AU2020427632B2

AU2020427632B2 - Kv3 modulators

Info

Publication number: AU2020427632B2
Application number: AU2020427632A
Authority: AU
Inventors: Giuseppe Alvaro; Agostino Marasco
Original assignee: Autifony Therapeutics Ltd
Current assignee: Autifony Therapeutics Ltd
Priority date: 2020-02-06
Filing date: 2020-02-06
Publication date: 2025-12-18
Anticipated expiration: 2040-02-06
Also published as: MX2022009702A; IL295027A; NZ790491A; CL2022002081A1; AU2020427632A1; KR20220139923A; CN115066424A; JP7522203B2; IL295027B1; JP2023523501A; CA3169057A1; WO2021156584A1; KR102937911B1; BR112022013339A2; PH12022551955A1; EP4100402A1; CN115066424B

Abstract

A compound of formula (I) and related aspects.

Description

WO wo 2021/156584 PCT/GB2020/050268

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KV3 MODULATORS Technical field

This invention relates to novel compounds, pharmaceutical compositions containing them and

their use in therapy, in particular in the prophylaxis or treatment of hearing disorders, including

hearing loss and tinnitus, as well as schizophrenia, substance abuse disorders, pain and Fragile

X syndrome.

Background to the invention

The Kv3 voltage-gated potassium channel family includes four members, Kv3.1, Kv3.2, Kv3.3,

and Kv3.4. Kv3 channels are activated by depolarisation of the plasma membrane to voltages

more positive than -20mV; furthermore, the channels deactivate rapidly upon repolarisation of the

membrane. These biophysical properties ensure that the channels open towards the peak of the

depolarising phase of the neuronal action potential to initiate repolarisation. Rapid termination of

the action potential mediated by Kv3 channels allows the neuron to recover more quickly to reach

sub-threshold membrane potentials from which further action potentials can be triggered. 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 subtypes are predominant in the CNS, whereas Kv3.4

channels are also found in skeletal muscle and sympathetic neurons (Weiser et al., 1994). Kv3.1-

3 channel subtypes are differentially expressed by sub-classes of interneurons in cortical and

hippocampal brain areas (e.g. Chow et al., 1999; Martina et al., 1998; McDonald et al., 2006;

Chang et al., 2007), in the thalamus (e.g. Kasten et al., 2007), cerebellum (e.g. Sacco et al., 2006;

Puente et al., 2010), and auditory brain stem nuclei (Li et al., 2001).

Tetraethylammonium (TEA) has been shown to inhibit the channels at low millimolar concentrations (Rudy et al., 2001), and blood-depressing substance (BDS) toxins from the sea

anemone, Anemonia sulcata (Diochot et al., 1998), have been shown to selectively inhibit Kv3

channels with high affinity (Yeung et al., 2005).

Kv3 channels are important determinants of the function of the cerebellum, a region of the brain

important for motor control (Joho et al., 2009). Characterisation of mice in which one or more of

the Kv3 subtypes has been deleted shows that the absence of Kv3.1 gives rise to increased

locomotor activity, altered electroencephalographic activity, and a fragmented sleep pattern (Joho

et al., 1999). The deletion of Kv3.2 leads to a reduction in seizure threshold and altered cortical

relectroencephalographic activity (Lau et al., 2000). Deletion of Kv3.3 is associated with mild

PCT/GB2020/050268

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ataxia and motor deficits (McMahon et al., 2004). Double deletion of Kv3.1 and Kv3.3 gives rise

to a severe phenotype characterised by spontaneous seizures, ataxia, and an increased

sensitivity to the effects of ethanol (Espinosa et al., 2001; Espinosa et al., 2008). A spontaneous

mutation in the Kv3.1 gene (KCNC1) causes progressive myoclonic epilepsy (Muona et al., 2014).

Mutations of the Kv3.3 gene (KCNC3) in humans have been associated with forms of spinocerebellar ataxia (SCA13) (Figueroa et al., 2010).

