of the present invention there is provided a compound of formula (I) or pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof for use in the treatment or prophylaxis of schizophrenia. Schizophrenia includes the subtypes: paranoid type (295.30), disorganized type (295.10), catatonic type (295.20), undifferentiated type (295.90) and residual type (295.60); schizophreniform disorder (295.40); schizoaffective disorder (295.70) including the subtypes bipolar type and depressive type; delusional disorder (297.1) including the subtypes erotomanic subtype, grandiose subtype, jealous subtype, persecutory subtype, somatic subtype, mixed type and unspecified type; brief psychotic disorder (298.8); general psychotic disorder (297.3); psychotic disorder due to common medical condition, including the subtypes With Delusions and With Hallucinations; substance-induced psychotic disorder, including the subtypes With Delusions (293.81) and With Hallucinations (293.82); and psychotic disorder, unspecified (298.9).

The compounds of formula (I) or pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prophylaxis of depression and mood disorders, including major depressive episode, manic episode, mixed episode and hypomanic episode; depressive disorders, including major depressive disorder, dysthymic disorder (300.4), depressive disorder, unspecified (311); bipolar disorders, including bipolar I disorder, bipolar II disorder (recurrent major depressive episodes with hypomanic episodes) (296.89), cyclothymic disorder (301.13) and bipolar disorder, unspecified (296.80); other mood disorders, including mood disorder due to a common medical condition (293.83), including the subtypes with depressive features, with major depressive-like episode, with manic features, and with mixed features, substance-induced mood disorder (including the subtypes with depressive features, with manic features, and with mixed features), and mood disorder, unspecified (296.90); seasonal affective disorder.

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prophylaxis of epilepsy (including, but not limited to, localized epilepsies, generalized epilepsies, epilepsies with generalized and localized seizures, and the like), seizures associated with Lennox-Gastaut syndrome, seizures as a complication of a disease or condition (such as seizures associated with encephalopathy, phenylketonuria, juvenile Gaucher disease, Lundborg's progressive myoclonic epilepsy, stroke, head injury, stress, hormonal changes, drug use or withdrawal, alcohol use or withdrawal, sleep deprivation, fever, infection, and the like), essential tremor, restless legs syndrome, partial and generalized seizures (including tonic, clonic, tonic-clonic, atonic, myoclonic, petit mal), secondary generalized seizures, temporal lobe epilepsy, petit mal epilepsy (including childhood, juvenile, myoclonic, photosensitive and pattern sensitive), severe epileptic encephalopathies (including hypoxia-related and Rasmussen syndrome), febrile seizures, partial continuous epilepsy, progressive myoclonic epilepsy (including Unverricht-Lundborg disease and Lafora disease), post-traumatic seizures/epilepsy including those associated with head injury, simple reflex epilepsies (including photosensitive, somatosensory and proprioceptive, audiogenic and vestibular), metabolic disorders frequently associated with epilepsy such as pyridoxine-dependent epilepsy, kinky hair syndrome in Menkes disease, Krabbe disease, epilepsy due to alcohol and drug abuse (eg, cocaine), cortical malformations associated with epilepsy (eg, double cortex syndrome or subcortical band heterotopia), chromosomal abnormalities associated with seizures or epilepsy such as partial monosomy (15Q)/Angelman syndrome).

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prevention of disorders associated with the intake of psychoactive substances, including disorders caused by the use of psychoactive substances, such as dependence on psychoactive substances, addiction to psychoactive substances and substance abuse; induced by the intake of psychoactive substances

- 17 049235 conditions such as substance intoxication, substance withdrawal syndrome, substance-induced delirium, substance-induced persistent dementia, substance-induced persistent amnestic disorder, substance-induced psychotic disorder, substance-induced mood disorder, anxiety disorder, substance-induced sexual dysfunction, substance-induced sleep disorder and hallucinogenic persistent perception disorder (flashbacks); alcohol use disorders such as alcohol dependence (303.90), alcohol abuse (305.00), alcohol intoxication (303.00), alcohol withdrawal syndrome (291.81), alcohol intoxication delirium, alcohol withdrawal delirium, alcohol-induced persistent dementia, alcohol-induced persistent amnestic disorder, alcohol-induced psychotic disorder, alcohol-induced mood disorder, alcohol-induced anxiety disorder, alcohol-induced sexual dysfunction, alcohol-induced sleep disorder, and alcohol-induced related disorder, unspecified (291.9); amphetamine (or amphetamine-like substance) use disorders such as amphetamine dependence (304.40), amphetamine abuse (305.70), amphetamine intoxication (292.89), amphetamine withdrawal syndrome (292.0), amphetamine intoxication delirium, amphetamine-induced psychotic disorder, amphetamine-induced mood disorder, amphetamine-induced anxiety disorder, amphetamine-induced sexual dysfunction, amphetamine-induced sleep disorder, and amphetamine-induced related disorder, unspecified (292.9); caffeine-related disorders such as caffeine intoxication (305.90), caffeine-induced anxiety disorder, caffeine-induced sleep disorder, and caffeine-induced related disorder, unspecified (292.9); cannabinoid-related disorders such as cannabinoid dependence (304.30), cannabinoid abuse (305.20), cannabinoid intoxication (292.89), cannabinoid intoxication with delirium, cannabinoid-induced psychotic disorder, cannabinoid-induced anxiety disorder, and cannabinoid-related disorder, unspecified (292.9); cocaine use disorders such as cocaine dependence (304.20), cocaine abuse (305.60), cocaine intoxication (292.89), cocaine withdrawal syndrome (292.0), cocaine intoxication delirium, cocaine-induced psychotic disorder, cocaine-induced mood disorder, cocaine-induced anxiety disorder, cocaine-induced sexual dysfunction, cocaine-induced sleep disorder, and cocaine-induced related disorder, unspecified (292.9); hallucinogen-related disorders such as hallucinogen dependence (304.50), hallucinogen abuse (305.30), hallucinogen intoxication (292.89), hallucinogen-induced persistent perception disorder (flashbacks) (292.89), hallucinogen-induced intoxication delirium, hallucinogen-induced psychotic disorder, hallucinogen-induced mood disorder, hallucinogen-induced anxiety disorder, and hallucinogen-induced related disorder, unspecified (292.9); inhalant-related disorders such as inhalant dependence (304.60), inhalant abuse (305.90), inhalant intoxication (292.89), inhalant-induced intoxication delirium, inhalant-induced persistent dementia, inhalant-induced psychotic disorder, inhalant-induced mood disorder, inhalant-induced anxiety disorder, and inhalant-induced related disorder, unspecified (292.9); nicotine-related disorders such as nicotine dependence (305.1), nicotine withdrawal syndrome (292.0), and nicotine-induced related disorder, unspecified (292.9); opioid use disorders such as opioid dependence (304.00), opioid abuse (305.50), opioid intoxication (292.89), opioid withdrawal syndrome (292.0), opioid-induced intoxication delirium, opioid-induced psychotic disorder, opioid-induced mood disorder, opioid-induced sexual dysfunction, opioid-induced sleep disorder, and opioid-induced related disorder unspecified (292.9); disorders related to phencyclidine (or phencyclidine-like substances) such as phencyclidine dependence (304.60), phencyclidine abuse (305.90), phencyclidine intoxication (292.89), phencyclidine-induced intoxication delirium, phencyclidine-induced psychotic disorder, phencyclidine-induced mood disorder, phencyclidine-induced anxiety disorder and phencyclidine-induced related disorder, unspecified (292.9); disorders related to sedatives, hypnotics, or anxiolytics such as sedative, hypnotic, or anxiolytic dependence (304.10), sedative, hypnotic, or anxiolytic abuse (305.40), sedative, hypnotic, or anxiolytic intoxication (292.89), sedative, hypnotic, or anxiolytic withdrawal syndrome (292.0) due to sedatives, hypnotics, or anxiolytics intoxication delirium due to sedatives, hypnotics, or anxiolytics withdrawal delirium due to sedatives, hypnotics, or anxiolytics persistent dementia due to

- 18 049235 sedatives, hypnotics or anxiolytics persistent amnestic disorder due to sedatives, hypnotics or anxiolytics psychotic disorder due to sedatives, hypnotics or anxiolytics mood disorder due to sedatives, hypnotics or anxiolytics anxiety disorder due to sedatives, hypnotics or anxiolytics sexual dysfunction due to sedatives, hypnotics or anxiolytics sleep disorder and sedatives, hypnotics or anxiolytics related disorder, unspecified (292.9); multiple substance use disorder such as multiple substance dependence (304.80); and other (or unknown) disorders associated with the use of substances such as anabolic steroids, nitrate volatile substances and nitric oxide.

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prevention of anxiety disorders, including panic attack; panic disorder, including panic disorder without agoraphobia (300.01) and panic disorder with agoraphobia (300.21); agoraphobia; agoraphobia without history of panic disorder (300.22), specific phobia (300.29, formerly called simple phobia, including the subtypes animal phobia, natural phenomena phobia, blood/injection/injury phobia, specific situation phobia, and other types), social phobia (social anxiety disorder, 300.23), obsessive-compulsive disorder (300.3), posttraumatic stress disorder (309.81), acute stress disorder (308.3), generalized anxiety disorder (300.02), anxiety disorder due to a common medical condition (293.84), substance-induced anxiety disorder, separation anxiety disorder (309.21), adjustment disorder with anxiety (309.24), and anxiety disorder unspecified (300.00).

The compounds of formula (I) or pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prophylaxis of sleep disorders, including primary sleep disorders such as dyssomnias such as primary insomnia (307.42), primary hypersomnia (307.44), narcolepsy (347), breathing-related sleep disorder (780.59), circadian rhythm sleep disorder (307.45) and dyssomnia, unspecified (307.47); primary sleep disorders such as parasomnias such as night terror disorder (307.47), sleep terror disorder (307.46), sleepwalking (307.46) and parasomnia, unspecified (307.47); sleep disorders associated with another mental disorder, such as insomnia associated with another mental disorder (307.42) and hypersomnia associated with another mental disorder (307.44); sleep disorder due to a general medical condition, in particular sleep disorders associated with medical conditions such as neurological disorders, neuropathic pain, restless legs syndrome, cardiac and pulmonary diseases; and substance-induced sleep disorder, including the subtypes insomnia type, hypersomnia type, parasomnia type and mixed type; sleep apnea and jet lag disorder.

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prevention of hyperacusis and auditory perception disorders, including fragile X syndrome and autism.

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prevention of impulse control disorders, including: intermittent explosive disorder (312.34), kleptomania (312.32), pathological gambling (312.31), pyromania (312.33), trichotillomania (312.39), impulse control disorder unspecified (312.3), eating disorders, compulsive buying disorder, compulsive sexual behavior and pathological hoarding.

The compounds of formula (I) or pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prophylaxis of sexual dysfunctions, including sexual desire disorders such as hypoactive sexual desire disorder (302.71) and sexual aversion disorder (302.79); sexual arousal disorders such as female sexual arousal disorder (302.72) and male erectile disorder (302.72); orgasmic disorders such as female orgasmic disorder (302.73), male orgasmic disorder (302.74) and premature ejaculation (302.75); painful intercourse disorder such as dyspareunia (302.76) and vaginismus (306.51); sexual dysfunction, unspecified (302.70); paraphilias such as exhibitionism (302.4), fetishism (302.81), frotteurism (302.89), pedophilia (302.2), sexual masochism (302.83), sexual sadism (302.84), transvestic fetishism (302.3), voyeurism (302.82), and paraphilia, unspecified (302.9); gender identity disorders such as gender identity disorder of children (302.6) and gender identity disorder of adolescents and adults (302.85); and sexual disorder, unspecified (302.9).

Compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (e.g. with

- 19 049235 or) and/or their derivatives can be used to treat or prevent personality disorders, including the subtypes: paranoid personality disorder (301.0), schizoid personality disorder (301.20), schizotypal personality disorder (301.22), antisocial personality disorder (301.7), borderline personality disorder (301.83), histrionic personality disorder (301.50), narcissistic personality disorder (301.81), avoidant personality disorder (301.82), dependent personality disorder (301.6), obsessive-compulsive personality disorder (301.4) and personality disorder, unspecified (301.9).

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prophylaxis of attention deficit hyperactivity disorder, including the subtypes: attention deficit hyperactivity disorder, combined type (314.01), attention deficit hyperactivity disorder with predominant attention deficit (314.00), attention deficit hyperactivity disorder, hyperactive-impulsive type (314.01) and attention deficit hyperactivity disorder, unspecified (314.9); hyperkinetic disorder; social behavior disorders such as conduct disorder, including the subtypes such as childhood-onset (321.81), adolescent-onset (312.82) and unspecified (312.89), oppositional defiant disorder (313.81) and social behavior disorder, unspecified; and tics such as Tourette's syndrome (307.23).

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prevention of autism spectrum disorders, including autism (299.00), Asperger syndrome (299.80), Rett disease (299.80), childhood disintegrative disorder (299.10) and developmental disorder not otherwise specified (299.80, including atypical autism).

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used for the treatment or prevention of eating disorders such as anorexia nervosa (307.1), including the subtypes: restricting type and type with binge eating and artificial elimination of food; bulimia nervosa (307.51), including the subtypes: type with artificial elimination of food and type without artificial elimination of food; obesity; compulsive eating disorder; binge eating disorder; and eating disorder, unspecified (307.50).

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof can be used to improve cognitive ability, including the treatment of cognitive impairment in other diseases such as schizophrenia, bipolar disorder, depression, other psychiatric disorders and psychotic conditions associated with cognitive impairment, e.g. Alzheimer's disease. Alternatively, the compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates thereof can be used to prevent cognitive impairment, e.g. which can be associated with diseases such as schizophrenia, bipolar disorder, depression, other psychiatric disorders and psychotic conditions associated with cognitive impairment, e.g. Alzheimer's disease.

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (eg salts) and/or derivatives thereof can be used for the treatment or prevention of ataxia, including ataxia, in particular spinocerebellar ataxia, in particular ataxia associated with mutations R420H, R423H or F448L.

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (eg salts) and/or derivatives thereof can be used for the treatment or prevention of pain, including nociceptive, neuropathic, inflammatory or mixed pain.

Nociceptive pain is a normal response to damage or injury to tissues such as skin, muscles, internal organs, joints, tendons or bones. Examples of nociceptive pain that form part of the invention include somatic pain: musculoskeletal (joint pain, myofascial pain) or cutaneous, which is often well localized; or visceral pain: in hollow organs or smooth muscles.

Neuropathic pain is pain initiated or caused by a primary lesion or disease of the somatosensory nervous system. Sensory abnormalities range from deficits perceived as paresthesia (numbness) to hypersensitivity (hyperalgesia or allodynia) and dysesthesia (tingling and other sensations). Examples of neuropathic pain that form part of the invention include, but are not limited to, diabetic neuropathy, postherpetic neuralgia, spinal cord injury pain, phantom pain (post-amputation), and central pain after stroke.

Other causes of neuropathic pain include injury, chemotherapy, and heavy metal exposure.

Inflammatory pain results from activation and sensitization of the nociceptive pain pathway by a variety of mediators released at the site of tissue inflammation. Mediators that play a key role in inflammatory pain include proinflammatory cytokines such as interleukin-1 (IL-1) alpha, IL-1-beta, IL-6, and tumor necrosis factor (TNF) alpha, chemokines, reactive oxygen species, vasoactive amines, lipids, ATP, acids, and other factors released by infiltrating leukocytes, vascular endothelial cells, or tissue-resident mast cells. Examples of causes of inflammatory pain that form part of the invention include appendicitis, rheumatoid arthritis, inflammatory bowel disease, and herpes zoster.

Mixed pain refers to pain conditions or disorders that are not easily classified. At present, the understanding of the mechanisms underlying them is still poorly developed, although specific treatments for these disorders are well established; they include cancer pain, migraine and other primary headaches, and widespread pain such as fibromyalgia.

Accordingly, specific pain indications that may be mediated by a Kv3.1 and/or Kv3.2 and/or Kv3.3 channel modulator are neuropathic pain and/or inflammatory pain.

Pain is a subjective condition and in clinical settings is typically measured by self-reporting by the patient. Therefore, it can be difficult to measure and quantify pain thresholds. For chronic pain, a subjective 11-point rating scale is commonly used, with 0 representing no pain and 10 representing the worst pain imaginable. Subjects typically record their worst pain over a specified period, usually 24 hours. A minimum mean baseline score is also recorded and the response to medication is measured relative to baseline, for example, a reduction in pain of at least 10%, 20%, 30%, 40% or 50% compared to the baseline score.

Because individual responses to medications may vary, not all individuals may experience a reduction in pain compared to the baseline assessment. Therefore, appropriately, a reduction is observed in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or all individuals studied.

Thus, in one embodiment of the invention, a reduction in pain of at least 10%, 20%, 30%, 40% or 50% of the baseline score is observed upon administration to a subject in need thereof of a Kv3.1/Kv3.2/Kv3 modulator, such as a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof.

The administration of the Kv3.1/Kv3.2/Kv3.3 modulator may be performed prior to the expected onset of pain or after the onset of pain. In cases where the development of a disease or disorder is expected to result in an increase in pain experienced by the subject, a Kv3.1/Kv3.2/Kv3.3 modulator, such as a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof, may be administered. In cases where the subject is already experiencing pain, a Kv3.1/Kv3.2/Kv3.3 modulator, such as a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof, may be administered to the subject in need thereof.

The treatment of a subject in need thereof may be continued for as long as required, such as 1 day, 1 week, 2 weeks, 3 weeks, 1 month, 6 months, 1 year, more than 1 year, more than 2 years, more than 5 years or more than 10 years. Therefore, in one embodiment of the present invention, a therapeutically effective amount of a Kv3.1/Kv3.2/Kv3.3 modulator, such as a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof, is administered to a subject in need thereof for 1 day to 1 month, 1 week to 3 months, 1 month to 6 months, 3 months to 1 year or more than 1 year.

The reduction in pain in a subject may be measured by assessing the response to external stimuli, such as mechanical or thermal (e.g., cold) stimuli (e.g., as described in the Experimental Section). The reduction may be considered either as a percentage change (calculated by measuring pre- and post-dose thresholds for the lesioned painful site with an unlesioned painful site, e.g., as described in more detail in the Data Analysis section of the Experimental Section) or by measuring the withdrawal threshold at the lesioned painful site. Preferably, a reciprocal percentage calculation is used.