Bipolar disorder, schizophrenia, anxiety, and epilepsy are serious disorders of the central nervous

system that have been associated with reduced function of inhibitory interneurons and gamma-

amino butyric acid (GABA) transmission (Reynolds et al., 2004; Benes et al., 2008; Brambilla et

al., 2003; Aroniadou-Anderjaska et al., 2007; Ben-Ari, 2006). Parvalbumin positive basket cells

that express Kv3 channels in the cortex and hippocampus play a key role in generating feedback

inhibition within local circuits (Markram et al., 2004). Given the relative dominance of excitatory

synaptic input over inhibitory input to glutamatergic pyramidal neurons in these circuits, fast-firing

of interneurons supplying inhibitory input is essential to ensure balanced inhibition. Furthermore,

accurate timing of inhibitory input is necessary to sustain network synchronisation, for example,

in the generation of gamma frequency field potential oscillations that have been associated with

cognitive function (Fisahn et al., 2005; Engel et al., 2001). Notably, a reduction in gamma

oscillations has 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 had not been taking antipsychotic drugs for at least 2 months

before death (Yanagi et al., 2014). Consequently, positive modulators of Kv3 channels might be

expected to enhance the firing capabilities of specific groups of fast-firing 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 circadian pacemaker in the CNS (Schulz et al., 2009).

Voltage-gated ion channels of the Kv3 family are expressed at high levels in auditory brainstem

nuclei (Li et al., 2001) where they permit the fast firing of neurons that transmit auditory

information from the cochlear to higher brain regions. Phosphorylation of Kv3.1 and Kv3.3

channels in auditory brainstem neurons is suggested to contribute to the rapid physiological

adaptation to sound levels that may play a protective role during exposure to noise (Desai et al.,

2008; Song et al. 2005). Loss of Kv3.1 channel expression in central auditory neurons is

observed in hearing impaired mice (von Hehn et al., 2004); furthermore, a decline in Kv3.1

expression may be associated with loss of hearing in aged mice (Jung et al. 2005), and loss of

Kv3 channel function may also follow noise-trauma induced hearing loss (Pilati et al., 2012).

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Furthermore, pathological plasticity of auditory brainstem networks is likely to contribute to

symptoms that are experienced by many people suffering from hearing loss of different types.

Recent studies have shown that regulation of Kv3.1 channel function and expression has a major

role in controlling auditory neuron excitability (Kaczmarek et al., 2005; Anderson et al., 2018; Glait

et al., 2018; Olsen et al., 2018, Chambers et al., 2017), suggesting that this mechanism could

account for some of the plastic changes that give rise to tinnitus. Tinnitus may follow noise-

induced hearing loss as a result of adaptive changes in central auditory pathways from brainstem

to 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 that may control

the function of these circuits.

It is known that Kv3.1 and/or Kv3.2 modulators have utility in the treatment of pain

(WO2017/098254). In the broadest sense, pain can be grouped in to acute pain and chronic pain.

Acute pain is defined as pain that is self-limited and generally requires treatment for no more than

up to a few weeks, for example postoperative or acute musculoskeletal pain, such as fractures

(US Food and Drug Administration, 2014). Chronic pain can be defined either as pain persisting

for longer than 1 month beyond resolution of the initial trauma, or pain persisting beyond three

months. There is often no clear cause of chronic pain, and a multitude of other health problems

such as fatigue, depression, insomnia, mood changes and reduction in movement, often

accompany chronic pain.

Chronic pain can be sub-divided in to the following groups: neuropathic pain, chronic

musculoskeletal pain and miscellaneous chronic pain. Neuropathic pain usually accompanies

tissue injury and is initiated or caused 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 diseases such as osteoarthritis and

chronic lower back pain and can occur following damage to muscle tissue as well as trauma to

area for example, fractures, sprains and dislocation. Miscellaneous chronic pain an 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 tendinitis.

Chronic pain is a highly heterogeneous condition that remains amongst the most troublesome

and difficult to manage of clinical indications (McCarberg et al., 2008; Woolf, 2010; Finnerup et

al., 2015). Despite years of research and drug development, there has been little progress in

identifying treatments that can match the opioids for efficacy without significant side effects and

risk of dependence. Voltage-gated ion channels have been important targets for the management

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of specific pain indications, in particular neuropathic pain states. Furthermore, genetic mutations

in specific ion channels have been linked to some chronic pain disorders (Bennett et al., 2014).

Examples of voltage-gated ion channels that are being explored as pharmaceutical targets

include: Sodium channels (in particular NaV1.7) - Sun et al., 2014; Dib-Hajj et al., 2013; N-type

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 the C-fibres 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 demonstration of causality is still lacking

(Cervero, 2009).

Drugs targeting hyperexcitability, such as sodium channel blockers (e.g. CNV1014802,

lamotrigine, carbamazepine, and local anaesthetics), Kv7 positive modulators (e.g. flupertine and

retigabine), and N-type calcium channel modulators (e.g. gabapentin, which interacts with the a2d

subunit of the N-type calcium channel, and ziconitide, derived from a cone snail toxin) show

efficacy in models of inflammatory and/or neuropathic pain. However, amongst these drugs, there

is mixed evidence for clinical efficacy, for example, balancing efficacy and increased burden of

side effects on the central nervous system. The disparity between efficacy in animal models and

efficacy in humans is likely to be due to a range of factors, but in particular, drug concentration

achievable in humans (due to poor tolerability) and heterogeneity of human pain conditions are

likely to be the main culprits. For pain indications, there is also a need to identify targets through

which pain relief can be achieved with reduced tolerance or tachyphylaxis and reduced abuse

liability and/or risk of dependence.

Thus, improving the pharmacological management of pain is focused on mechanisms that can

deliver good efficacy with a reduced side-effect burden, reduced tolerance or tachyphylaxis, and

reduced abuse liability and/or risk of dependence.

Recently, Kv3.4 channels have become a target of interest for the treatment of chronic pain. Kv3.4

channels are expressed on neurons of the dorsal root ganglia (Ritter et al., 2012; Chien et al.,

2007), where they are predominantly expressed on sensory C-fibres (Chien et al., 2007). Kv3

channels are also expressed by specific subsets of neurons in the spinal cord. Specifically,

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Kv3.1b (Deuchars et al., 2001; Brooke et al., 2002), Kv3.3 (Brooke et al., 2006), and Kv3.4

subunits (Brooke et al., 2004) have been identified in rodent spinal cord, although not always in

association with circuits involved with sensory processing. It is likely that Kv3 channels shape

the firing properties of spinal cord neurons, including motoneurons.

In addition recent studies showed the Kv3.4 channels expressed in DRG nociceptors have a

significant impact on glutamatergic synaptic transmission (Muqeem et al., 2018). animal model

data suggest a down-regulation of Kv3.4 channel surface expression in DRG neurons following

spinal cord injury associated with hypersensitivity to painful stimuli (Ritter et al., 2015; Zemel et

al., 2017; Zemel et al., 2018). Similarly, it has been observed that there is a down-regulation of

Kv3.4 expression in DRGs of rodents following spinal cord ligation (Chien et al., 2007). This latter

study also showed that intrathecal administration to rats of an antisense oligonucleotide to

supress the expression of Kv3.4 led to hypersensitivity to mechanical stimuli. It has been shown

that Kv3.4 channel inactivation could be influenced by protein kinase C-dependent

phosphorylation of the channels, and that this physiological mechanism might allow DRG neurons

to alter their firing characteristics in response to painful stimuli (Ritter et al., 2012). These studies

suggest a causal relationship between the emergence of mechanical allodynia and reduced Kv3.4

channel expression or function. No evaluation of Kv3.1, Kv3.2, or Kv3.3 expression in SC or DRG

neurons was conducted in any of these studies, and expression of these two subtypes has not

been explicitly demonstrated on DRG neurons (although as mentioned above, they are abundant

within specific regions of the spinal cord). The in vivo studies reported above provide a rationale

for modulation of Kv3.4 as a novel approach to the treatment of certain neuropathic pain states.

Dementia with Lewy Bodies (DLB) and Parkinson's disease (PD) are serious neurodegenerative

disorders that are associated with the accumulation of the protein, alpha-synuclein in Lewy

bodies, which leads to loss of connectivity and neuronal cell death. Symptoms of DLB include

progressive cognitive deficits, in particular difficulties with planning and attention. Visual

hallucinations are also common, occurring in approximately 60% of patients. PD is associated

initially with motor deficits, primarily due to loss of dopamine neurons. While there are currently

no studies directly linking Kv3 channels to DLB or PD, the location and role of Kv3 channels, in

particular Kv3.1, in cortical and basal ganglia circuits suggests that modulators of these channels

could improve symptoms of DLB or PD, either alone, or in combination with current treatments,

such as acetyl-cholinesterase inhibitors for DLB or L-DOPA for PD.