Therefore, in one embodiment of the invention, sensitivity to pain (such as neuropathic pain or inflammatory pain) is eliminated by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more than 90% upon administration of a therapeutically effective amount of a Kv3.1/Kv3.2/Kv3.3 modulator, such as a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof. Suitably, sensitivity to pain is eliminated by more than 80% or more than 90%.

Subjects receiving a Kv3.1/Kv3.2/Kv3.3 modulator may experience additional benefits such as one or more of improved function, mood, sleep, quality of life, reduced time off work.

In a specific embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof

- 21 049235 and/or solvates (eg salts) and/or derivatives thereof can be used for the treatment or prevention of neuropathic pain.

In a specific embodiment, the compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (eg salts) and/or derivatives thereof can be used for the treatment or prevention of inflammatory pain.

In a specific embodiment, the compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates (eg salts) and/or derivatives thereof can be used for the treatment or prevention of mixed pain.

In one embodiment, a compound of formula (I) is provided for use in the prevention of acute noise-induced hearing loss.

In one embodiment, a method for preventing acute noise-induced hearing loss is provided, comprising administering to a subject in need thereof a compound of formula (I).

In one embodiment, the use of a compound of formula (I) in the manufacture of a medicament for the prevention of acute noise-induced hearing loss is proposed.

Acute noise-induced hearing loss may be precipitated by events such as exposure to loud noise or a blast wave. In these cases, where a future event is expected to result in acute noise-induced hearing loss, the compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof may be administered prior to such an event to prevent or reduce acute noise-induced hearing loss. Administration of compound (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof may prevent any acute noise-induced hearing loss or may reduce the severity of acute noise-induced hearing loss or may alleviate other symptoms that occur with acute noise-induced hearing loss, such as tinnitus.

Acute hearing loss is characterized as hearing loss that occurs rapidly over a period of hours or days. For example, hearing loss may occur within minutes, hours, or days (e.g., over a period of up to 1 day, such as 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days). Acute hearing loss is usually caused by exposure to a loud sound or blast wave. Hearing loss caused by exposure to a loud sound or blast wave is referred to herein as noise-induced hearing loss. Thus, acute noise-induced hearing loss is hearing loss that occurs rapidly over a period of hours or days due to exposure to a loud sound or blast wave.

Important symptoms of acute hearing loss include:

1) a shift in the auditory threshold, that is, an increase in the minimum level of pure tone sound that can be heard without the presence of other sounds;

2) tinnitus; and

3) impairment of central auditory processing, such as impaired auditory temporal processing and/or speech comprehension.

The loud noise or explosion may be at least 90 dB, such as at least 100 dB, at least 110 dB, at least 120 dB, or at least 130 dB.

In one embodiment, the administration of the compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is started prior to the event expected to cause acute noise-induced hearing loss. For example, the administration of the compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof can be started up to 2 weeks, such as 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes or 15 minutes prior to the event expected to cause acute noise-induced hearing loss. The compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof can be administered multiple times prior to the event expected to cause acute noise-induced hearing loss.

In one embodiment, a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is administered in advance of potential exposure to a noise or blast that is expected to cause acute noise-induced hearing loss, to prevent or reduce the development of persistent tinnitus; to prevent or reduce the development of persistent auditory threshold shift; or to prevent or reduce the development of persistently impaired central auditory processing, including, for example, temporary auditory processing and/or speech understanding.

It should be understood that advance administration may be carried out in circumstances where the subject is expected to be at risk of exposure to noise or blast that is expected to cause acute noise-induced hearing loss, and is not limited to those circumstances where such exposure ultimately occurs.

In one embodiment, the administration of a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is commenced during an event that is expected to cause acute noise-induced hearing loss. The compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof may be administered multiple times during an event that is expected to cause

- 22 049235 Acute noise-induced hearing loss.

In one embodiment, a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is initially administered during a noise or blast that is expected to cause acute noise-induced hearing loss, to prevent or reduce the development of persistent tinnitus; to prevent or reduce the development of persistent auditory threshold shift; or to prevent or reduce the development of persistently impaired central auditory processing, including, for example, temporary auditory processing and/or speech understanding.

In one embodiment, administration of a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is commenced following an event that is expected to cause acute noise-induced hearing loss.

Thus, in one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is initially administered following a noise or blast that is expected to cause acute noise-induced hearing loss, to prevent or reduce the development of persistent tinnitus; to prevent or reduce the development of persistent auditory threshold shift; or to prevent or reduce the development of permanently impaired central auditory processing, including, for example, temporary auditory processing and/or speech understanding.

When a compound of formula (I) is administered following an event that is expected to cause acute noise-induced hearing loss, such administration is typically performed during the acute phase, i.e., before the hearing loss has become established.

In one embodiment, the administration of the compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof may be started up to 2 months after the event that is expected to cause acute noise-induced hearing loss, such as up to 1 month, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, up to 30 minutes or up to 15 minutes after the event that may cause acute noise-induced hearing loss. The compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof may be administered multiple times after the event that is expected to cause acute noise-induced hearing loss.

The compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof may be administered over a period of up to 7 days (e.g. up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days or up to 7 days), over a period of 1-2 weeks (e.g. 7-8 days, 7-9 days, 7-10 days, 7-11 days, 7-12 days, 7-13 days or 7-14 days), over a period of 2-4 weeks (e.g. 2-3 weeks or 2-4 weeks) or over a period of 1-2 months (e.g. 4-6 weeks or 4-8 weeks).

The compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof may be initially administered 1 day before, such as 2 days before, 3 days before, 5 days before, 1 week before, 2 weeks before or 1 month before the noise or explosion that is expected to cause acute noise-induced hearing loss, wherein administration that is started at any time before the onset of exposure to the noise or explosion that is expected to cause acute noise-induced hearing loss is typically continued for up to 2 months after exposure to the noise or explosion that is expected to cause acute noise-induced hearing loss, such as for a period of up to 1 month after, up to 3 weeks after, up to two weeks after, up to 1 week after, up to 5 days after, up to 3 days after, up to 2 days after or up to 1 day after.

In one embodiment, there is provided a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof for use in preventing or reducing the development of a permanent auditory threshold shift, wherein the permanent auditory threshold shift is reduced by at least 10 dB, such as at least 15 dB, at least 20 dB, at least 30 dB, at least 40 dB or completely.

Pharmaceutical compositions.

For use in therapy, the compounds of the invention are typically administered in the form of a pharmaceutical composition. The present invention also provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate (e.g. salt) and/or derivative thereof and a pharmaceutically acceptable carrier or excipient.

In one embodiment, there is provided a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate (e.g. a salt) and/or derivative thereof, for use in the treatment or prevention of a disease or disorder selected from the group consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse, anxiety disorders, sleep disorders, hyperacusis and auditory perception disorders, Meniere's disease, balance disorders and inner ear disorders, impulse control disorders, personality disorders, attention deficit hyperactivity disorder, autism spectrum disorders, eating disorders, cognitive impairment, ataxia, pain such as neuropathic pain, inflammatory pain and mixed pain, dementia with Lewy bodies and Parkinson's disease.

In a further embodiment, a method for preventing or treating a disease or disorder is provided.

- 23 049235 a disorder selected from the group consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse, anxiety disorders, sleep disorders, hyperacusis and auditory perception disorders, Meniere's disease, balance disorders and inner ear disorders, impulse control disorders, personality disorders, attention deficit hyperactivity disorder, autism spectrum disorders, eating disorders, cognitive impairment, ataxia, pain such as neuropathic pain, inflammatory pain and mixed pain, dementia with Lewy bodies and Parkinson's disease, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate (e.g. salt) and/or derivative thereof.

The present invention also provides the use of a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate (e.g. salt) and/or derivative thereof in the manufacture of a medicament for the treatment or prevention of a disease or disorder selected from the group consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse, anxiety disorders, sleep disorders, hyperacusis and auditory perception disorders, Meniere's disease, balance disorders and inner ear disorders, impulse control disorders, personality disorders, attention deficit hyperactivity disorder, autism spectrum disorders, eating disorders, cognitive impairment, ataxia, pain such as neuropathic pain, inflammatory pain and mixed pain, dementia with Lewy bodies and Parkinson's disease.

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates and/or derivatives thereof may be administered by any convenient route, for example by oral, parenteral, buccal, sublingual, intranasal, rectal or transdermal administration, and the pharmaceutical compositions are adapted accordingly. Other possible routes of administration include intratympanic and intracochlear.

The compounds of formula (I) or their pharmaceutically acceptable salts and/or solvates and/or derivatives thereof, which are active when administered orally, can be prepared in the form of liquids or solids, for example as syrups, suspensions, emulsions, tablets, capsules or lozenges.

A liquid preparation generally consists of a suspension or solution of the active ingredient (such as a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate (e.g. salt) and/or derivative thereof) in a suitable liquid carrier(s), for example an aqueous solvent such as water, ethanol or glycerol, or a non-aqueous solvent such as polyethyleneglycol or oil. The preparation may also contain a suspending agent, a preservative, a flavouring and/or a colouring agent.

The composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) commonly used for the preparation of solid preparations, such as magnesium stearate, starch, lactose, sucrose and cellulose.

A capsule composition may be prepared using conventional encapsulation techniques, for example granules containing the active ingredient (such as a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate (e.g. salt) and/or derivative thereof) may be prepared using conventional carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension may be prepared using any suitable pharmaceutical carrier(s) such as aqueous gums, celluloses, silicates or oils, and this dispersion or suspension is then filled into a soft gelatin capsule.

Typical parenteral compositions consist of a solution or suspension of the active ingredient (such as a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate (e.g. salt) and/or derivative thereof) in a sterile aqueous vehicle or a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil or sesame oil. Alternatively, the solution may be lyophilized and then reconstituted with a suitable solvent immediately prior to administration.

Compositions for intranasal administration may conveniently be prepared as aerosols, drops, gels and powders. Aerosol preparations typically comprise a solution or finely divided suspension of the active ingredient in a pharmaceutically acceptable aqueous or non-aqueous solvent and are typically presented in single or multi-dose quantities in sterile form in a sealed container, which may be in the form of a cartridge or a refill for use with an atomizer. Alternatively, the sealed container may be a disposable metering device such as a single-dose nasal inhaler or an aerosol dispenser fitted with a metering valve. If the dosage form includes an aerosol dispenser, it will contain a propellant, which may be a compressed gas such as air or an organic propellant such as a fluorochlorocarbon or hydrofluorocarbon. Aerosol dosage forms may also be in the form of pump sprayers.

Formulations suitable for buccal or sublingual administration include tablets

- 24 049235 lozenges, troches and pastilles in which the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth or gelatin and glycerol.

Compositions for rectal administration are usually in the form of suppositories containing a conventional suppository base such as cocoa butter.

Compositions suitable for transdermal administration include ointments, gels and patches. In one embodiment, the composition is in a unit dosage form such as a tablet, capsule or ampoule.

The composition may contain from 0.1% to 100% by weight, such as from 10 to 60% by weight, of the active substance, depending on the route of administration. The composition may contain from 0% to 99% by weight, such as from 40% to 90% by weight, of the carrier, depending on the route of administration. The composition may contain from 0.05 mg to 1000 mg, such as from 1.0 mg to 500 mg, of the active substance, depending on the route of administration. The composition may contain from 50 mg to 1000 mg, such as from 100 mg to 400 mg of the carrier, depending on the route of administration. The dose of the compound used in the treatment of the above disorders will vary in the usual manner depending on the severity of the disorders, the weight of the patient and other similar factors. However, as a general guide, suitable unit doses may range from 0.05 mg to 1000 mg, more preferably from 1.0 mg to 500 mg, and such unit doses may be administered more than once daily, such as twice or three times daily. Such therapy may be continued for several weeks or months.

The dose administered to the subject is usually a safe and effective dose, that is, it represents an acceptable balance between the desired benefits and undesirable side effects.

In a further aspect the present invention provides a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof (eg a combination comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof) together with an additional pharmaceutically acceptable active ingredient or ingredients.

The present invention provides a compound of formula (I) for use in combination with an additional pharmaceutically acceptable active ingredient or ingredients.

When the compounds are used in combination with other therapeutic agents, the compounds can be administered either sequentially or simultaneously by any convenient route. Alternatively, the compounds can be administered separately.

The combinations mentioned above may conveniently be presented for use in the form of a pharmaceutical preparation, and thus pharmaceutical preparations comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient form a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical preparations. The individual components of the combinations may also be administered separately by the same route or by different routes.

When a compound of formula (I) or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent active against the same disease state, the dose of each compound may differ from the dose when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

Suitably, the compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is administered orally.

Suitably, the compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is administered at a dose of 2 to 400 mg per day, such as 2 to 300 mg per day, especially 5 to 250 mg per day.

Suitably, the compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is administered once or twice daily.

Suitably, the compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is administered for at least three months.

Preferably, the compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is administered orally once or twice daily at a dose of 2 to 400 mg per day, for example 2 to 300 mg per day, especially 5 to 250 mg per day.

The human may be an adult, such as between the ages of 18 and 65. Alternatively, the human may be 66 years of age or older. The compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof may be administered to a human under the age of 18, such as between the ages of 4 and 17. Administration to a human under the age of 18 may be of particular importance in the context of progressive myoclonic epilepsy and fragile X syndrome.

For convenience and to aid in patient compliance, delivery technologies such as patches or implants may be used to deliver a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof over an extended period of time, such as at least one week or at least 4 weeks.

- 25 049235

Experimental section.

The present invention is illustrated by the compounds described below. The following examples describe laboratory syntheses of specific compounds of the invention and are not intended to limit the scope of the invention in any way with respect to the compounds or methods. It should be understood that although certain reagents, solvents, temperatures and time periods are used, there are many possible equivalent alternatives that can be used to obtain similar results. It is intended that the present invention include such equivalents.

Analytical equipment.

The starting materials, reagents and solvents were obtained from commercial suppliers and were used without further purification unless otherwise stated. Unless otherwise stated, all compounds with chiral centers are racemic. Where reactions are described as being carried out in the same manner as reactions described in more detail above, the general reaction conditions used were essentially the same. The work-up conditions used were standard in the art but could be adapted for different reactions. The starting material was not necessarily obtained from the mentioned batch. The synthesized compounds may have different degrees of purity, for example in the range from 85% to 99%. In some cases, molar and yield calculations have been adjusted to accommodate this.

HPLC/mass spectra (HPLC-MS) were recorded on an Agilent 1100 Series LC/MSD mass spectrometer coupled to an Agilent 1100 Series HPLC instrument operating in positive electrospray ionization mode and under acid gradient conditions.

Quality control (3-minute method): LC/EDMA+MS under acidic conditions was performed on a Zorbax SB C18 column (1.8 μm, 3x50 mm). Mobile phase: A: (H2O plus 0.05% TFA by volume)/B: (CH3CN plus 0.05% TFA by volume). Gradient: t = 0 min 0% (B), from 0 to 95% (B) in 2.5 min, 95% (B) in 0.2 min, from 95 to 100% (B) in 0.2 min, 100% (B) for 0.4 min, from 100% to 0% (B) in 0.1 min. Finish time 4 min. Column T = 60°C. Flow rate: 1.5 mL/min. ER+ mass range: (100-1000 amu, F=60). UV detection wavelengths: DAD 1A = 220.8, DAD 1B = 254.8. The use of this methodology is designated QC_3_MIN in the analytical characteristics of the described compounds.

Chiral control: LC/EDMA+MS under acidic conditions was performed on a CHIRALCEL® OD-H (250 x 4.6 mm, 5 μm). Mobile phase: A: ( _H2O plus 0.05% TFA by volume)/B: (CH3CN plus 0.05% TFA by volume). Gradient: t = 0-6 min 35% (B), t = 6-40 min from 35% to 50% (B), t = 40-45 min from 50% to 70% (B), t = 45-50 min from 70% to 35% (B), t = 50-55 min 35% (B). End time 60 min. Column T = 40°C. Flow rate: 1.0 mL/min. UV detection wavelengths: DAD 1A = 220.8, DAD 1B = 254.8.

Proton magnetic resonance (NMR) spectra were recorded on either Varian instruments at 300, 400, 500, or 600 MHz or Bruker instruments at 400 MHz. Chemical shifts are expressed in ppm (δ) using the residual solvent line as an internal standard. Split patterns are designated as s (singlet), br.s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), and m (multiplet). NMR spectra were recorded at temperatures ranging from 25 to 60°C.

The data from the 2D NOESY NMR experiments were collected with a mixing time of 500 ms using a spectral width of 3355 Hz for both f1 and f2. A total of 256 increments were collected, processed to 1K using linear prediction, with 8 scans each. The data were processed with a sine bell shift in both dimensions and with 1b=0.3 Hz at f1. In some preparations, purification was performed using Biotage automated flash chromatography (SP1 and SP4) or Flash Master Personal systems.

Flash chromatographies were performed on 230–400 mesh silica gel (provided by Merck AG Darmstadt, Germany) or 300–400 mesh silica gel (provided by Sinopharm Chemical Reagent Co., Ltd.), Varian Mega Be-Si pre-packaged cartridges, Biotage pre-packaged silica cartridges (e.g. Biotage SNAP cartridge).

- 26 049235

Abbreviations:

AIBN azobisisobutyronitrile BuLi butyllithium CDC1 ₃ deuterated chloroform CC1 ₄ carbon tetrachloride D2O deuterated water DCM dichloromethane DIPEA N,N-diisopropylethylamine DMAP 4-dimethylaminopyridine DMF N,N-dimethylformamide DMSO dimethyl sulfoxide DMSO-t/b deuterated dimethyl sulfoxide Et20 diethyl ether EtOAc ethyl acetate h watch HATU hexafluorophosphate (O-7-azabenzotriazol-1-yl)-X,X,X’,X’- tetramethyluronium) NS1 hydrogen chloride K2CO3 potassium carbonate MeCN/ _CH3CN acetonitrile MeOH methanol MOM methyloxymethyl NaH sodium hydride _{Na2SO4 ​} sodium sulfate _{Na2CO3 ​} sodium carbonate NaOH sodium hydroxide NaOMe sodium methoxide NMR nuclear magnetic resonance K.T. room temperature T3P propylphosphonic anhydride MTBE methyl threot-butyl ether TBTU benzotriazol-1-yl-Ν,Ν,Ν',Ν'- tetrafluoroborate tetramethyluronium TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran THP tetrahydropyran May. weight

Examples of connections.

Intermediate connection 1.