Patent applications WO2011/069951, WO2012/076877, WO2012/168710, WO2013/175215, WO2013/083994, WO2013/182850, WO2017/103604, WO2018/020263 and WO2018/109484

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disclose compounds which are modulators of Kv3.1 and Kv3.2. Further, the utility of such

compounds is demonstrated in animal models of seizure, hyperactivity, sleep disorders,

psychosis, hearing disorders and bipolar disorders.

Patent application WO2013/182851 discloses modulation of Kv3.3 channels by certain

compounds.

Patent application WO2013/175211 discloses that modulation of Kv3.1, Kv3.2 and/or Kv3.3

channels has been found to be beneficial in preventing or limiting the establishment of a

permanent hearing loss resulting from acute noise exposure. The benefits of such prevention may

be observed even after administration of the Kv3.1, Kv3.2 and/or Kv3.3 modulator has ceased.

Patent application WO2017/098254 discloses that modulation of Kv3.1, Kv3.2 and/or Kv3.3

channels has been found to be beneficial in the prophylaxis or treatment of pain, in particular

neuropathic or inflammatory pain.

Patent application WO2019/222816 discloses 'meta-linked' pyridinyl compounds of the general

formula:

R'

o

X N NH R°

R°

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

Patent application WO2020/000065 discloses "meta-linked" diazine and triazine compounds of

the general formula:

I'V

0 A Ri NY NM

w 8°

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

There remains a need for the identification of alternative modulators of Kv3. 1, Kv3.2 and/or Kv3.3,

in particular modulators of Kv3.1 and/or Kv3.2. Such modulators may demonstrate high in vivo

potency, channel selectivity, an improved safety profile, or desirable pharmacokinetic parameters,

for example high brain availability and/or low clearance rate that reduces the dose required for

therapeutic effect in vivo. Alternative modulators may provide a benefit through having distinct

PCT/GB2020/050268

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metabolites from known modulators. Compounds which have balanced Kv3.1, Kv3.2 and/or

Kv3.3 modulatory properties may be desirable e.g. compounds with modulate Kv3.1 and Kv3.2

to the same, or a similar extent. For certain therapeutic indications, there is also a need to identify

compounds with a different modulatory effect on Kv3.1, Kv3.2 and/or Kv3.3 channels, for

example, compounds that alter the kinetics of channel gating or channel inactivation, and which

may behave in vivo as negative modulators of the channels.

Summary of the invention

The present invention provides a compound of formula (I):

O N O R R R2 N N NH o R3 R O O R5(I) R4 R5

wherein:

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, are a spirocyclopropyl ring;

R4 is methyl or ethyl;

R5 is H or methyl;

or R4 and R5, together with the carbon atom to which they are attached, form a C3-C4 spiro

carbocyclyl.

A 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 thereof and/or derivative thereof. In one embodiment of the invention a compound

of formula (I) is provided in the form of a pharmaceutically acceptable salt.

The compounds of formula (I) may be used as medicaments, in particular for use in the

prophylaxis or treatment of hearing disorders, including hearing loss and tinnitus, as well as

schizophrenia, substance abuse disorders, pain or Fragile X syndrome.

Further, there is provided a method for the prophylaxis or treatment of hearing disorders, including

hearing loss and tinnitus, as well as hearing disorders, including hearing loss and tinnitus, as well

as schizophrenia, substance abuse disorders, pain or Fragile X syndrome.

Compounds of formula (I) may be used in the manufacture of a medicament for the prophylaxis 21 Nov 2025

or treatment of hearing disorders, including hearing loss and tinnitus, as well as schizophrenia, substance abuse disorders, pain or Fragile X syndrome.

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

Also provided are processes for preparing compounds of formula (I) and novel intermediates of 2020427632

use in the preparation of compounds of formula (I). 10 Additionally provided are prodrug derivatives of the compounds of formula (I).

In another aspect, the present invention provides a use of a compound of formula (I):

(I) 15 wherein: 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, are a spirocyclopropyl ring; R4 is methyl or ethyl; 20 R5 is H or methyl; or R4 and R5, together with the carbon atom to which they are attached, form a C3-C4 spiro carbocyclyl; or a pharmaceutically acceptable salt and/or solvate thereof, in the manufacture of a medicament wherein: 25 i) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered at 2 to 400 mg per day; or ii) wherein the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered for a period of at least three months; or iii) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate 30 thereof is administered orally at 1 to 500 mg per day once or twice per day; or iv) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered to an adult human subject.