2-Bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazine

Vg

- 27 049235

A mixture of 7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-ol (Intermediate 156 in WO2012076877; 1.11 g, 6.30 mmol), 2,5-dibromopyrazine (1.5 g, 6.30 mmol) and potassium carbonate (1.31 g, 9.46 mmol) in N,N-dimethylformamide (14 mL) was stirred at 120 °C for 3 h. After cooling, the reaction mixture was diluted with MTBE (100 mL) and washed with brine (50 mL). The phases were separated and the aqueous layer was washed with MTBE (100 mL) and EtOAc (100 mL). All organic phases were collected, dried over Na ₂ SO ₄ , filtered and evaporated. The residue was purified by flash chromatography (Biotage System) on silica gel using SNAP 100g as a column and cyclohexane:ethyl acetate 100:0 to 90:10 as eluent to give 2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4yl)oxypyrazine (1.8 g) as a white solid.

LC/MS: QC_3_MIN: Rt 2.705 min; m/z 333 & 335 [M+H]+.

The following compounds were prepared using the above methodology substituting the appropriate phenol for 7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-ol. The final products were purified by flash chromatography (silica cartridge; cyclohexane/EtOAc or other suitable solvent system).________________________________________

Intermediate connection Structure Name Phenol zhhms 2 Vg 2-bromo-5spiro[2Hbenzofuran-3,Gcyclopropane]-4yloxy-pyrazine spiro[2Hbenzofuran-3,1'cyclopropan]-4-ol (intermediate 85 in WO2012076877) LC/MS: QC 3 _MIN: Rt 2.575 min; m/z 319 & 321 [M+H]+. 3 Yh Вг 2-bromo-5-[(3,3,7-trimethyl-2Hbenzofuran-4yl)oxy]pyrazine 3,3,7-trimethyl-2Hbenzofuran-4-ol (intermediate 184 in WO2012076877) LC/MS: QC 3 MIN: Rt 2.365 min; m/z 335 & 337 [M+H]+. 4 Vg 2-bromo-5-[(3,3-dimethyl-2H-benzofuran-4yl)oxy]pyrazine 3,3-d-methyl-2Hbenzofuran-4-ol (intermediate 50 in WO2012076877) LC/MS: QC 3 MIN: Rt 2.632 min; m/z 321 & 323 [M+H]+.

Intermediate connection 5, path 1.

-(5-Chloropyrazin-2-yl)-5,5-dimethylimidazolidine-2,4-dione.

To a solution of bis(trichloromethyl) carbonate (950 mg, 3.20 mmol) in ethyl acetate (30 ml) at 0 °C was added dropwise a solution of 5-chloropyrazin-2-amine (0.75 g, 5.79 mmol)/N,N-diisopropylethylamine (6.05 ml, 34.74 mmol) in ethyl acetate (12 ml) and the reaction mixture was stirred for 15 min at the same temperature. While maintaining the reaction mixture at 0 °C, a vacuum was applied (5 min) to remove excess phosgene. A solution of 4-(dimethylamino)pyridine (710 mg, 5.81 mmol) in ethyl acetate (8 ml)/dichloromethane (2 ml) was added and the reaction mixture was stirred for 5 min at the same temperature. Then methyl 2-amino-2-methylpropanoate hydrochloride (1.4 g, 9.1 mmol) was added at 0 °C and the reaction mixture was stirred for 30 min at the same temperature. The reaction mixture was quenched with 0.2 N HCl (100 mL) and the two phases were separated. The organic layer was washed with brine (100 mL), dried over Na ₂ SO ₄ , filtered and evaporated to give the intermediate urea.

The obtained urea was dissolved in dichloromethane (20 ml) and sodium methoxide (315 mg, 5.83 mmol) was added at 0 °C. The reaction mixture was stirred for 15 min at the same temperature; the reaction mixture was quenched with a saturated solution of NH4Cl, ensuring that pH 3-4 was reached. The mixture was extracted with ethyl acetate (50 ml); the phases were separated and the organic layer was washed with brine (50 ml), dried over Na2SO4, filtered and evaporated. The residue was purified by reverse phase flash chromatography (Biotage system) on phase C-18 using SNAP 30g as a column and a mixture of water:acetonitrile from 95:5 to 40:60 as an eluent. The appropriate fractions were combined and evaporated to dryness to give 3-(5-chloropyrazin-2-yl)-5,5-dimethylimidazolidine-2,4-dione (220 mg) as a pale

- 28 049235 brown solid.

LC/MS: QC_3_MIN: Rt 1.649 min; m/z 241 & 243 [M+H]+.

The following compounds were prepared using the above methodology substituting the corresponding amino ester hydrochloride for 2,2-dimethylglycine methyl ester hydrochloride. The final products were purified by flash chromatography (silica cartridge; cyclohexane/EtOAc or other suitable solvent system) or triturated with an appropriate solvent or crystallized from an appropriate solvent.

Intermediate connection Structure Name Amino ester hydrochloride zhhms 6 a n ^N =< O N— 5R)-3-(5- chloropyrazin-2-yl)- 5-ethyl-5-methylimidazolidine-2,4- dione Methyl(2R)-2-αmino-2-methylbutanoate hydrochloride LC/MS: QC 3 MIN: Rt 1.546 min; m/z 255 & 257 [M+N]+.

Intermediate junction 5, path 2.

3-(5-Chloropyrazin-2-yl)-5,5-dimethylimidazolidine-2,4-dione.

To a solution of 5-chloropyrazin-2-amine (500 mg, 3.86 mmol) and 2-amino-2-methylpropanoic acid hydrochloride (646 mg, 4.63 mmol) in acetonitrile (10 mL) was added slowly at room temperature a solution of at least 50 wt% propylphosphonic anhydride in ethyl acetate (3.68 g, 5.78 mmol). The reaction mixture was stirred at 80 °C for 6 h. The reaction mixture was diluted with ethyl acetate (10 mL) and 1 N HCl was added. aqueous NaOH solution, allowing the pH to reach approximately 8. The two phases were separated and the organic phase was washed with brine (10 ml), dried over Na ₂ SO ₄ , concentrated in vacuo and the crude product was purified by flash chromatography on silica gel (BIOTAGE SYSTEM) using SNAP 25g as a column and DCM:MEOH 99/1 to 90/10 as eluent to give 2-amino-N-(5-chloropyrazin-2-yl)-2-methylpropanamide (190 mg) as a yellow solid.

LC/MS: QC_3_MIN: Rt 1.181 min; m/z 215 & 217 [M+H]+.

To a solution of 2-amino-N-(5-chloropyrazin-2-yl)-2-methylpropanαamideα (190 mg, 0.88 mmol) and triethylamine (268 mg, 2.6555 mmol) in dichloromethane (5 ml) at 0°C was slowly added a solution of bis(trichloromethyl)carbonate (105.07 mg, 0.3541 mmol) in dichloromethane (4 ml) and the reaction mixture was stirred for 30 min at the same temperature. The reaction mixture was diluted with DCM (10 ml), washed with aqueous 0.2 N HCl (10 ml) and brine (10 ml). The organic phases were concentrated in vacuo and the crude product was purified by flash chromatography on silica gel (Biotage system) using SNAP 25g as a column and Chexane/EtOAc 80/20 to 0/100 as eluent to give 3-(5-chloropyrazin-2-yl)-5,5-dimethylimidazolidine-2,4-dione (130 mg) as a white solid.

LC/MS: QC_3_MIN: Rt 1.598 min; m/z 241 & 243 [M+H]+.

Intermediate connection 7.

tert-Butyl N-[(1R)-1-carbamoylpropyl]carbamate.

A mixture of [dimethylamino-(3-oxidotriazolo[4,5-b]pyridin-3-ium-1yl)methylene]dimethylammonium tetrafluoroborate (1.1084 g, 3.4415 mmol), N,N-diisopropylethylamine (0.7939 g, 6.1431 mmol) and (2R)-2-(tert-butoxycarbonylamino)butanoic acid (0.5000 g, 2.4601 mmol) in anhydrous N,N-dimethylformamide (8 mL) was stirred at room temperature for 10 min. Hexamethyldisilazane (0.5960 g, 3.6928 mmol) was added and the mixture was stirred for 18 h.

The reaction mixture was partitioned between MTBE (30 mL) and brine (20 mL). The organic layer was dried over sodium sulfate, filtered, and the solvent was removed. The resulting oil was triturated with MTBE (3 mL), and the resulting precipitate was washed with MTBE and dried in vacuo to give tert-butyl N[(1R)-1-carbamoylpropyl]carbamate (0.3000 g, 1.4833 mmol, 60.294%) as a white solid.

LC/MS: QC_3_MIN: m/z 147 [M-tBu+H]+.

- 29 049235

The following compounds were prepared using the above methodology with the replacement of (2R)2-(tert-butoxycarbonylamino)butanoic acid with the appropriate protected amino acid.

Intermediate connection Structure Name Amino acid zhhms 8 NH ₂ J ^{n ο::} \/Νγ ° Ί— ⁰ tert-butyl-N-[(1R)1-carbamoyl-1-methylpropyl] carbamate (A^AAtert-butoxycarbonylamino)-2-methylbutanoic acid LC/MS: QC3MIN: m/z 455 [2M+Na]+.

Intermediate 9 (path 1).

tert-Butyl-N-[(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2yl]carbamoyl]propyl]carbamate.

A mixture of 2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazine (Intermediate 1; 50 mg; 0.15 mmol), tert-butyl N-[(1R)-1-carbamoylpropyl]carbamate (Intermediate 7; 46 mg; 0.23 mmol), tris(dibenzylideneacetone)dipalladium(0) (10.3 mg; 0.011 mmol), dicyclohexyl [2-(2,4,6-triisopropylphenyl)phenyl]phosphane (XPhos) (5.4 mg; 0.011 mmol) and cesium carbonate (73 mg; 0.22 mmol) in 1,4-dioxane (2 ml) was stirred under nitrogen at 80°C for 3 hours.

The reaction mixture was partitioned between ethyl acetate and brine. The organic layer was separated, dried over sodium sulfate, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel (Biotage system) using a SNAP 10g column and cyclohexane and EtOAc from 100/0 to 0/100 as eluent. The appropriate fractions were combined and evaporated to dryness to give tert-butyl N-[(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2yl]carbamoyl]propyl]carbamate (10 mg).

LC/MS: QC_3_MIN: Rt 2.696 min; m/z 455 [M+H]+.

Intermediate 9 (path 2).

tert-Butyl-N-[(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2yl]carbamoyl]propyl]carbamate.

To a mixture of 2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazine (Intermediate 1; 16 g; 48.0 mmol), tert-butyl N-[(1R)-1-carbamoylpropyl]carbamate (Intermediate 7; 10 g; 49.4 mmol), cesium carbonate (24.16 g; 74.17 mmol) in 1,4-dioxane (150 ml) after purging with argon were added diacetoxypalladium (0.555 g; 2.47 mmol) and (5-diphenylphosphanyl-9,9-dimethylxanthen-4-yl)diphenylphosphane (2.15 g; 3.71 mmol). A three-time vacuum-argon cycle was applied and the reaction mixture was stirred at 95 °C for 1.5 h. The reaction mixture was cooled using an external ice bath and then filtered under vacuum to remove cesium carbonate. The filtrate was collected, diluted with EtOAc (150 mL) and washed with aqueous saturated NH ₄ Cl (100 mL) and then with aqueous saturated NaCl (100 mL), dried over sodium sulfate, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel (Biotage system) using a 2x SNAP 100g column (200 g silica) and cyclohexane/EtOAc from 0 to 40% as eluent to give tert-butyl N-[(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'cyclopropan]-4-yl)oxypyrazin-2-yl]carbamoyl]propyl]carbamate (16.8 g) as a yellow solid.

The following compounds were prepared using the above methodology (route 1 or route 2) replacing 2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazine (Intermediate 1) with the appropriate bromopyrazine. The final products were purified by flash chromatography (silica cartridge; cyclohexane/EtOAc or other suitable solvent system).

- 30 049235

Intermediate connection Structure Name Brompyrazine zhhms 10 N—χ V—O ^H N from/?eot-butyl-X-[(1R)1-[(5-spiro[2Hbenzofuran-3,Gcyclopropane]-4yloxypyrazin-2yl)carbamoyl]propyl]carbamate 2-bromo-5spiro[2Hbenzofuran-3,Gcyclopropane]-4yloxy-pyrazine (intermediate 2) LC/MS: QC 3 _MIN: Rt 2.246 min; m/z 441 [M+H]+. AND o/?eot-butyl-X-[(1R)1-[[5-[(3,3,7trimethyl-2Hbenzofuran-4yl)oxy]-pyrazin-2yl]carbamoyl]propyl] carbamate 2-bromo-5[(3,3,7-trimethyl-2Hbenzofuran-4yl)oxy]pyrazine (intermediate 3) LC/MS: QC 3 MIN: Rt 2.309 min; m/z 457 [M+H]+. 12 otreot-butyl-N-[(1R)1-[[5-[(3,3-dimethyl2H-benzofuran-4yl)oxy]pyrazin-2yl]carbamoyl]propyl] carbamate 2-bromo-5-[(3,3-dimethyl-2Hbenzofuran-4yl)oxy]pyrazine (intermediate 4) LC/MS: QC 3 MIN: Rt 2.366 min; m/z 443 [M+H]+.

Intermediate connection 13.

(2R)-2-Amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)-oxypyrazin-2yl]butanamide.

A mixture of tert-butyl N-[(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin2-yl]carbamoyl]propyl]carbamate (Intermediate 9; 16 mg; 0.035 mmol) and 2,2,2-trifluoroacetic acid (0.50 ml; 6.53 mmol) in dichloromethane (2 ml) was stirred at room temperature for 2 hours.

The reaction mixture was diluted with dichloromethane (20 mL) and saturated _NaHCO3 (aq) was added until pH 8. The phases were separated and the organic layer was washed _with brine (20 mL), dried over _Na2SO4 , filtered and evaporated to give (2R)-2-amino-N-[5-(7-methylspiro[2Hbenzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]butanamide (13 mg), which was used in the next step without further purification.

LC/MS: QC_3_MIN: Rt 2.009 min; m/z 355 [M+H]+.

The following compounds were prepared using the above methodology replacing tert-butyl N-[(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2yl]carbamoyl]propyl]carbamate (Intermediate 9) with the corresponding Boc-amine.

- 31 049235

Intermediate Structure Name Vos-amii zhhms hot.connect. 14 / ^- Ho (2K)-2-amino-X- tert-butyl-N- [(1R)-1 - [(5- LC/MS: Kb (5-spiro[2H- spiro[2H-benzofuran- QC 3 MIN: N— benzofuran-3, G- 3,1 ’-cyclopropane]-4-yl- Rt 1,675 ⁿ Xνη ₂ 'C cyclopropane] -4- oxypyrazin-2-yl)- min; \ yloxypyrazine- carbamoyl] propyl] - m/z 342 2-yl)butanamide carbamate (intermediate) [M+N]+ connection 10) 15 (2R)-2-aMHHO-N- tert-buty l-N -[(lR)-l-[[5- LC/MS: Kb „ [5-[(3,3,7- [(3,3,7-trimethyl-2H- QC3MIN: trimethyl-2H- benzofuran-4-yl)-oxy]- Rt 1,756 benzofuran-4- pyrazin-2-yl] carbamoyl] - min; yl)oxy]- propyl] carbamate m/z 357 pyrazin-2-yl]- (intermediate [M+N]+ butanamide connection I) 16 Z O (2R)-2-aMHHO-N- tert-buty l-N -[(lR)-l-[[5- LC/MS: Kb [5-[(3,3-dimethyl- [(3,3 -d imethyl-2H- QC3MIN: N—Ч 2H-benzofuran- benzofuran-4-yl)oxy]- Rt 1,673 ⁿ nh ₂ 4-yl)oxy]- pyrazin-2-yl] carbamoyl] - min; \ pyrazin-2-yl]- propyl] carbamate m/z 343 butanamide (intermediate [M+N]+ connection 12)

Intermediate connection 17.

(5R)-5-Ethyl-5-methyl-imidazolidine-2,4-dione.

A mixture of tert-butyl N-[(1R)-1-carbamoyl-1-methyl-n-propyl]carbamate.

(Intermediate 8; 100 mg; 0.4624 mmol) and potassium carbonate (191.71 mg; 1.3871 mmol) in 1-butanol (5 mL) were stirred under nitrogen at 95 °C overnight. After cooling, potassium carbonate was filtered off and the reaction mixture was diluted with ethyl acetate (30 mL) and washed with aqueous 0.1 N HCl (30 mL) and then with brine (30 mL). The phases were separated and the organic layer was collected, dried over Na ₂ SO ₄ , filtered and evaporated to give (5R)-5-ethyl-5-methylimidazolidine-2,4-dione (60 mg; 0.4221 mmol; 91.283%).

LC/MS: QC_3_MIN: m/z 285 [2M+H]+.

Intermediate connection 18.

tert-Butyl-N-(1-carbamoylcyclobutyl)carbamate.

Intermediate 18 was prepared using the methodology described for Intermediate 7, replacing (2R)-2-(tert-butoxycarbonylamino)butanoic acid with 1-(tert-butoxycarbonylamino)-cyclobutanecarboxylic acid.

LC/MS: QC_3_MIN: m/z 159 [M-tBu+H]+.

Intermediate connection 19.

tert-Butyl N-[1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)-oxypyrazin-2yl]carbamoyl]cyclobutyl]carbamate.

- 32 049235

A mixture of 2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazine (Intermediate 1; 50 mg; 0.1501 mmol), tert-butyl N-(1-carbamoylcyclobutyl)carbamate (Intermediate 18; 64 mg; 0.2987 mmol), potassium carbonate (62 mg; 0.4486 mmol), copper(I) iodide (2.9 mg; 0.0152 mmol) and N,N'-dimethylethane-1,2-diamine (0.0065 ml; 0.0601 mmol) in 1-butanol (1 ml) was stirred under nitrogen at 95°C for 4 hours. After cooling, the reaction mixture was diluted with ethyl acetate (30 ml) and washed with aqueous 0.1 M HCl (30 ml) and then with brine (30 ml). The phases were separated and the organic layer was collected, dried over Na ₂ SO ₄ , filtered and evaporated. The residue was purified by flash chromatography (Biotage System) on silica gel using SNAP 10g as a column and cyclohexane:ethyl acetate from 100:0 to 30:70 as eluent to give tert-butyl N-[1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2yl]carbamoyl]cyclobutyl]carbamate (18 mg).

LC/MS: QC_3_MIN: Rt 2.675 min; m/z 467 [M+H]+.

Intermediate connection 20.

-Amuho-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)-oxypyrazin-2yl]cyclobutanecarboxamide.

Intermediate 20 was prepared using the methodology described for Intermediate 13, replacing tert-butyl N-[(III)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]carbamoyl]propyl]carbamate (Intermediate 9) with tert-butyl N-[1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2yl]carbamoyl]cyclobutyl]carbamate (Intermediate 19).

LC/MS: QC_3_MIN: Rt 1.979 min; m/z 367 [M+H]+.

Intermediate connection 21.

tert-Butyu-Shch 1 -carbamoylcyclopropyl)carbamate.

Intermediate 21 was prepared using the methodology described for Intermediate 7, replacing (2R)-2-(tert-butoxycarbonylamino)butanoic acid with 1-(tert-butoxycarbonylamino)cyclopropanecarboxylic acid.

Intermediate connection 22.

tert-Butyl-I-[1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)-oxypyrazin-2yl]carbamoyl]cyclopropyl]carbamate.

A mixture of dicyclohexyl [2-(2,4,6-triisopropylphenyl)phenyl]phosphane (12 mg; 0.0252 mmol), tert-butyl N-(1-carbamoylcyclopropyl)carbamate (Intermediate 21; 67 mg; 0.3346 mmol), tris(dibenzylideneacetone)dipalladium(0) (22 mg; 0.0240 mmol), 2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazine (Intermediate 1; 79.518 mg; 0.2387 mmol) and cesium carbonate (116 mg; 0.3560 mmol) in 1,4-dioxane (1 ml) was stirred under nitrogen at 95°C for 2 hours. Additional tert-butyl-I-(1-carbamoylcyclopropyl)carbamate (intermediate) was added.

- 33 049235 creeping compound 21; 67 mg; 0.3346 mmol) and tris(dibenzylideneacetone)dipalladium(0) (22 mg; 0.0240 mmol) and the reaction mixture was stirred at 95 °C under nitrogen for another 2 h, followed by additional addition of dicyclohexyl-[2-(2,4,6-triisopropylphenyl)phenyl]phosphane (12 mg; 0.0252 mmol), tris(dibenzylideneacetone)dipalladium(0) (22 mg; 0.0240 mmol) and cesium carbonate (58 mg) and the mixture was stirred under nitrogen for another 2 h. The reaction mixture was then quenched with water (10 mL), NH ₄ Cl (10 mL) and extracted with ethyl acetate (20 mL). The organic layer was then washed with brine (15 mL), dried _{over Na2SO4} , filtered, then concentrated in vacuo. The crude product was purified by flash chromatography (Biotage System) on silica gel using SNAP 10 g as a column and cyclohexane:ethyl acetate from 90:10 to 70:30 as an eluent to give tert-butyl N-[1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2yl]carbamoyl]cyclopropyl]carbamate (55 mg) as a yellow solid.

LC/MS: QC_3_MIN: Rt 2.634 min; m/z 453 [M+H]+.

Intermediate connection 23.

1-Amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)-oxypyrazin-2yl]cyclopropanecarboxamide.

tert-Butyl N-[1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)-oxypyrazin-2yl]carbamoyl]cyclopropyl]carbamate (Intermediate 22; 55 mg; 0.1215 mmol) was dissolved in dichloromethane (4 ml) and cooled to 0 °C. 2,2,2-Trifluoroacetic acid (1154.7 mg; 10.026 mmol) (0.8 ml) was added dropwise and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then cooled to 0 °C and NaHCO ₃ was added until pH 8. The mixture was then allowed to warm to room temperature and extracted with DCM (10 ml). The organic layer _{was dried over Na2SO4 ,} filtered and concentrated in vacuo to give 1-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]cyclopropanecarboxamide (40 mg) as a yellow oil.

LC/MS: QC_3_MIN: Rt 1.935 min; m/z 353 [M+H]+.

Intermediate connection 24.

1,3-Dibenzyloxy-2-bromobenzene.

To a solution of 2-bromobenzene-1,3-diol (20 g, 105.8 mmol) in acetone (200 mL) was added potassium carbonate (43.87 g, 317.4 mmol) followed by benzyl bromide (40.72 g, 238.1 mmol) (28 mL) and the reaction mixture was heated under reflux for 1.5 h. After cooling, the reaction mixture was filtered under vacuum and the filtrate was concentrated to dryness. The residue was diluted with ethyl acetate (100 mL) and washed with water (100 mL) and then brine (100 mL). The phases were separated and the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The residue was suspended in isopropanol (8 volumes) and the mixture was heated at 80°C and stirred for 1 hour at this temperature (until a clear solution was obtained). The mixture was then allowed to warm to room temperature (over 1 hour) and the resulting suspension was filtered. The solid was washed with ice-cold isopropanol and then dried to give the title compound 1,3-dibenzyloxy-2-bromobenzene (34 g) as a pale pink solid.

LC/MS: QC_3_MIN: Rt 2.688 min.

Intermediate connection 25.

Bromo-(1-methoxycarbonylcyclopropyl)zinc.

- 34 049235

Activated zinc powder (6.84 g, 104.6 mmol) was added to a two-necked round-bottomed flask and the powder was heated under vacuum. The system was placed under argon and anhydrous tetrahydrofuran (58 ml) was added. Then 1,2-dibromoethane (2.18 g, 11.62 mmol) was added and the mixture was heated under reflux. Chlorotrimethylsilane (505 mg, 4.65 mmol) was added in one portion and the mixture was continued to stir at reflux temperature. A solution of methyl 1-bromocyclopropyl carboxylate (10.4 g, 58.1 mmol) in anhydrous tetrahydrofuran (12 ml) was slowly added at the same temperature and the reaction mixture was heated under reflux for 1.5 h. The reaction mixture was cooled to room temperature and allowed to stand for zinc precipitation to yield 70 ml of a 0.83 M (theoretical amount) solution of bromo-(1methoxycarbonylcyclopropyl)-zinc in THF, which was used in the next step without further treatment.

Intermediate connection 26.

Methyl 1-(2,6-dibenzyloxyphenyl)cyclopropanecarboxylate.

about I

To a solution of 1,3-dibenzyloxy-2-bromobenzene (Intermediate 24; 16 g; 43.33 mmol) and bis(tri-tert-butylphosphine)palladium(0) (221 mg; 0.43 mmol) in N,N-dimethylformamide (150 ml), preheated to 70°C, was added a 0.83 M (theoretical amount) solution of bromo-(1-methoxycarbonylcyclopropyl)zinc in THF (Intermediate 25; 60 ml) (via catheter) and the reaction mixture was stirred at the same temperature for 40 min. After cooling, the reaction mixture was concentrated in vacuo to approximately 30 ml and the residue was diluted with ethyl acetate (450 ml) and washed twice with 1 N The mixture was washed with 100 mL of aqueous HCl (2 x 100 mL) and then with ice-cold brine three times (3 x 100 mL). The phases were separated and the organic layer was filtered under vacuum on a Gooch filter assembly of cellulose paper and washed with ethyl acetate. The filtrate was dried over Na ₂ SO ₄ , filtered and evaporated to give the title compound methyl 1-(2,6-dibenzyloxyphenyl)cyclopropanecarboxylate (15.5 g), which was used in the next step without further purification.

LC/MS: QC_3_MIN: Rt 2.606 min; m/z 389 [M+H]+.

Intermediate connection 27.

4-Hydroxyspiro[benzofuran-3,1'-cyclopropane]-2-one. OH

The reaction was carried out in three different runs using approximately 20 g of starting material in each.

General procedure: To a mixture of methyl 1-(2,6-dibenzyloxyphenyl)cyclopropanecarboxylate (intermediate 26; 20.4 g; 52.52 mmol) and 5 wt% palladium on carbon (1.02 g) in ethanol (200 mL) was added ammonium formate (16.56 g; 262.6 mmol) and the reaction mixture was stirred at 80 °C for 1 h. After cooling, the catalyst was filtered through a pad of cellulose and the filtrate was concentrated in vacuo to approximately 20 mL.

The residues obtained from 3 cycles were combined and diluted with ethyl acetate (400 mL) and washed twice with water (2 x 300 mL). The two phases were separated and the organic phase was washed with brine (300 mL), dried over Na ₂ SO ₄ and concentrated in vacuo to give 4-hydroxyspiro[benzofuran-3,1'cyclopropan]-2-one (27.55 g) (containing approximately 10-15% of the uncyclized methyl 1-(2,6-dihydroxyphenyl)cyclopropanecarboxylate intermediate), which was used in the next step without further purification.

LC/MS: QC_3_MIN: Rt 1.707 min.

Intermediate connection 28.

4-Benzyloxyspiro[benzofuran-3,1'-cyclopropan]-2-one.

- 35 049235

To a solution of 4-hydroxyspiro[benzofuran-3,1'-cyclopropan]-2-one (Intermediate 27; 28.5 g; 161.8 mmol) (containing approximately 10-15% of the uncyclized methyl 1-(2,6-dihydroxyphenyl)cyclopropanecarboxylate intermediate) in acetonitrile (200 ml)/tetrahydrofuran (50 ml) was added potassium carbonate (33.54 g; 242.7 mmol) and the reaction mixture was stirred at 70 °C for 1.5 h. The reaction mixture was then cooled to room temperature and benzyl bromide (27.67 g; 161.8 mmol) was slowly added. The reaction mixture was stirred at 60 °C for 5 h. After cooling, the reaction mixture was filtered under vacuum and the solid discarded, the filtrate was concentrated to 50 ml, diluted with ethyl acetate (250 ml) and washed twice with brine (2 x 100 ml). The phases were separated and the organic layer was dried over Na ₂ SO ₄ , filtered and evaporated to give the title compound 4-benzyloxyspiro[benzofuran-3,1'-cyclopropan]-2-one (42.4 g), which was used in the next step without further purification.

LC/MS: QC_3_MIN: Rt 2.389 min; m/z 267 [M+H]+.

Intermediate connection 29.

3-Benzyloxy-2-[1-(hydroxymethyl)cyclopropyl]phenol.

⁰

G 1

To a solution of 4-benzyloxyspiro[benzofuran-3,1'-cyclopropan]-2-one (Intermediate 28; 42.4 g; 159.2 mmol) in anhydrous tetrahydrofuran (300 ml) was slowly added 1 M lithium aluminium hydride in THF (79.6 ml; 79.6 mmol) at 0 °C and the reaction mixture was stirred at the same temperature for 30 min. The reaction mixture was quenched with ice, water (400 ml) and 1 M aqueous HCl (160 ml) and then diluted with ethyl acetate (700 ml). The phases were separated and the aqueous layer was re-extracted with ethyl acetate (500 ml). The combined organic phases were washed with brine (600 ml), dried over Na ₂ SO ₄ , filtered and evaporated to give the title compound 3-benzyloxy-2-[1-(hydroxymethyl)cyclopropyl]phenol (43 g), which was used in the next step without further purification.

LC/MS: QC_3_MIN: Rt 2.148 min; m/z 271 [M+H]+, m/z 293 [M+Na]+, m/z 253 [M-OH]+.

Intermediate connection 30.

4-Benzyloxyspiro[2H-benzofuran-3,1'-cyclopropane].

To a solution of 3-benzyloxy-2-[1-(hydroxymethyl)cyclopropyl]phenol (Intermediate 29, 43 g, 159.1 mmol) in dimethyl carbonate (430 ml) was slowly added potassium tert-butoxide (35.7 g, 318.1 mmol) and the reaction mixture was stirred at 85 °C for 3.5 h. The reaction mixture was cooled to room temperature, concentrated in vacuo to 150 ml, diluted with MTBE (400 ml) and washed with water (400 ml). The phases were separated and the aqueous layer was re-extracted with MTBE (250 ml). The combined organic layers were washed with brine (350 mL), dried over Na2SO4, filtered and concentrated to give the title compound 4-benzyloxyspiro[2Hbenzofuran-3,1'-cyclopropane] (40 g), which was used in the next step without further purification.

LC/MS: QC_3_MIN: Rt 2.457 min; m/z 253 [M+H]+.

Intermediate 31 (intermediate 85 in WO2012/076877).

-Spiro[2H-benzofuran-3,1'-cyclopropane]-4-ol.

The interaction was carried out in two cycles using 20 g of the starting material in each.

To a mixture of 4-benzyloxyspiro[2H-benzofuran-3,1'-cyclopropane] (Intermediate 30; 20 g; 79.27 mmol) and ammonium formate (24.99 g; 396.34 mmol) in ethanol (160 mL) was added 5 wt% palladium on carbon (2.0 g) and the reaction mixture was stirred at 80 °C for 10 min. After cooling, the catalyst was filtered through a pad of cellulose and the filtrate was concentrated in vacuo to approximately 20 mL. The residues obtained from the two reactions were combined and the mixture

- 36 049235 was diluted with ethyl acetate (300 ml) and washed three times with water (3 x 200 ml) and then with brine (200 ml). The two phases were separated and the organic phase was dried over Na ₂ SO ₄ and concentrated in vacuo. The residue was purified by flash chromatography (Biotage System) on silica gel using cyclohexane:ethyl acetate from 99:1 to 85:15 as eluent to give spiro[2H-benzofuran-3,1'cyclopropan]-4-ol (17.75 g) as a white solid.

LC/MS: QC_3_MIN: Rt 1.723 min; m/z 163 [M+H]+.

Intermediate connection 32.

tert-Butyl N-[(1S)-1-carbamoylpropyl]carbamate.

The title compound was synthesized using the same methodology used for the synthesis of Intermediate 7, replacing (2R)-2-(tert-butoxycarbonylamino)butanoic acid with (2S)-2-(tert-butoxycarbonylamino)butanoic acid.

LC/MS: QC_3_MIN: m/z 147 [M-tBu+H]+, m/z 427 [2M+Na]+.

Intermediate connection 33.

tert-Butyl N-[(1S)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2yl] carbamoyl] propyl] carbamate.

The title compound was synthesized according to the methodology of Route 1 used for the synthesis of Intermediate 9, replacing tert-butyl N-[(1R)-1carbamoylpropyl]carbamate (Intermediate 7) with tert-butyl N-[(1S)-1carbamoylpropyl]carbamate (Intermediate 32).

LC/MS: QC_3_MIN: Rt 2.65 min; m/z 455 [M+H]+.

Intermediate connection 34.

(2S)-2-Amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)-oxypyrazin-2yl]butanamide.

NH

The title compound was synthesized according to the same procedure as used for the synthesis of Intermediate 13, replacing tert-butyl N-[(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]carbamoyl]propyl]carbamate (Intermediate 9) with tert-butyl N-[(1S)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]carbamoyl]propyl]carbamate (Intermediate 33).

LC/MS: QC_3_MIN: Rt 1.98 min; m/z 355 [M+H]+.

Example 1, path 1.

5,5-Dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)-oxypyrazin-2yl]imidazolidin-2,4-dione.

To a solution of 2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazine (Intermediate 1; 30 mg; 0.069 mmol) in N,N-dimethylacetamide (1 ml) was added 5.5

- 37 049235 dimethylimidazolidine-2,4-dione (44.4 mg, 0.345 mmol) and copper(I) oxide (5 mg, 0.035 mmol). The flask was flushed with nitrogen gas and left to stir overnight at 135°C. The reaction mixture was diluted with EtOAc (10 mL) and washed first with aqueous saturated ammonium chloride solution (20 mL) and then with brine (20 mL). The organic layer was collected, dried over sodium sulfate and evaporated to dryness. The residue was then purified by flash column chromatography using cyclohexane:ethyl acetate 80:20 to 40:60 as eluent to give 5,5-dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione (17 mg) as a white solid.

¹ H-NMR (400 MHz; DMSO-d6): δ ppm 8.72 (bs, 1H), 8.51 (d, 1H), 8.30 (d, 1H), 6.95 (dd, 1H), 6.53 (d, 1H), 4.46 (s, 2H), 2.14 (s, 3H), 1.42 (s, 6H), 1.07-1.14 (m, 2H), 0.89-0.95 (m, 2H).

The following compounds were prepared using the above methodology substituting the appropriate bromopyrazine for 2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazine (Intermediate 1) and the appropriate hydantoin for 5,5-dimethylimidazolidine-2,4-dione. The final products were purified by flash chromatography (silica cartridge; cyclohexane/EtOAc or other suitable solvent system) and/or reverse chromatography (C-18 cartridge; water/acetonitrile or other suitable solvent system).

Note. Structure Name Brompyrazine Hydantoin LCMS/NMR 2 8% N— 3-[5-[(3,3-dimethyl2H-benzofuran-4yl)oxy]-pyrazin 2-yl]-5,5-dimethyl imide azo lidin-2,4dione 2-bromo-5-[(3,3-dimethyl-2Hbenzofuran-4yl)oxy]pyrazine (intermediate 4) 5,5-dimethylimidazolide in-2,4dione LC/MS: QC 3 MIN: Rt 2.288 min; m/z 369 [M+H]+. Ή-NMR (500 MHz; DMSO-d6): δ ppm 8.73 (bs, 1H), 8.60 (d, 1H), 8.32 (d, 1H), 7.17 (dd, 1H), 6.70 (d, 1H), 6.66 (d, 1H), 4.23 (s, 2H), 1.42 (s, 6H), 1.28 (s, 6H). 3 cf d XX ζ o (5C)-5-ethyl-5methyl-3-(5spiro[2Hbenzofuran-3,1 cyclopropane]-4 yloxypyrazin-2 yl)imide azolidin-2, 4-dione 2-bromo-5-cyclo[2Hbenzofuran-3,Hcyclopropane]-4-yloxy-pyrazine (Intermediate 2) (5K)-5-ethyl-5-methyl- imide azolidine-2,4-dione (Intermediate 17) LC/MS: QC 3 MIN: Rt 2.228 min; m/z 381 [M+H]+.

Example 1, path 2.

5,5-Dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)-oxypyrazin-2yl]imidazolidine-2,4-dione.