8a

In a further aspect, the present invention provides a use of a compound of formula (I): 21 Nov 2025

(I) wherein: R1 is H or methyl; 2020427632

5 R2 and R3 are both methyl, or R2 and R3, together with the carbon atom to which they are attached, are a spirocyclopropyl ring; R4 is methyl or ethyl; R5 is H or methyl; or R4 and R5, together with the carbon atom to which they are attached, form a C3-C4 spiro 10 carbocyclyl; or a pharmaceutically acceptable salt and/or solvate thereof, in the manufacture of a medicament wherein the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered to a human subject of less than 18 years of age. 15 In a further aspect, the present invention provides a method for the prophylaxis or treatment of a disease or disorder selected from the group consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse disorders, anxiety disorders, sleep disorders, hyperacusis and disturbances of loudness perception, Ménière's disease, 20 disorders of balance, and disorders of the inner ear, impulse control disorder, personality disorders, attention-deficit/hyperactivity disorder, autism spectrum disorders, eating disorders, cognition impairment, ataxia, pain such as neuropathic pain, inflammatory pain and miscellaneous pain, Lewy body dementia and Parkinson’s disease which comprises administering to a subject in need thereof an effective amount of a compound of formula (I):

25 (I) wherein: 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, are a spirocyclopropyl ring;

8b

R4 is methyl or ethyl; 21 Nov 2025

R5 is H or methyl; or R4 and R5, together with the carbon atom to which they are attached, form a C3-C4 spiro carbocyclyl; 5 or a pharmaceutically acceptable salt and/or solvate thereof wherein: i) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered at 2 to 400 mg per day; or ii) wherein the compound of formula (I), or a pharmaceutically acceptable salt and/or 2020427632

solvate thereof is administered for a period of at least three months; or 10 iii) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered orally at 1 to 500 mg per day once or twice per day; or iv) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered to an adult human subject.

15 In a further aspect, the present invention provides a compound which is selected from: (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2- yl]imidazolidine-2,4-dione:

; and (5R)-5-ethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-yl)imidazolidine-2,4- 20 dione:

. In a further aspect, the present invention provides a salt of a compound, the salt being selected from: a salt of (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4- 25 yl)oxypyrazin-2-yl]imidazolidine-2,4-dione:

8c 21 Nov 2025

; and a salt of (5R)-5-ethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2- yl)imidazolidine-2,4-dione: 2020427632

. 5 In a further aspect, the present invention provides a pharmaceutically acceptable salt of a compound, the pharmaceutically acceptable salt being selected from: a pharmaceutically acceptable salt of (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'- cyclopropane]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione:

10 ; and a pharmaceutically acceptable salt of (5R)-5-ethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]- 4-yloxypyrazin-2-yl)imidazolidine-2,4-dione:

.

15 A reference herein to a patent document or other matter which is given as prior art is not to be taken as admission that the document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

8d

Unless the context requires otherwise, where the terms “comprise”, “comprises”, “comprised” or 21 Nov 2025

“comprising” are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof. 5 Detailed description of the invention

The present invention provides compounds of formula (I): 2020427632

(I) 10 wherein: 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, are a spirocyclopropyl ring; R4 is methyl or ethyl; 15 R5 is H or methyl; or R4 and R5, together with the carbon atom to which they are attached, form a C3-C4 spiro carbocyclyl; or a pharmaceutically acceptable salt and/or solvate and/or derivative thereof.

20 Embodiments set out below relating to relative stereochemistry and the nature of groups, including R1, R2, R3, R4, R5, are envisaged as being independently, fully combinable with one another where appropriate to the circumstances (i.e. where chemically sensible) to form further embodiments of the invention. Such embodiments apply equally to intermediates which may be

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of use in the synthesis of a compound of formula (I) e.g. 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, R1 is H. In a second embodiment R1 is methyl.

In one embodiment, R2 is methyl and R3 is methyl. In another embodiment, R2 and R3 are a spiro

cyclopropyl such that that the following moiety is formed:

1 R1 R o

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. methyl).