To a solution of 3-(5-chloropyrazin-2-yl)-5,5-dimethylimidazolidine-2,4-dione (Intermediate 5; 20 mg; 0.083 mmol) and 7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-ol (Intermediate 156 in WO2012076877; 22 mg; 0.125 mmol) in acetonitrile (1 ml) was added potassium carbonate (17.2 mg; 0.12 mmol). The reaction mixture was stirred overnight at 60°C and then for 3 h at 80°C. The reaction mixture was concentrated in vacuo and the crude product was purified by flash chromatography on silica gel (BIOTAGE SYSTEM) using SNAP 10g as a column and Chexane/EtOAc 80/20 to 20/80 as eluent. The fraction was still contaminated with impurities and was purified by reverse chromatography using SNAP C-18 as a column and H2O/ACN 95/5 to 5/95 as eluent to give 5,5-dimethyl-3-[5-(7-38 049235 methylspiro)[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione (9.4 mg) as a white solid.

LC/MS: QC_3_MIN: Rt 2.224 min; m/z 381 [M+H]+.

The following compounds were prepared using the above methodology substituting the appropriate phenol for 7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-ol and using 3(5-chloropyrazin-2-yl)-5,5-dimethylimidazolidine-2,4-dione (Intermediate 5) or substituting the appropriate chloropyrazine intermediate. The final products were purified by flash chromatography (silica cartridge; cyclohexane/EtOAc or other suitable solvent system) and/or reverse chromatography (C-18 cartridge; water/acetonitrile or other suitable solvent system).

Note:

Structure

Name

Chloropyrazine intermediate

LCMS/NMR

5,5-Dimethyl-3-(5-spiro[2H-benzofuran-3,G-cyclopropane]-4-yloxypyrazin-2yl)imide azolidine-2,4-dione (5R)-5-ethyl-5-methyl-3-[5-(7methylspiro[2H-benzofuran-3,G-cyclopropane]4-yl)oxypyrazin-2yl]imide azolidine-2,4-dione (5R)-3-[5-[(3,3-dimethyl-2Hspiro[2Hbenzofuran-3,1'-cyclopropane]-4-ol (Intermediate 3-(5-chloropyrazin-2-yl)5,5-dimethyl LC/MS: QC 3 MIN: Rt 2.085 min;

m/z 367 [M+H]+. Ή-NMR (500 MHz; DMSO-d6): δ ppm

imide azo-lide in2,4-dione compound (Intermediate85bWO

2012/076877)

7-methylspiro[2H-benzofuran-3,Gcyclopropan]4-ol (Intermediate compound

156 in WO

2012/076877)

3,3-dimethyl-2H-benzobenzofuran-4yl)oxy]pyrazin-2-yl]5-ethyl-5-methylfuran-4 -ol (Intermediate compound

50bWO

2012/076877) connection

8.73 (bs, 1H), 8.54 (d, 1H), 8.32 (d, 1H), 7.11 (dd, 1H), 6.71 (d, 1H), 6.62 (d, 1H),

5)

4.46 (s, 2H), 1.42 (s,

6N), 1.12-1.16 (w,

2N), 0.92-0.97 (t, 5N).

LC/MS: QC 3 MIN: Rt 2.361 min;

(5R)-3-(5-xnoppyrazin-2-yl)5 -ethyl-5 -methylimide azo Lidin2,4-dione (Intermediate compound

6) m/z 395 [M+H]+.

Ή-NMR (500 MHz;

DMSO-d6): δ ppm

8.64 (bs, 1H), 8.48 (d,

1H), 8.25 (d, 1H),

6.91 (dd, 1H), 6.49 (d,

1H), 4.42 (s, 2H), 2.11 (s, 3H), 1.71-1.79 (m,

1H), 1.60-1.68 (w,

1H), 1.38 (s, ЗН),

1.02-1.09 (m, 2H),

0.82-0.92 (m, 5H).

(5R)-3-(5-chloropyrazin-2-yl)5-ethyl-5-methylimidazolidineLC/MS: QC 3 MIN:

Rt 2,008 min;

m/z 383 [M+H]+.

2,4-dione (Intermediate- 39 049235

azo imide lidin- 2,4-dione connection 6) ₇ 5,5-dimethyl-3[5-[(3,3,7trimethyl-2Hbenzofuran-4yl)oxy]- pyr azin-2-yl]imide azo Lidin- 2,4-dione 3,3,7-trimethyl2H-benzofuran-4-ol (Intermediate 184 in WO 2012/076877) 3-(5-chloropyr azin-2-yl)5,5-dimethylimide azolidine2,4-dione (Intermediate 5) LC/MS: QC 3 MIN: Rt 2.025 min; m/z 383 [M+H]+. r, h (5C)-5-ethyl-5methyl-3-[5[(3,3,7trimethyl-2Hbenzofuran-4yl)oxy]pyrazin-2-yl]imide azo Lidine2,4-dione 3,3,7-trimethyl2H-benzofuran-4-ol (Intermediate 184 in WO 2012/076877) (5R)-3-(5-xnopyr azin-2-yl)5-ethyl-5-methyl- imide azo Lidin2,4-dione (Intermediate 6) LC/MS: QC3MIN: Rt 2.111 min; m/z 397 [M+H]+.

Example 9 (path 1).

(5R)-5-Ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)-oxypyrazin-2yl]imidazolidine-2,4-dione.

A mixture of (2R)-2-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2yl]butanamide (Intermediate 13; 13 mg; 0.037 mmol) and N,N-diethylethanamine (11 mg; 0.11 mmol) in dichloromethane (2 ml) was cooled to 0 °C. A solution of bis(trichloromethyl)carbonate (4.5 mg; 0.015 mmol) in dichloromethane (0.5 ml) was added dropwise and the reaction mixture was stirred for 1 hour at the same temperature. Additional bis(trichloromethyl)carbonate (1.5 mg) in dichloromethane (0.5 ml) was added and stirring was continued for 30 min. The mixture was allowed to warm to room temperature. The reaction mixture was diluted with dichloromethane (20 mL) and the organic phase was washed with aqueous 0.1 N HCl (20 mL) and then with brine (20 mL). The phases were separated and the organic layer was dried over Na ₂ SO ₄ , filtered and evaporated. The residue was purified by reverse phase chromatography using a SNAP C18 column eluting with water:acetonitrile from 90:10 to 0:100. The appropriate fractions were combined and evaporated to dryness to give (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4yl)oxypyrazin-2-yl]imidazolidine-2,4-dione (7.5 mg) as a white solid.

LC/MS: QC_3_MIN: Rt 2.305 min; m/z 381 [M+H]+. Enantiomeric purity was confirmed to be greater than 95% using the chiral control method.

Example 9 (path 2).

(5R)-5-Ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)-oxypyrazin-2yl]imidazolidine-2,4-dione.

To a solution of (2R)-2-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin2-yl]butanamide (Intermediate 13; 21 g; 59.26 mmol) in ethyl acetate (500 ml) was added 11'-carbonyldiimidazole (10.57 g; 65.18 mmol) in 5 portions of 2 g each and stirred at room temperature.

- 40 049235 temperature for 4 hours. The reaction mixture was quenched with ice and 0.2 N aqueous HCl (250 mL) was added. The two phases were separated and the organic layer was washed with 0.2 N aqueous HCl (250 mL) and brine (200 mL), then dried over sodium sulfate, filtered and evaporated to dryness. The crude product was divided into 4 aliquots of approximately 4.2 g each and each aliquot was purified by flash chromatography on silica gel using SNAP (100G) as the column and cyclohexane/ethyl acetate 80/20 to 20/80 as the eluent. The desired fractions from each run were collected and the solvent was evaporated to a dry residue. The resulting light yellow solid was suspended in cyclohexane/ethyl acetate (1/1; 3 vol) solution (90 ml) and stirred for 2 h at 50°C. The mixture was then allowed to cool to room temperature and filtered under vacuum. The wet cake was washed with ice-cold cyclohexane (15 ml) and the solid was collected and dried to give the title compound (5R)-5-ethyl-3-[5-(7-methylcyclo[2Hbenzofuran-3,1')-cyclopropan]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione (13.6 g) as a white solid.

¹ H-NMR (500 MHz; DMSO-d6): δ ppm 8.69 (bs, 1H), 8.52 (d, 1H), 8.26 (d, 1H), 6.94 (d, 1H), 6.53 (d, 1H), 4.46 (s, 2H), 4.26-4.30 (m, 1H), 2.14 (s, 3H), 1.77-1.86 (m, 1H), 1.65-1.76 (m, 1H), 1.07-1.12 (m, 2H), 0.90-0.99 (m, 5H).

The following compounds were prepared using the above methodology (either route 1 or route 2) replacing (2R)-2-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4yl)oxypyrazin-2-yl]butanamide (Intermediate 13) with the appropriate butanamide. The final products were purified by flash chromatography (silica cartridge; cyclohexane/EtOAc or other suitable solvent system) and/or reverse chromatography (C-18 cartridge; water/acetonitrile or other suitable solvent system).

Name

Butanamide

LCMS/NMR (5P)-5-ethyl-3-(5spiro[2Hbenzofuran-3,1'cyclopropane]-4-yloxypyrazin-2-yl)imide azo 2,4-dione (2R)-2-amino-N-(5spiro[2Hbenzofuran-3,1'cyclopropane]-4-yloxypyrazin-2-yl)butanamide (Intermediate 14)

LC/MS: QC 3 MIN: Rt 2.081 min; m/z 367 [M+H]+.

Enantiomeric purity was confirmed to be greater than 95% using chiral monitoring. Ή-NMR (500 MHz; DMSOd6): 5 ppm 8.70 (bs, 1H), 8.55 (d, 1H), 8.27 (d, 1H), 7.11 (dd, 1H), 6.71 (dd, 1H), 6.62 (dd, 1H), 4.46 (s, 2H), 4.27-4.31 (br, 1H), 1.76-1.87 (m, 1H), 1.65-1.76 (m, 1H), 1.11-1.17 (br, 2H), 0.92-0.98 (m, 5H).

(5R)-3-[5-[(3,3-dimethyl-2Hbenzofuran-4yl)oxy]pyrazine-2(2R)-2-amino-N-[5 [(3,3-dimethyl-2Hbenzofuran-4yl)oxy]pyrazine-2LC/MS: QC 3 MIN: Rt 2.142 min; m/z 369 [M+H]+.

- 41 049235

yl]-5-ethyl- imide azolidine-2,4- dione yl]butanamide (Intermediate 16) 12 7—ί Ζ—Ν y ^{4 Ν} =< _X O O^S^NH (5K)-5-ethyl-3-[5[(3,3,7-trimethyl-2H-benzofuran-4yl)oxy]pyrazin-2 yl]-imide azo Lidin-2,4-dione (2R)-2-amino-N-[5[(3,3,7-trimethyl-2H-benzofuran-4yl)-oxy]pyrazin2-yl]butanamide (Intermediate 15) LC/MS: QC 3 MIN: Rt 2.111 min; m/z 383 [M+H]+. Ab ^N \ ,0 N— 7-[5-(7- methylspiro [2Hbenzofuran-3, Gcyclopropane] -4yl)oxypyrazin-2yl] -5,7-diazaspiro[3,4]octane-6,8-dione 1-amino-N-[5-(7 methylspiro[2Hbenzofuran-3,1'cyclopropane]-4 yl)oxypyrazin-2 yl]cyclobutanecarboxamide (Intermediate 20) LC/MS: QC3MIN: Rt 2.309 min; 393 m/z [M+H]+. 14 AND 6-[5-(7- methylspiro [2Hbenzofuran-3,Gcyclopropane] -4yl)oxypyrazin-2yl] -4,6diazaspiro [2.4]heptane-5,7-dione 1-amino-N-[5-(7 methylspiro[2Hbenzofuran-3,1'cyclopropane]-4 yl)oxypyrazin-2 yl]cyclopropanecarboxamide LC/MS: QC 3 MIN: Rt 2.236 min; 379 m/z [M+H]+.

Example 15.

(3S)-5-Ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)-oxypyrazin-2yl]imidazolidine-2,4-dione.

The title compound was synthesized according to the methodology of route 1 used for the synthesis of intermediate 9, replacing (2R)-2-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]butanamide (Intermediate 13) with (2S)-2-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]butanamide (Intermediate 34).

LC/MS: QC_3_MIN: Rt 2.29 min; m/z 381 [M+H]+.

Biological examples.

Biological example 1: measuring the modulation of Kv3.1, Kv3.2 and Kv3.3 channels.

The ability of the compounds of the invention to modulate the Kv3.3/Kv3.2/Kv3.1 voltage-gated potassium channel subtypes can be determined using the following assay. Similar methods can be used to examine the ability of the compounds of the invention to modulate other channel subtypes.

Cell biology.

To evaluate the effects of the compounds on human Kv3.3 (hKv3.3) channels, a stable cell line expressing human Kv3.3 channels was generated by transfecting Chinese hamster ovary (CHO)-K1 cells with the pBacMire_KCNC-3 vector. The cells were cultured in DMEM/F12 (Gibco) supplemented with 10% embryonic

- 42 049235 natural bovine serum (Gibco), 1X nonessential amino acids (Invitrogen) and 400 μg/ml geneticin (G418). Cells were grown and maintained at 37°C in a humidified environment containing 5% CO2 in air.

To evaluate the effects of compounds on human Kv3.2 (hKv3.2) channels, a stable cell line expressing human Kv3.2 (hKv3.2) channels was generated by transfecting CHO-K1 cells with the pCIH5-hKv3.2 vector. Cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (Gibco), 1X nonessential amino acids (Invitrogen), and 500 μg/ml hygromycin-B (Invitrogen). Cells were grown and maintained at 37°C in a humidified environment containing 5% CO2 in air.

To assess the effects of compounds on human Kv3.1 (hKv3.1) channels:

The human embryonic kidney (HEK)-hKv3.1 cell line was generated by transfecting HEK-293 cells with the human Kv3.1 expression vector (NM_004976.4). The cells were cultured in MEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1% penicillin-streptomycin, and 0.6 mg/ml geneticin (G418). HEK-hKv3.1b cells were amplified in a T175 ^cm2 flask at 37°C with 5% CO2 using MEM amplification medium containing the selective antibiotic G418 (0.6 mg/ml). Cells were detached every 3-4 days using Dulbecco's phosphate-buffered saline (DPBS) to wash the flask twice, then TrypLE to remove cells, and replated at a density of 2-4x106 cells/flask.

Preparing cells for IonWorks Quattro™ experiments.

On the day of the experiment, the cells were removed from the incubator and the culture medium was discarded. The cells were washed with 5 ml of Dulbecco's PBS (DPBS) containing no calcium and magnesium and detached by adding 3 ml of Versene (Invitrogen, Italy) followed by a brief incubation at 37°C for 5 min. The flask was shaken to separate the cells and 10 ml of DPBS containing calcium and magnesium was added to obtain a cell suspension. This cell suspension was then placed in a 15 ml centrifuge tube and centrifuged for 2 min at 1200 rpm. After centrifugation, the supernatant was discarded and the cell pellet was resuspended in 4 ml of DPBS containing calcium and magnesium using a 5 ml pipette to disrupt the pellet. The volume of the cell suspension was then adjusted to obtain a cell concentration of approximately 3 million cells per ml for the assay.

All solutions added to cells were preheated to 37°C.

Electrophysiology.

Ionworks.

Experiments were performed at room temperature using IonWorks Quattro™ flat-array electrophysiology technology (Molecular Devices Corp.) via a PatchPlate™ PPC. Stimulation protocols and data acquisition were performed using a microcomputer (Dell Pentium 4). The interelectrode resistance of the flat electrodes (Rp) was determined by applying 10 mV potential steps across each well. These measurements were performed before adding cells. After adding cells and creating isolation, isolation was tested by applying a potential step from -80 mV to -70 mV for 160 ms. Amphotericin B solution was then added to the inside of the well side of the electrode to gain access to the cell interior. Cells were maintained at -70 mV. Leak subtraction was performed in all experiments by applying 50 ms hyperpolarizing (10 mV) lead pulses to induce leak currents, followed by a 20 ms period at resting potential before the test pulses.

For hKv3.2 and hKv3.1 assays, starting from a baseline potential of -70 mV, a first test pulse at -15 mV was applied for 100 ms, followed by 100 ms at -70 mV and a second pulse at +40 mV for 50 ms. Cells were then held for 100 ms at -100 mV and another pulse from -70 mV to +40 mV (50 ms duration) was applied, followed by voltage clamping at -40 mV for 200 ms.

For hKv3.3 assays, starting from a baseline potential of -70 mV, a first test pulse at 0 mV was applied for 500 ms, followed by a second pulse of 100 ms at -70 mV to 40 mV for 200 ms. These longer test pulses are used to study hKv3.3 channel inactivation. The test pulse protocol can be performed in the absence (pre-read) and presence (post-read) of the test compound. The pre- and post-reads can be separated by adding the compound followed by a 3-min incubation.

Solutions and medicines.

The intracellular solution contained the following (in mM): K-gluconate 100, KCl 54, _MgCl2 3.2, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) 5, with pH adjusted to 7.3 with KOH. Amphotericin B solution was prepared as a 50 mg/mL stock solution in DMSO and diluted to a final working concentration of 0.1 mg/mL in the intracellular solution. The external solution was Dulbecco's phosphate-buffered saline (DPBS) and contained the following (in mM): CaCl2 _0.90 , _{KCl 2.67} , KH2PO4 1.47, MgCl- _6H2O 0.493, NaCl 136.9, _{Na3PO4 8.06} , with a pH of 7.4.

- 43 049235

The compounds used in the invention (or reference compounds such as N-cyclohexylN-[(7,8-dimethyl-2-oxo-1,2-dihydro-3-quinolinyl)methyl]-N'-phenylurea) were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM. These solutions were further diluted with DMSO using a Biomek FX (Beckman Coulter) in a 384 compound plate. Each dilution (1 μl) was transferred to another compound plate and an external solution containing 0.05% pluronic acid (66 μl) was added. 3.5 μl from each plate containing a compound of the invention was added and incubated with the cells during the IonWorks Quattro™ experiment. The final dilution for the assay was 200, and final compound concentrations ranged from 50 μM to 50 nM.

Data analysis.

Instrument readings were analyzed and collected using both insulation resistance (>20 MΩ) and maximum current amplitude (>500 pA at a 40 mV potential difference step) in the absence of compound to exclude inappropriate cells from further analysis. For hKv3.2 and hKv3.1 assays, paired comparisons of pre-drug and post-drug evoked current values measured at -15 mV were used to determine the effect of positive modulation for each compound. Kv3 channel-mediated outward currents were determined by measuring the mean current amplitude during the final 10 ms of the -15 mV voltage pulse minus the mean baseline current at -70 mV over the 10 ms period immediately preceding the -15 mV step. These Kv3 channel currents after test compound addition were then compared with the currents recorded before compound addition. Data were normalized to the maximal effect of the reference compound (50 μM N-cyclohexyl-N-[(7,8-dimethyl-2-oxo-1,2-dihydro-3quinolinyl)methyl]-N'-phenylurea) and to the effect of the vehicle control (0.5% DMSO). These normalized data were analyzed using ActivityBase or Excel software. The concentration of compound required to increase currents by 50% of the maximal increase elicited by the reference compound (EC ₅₀ ) was determined by fitting the concentration-response data using a four-parameter logistic function in ActivityBase. For hKv3.3 assays, pairwise comparisons of evoked currents in the pre-drug and post-drug intervals were measured for a 0 mV step, taking into account the peak current and the decay (inactivation) of the current over the duration of the 0 mV test pulse (500 ms).

N-Cyclohexyl-N-[(7,8-dimethyl-2-oxo-1,2-dihydro-3-quinolinyl)methyl]-N'-phenylurea was obtained from ASINEX (registration number: 552311-06-5).____________________________

Note. Connection Kv3.1 pEC50 Kv3.1 maxK% Link/LCMS RE1 dd 4.78 105 Example 57 WO2011/069951 RE2 gague 5.25 118 Example 45 WO2011/069951 RE3 VC rare 4.89 79 LC/MS: QC 3 MIN: Rt 2.376 min; m/z 396 [M+H]+. RE4 rarl _f A^ ^{f r} F <4.3 24 LC/MS: QC 3 MIN: Rt 2.346 min; m/z 397 [M+H]+.

As shown in the RE1-RE4 assay, inclusion of the pyrazine ring can negatively impact the pEC50 and maxR values of Kv3.1 modulators.

- 44 049235

Note. Connection Kν3.1 pEC50 Kv3.1 maxK% Link/LCMS RE5 YL. 5.14 158 Example 58 WO2012/076877 RE6 Jm 5.58 144 Example 70 WO2012/076877 RE7 o^n 5.56 130 Example 3 WO2017/103604 RE8 4.98 42 LC/MS: QC3MIN: Rt 2.224 min; m/z 381 [M+H]+. RE9 <4.3 16 LC/MS: QC3MIN: Rt 2.043 min; m/z 381 [M+H]+. LC/MS: <4.3 99 QC3MIN: Rt L ^and ' ¹ ** < n 2.29 min; m/z 381 [M+H]+. 1 + 5.47 164 Example 1

⁺ n=10. For n=18, pEC50 was 5.56 and MakcR% was 152.

As shown in testing RE5-RE9 compared to Example 1, the inclusion of the para-pyrazine ring in Example 1 unexpectedly results in a high pEC50 value and a high maxR value in the Kv3.1 assay. RE10 shows that the meta-pyrazine central ring significantly reduces the pEC50 value and maxR value compared to the para-pyrazine of Example 1.

Example Kv3.1 pEC50 Kv3.1 maxP% 1 ⁺ 5.47 164 2 4.68 149 3 5.15 205 4 5D7 170 5 5.69 149 6 4.75 165 7 5,12 134

Example Kv3.1 pEC50 Kv3.1 maxI% 8 5.29 119 9* 5.88 172 10 ⁵ 5.45 153 AND 4.89 165 12 5.56 118 13 5.09 165 14 5.51 145 15 5,10 136

⁺ n=10. For n=18, pEC50 was 5.56 and maxR% was 152 * n=4. For n=22, pEC50 was 5.90 and maxR% was 146 $ n=2. For n=26, pEC50 was 5.63 and maxR% was 147.

- 45 049235

Note. Connection Kv3.1 pEC50 Kv3.1 maxI% Link/LCMS RE11 6.1 152 Example 62 WO2012/076877 RE12 5,6 149 Example 4 WO2017/102604 9 5.90 146 Example 9

Note. Connection Kv3.1 pEC50 Kv3.1 maxI% Link/LCMS RE13 Su 6.1 149 Example 15 WO2012/076877 RE14 5.2 150 Example 6 WO2017/102604 10 5.63 147 Example 10

All tested examples of compounds of formula (I) are shown above and demonstrate good properties in the Kv3.1 assay, expressed as pEC50 and maxB. Previous disclosures of Kv3.1 data for reference compounds may differ slightly due to a smaller number of measurements.

A secondary analysis of the data from the hKv3.1, hKv3.2 and hKv3.3 analyses described above can be used to investigate the effects of coupling on the rate of current rise from the onset of the depolarizing voltage pulses. The magnitude of the coupling effect can be determined from the time constant (Tau _act ) obtained from a nonlinear fit using the equation below to the rise of the Kv3.1, Kv3.2 and Kv3.3 currents after the onset of the -15 mV depolarizing voltage pulse.

Y = (Y0 - Ymax) * exp(-K*X) + Ymax, where:

Y0 represents the current value at the beginning of the depolarizing voltage pulse;

Umax is the current value when the plateau is reached;

K is the rate constant, and

Tau _act is the activation time constant, which is the reciprocal of K.

Similarly, the effect of coupling on the decay time of the Kv3.1, Kv3.2 or Kv3.3 currents during channel closure at the end of -15 mV depolarizing voltage pulses can be investigated. In this latter case, the magnitude of the coupling effect on channel closure can be determined from the time constant (Tau _deact ) of the nonlinear approximation of the current decay (trace current) immediately after the end of the depolarizing voltage pulse.

Kv3.1, Kv3.2, and Kv3.3 channels must be activated and deactivated very rapidly to allow neurons to fire action potentials at high frequencies (Rudy et al., 2001). Slowing down activation would likely delay the onset of action potential repolarization; slowing down deactivation would result in hyperpolarizing currents, which would reduce the excitability of the neuron and delay the time before the neuron could fire an additional action potential. Together, these two slowing effects on channel activation and deactivation would likely result in a reduction, rather than a facilitation, of the ability of neurons to fire at high frequencies. Thus, compounds that have this slowing effect on Kv3.1 channels

-46049235 and/or Kv3.2 and/or Kv3.3, will effectively behave as negative channel modulators, leading to a slowing of neuronal firing. This latter effect has been demonstrated for some of the compounds disclosed in WO2011/069951, where a marked increase in Tauact can be observed from recordings taken from fast-firing interneurons in the rat cerebral cortex using in vitro electrophysiological methods. Addition of the respective compounds reduces the ability of neurons to fire in response to trains of depolarizing pulses at 300 Hz.

Therefore, although some compounds can be identified as acting as positive modulators in recombinant cell assays, those compounds that markedly increase Tauact may reduce the ability of neurons in native tissues to fire at high frequencies.

Biological Example 2: Determination of binding in blood and brain tissue.

Materials and methods.

Whole blood from Sprague Dawley rats collected during the week of the K3-EDTA anticoagulant experiment was diluted 1:1 (v/v) in isotonic phosphate buffer. Whole brain from Sprague Dawley rats stored frozen at -20°C was thawed and homogenized in artificial cerebrospinal fluid (CSF) 1:2 (w/v).

An appropriate amount of test compound was dissolved in DMSO to give a 10 mM solution. Further dilutions were then prepared to give a 166.7 μM working solution using 50% acetonitrile in MilliQ water. This working solution was used to spike the blood sample to give a final concentration of 0.5 μM in whole blood. Similarly, this working solution was used to spike the brain samples to give a final concentration of 5 μM in whole brain. Control samples (n=3) were immediately extracted from these blood and brain preparations and used to calculate the initial recoveries of the test samples.

150 μl of compound-free buffer (isotonic phosphate buffer for blood or artificial cerebrospinal fluid buffer for brain) were dispensed into one half of the well and 150 μl of the prepared sample (blood or brain) was loaded into the other half of the well, where the two halves were separated by a semipermeable membrane. After an equilibration period of 5 hours at 37°C, 50 μl of the dialyzed sample (blood or brain) was added to 50 μl of the corresponding compound-free buffer and vice versa for the buffer, so that the volume of buffer for the sample (blood or brain) remains the same. The samples were then extracted by protein precipitation with 300 μL of acetonitrile containing rolipram (positive ionization mode control) or diclofenac (negative ionization mode control) as internal standards and centrifuged for 10 min at 3000 rpm. The supernatants were collected (100 μL), diluted with 27% AcN in MilliQ water (200 μL) and then injected into an HPLC-MS/MS or UPLC-MS/MS system to determine the concentration of the test compound present.

Analysis.

Binding in blood and brain tissue was then determined using the following formulas:

Afu = Buffer/Blood or Afu = CSF/Brain, where Afu is the apparent unbound fraction; Buffer is the analyte/internal standard ratio determined in the buffer compartment; Blood is the analyte/internal standard ratio determined in the blood compartment; Brain is the analyte/internal standard ratio determined in the brain compartment.

Fucr = 1/D_______

[(1/Afu- 1)+1/D], where: fucr is the corrected unbound fraction; D is the sample dilution factor (D=2 for blood and D=3 for brain). Then:

% Binding = (Ι-fucr) x 100% Unbound = 100% bound

Determination of the Brain/Blood ratio (Kbb).

For compounds that readily cross the blood-brain barrier (BBB), unbound concentrations in blood and brain will be equivalent under steady state distribution conditions. Therefore, the Kbb value can be calculated as:

Ri(blood)/Ri(brain), which is expected to be equivalent to the ratio of brain to blood concentrations (Ct(brain)/Ct(blood)) if efflux pump transporters are not involved.

Results.

The compounds of Examples 1, 9 and 10, as well as some reference compounds, were tested using the above methodology to determine the unbound fraction in the brain. The results were as follows:

- 47 049235

*Supernatant diluted with 18% AcN in water.

The pyrazine compounds of the invention demonstrated an increased unbound fraction in the brain compared to the reference pyridine compounds.

Biological example 3: Determination of pharmacokinetic parameters in vivo.

Materials and methods.

Adult male rats (Charles River, Italy) were administered the test compound orally at a dose of 1 mg/kg (5 ml/kg, in 5% v/v DMSO, 0.5% w/v HPMC in water) and intravenously at a dose of 0.5 mg/kg (2 ml/kg in 5% v/v DMSO, 40% w/v PEG400 in saline). After oral administration, blood samples were collected under deep isofluorane anesthesia from the portal vein and heart of each rat (1 rat per time point). After intravenous administration, serial blood samples were collected from the lateral tail vein of each rat. Another group of rats (n=1 for each test compound) received a single intravenous injection of the PgP transport inhibitor elacridar (3 mg/kg) shortly before oral administration of the test compound at a dose of 1 mg/kg as above. Blood and brain samples were collected 0.5 h post-dose for these animals. In all cases,

-48049235 blood samples were collected in tubes containing potassium EDTA.

Blood and brain samples can be analyzed for test compound concentration using a method based on acetonitrile protein precipitation followed by HPLC/MS-MS analysis using an optimized analytical method.

Analysis.

The concentrations of the test compound in blood (expressed in ng/mL) and brain (expressed in ng/g) at different time points after oral or intravenous administration were analyzed using a non-compartmental pharmacokinetic model using WinNonLin Professional version 4.1. The following parameters were obtained:

Intravenous dosing: maximum concentration over time (Cmax), integrated concentration over time (AUC), clearance (Clb), volume of distribution (Vss) and half-life (t1/2).

Oral dosing: Cmax, time to maximum concentration (Tmax), AUC, bioavailability (F%), fraction absorbed (Fa%), blood-brain ratio (AUC BB), and fold change in AUC BB in the presence of elacridar.

The compounds of the invention can be expected to exhibit good availability in brain tissue.

Biological example 4: In vitro study of metabolic stability in human hepatocytes.

Methodology.

The objective of this study was to determine the metabolic stability of cryopreserved hepatocytes from humans of both sexes. Testosterone and 7-hydroxycoumarin were used as positive controls for phase I and phase II metabolism, respectively.

The incubation medium was prepared by combining William E medium, 1 M HEPES buffer and 200 mM L-glutamine in the following proportions: 88%, 10% and 2%, respectively (440 ml, 50 ml and 10 ml, respectively). The resulting medium was bubbled with carbogen (5% CO2, 95% _O2 ) for 30 min before use. Cryopreserved hepatocytes were thawed and suspended in the incubation medium pre-warmed to 37°C. The cells were centrifuged, resuspended in the medium and counted using a hemocytometer (Bürker chamber). Cell viability was measured using a trypan blue exclusion test.

The test compounds were separately dissolved in DMF to obtain 50 mM stock solutions, which were then diluted in water/acetonitrile 50/50 (v/v) to obtain the corresponding 50 μM working solutions. Testosterone and 7-hydroxycoumarin were dissolved in DMF to obtain a 50 mM testosterone solution and a 5 mM 7-hydroxycoumarin solution. These solutions were then diluted in incubation medium to obtain a 1 mM testosterone working solution and a 500 μM 7-hydroxycoumarin working solution.

µl of each working solution, i.e. 50 µM test compound, 1 mM testosterone and 500 µM 7-hydroxycoumarin, were added to 990 µl of cell suspensions containing 0.5 x 10 ⁶ cells to obtain final concentrations of 0.5 µM, 10 µM and 5 µM, respectively. The concentration of the organic solvent in each incubation was constant and was less than 1% (v/v).

Test compounds were individually incubated at 0.5 μM for 0, 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, and 180 min (12 time points) with cryopreserved human hepatocytes of both sexes at 37°C in a 24-well plate. At each time point, the robotic processor aspirated 50 μl of the incubation mixture from each well and dispensed it into a chilled 96-well plate containing 100 μl of acetonitrile with 150 ng/ml of the appropriate internal standard to stop the reaction. An aliquot of water (120 μl) was then added to equilibrate the organic solvent content to 37%. Samples were centrifuged (approximately 3500 g for 10 min) prior to LCMS/MS analysis.

Positive controls, testosterone and 7-hydroxycoumarin, were incubated separately (n=1) at 10 and 5 μM, respectively, for 0, 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, and 180 min (12 time points) with cryopreserved human hepatocytes of both sexes under the same conditions as described above for the test samples to demonstrate phase I and phase II metabolism in hepatocyte systems. At each time point, the robotic processor aspirated 50 μl of the incubation mixture from each well and dispensed it into a chilled 96-well plate containing 100 μl of acetonitrile with rolipram as an internal standard to stop the reaction. An aliquot of water (120 µL) was then added to equilibrate the organic solvent content to 37%. Samples were centrifuged (approximately 3500 g for 10 min) prior to LCMS/MS analysis.

Metabolic stability was calculated from the ratio of peak areas of the remaining test compound and the internal standard as a function of time.

The internal clearance (CLint) was determined from the first-order elimination rate constant k (min ^-1 ) (obtained using GraphPad by plotting the natural logarithm of the ratio of the peak area of the remaining test sample and the internal standard as a function of time), using

- 49 049235 using the actual incubation volume V (ml), the number of hepatocytes in the incubation M (million cells) and the number of hepatocellularity per g of liver Hn (120 for humans).

Mg liver

CLint values were expressed in ml/min/g liver.____________________________________

Example Connection o n Speed constant k (min ^-1 ) In vitro Clint (ml/min/g liver) RE5 ΈΧαΧ ° 0,002 0.31 RE11 O 0,02 3.58 9 H < ^Z 0,004 1.03 RE13 yr 0,009 2.16 10 0,003 0,70

Compounds of Examples 9 and 10 exhibit low clearance compared to the pyridine reference compounds RE11 and RE13.

Biological example 5: Ames test.

Methodology.

The objective of this in vitro study was to evaluate the potential of the test samples to induce gene mutations in vitro in the bacterial strains Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli WP2 uvrA (pKM101); the testing methodology was based on established bacterial mutagenicity testing methods and the assays were performed in the presence and absence of an exogenous mammalian oxidative metabolism system (S9 mixture).

The study was designed in accordance with national and international guidelines to meet regulatory requirements for toxicity testing of new drugs. The study design complies with the following testing guidelines:

ICH Guideline M3(R2) for the design of nonclinical safety studies for the conduct of large-scale studies and the approval of pharmaceutical products (CPMP/ICH/286/95, June 2009).

ICH Topic S2 (R1) Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use. June 2012.

Bacterial strains.

The following bacterial strains were used:

- 50 049235

Genus Strain Genotype S. typhimurium TA1535 hisG46 rfa Δ uvrB S. typhimurium TA1537 hisC3076 rfa Δ uvrB S. typhimurium TA98 hisD3052 rfa Δ uvrB (pKMIOl) S. typhimurium TAI 00 hisG46 rfa Δ uvrB (pKMIOl) E. coli WP2 uvrA (pKMIOl) TrpE Ochre uvrA (pKMIOl) Source Molecular Toxicology Incorporated, Boone, NC, USA (MolTox™) Growth phase Late logarithmic phase

Strains TA1535, TA100 and WP2 uvrA pKM101 exhibit point mutations. Strains TA1537 and TA98 exhibit frameshift mutations.

Bacterial inocula were used to prepare fresh cultures in 10 ml of nutrient broth (NB2 containing ampicillin for pKM101-containing S. typhimurium strains, strains TA98 and TA100, and E. coli WP2 uvrA (pKM101) to maintain the plasmid copy number). Bacteria were grown for 10-12 h in a shaking incubator at 37±2°C to obtain ^1-2x109 cells/ml.

The bacterial suspension was added to the top layer of agar (containing trace amounts of amino acids required for auxotrophy) in a volume of 100 µl.

Oxidative metabolism system of mammals.

Phenobarbital/5,6-benzoflavone-induced rat liver postmitochondrial fraction (S9) from Molecular Toxicology Incorporated, USA (MolTox™) was used as an exogenous oxidative metabolism system. Batches of S9 fraction stored as frozen aliquots at approximately -80°C were thawed immediately before use. The S9 mixture was prepared by adding S9 (10% v/v) to a NADPH generating system that included NADP (3.15 mg/mL), glucose-6-phosphate (1.5 mg/mL), and 2% v/v saline containing _MgCl2 (81.3 mg/mL) and KCl (123 mg/mL) in phosphate buffer pH 7.4. For treatment in the presence of S9 mixture, S9 mixture was used in a final volume of 500 µl/plate. For treatment in the absence of S9 mixture, an equivalent volume of sterile phosphate buffer pH 7.4 was added instead of S9 mixture.

Positive control drugs.