In embodiments wherein R4 and R5 are different, they may have the following stereochemical

arrangement:

O NH R4 N R5

O 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 embodiments wherein R4 and R5 are different, they may alternatively have the following

stereochemical arrangement:

O / NH R5 N R IIIRA

and R5 is methyl.

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

spirocyclopropyl.

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

form a 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]-4-yl)oxypyrazin-2

yl]imidazolidine-2,4-dione;

B-[5-[(3,3-dimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5,5-dimethyl-imidazolidine-2,4-

dione;

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

yl)imidazolidine-2,4-dione;

5,5-dimethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-

yl)imidazolidine-2,4-dione;

5R)-5-ethyl-5-methyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cy

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-2,4-dione;

6,5-dimethyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]imidazolidine-2,4-

dione;

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

yl]imidazolidine-2,4-dione;

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

yl]imidazolidine-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;

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

dione;

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

diazaspiro[3.4octane-6,8-dione;

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

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

6-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]-4,6-

diazaspiro[2.4]heptane-5,7-dione;

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

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

yl]imidazolidine-2,4-dione;

When the compound contains a C1-3alkyl group, whether alone or forming part of a larger group,

the alkyl group may be straight chain, branched or cyclic. Examples of C1-3alkyl are methyl, ethyl,

in-propyl, isopropyl and cyclopropyl. Reference to "propyl" includes in-propyl, isopropyl and

cyclopropyl.

The term 'halo' or 'halogen' as used herein, refers to a fluorine, chlorine, bromine or iodine atom.

Particular examples of halo are fluorine, chlorine and bromine, such as chlorine or bromine.

The term 'C3-4 spiro carbocyclyl' 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 via a spirocentre such that the secondary carbon is one of the 3

to 4 carbon atoms in the cyclic ring as follows:

3

2 3 3 2 4 y y , 1 , 1 , , , C3 spiro carbocyclyl C4 spiro carbocyclyl

It will be appreciated that for use in medicine the salts of the compounds of formula (I) should 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 e.g. hydrochloric, hydrobromic,

sulphuric, nitric or phosphoric acid and organic acids e.g. succinic, maleic, acetic, fumaric, citric,

tartaric, benzoic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid. Non-

pharmaceutically acceptable salts may be used, for example, in the isolation of compounds of

formula (I) and are included within the scope of this invention.

Certain of the compounds of formula (I) may form acid addition salts with one or more equivalents

of the acid. The present invention includes within its scope all possible stoichiometric and non-

stoichiometric forms.

The compounds of formula (I) may be prepared in crystalline or non-crystalline form and, if

crystalline, may optionally be solvated, e.g. as the hydrate. This invention includes within its

scope stoichiometric solvates (e.g. hydrates) as well as compounds containing variable amounts

of solvent (e.g. water).

It will be understood that the invention includes pharmaceutically acceptable derivatives of

compounds of formula (I) and that these are included within the scope of the invention.

As used herein "pharmaceutically acceptable derivative" includes any pharmaceutically

acceptable ester or salt of such ester of a compound of formula (I) which, upon administration to

the recipient is capable of providing (directly or indirectly) a compound of formula (I) or an active

metabolite or residue thereof.

A pharmaceutically acceptable prodrug may be formed by functionalising the secondary nitrogen

of the hydantoin, for example with a group "L" as illustrated below (wherein R4 and R5 are as

described above):

O N N N -L

o R Rs R In one embodiment of the invention, a compound of formula (I) is functionalised via the secondary

nitrogen of the hydantoin with a group L, wherein L is selected from:

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

b) -PO(O)2 .2M+, c) -PO(O) .D2+, wherein D2+ is a pharmaceutically acceptable divalent counterion, wo 2021/156584 WO PCT/GB2020/050268 PCT/GB2020/050268

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d) -CH(RX)-PO(OH)OM, wherein RX is hydrogen or C1-3 alkyl,

e) -CH(RX)-PO(O)2*2M, f) -CH(R^)-PO(O')2.DD*,

g) -SOM+, h) -CH(R*)-SO*M*, and

i) -CO-CH2CH2-CM.

It is to be understood that the present invention encompasses all isomers of formula (I) and their

pharmaceutically acceptable derivatives, including all geometric, tautomeric and optical forms,

and mixtures thereof (e.g. racemic mixtures). Where additional chiral centres are present in

compounds of formula (I), the present invention includes within its scope all possible

diastereoisomers, including mixtures thereof. The different isomeric forms may be separated or

resolved one from the other by conventional methods, or any given isomer may be obtained by

conventional synthetic methods or by stereospecific or asymmetric syntheses.