The following positive controls (supplied by MolTox™ via Trinova Biochem GmbH, Giessen, Germany and Sigma Aldrich, Milan, Italy) were used and prepared as follows: ______________________________________________________________________

Bacterial strain Positive control Concentration (mcg/tablet) Carrier (solvent) Mixture S9 TA98 2-Nitrofluorene (2NF) 2 Dimethyl sulfoxide (DMSO) No TA1535, TA100 Sodium azide (NaAz) 2 _H2O No TA1537 ICR-191 1 DMSO No WP2 uvrA (pKMIOl) 4-Nitroquinoline-1 oxide (4NQO) 1 DMSO No TA98 Benz[a]pyrene (B[a]P) 1.25 DMSO Yes TA1535, TA1537, TA100, WP2 uvrA (pKMIOl) 2-Aminoanthracene (2AAN) 5 DMSO Yes

Positive controls were prepared from frozen (approximately -20°C) stock solutions and stored at ambient temperature during use.

- 51 049235

Test samples.

The test consisted of quadruplicate plates for vehicle (DMSO) controls and 2 duplicate plates for test sample and positive controls processed in the absence and presence of S9 mixture. A range of test sample concentrations were tested, starting from 5 μg/plate to 5000 μg/plate, as follows:

Genus Strain Test sample concentrations (µg/cup) Mixture S9 S. typhimurium TA1535, TA1537, TA98 and TA100 5, 15, 50, 150, 500, 1500 and 5000 No E. coli WP2 uvrA (pKMIOl) 8. typhimurium TA1535, TA1537, TA98 and TA100 Yes E. coli WP2 uvrA (pKMIOl)

Vehicle, test sample and positive control preparations were added to the plates at a volume of 100 µl/plate.

Processing of plates and incubation.

Trace amounts of histidine and biotin or tryptophan were added to the overlay agar, aliquoted (2 ml/plate) and maintained at 46 ± 2°C. The appropriate bacterial suspension was added to 2 ml of overlay agar, followed by the addition of the test sample or vehicle/positive control solutions and sterile phosphate buffer pH 7.4 or S9 mixture. This mixture, after final processing, was poured onto minimal agar plates (Vogel-Bonner plates) and incubated in the dark for approximately 64 hours at 37 ± 2°C.

Colony counting and plate analysis.

At the end of the incubation period, plates were assessed (by visual inspection) for precipitation of the test sample. Plates were electronically assessed for bacterial colony formation using a ProtoCOL3 Synbiosis colony counter. Where precipitation of the test sample occurred, bacterial colony counts for each strain were performed manually and stopped at the lowest treatment concentration that did not interfere with manual counting.

Colony counts were followed by inspection of plates for signs of toxicity (i.e. decreased growth/reduction in background lawn, presence of pinhead colonies/pseudo-revertant colonies, and/or decreased colony counts).

If the data for any treatment concentration show a response at least 2-fold above the parallel vehicle control value for TA98, TA100, and WP2 uvrA (pKM101), or at least 3-fold above the parallel vehicle control value for TA1535 and TA1537, in combination with a dose-dependent response, the result should be considered positive. Results that only partially satisfy these criteria, or where the data for any strain show a dose-dependent response but do not exceed the 2- or 3-fold threshold as detailed, are considered equivocal.

The following acceptance criteria were used:

1. The highest concentration tested should be 5000 µg/plate or limited by the solubility of the test sample in the vehicle.

2. If the solubility of the test sample is a limiting factor, the maximum concentration selected for plate counting will be the lowest concentration at which precipitation of the test sample is observed on the treated plates at the end of the incubation period and which does not interfere with the count.

If toxicity is the limiting factor, the maximum concentration determined for a gene mutation will be the lowest concentration at which evidence of significant bacterial toxicity is observed during plate counting.

- 52 049235

Results.

Example Connection Ames test result Aniline Ames test result RE5 u Non-mutagenic Not tested Not tested RE6 °y-N „ Гт * /=\ xL JJ о ГЛ ° °хУ\7 Non-mutagenic .O / c Mutagenic for TA1535 in the presence of RE11 li < Non-mutagenic metabolic activation at 150 mcg/cup 1 o _x V q xg ν Non-mutagenic / ΥΛα ch Non-mutagenic 9 ΙΧίϊΤ Non-mutagenic 10 Non-mutagenic p.<g Non-mutagenic

The aniline associated with RE6/RE11, which has been shown to degrade under certain conditions, was found to be mutagenic. This finding poses risks to the future development of RE6/RE11 as well as to compounds that can produce related anilines (e.g., (5R)-5-ethyl-3-(6-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxy-3-pyridyl)imidazolidine-2,4-dione, i.e., RE13). Compounds that can be distinguished on the basis of their associated anilines are preferred.

The anilines for the compounds of Examples 1, 9 and 10 are not mutagenic, which may be expected to apply to other compounds of the invention that can produce related anilines.

Additional animal models.

Patent applications WO2011/069951, WO2012/076877, WO2012/168710, WO2013/083994, WO2013/175215 and WO2013/182851 (all incorporated by reference for the purpose of illustrating the potential utility of the compounds and providing animal models for testing the compounds) demonstrate the activity of compounds that are modulators of Kv3.1 and Kv3.2 in animal models of seizures, hyperactivity, sleep disorders, psychosis, hearing impairment and bipolar disorders.

Patent application WO2013/175211 (incorporated by reference for the purpose of illustrating the potential utility of the compounds and providing animal models for testing the compounds) demonstrates the efficacy of a compound that is a modulator of Kv3.1 and Kv3.2 in a chinchilla model of acute noise-induced hearing loss, and also evaluates the efficacy of the compound in a model of central auditory processing deficit and in a model of tinnitus.

Glait et al 2018, Anderson et al 2018 and Chamber et al 2018 demonstrated the efficacy of Kv3.1 and Kv3.2 modulator in hearing-related models.

Patent application WO2017/098254 (incorporated by reference to illustrate the potential utility of the compounds and to provide animal models for testing the compounds) demonstrates the efficacy of a compound that is a modulator of Kv3.1 and Kv3.2 in models of neuropathic and inflammatory pain.

Throughout the description and the following claims, unless the context otherwise requires, the word "comprise" and such variations as "comprises" and "comprising" are to be understood as implying the inclusion of the stated integer, step, group of integers, or group of steps, but not the exclusion of any other integer, step, group of integers, or group of steps.

The application of which this description and claims form a part may be used as a basis for priority in relation to any subsequent application. Claims

- 53 049235 of such subsequent application may be directed to any feature or combination of features described in this document. They may be presented as a product, composition, method or claim for use and may include, by way of example and without limitation, the claims that follow.

Points of the invention:

Item 1 - Compound of formula (I):

Where:

Ri is H or methyl;

R2 and _R3 both represent methyl, or R2 and _R3 , together with the carbon atom to which they are attached, represent a spirocyclopropyl ring;

R4 represents methyl or ethyl;

R5 is H or methyl;

or R4 and R5, together with the carbon atom to which they are attached, form _C3 - _C4 spirocarbocyclyl;

or its salt and/or solvate and/or derivative.

Item 2 - The compound according to item 1, wherein R1 is H.

Item 3 - A compound according to item 1, wherein R ₁ is methyl.

Item 4 - A compound according to any one of items 1-3, wherein R2 and _R3 represent a spirocyclopropyl ring.

Item 5 - A compound according to any one of items 1-3, wherein R2 is methyl and _R3 is methyl.

Item 6 - A compound according to any one of items 1-5, wherein R4 is methyl.

Item 7 - A compound according to any one of items 1-5, wherein R4 is ethyl.

Item 8 - A compound according to any one of items 1-7, wherein R5 is H.

Item 9 - A compound according to any one of items 1-7, wherein R5 is methyl.

Item 10 - A compound according to any one of items 1-9, wherein when R4 and R5 are different, they have the following stereochemical configuration:

Item 11 - A compound according to any one of items 1-9, wherein when R ₄ and R ₅ are different, they have the following stereochemical configuration:

Item 12 - A compound according to any one of items 1 to 5, wherein R4 and R5, together with the carbon atom to which they are attached, form spirocyclopropyl.

Item 13 - A compound according to any one of items 1 to 5, wherein R ₄ and R ₅ , together with the carbon atom to which they are attached, form spirocyclobutyl.

Item 14 - A compound according to item 1, selected from the group consisting of:

- 54 049235

5,5-dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]imidazolidin-2,4-dione;

-[5 - [(3,3-d imethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5,5-dimethylimidazolidine-2,4-dione;

(5R)-5-ethyl-5-methyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4yloxypyrazin-2-yl)azolidin-2,4-dione imide;

5,5-dimethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2yl)imide in-2,4-dione azolide;

(5R)-5-ethyl-5-methyl-3-[5-(7-methylpyro[2H-benzofuran-3,Γ-cyclopropane]-4yl)oxypyrazin-2-yl]imidazolidine-2,4-dione;

(5R)-3 -[5 - [(3,3-d imethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5 -ethyl-5 methyl-imidazolidine-2,4-dione;

5,5-dimethyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]imidazolidin-2,4-dione;

(5R)-5-ethyl-5-methyl-3-[5-[(3,3,7-trimethyl-2H-benzopyran-4-yl)oxy]pyrazin-2-yl]imide azolide in-2,4-dione;

(5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]imidazolidin-2,4-dione;

(5R)-5-ethyl-3-(5-cypo[2H-benzopyran-3,Γ-cyclopropane]-4-yloxypyrazin-2yl)imide azolide in-2,4-dione;

(5R)-3 -[5 - [(3,3-d imethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5 -ethylimidazolide in-2,4-dione;

(5R)-5-ethyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]imidazolidin-2,4-dione;

7-[5-(7-methylspiro[2H-benzofuran-3,N-cyclopropan]-4-yl)oxypyrazin-2-yl]5,7-diazaspiro[3.4]octane-6,8-dione;

6-[5-(7-methylspiro[2H-benzofuran-3,G-cyclopropan]-4-yl)oxypyrazin-2-yl]4,6-diazaspiro[2.4]heptane-5,7-dione;

(58)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]imidazolidin-2,4-dione;

or its salt and/or solvate and/or derivative thereof.

Item 15 - The compound according to item 1, which is: 5,5-dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,N-cyclopropan]-4-yl)-oxypyrazin-2-yl]imidazolidine-2,4-dione.

Item 16 - A compound according to item 1, which is: 3-[5-[(3,3-dimethyl-2Hbenzofuran-4-yl)oxy]pyrazin-2-yl]-5,5-dimethylimidazolidine-2,4-dione.

Item 17 - The compound according to item 1, which is: (5R)-5-ethyl-5-methyl-3-(5spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-yl)imidazolidine-2,4-dione.

Item 18 - The compound according to item 1, which is: 5,5-dimethyl-3-(5-spiro[2Hbenzofuran-3,1'-cyclopropan]-4-yloxypyrazin-2-yl)imidazolidine-2,4-dione.

Item 19 - The compound according to item 1, which is: (5R)-5-ethyl-5-methyl-3-[5-(7methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione.

Item 20 - A compound according to item 1, which is: (5R)-3-[5-[(3,3-dimethyl-2Hbenzofuran-4-yl)oxy]pyrazin-2-yl]-5-ethyl-5-methyl-imidazolidine-2,4-dione.

Item 21 - The compound according to item 1, which is: 5,5-dimethyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-imidazolidine-2,4-dione.

Item 22 - The compound according to item 1, which is: (5R)-5-ethyl-5-methyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]imidazolidine-2,4-dione.

Item 23 - The compound according to item 1, which is: (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)-oxypyrazin-2-yl]imidazolidine-2,4-dione.

- 55 049235

Item 24 - A compound according to item 1, which is: (5R)-5-ethyl-3-(5-spiro[2Hbenzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-yl)imidazolidine-2,4-dione.

Item 25 - A compound according to item 1, which is: (5R)-3-[5-[(3,3-dimethyl-2Hbenzofuran-4-yl)oxy]pyrazin-2-yl]-5-ethyl-imidazolidine-2,4-dione.

Item 26 - The compound according to item 1, which is: (5R)-5-ethyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]imidazolidine-2,4-dione.

Item 27 - The compound according to item 1, which is: 7-[5-(7-methylspiro[2Hbenzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]-5,7-diazaspiro[3.4]octane-6,8-dione.

Item 28 - The compound according to item 1, which is: 6-[5-(7-methylspiro[2Hbenzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]-4,6-diazaspiro[2.4]heptane-5,7-dione.

Item 29 - The compound according to item 1, which is: (58)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)-oxypyrazin-2-yl]imidazolidine-2,4-dione.

Item 30 - A compound of formula (I) according to any one of items 1-29 or a pharmaceutically acceptable salt and/or solvate thereof.

Item 31 - A compound according to any one of items 1-30 for use as a medicine.

Item 32 - A compound according to item 31 for use in the prevention or treatment of a disease or disorder selected from the group consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse, anxiety disorders, sleep disorders, hyperacusis and auditory perception disorders, Meniere's disease, balance disorders and inner ear disorders, impulse control disorder, personality disorders, attention deficit hyperactivity disorder, autism spectrum disorders, eating disorders, cognitive impairment, ataxia, pain such as neuropathic pain, inflammatory pain and mixed pain, dementia with Lewy bodies and Parkinson's disease.

Item 33 - A compound according to item 31 for use in the prevention or treatment of schizophrenia.

Item 34 - A compound according to item 31 for use in the prevention or treatment of hearing disorders.

Item 35 - A compound according to item 31 for use in the prevention or treatment of pain.

Item 36 - The compound according to item 31 for use in the treatment of fragile X syndrome.

Item 37 - A method for preventing or treating a disease or disorder selected from the group consisting of hearing impairment, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse, anxiety disorders, sleep disorders, hyperacusis and auditory processing disorders, Meniere's disease, balance disorders and inner ear disorders, impulse control disorder, personality disorders, attention deficit hyperactivity disorder, autism spectrum disorders, eating disorders, cognitive impairment, ataxia, pain such as neuropathic pain, inflammatory pain and mixed pain, dementia with Lewy bodies and Parkinson's disease, comprising administering to a subject in need thereof an effective amount of a compound according to any one of items 1-30.

Item 38 - A method for the prevention or treatment of schizophrenia comprising administering to a subject in need thereof a compound according to any one of items 1-30.

Item 39 - A method for preventing or treating hearing disorders comprising administering to a subject in need thereof a compound according to any one of items 1-30.

Item 40 - A method for preventing or treating pain, comprising administering to a subject in need thereof a compound according to any one of items 1-30.

Item 41 - A method of treating fragile X syndrome comprising administering to a subject in need thereof a compound according to any one of items 1-30.

Item 42 - Use of a compound according to any one of items 1-30 in the manufacture of a medicament for the prevention or treatment of a disease or disorder selected from the group consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse, anxiety disorders, sleep disorders, hyperacusis and auditory perception disorders, Meniere's disease, balance disorders and inner ear disorders, impulse control disorder, personality disorders, attention deficit hyperactivity disorder, autism spectrum disorders, eating disorders, cognitive impairment, ataxia, pain such as neuropathic pain, inflammatory pain and mixed pain, dementia with Lewy bodies and Parkinson's disease.

Item 43 - Use of a compound according to any of items 1-30 in the manufacture of a medicament for the prevention or treatment of schizophrenia.

Item 44 - Use of a compound according to any of items 1-30 in the manufacture of a medicament for the prevention or treatment of hearing disorders.

- 56 049235

Item 45 - Use of a compound according to any of items 1-30 in the manufacture of a medicament for the prevention or treatment of pain.

Item 46 - Use of a compound according to any of items 1-30 in the manufacture of a medicament for the treatment of fragile X syndrome.

Item 47 - A pharmaceutical composition comprising a compound according to any of items 130 and a pharmaceutically acceptable carrier or excipient.

Item 48 - A compound according to any one of items 1-30 for use in combination with an additional pharmaceutically acceptable active ingredient.

Item 49 - Compound of formula (II) or (XVI):

^R 3 (II), where R1, R2 and R ₃ are as defined in paragraph 1, X is a halogen such as Br.

Item 50 - Compound of formula (XVI):

where R _b R2 and R ₃ are as defined in claim 1. Claim 51 - A compound of formula (IV):

where R ₄ and R 5 are as defined in paragraph 1, Y is a halogen such as Cl.

Item 52 - A derivative of a compound of formula (I) or a salt and/or solvate thereof according to any one of items 1-30, functionalized via a secondary nitrogen atom of a hydantoin or via a secondary nitrogen atom of a triazolone with a group L selected from the groups consisting of:

a) -PO(OH)O ^- -M ⁺ , where M ⁺ is a pharmaceutically acceptable monovalent counterion,

b) -PO(O ^- )2-2M ⁺ ,

c) -PO(O ^- ) ₂ -D ²⁺ , where D ²⁺ is a pharmaceutically acceptable divalent counterion,

d) -CH(R ^X )-PO(OH)O ^- -M ⁺ , where R ^X is hydrogen or C _1-3 alkyl,

e) -CH(R ^x )-PO(O ^- )2-2M ⁺ ,

e) -CH(R ^X )-PO(O ^- ) ₂ -D ²⁺ ,

g) -SO3--M+,

c) -CH(R ^X )-SO3 ^- -M ⁺ , and

i) -CO-CH2CH2-CO2-M+.

Item 53 - A compound according to any of items 1-36, which is in a natural isotopic form.

Item 54 - A compound, method, use, composition or derivative according to any of items 1-48, 52 or 53 for oral administration.

Item 55 - A compound, method, use, composition or derivative according to any of items 1-48 or 52-54 for administration in an amount of 2 to 400 mg per day, such as 2 to 300 mg per day, especially 5 to 250 mg per day.

Item 56 - A compound, method, use, composition or derivative according to any of items 1-48 or 52-55 for administration once or twice daily.

Item 57 - Compound according to item 56 for administration once daily.

Item 58 - Compound according to item 56 for administration twice daily.

Item 59 - A compound, method, use, composition or derivative according to any of items 1-48 or 52-58 for administration for at least three months.

Item 60 - A compound, method, use, composition or derivative according to any of items 1-48 or 52-58 for administration to a human.

Item 61 - A compound, method, use, composition or derivative according to item 60 for

- 57 049235 administration to an adult, for example aged 18 to 65 years.

Item 62 - A compound, method, use, composition or derivative according to item 60 for administration to a human aged 66 years or older.

Item 63 - A compound, method, use, composition or derivative according to item 60 for administration to a human under 18 years of age, such as between 4 and 17 years of age.

Item 64 - A compound, method, use, composition or derivative according to any of items 1-48, 52, 53 or 59-63, wherein the compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof is delivered via a patch or implant.

Links.

All publications, including, without limitation, patents and patent applications, cited in this specification are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference herein as if fully set forth.

Anderson LA et al. Increased spontaneous firing rates in auditorium midbrain following noise exposure are specifically abolished by a Kv3 channel modulator. Hear Res. 2018

Aug;365:77-89

Aroniadou-Anderjaska V et al. Mechanisms regulating GABAergic inhibitory transmission in the basolateral amygdala: implications for epilepsy and anxiety disorders.

Amino Acids 2007 Aug;32:305-315.

Baranauskas G, Nistri A. Sensitization of pain pathways in the spinal cord: cellular mechanisms. Prog. Neurobiol. 1998 Feb;54(3):349-65.

Baron R et al. Peripheral input and its importance for central sensitization. Ann. Neurol.

2013 Nov;74(5):630-6.

- 58 049235

Ben-Ari Y. Seizure Beget Seizure: The Quest for GABA as a Key Player. Crit. Rev. Neurobiol. 2006;18(l-2): 135-144.

Benes FM et al. Circuitry-based gene expression profiles in GABA cells of the trisynaptic pathway in schizophrenics versus bipolars. PNAS 2008 Dec; 105(52):20935–20940.

Bennett DL, Woods CG. Painful and painless channelopathies. Lancet Neurol. 2014 Jun;13(6):587-99.

Berge S et al. Pharmaceutical Salts. J. Pharm. Sci. 1977;66;1-19.

Brambilla P et al. GABAergic dysfunction in mood disorders. Mol. Psych. 2003 Apr;8:721-737.

Brooke RE et al. Spinal cord interneurones labeled transneuronally from the adrenal gland by a GFP-herpes virus construct contain the potassium channel subunit Kv3.1b. Auton. Neurosci. 2002 Jun;98(l-2):45-50.

Brooke RE et al. Association of potassium channel Kv3.4 subunits with pre- and postsynaptic structures in brainstem and spinal cord. Neuroscience 2004;126(4): 1001-10.

Brooke RE et al. Immunohistochemical localization of the voltage gated potassium ion channel subunit Kv3.3 in the rat medulla oblongata and thoracic spinal cord. Brain Res. 2006 Jan;1070(l): 101-15.

Cervero F. Spinal cord hyperexcitability and its role in pain and hyperalgesia. Exp. Brain Res. 2009 Jun;196(l): 129-37.

Chambers AR et al. Pharmacological modulation of Kv3.1 mitigates auditory midbrain temporal processing deficits following auditory nerve damage. Sci Rep. 2017 Dec 13;7(1): 17496

Chang S.Y. et al. Distribution ofKv3.3 Potassium Channel Subunits in Distinct Neuronal Populations of Mouse Brain. J. Comp. Neuro. 2007 Feb;502:953-972.

Chien LY et al. Reduced expression of A-type potassium channels in primary sensory neurons induces mechanical hypersensitivity. J. Neurosci. 2007 Sep;27(37):9855-65.

Chow A et al. K ⁺ Channel Expression Distinguishes Subpopulations of Parvalbumin and Somatostatin-Containing Neocortical Interneurons. J. Neurosci. 1999 Nov;19(21):93329345.

Desai R et al. Protein Kinase C Modulates Inactivation of Kv3.3 Channels. J Biol. Chem. 2008;283;22283-22294.

- 59 049235

Deuchars SA et al. Properties of interneurones in the intermediolateral cell column of the rat spinal cord: role of the potassium channel subunit Kv3.1. Neuroscience 2001;106(2):433-46.

Devulder J. Flupirtine in pain management: pharmacological properties and clinical use. CNS Drugs 2010 Oct;24(10):867-8L

Dib-Hajj SD et al. The Na(V)1.7 sodium channel: from molecule to man. Nat. Rev. Neurosci. 2013 Jan;14(l):49-62.

Diochot S et al. Sea Anemone Peptides with a Specific Blocking Activity against the Fast Inactivating Potassium Channel Kv3.4. J Biol. Chem. 1998 Mar;273(12);6744-6749.

Engel AK et al. Dynamic Predictions: Oscillations and Synchrony in Top-Down Processing. Nat. Rev. Neurosci. 2001 Oct;2(10):704-716.

Espinosa F et al. Alcohol Hypersensitivity, Increased Locomotion, and Spontaneous Myoclonus in Mice Lacking the Potassium Channels Kv3.1 and Kv3.3. J. Neurosci. 2001 Sep;21(17):6657-6665.

Espinosa F et al. Ablation of Kv3.1 and Kv3.3 Potassium Channels Disrupts Thalamocortical Oscillations In Vitro and In Vivo. J. Neurosci. 2008 May;28(21):5570-5581.

Figueroa K et al. KCNC3: phenotype, mutations, channel biophysics - a study of 260 familial ataxia patients. Human Mutation. 2010;31; 191-196.

Finnerup NB et al. Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. Lancet Neurol. 2015 Feb;14(2): 162-73.

Fisahn A. Kainate receptors and rhythmic activity in neuronal networks: hippocampal gamma oscillations as a tool. J. Physiol. 2005 Oct;561(l):65-72.

Glait L et al. Effects of AUT00063, a Kv3.1 channel modulator, on noise-induced hyperactivity in the dorsal cochlear nucleus. Hear Res. 2018 Apr;361:36-44

Greene TW, Wuts, PG. Greene's Protective Groups in Organic Synthesis, 2006, 4th Edition, John Wiley & Sons, Inc., Hoboken, NJ, USA.

Joho RH et al. Increased γ- and Decreased δ-Oscillations in a Mouse Deficient for a Potassium Channel Expressed in Fast-Spiking Interneurons. J. Neurophysiol. 1999 Jun;82:1855-1864.

Joho RH, Hurlock EC. The Role of Kv3-type Potassium Channels in Cerebellar Physiology and Behavior. Cerebellum 2009 Feb;8:323-333.

Jung D et al. Age-related changes in the distribution of Kvl. 1 and Kv3.1 in rat cochlear nuclei. Neurol. 7?ey2005;27;436-440.

- 60 049235

Kasten MR et al. Differential regulation of action potential firing in adult murine thalamocortical neurons by Kv3.2, Kvl, and SK potassium and N-type calcium channels. J. Physiol. 2007;584(2):565-582.

Kaczmarek L et al. Regulation of the timing of MNTB neurons by short-term and longterm modulation of potassium channels. Hearing Res. 2005;206;133-145.

Lau D et al. Impaired Fast-Spiking, Suppressed Cortical Inhibition, and

Increased Susceptibility to Seizures in Mice Lacking Kv3.2 K ⁺ Channel Proteins. J. Neurosci. 2000 Dec;20(24):9071-9085.

Li W et al. Localization of Two High-Threshol Potassium Channel Subunits in the Rat Central Auditory System. J. Comp. Neuro. 2001 May;437:196-218.

Lu R et al. Slack channels expressed in sensory neurons control neuropathic pain in mice. J. Neurosci. 2015 Jan;35(3): 1125-35.

Markram H et al. Interneurons of the neocortical inhibitory system. Nat. Rev. Neurosci. 2004 Oct;5:793-807.

Martina M et al. Functional and Molecular Differences between Voltage-Gated K ⁺ Channels of Fast-Spiking Interneurons and Pyramidal Neurons of Rat Hippocampus. J. Neurosci. 1998 Oct;18(20):8111-8125.

McCarberg BH et al. The impact of pain on quality of life and the unmet needs of pain management: results from pain sufferers and physicians participating in an Internet survey. Am. J. Ther. 2008 Jul-Aug;15(4):312-20.

McDonald AJ, Mascagni F. Differential expression of Kv3.1b and Kv3.2 potassium channel subunits in interneurons of the basolateral amygdala. Neuroscience 2006;138:537-547.

McMahon A et al. Allele-dependent changes of olivocerebellar circuit properties in the absence of the voltage-gated potassium channels Kv3.1 and Kv3.3. Eur. J. Neurosci. 2004 Mar;19:3317-3327.

Mitchell 1 et al. Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound. ACSMed. Chem. Lett. 2015;6(6);655-659.

Muona M, et al. A recurrent de novo mutation in KCNC1 causes progressive myoclonus epilepsy. Nat Genet. 2015 Jan;47(l):39-46.

Muqeem T et al. Regulation of Nociceptive Glutamatergic Signaling by Presynaptic Kv3.4 Channels in the Rat Spinal Dorsal Hom JNeurosci. 2018 Apr 11;38(15):3729-3740

Olsen T et al. Kv3 K+ currents contribute to spike-timing in dorsal cochlear nucleus principal cells. Neuropharmacology 2018 May 1;133:319-333

- 61 049235

Pilati N et al.. Acoustic over-exposure triggers burst firing in dorsal cochlear nucleus fusiform cells. Hearing Research 2012;283;98-106.

Puente N et al. Precise localization of the voltage-gated potassium channel subunits Kv3.1b and Kv3.3 revealed in the molecular layer of the rat cerebellar cortex by a preembedding immunogold method. Histochem. Cell. Biol. 2010 Sep;134:403-409.

Reynolds GP et al. Calcium Binding Protein Markers of GABA Deficits in Schizophrenia - Post Mortem Studies and Animal Models. Neurotox. Res. 2004 Feb;6(l):5762.

Ritter DM et al. Modulation of Kv3.4 channel N-type inactivation by protein kinase C shapes the action potential in dorsal root ganglion neurons. J. Physiol. 2012 Jan;590(Pt 1):14561.

Ritter DM et al. Dysregulation of Kv3.4 channels in dorsal root ganglia following spinal cord injury. J. Neurosci. 2015 Jan;35(3): 1260-73.

Roberts L et al. Ringing Ears: The Neuroscience of Tinnitus. J. Neurosci. 2010:30(45);14972-14979.

Rudy B, McBain CJ. Kv3 channels: voltage-gated K ⁺ channels designed for highfrequency repetitive firing. TRENDS in Neurosci. 2001 Sep;24(9): 517-526.

Sacco T et al. Properties and expression of Kv3 channels in cerebellar Purkinje cells. Mol. Cell. Neurosci. 2006 Jul;33:170-179.

Schulz P, Steimer T. Neurobiology of Circadian Systems. CNS Drugs 2009;23(Suppl. 2):3-13.

Song P et al. Acoustic environment determines phosphorylation state of the Kv3.1 potassium channel in auditory neurons Nat. Neurosci. 2005 Oct;8(10): 1335-1342.

Spencer KM et al. Neural synchrony indexes disordered perception and cognition in schizophrenia. PNAS 2004 Dec;101(49): 17288-17293.

Sun S et al. Inhibitors of voltage-gated sodium channel Navi.7: patent applications since 2010. Pharm. Pat. Anal. 2014 Sep;3(5):509-21.

U.S. Department of Health and Human Services, Food and Drug Administration. Draft Guidance for Industry Analgesic Indications: Developing Drug and Biological Products: http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/uc m384691.pdf 2014 Feb.

- 62 049235 von Hehn C et al. Loss of Kv3.1 Tonotopicity and Alterations in cAMP Response Element-Binding Protein Signaling in Central Auditory Neurons of Hearing Impaired Mice. J. Neurosci. 2004;24: 1936-1940.

Weiser M et al. Differential Expression of Shaw-related K ⁺ Channels in the Rat Central Nervous System. J. Neurosci. 1994 Mar;14(3):949-972.

Wickenden AD, McNaughton-Smith G. Kv7 channels as targets for the treatment of pain. Curr. Pharm. Des. 2009;15(15): 1773-98.

Woolf CJ. What is this thing called pain? J. Clin. Invest. 2010 Nov;120(l 1):3742-4.

Woolf CJ. Central sensitization: implications for the diagnosis and treatment of pain. Pain 2011 Mar; 152(3 Suppl):S2-15.

Yanagi M et al. Kv3.1-containing K(+) channels are reduced in untreated schizophrenia and normalized with antipsychotic drugs. Mol Psychiatry. 2014. 19(5):573-9.

Yeung SYM et al. Modulation of Kv3 Subfamily Potassium Currents by the Sea Anemone Toxin BDS: Significance for CNS and Biophysical Studies. J. Neurosci. 2005 Mar;25(38):8735-8745.

Zamponi GW et al. The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential Pharmacol Rev. 2015 Oct;67(4): 82170.

Claims (35)

1. A compound or its salt, which is a compound of formula (I): where: R ₁ is H or methyl; R2 and _R3 both represent methyl, or R2 and _R3 , together with the carbon atom to which they are attached, represent a spirocyclopropyl ring; R4 represents methyl or ethyl; R5 is H or methyl; or R4 and R5, together with the carbon atom to which they are attached, form _C3 - _C4 spirocarbocyclyl; or its salt. 2. A compound or salt thereof according to claim 1, which is a pharmaceutically acceptable salt of a compound of formula (I). 3-[5-[(3,3-dimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5,5-dimethylimidazolidine-2,4-dione; (5R)-5-ethyl-5-methyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2yl)imidazolidine-2,4-dione; 3. A compound or salt thereof according to claim 1, which is a compound of formula (I). 4. A compound or salt thereof according to any one of claims 1 to 3, wherein R1 is H. 5,5-dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2yl]imidazolidin-2,4-dione: 5,5-dimethyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]imidazolidin-2,4dione; (5R)-5-ethyl-5-methyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]imidazolidin2,4-dione; (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2yl]imidazolidin-2,4-dione; (5R)-5-ethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-yl)imidazolidine-2,4-dione; (5R)-3-[5-[(3,3-dimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5-ethyl-imidazolidine-2,4-dione; (5R)-5-ethyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]imidazolidin-2,4-dione; 5,5-dimethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-yl)imidazolidin2,4-dione; - 63 049235 (5R)-5-ethyl-5-methyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2 yl] imidazolidine-2,4-dione; (5R)-3-[5-[(3,3-dimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5-ethyl-5-methyl-imidazolidine-2,4dione; 5,5-dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2yl]imidazolidine-2,4-dione; 5. A compound or salt thereof according to any one of claims 1 to 3, wherein R1 is methyl. 6-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]-4,6-diazaspiro[2.4]heptane-5,7-dione and (5S)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione or a pharmaceutically acceptable salt thereof. 6. A compound or salt thereof according to any one of claims 1 to 5, wherein R2 and _R3 represent spirocyclopropyl. 7-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]-5,7-diazaspiro[3.4]octane-6,8-dione; 7. A compound or salt thereof according to any one of claims 1 to 6, wherein R4 is methyl and R5 is methyl. 8. A compound or salt thereof according to any one of claims 1 to 6, wherein R4 is ethyl and R5 is H. 9. A compound or salt thereof according to claim 2 or 3, selected from the group consisting of: 10. The compound or salt thereof according to claim 1, which is 5,5-dimethyl-3-[5-(7-methylspiro[2Hbenzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione: or a pharmaceutically acceptable salt thereof. 11. The compound or salt thereof according to claim 10, which is the compound: 5,5-dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione: 12. The compound or salt thereof according to claim 10, which is a pharmaceutically acceptable salt: 13. The compound or salt thereof according to claim 1, which is (5R)-5-ethyl-3-[5-(7-methylspiro[2Hbenzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione: or a pharmaceutically acceptable salt thereof. 14. The compound or its salt according to claim 13, which is a compound: (5R)-5-ethyl-3-[5-(7 - 64 049235 methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]imidazolidin-2,4-dione: 15. A compound or salt thereof according to claim 13, which is a pharmaceutically acceptable salt of: (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropan]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione: 16. The compound or salt thereof according to claim 1, which is (5R)-5-ethyl-3-(5-spiro[2H-benzofuran3,1'-cyclopropane]-4-yloxypyrazin-2-yl)imidazolidine-2,4-dione: or a pharmaceutically acceptable salt thereof. 17. The compound or salt thereof according to claim 16, which is the compound: (5R)-5-ethyl-3-(5spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-yl)imidazolidine-2,4-dione: 18. The compound or salt thereof according to claim 16, which is a pharmaceutically acceptable salt: (5R)-5-ethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-yl)imidazolidine-2,4-dione: 19. The use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 2 to 18 in the manufacture of a medicament for the prevention or treatment of a disease or disorder in which a modulator of Kv3.1 and/or Kv3.2 and/or Kv3.3 is required. 20. The use according to claim 19, wherein the disease or disorder for which a Kv3.1 and/or Kv3.2 and/or Kv3.3 modulator is required is selected from the group consisting of bipolar disorder, schizophrenia, attention deficit hyperactivity disorder, epilepsy, seizure, hearing impairment, pain and sleep disorders. 21. A method for the prevention or treatment of a disease or disorder requiring a Kv3.1 and/or Kv3.2 and/or Kv3.3 modulator, comprising administering to a subject in need thereof an effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 2 to 18. 22. The method according to claim 21, wherein the disease or disorder for which a Kv3.1 modulator is required - 65 049235 and/or Kv3.2 and/or Kv3.3 is selected from the group consisting of bipolar disorder, schizophrenia, attention deficit hyperactivity disorder, epilepsy, seizure, hearing impairment, pain and sleep disorders. 23. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 2 to 18 and a pharmaceutically acceptable carrier or excipient. 24. Use according to claim 19 or 20, wherein the compound or its salt is administered orally. 25. Use according to claim 19, 20 or 24, wherein the compound or its salt is administered in an amount of 2 to 400 mg per day, for example 2 to 300 mg per day, especially 5 to 250 mg per day. 26. Use according to any one of claims 19, 20, 24 or 25, wherein the compound or its salt is administered once a day. 27. Use according to any one of claims 19, 20, 24 or 25, wherein the compound or its salt is administered twice daily. 28. The use according to any one of claims 19, 20 and 24-27, wherein the compound or its salt is administered for at least three months. 29. The method according to claim 21 or 22, wherein the compound or its salt is administered orally. 30. The method according to claim 21, 22 or 29, wherein the compound or its salt is administered in an amount of 2 to 400 mg per day, for example 2 to 300 mg per day, especially 5 to 250 mg per day. 31. The method according to any one of claims 21, 22, 29 or 30, wherein the compound or its salt is administered once a day. 32. The method according to any one of claims 21, 22, 29 or 30, wherein the compound or its salt is administered twice daily. 33. The method according to any one of claims 21, 22 and 29-32, wherein the compound or salt thereof is administered for at least three months. 34. Compound of formula (II): where R _b R ₂ and R ₃ are as defined in paragraph 1, X is halogen. 35. Compound of formula (XVI): where Ri, R ₂ and R ₃ are as defined in paragraph 1. 36. A compound of formula (IV): where R ₄ and R ₅ are as defined in paragraph 1, Y is halogen.