The present disclosure includes all isotopic forms of the compounds of the invention provided

herein, whether in a form (i) wherein all atoms of a given atomic number have a mass number (or

mixture of mass numbers) which predominates in nature (referred to herein as the "natural

isotopic form") or (ii) wherein one or more atoms are replaced by atoms having the same atomic

number, but a mass number different from the mass number of atoms which predominates in

nature (referred to herein as an "unnatural variant isotopic form"). It is understood that an atom

may naturally exist as a mixture of mass numbers. The term "unnatural variant isotopic form" also

includes embodiments in which the proportion of an atom of given atomic number having a mass

number found less commonly in nature (referred to herein as an "uncommon isotope") has been

increased relative to that which is naturally occurring e.g. to the level of >20%, >50%, >75%,

>90%, >95% or > 99% by number of the atoms of that atomic number (the latter embodiment

referred to as an "isotopically enriched variant form"). The term "unnatural variant isotopic form"

also includes embodiments in which the proportion of an uncommon isotope has been reduced

relative to that which is naturally occurring. Isotopic forms may include radioactive forms (i.e. they

incorporate radioisotopes) and non-radioactive forms. Radioactive forms will typically be

isotopically enriched variant forms.

An unnatural variant isotopic form of a compound may thus contain one or more artificial or

uncommon isotopes such as deuterium (2H or D), carbon-11 (11C), carbon-13 (Superscript(3)C), carbon-14

(14C), nitrogen-13 (13N), nitrogen-15 (15N), oxygen-15 (150), oxygen-17 (170), oxygen-18 (180),

phosphorus-32 (32P), sulphur-35 (35S), chlorine-36 (36CI), chlorine-37 (37CI), fluorine-18 (18F)

iodine-123 (1231), iodine-125 (1251) in one or more atoms or may contain an increased proportion

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of said isotopes as compared with the proportion that predominates in nature in one or more

atoms.

Unnatural variant isotopic forms comprising radioisotopes may, for example, be used for drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. Superscript(3)H, and carbon-

14, i.e. are particularly useful for this purpose in view of their ease of incorporation and ready

means of detection. Unnatural variant isotopic forms which incorporate deuterium i.e. 2H or D may

afford certain therapeutic advantages resulting from greater metabolic stability, for example,

increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some

circumstances. Further, unnatural variant isotopic forms may be prepared which incorporate positron emitting isotopes, such as Superscript(1)C, 18F, 150 and N, and would be useful in Positron Emission

Topography (PET) studies for examining substrate receptor occupancy.

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

In one embodiment, the compounds of the invention are provided in an unnatural variant isotopic

form. In a specific embodiment, the unnatural variant isotopic form is a form in which deuterium

(i.e. 2H or D) is incorporated where hydrogen is specified in the chemical structure in one or more

atoms of a compound of the invention. In one embodiment, the atoms of the compounds of the

invention are in an isotopic form which is not radioactive. In one embodiment, one or more atoms

of the compounds of the invention are in an isotopic form which is radioactive. Suitably radioactive

isotopes are stable isotopes. Suitably the unnatural variant isotopic form is a pharmaceutically

acceptable form.

In one embodiment, a compound of the invention is provided whereby a single atom of the

compound exists in an unnatural variant isotopic form. In another embodiment, a compound of

the invention is provided whereby two or more atoms exist in an unnatural variant isotopic form.

Unnatural isotopic variant forms can generally be prepared by conventional techniques known to

those skilled in the art or by processes described herein e.g. processes analogous to those

described in the accompanying Examples for preparing natural isotopic forms. Thus, unnatural

isotopic variant forms could be prepared by using appropriate isotopically variant (or labelled)

reagents in place of the normal reagents employed in the Examples. Since the compounds of

formula (I) are intended for use in pharmaceutical compositions it will readily be understood that

they are each preferably provided in substantially pure form, for example at least 60% pure, more

suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are on a

weight for weight basis). Impure preparations of the compounds may be used for preparing the

more pure forms used in the pharmaceutical compositions.

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Since the compounds of formula (I) are intended for use in pharmaceutical compositions it will

readily be understood that they are each preferably provided in substantially pure form, for

example at least 60% pure, more suitably at least 75% pure and preferably at least 85%,

especially at least 98% pure (% are on a weight for weight basis). Impure preparations of the

compounds may be used for preparing the more pure forms used in the pharmaceutical compositions.

In general, the compounds of formula (I) may be made according to the organic synthesis

techniques known to those skilled in this field, as well as by the representative methods set forth

below, those in the Examples and modifications thereof.

Patent applications WO2011/069951, WO2012/076877, WO2012/168710, WO2013/175215,

WO2013/083994, WO2013/182850, WO2017/103604, WO2018/020263 and WO2018/109484 provide methods for the synthesis of intermediates which may be of use in the production of

compounds of the present invention.

General Synthesis Schemes

The following schemes detail synthetic routes to compounds of the invention and intermediates

in the synthesis of such compounds. In the following schemes reactive groups can be protected

with protecting groups and deprotected according to established techniques well known to the

skilled person.

Compounds of formula (I), and salts and solvates thereof, may be prepared by the general

methods outlined hereinafter. In the following description, the groups R1, R2, R3, R4 and R5 have

the meanings as previously defined for compounds of formula (I) unless otherwise stated.

Scheme 1a

R1 o R1 O o R2 R2 NN HH N // R2 N o + + R3 R3 NN O / O NH step (i) o O R3 N - N R4 R5 R5 X o NH NH R4 R4 R5 R5

(II) (III) (I)

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step (i): Compounds of formula (I) can be prepared by metal catalysed cross coupling

reactions. In this reaction a halo-pyrazine derivative of formula (II) wherein typically X=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, e.g. in N,N-dimethylacetamide, with conventional heating or microwave

heating.

Scheme 1b

Y N R1 o N o R1 R2 N O OH - N + R2 O o R3 N R3 N - o O NH o step (i) o R3 N R4 R4 R5 o O NH R4 R5 (IV) (V) (I)

Compounds of formula (I), wherein R4 and R5 are not H, can be prepared by nucleophilic

aromatic substitution. In this reaction a halo-pyrazine derivative of formula (IV) wherein typically

Y=CI and a phenol of formula (V) are reacted in the presence of a suitable base such as

potassium carbonate in a suitable solvent, e.g. in N,N-dimethylformamide or in acetonitrile, with

conventional heating or microwave heating.

Scheme 1c

R1 o R1 O R1 N O R2 R2 N R2 N o O O R3 R3 O o NN R3 N R3 N = R3 N R3 N = - NH step (i) NH step (ii)

N O O H O N o O NH2 PG NH O NH R4 R5 R4 R4 R5 R5 R4 R4 R5

(VII) (VI) (I)

step (ii): Compounds of formula (I) can be prepared by cyclization of compounds of formula (VI)

in a suitable solvent e.g. dichloromethane with a carbonylating agent e.g. triphosgene

preferentially prediluted in the same solvent and added in a second time at 0°C in presence of a

suitable base e.g. 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 ethyl acetate in presence of a base such as

triethylamine or DIPEA.

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step (i): Compounds of formula (VI) can be prepared by deprotection of compounds of formula

(VII) wherein PG is a protecting group, suitably the protecting group is BOC, BOC may be

removed in acidic conditions e.g. TFA in a suitable solvent e.g. dichloromethane at

approximately 0°C to room temperature.

Scheme 1d R2 R2 O O R3 O R3 R3

R2 R1 o O R3 O N R1 R1 - O N // R1 R1 o O N O o N N N N N: N O O NH2 // H NH + o N + O NH R4 R5 HCI R5 HCI N step (i) R4 R4 step (ii) O : NH2 O / R5 R4 R4

(IX) (I) (XVI) (XVII)

step (ii): Compounds of formula (I) can be prepared by reaction of ureas of formula (XVII) and a

suitable base such as sodium methoxide in a suitable solvent such as methanol at temperature

ranging from 0°C to room temperature.

step (i): Ureas of formula (XVII) can be prepared by reaction of anilines of formula (XVI) and

amino esters (such as the hydrochloride salt) of formula (IX) in a suitable solvent e.g.

dichloromethane or ethyl acetate with a carbonylating agent e.g. triphosgene preferentially

prediluted in the same solvent in presence of a suitable base e.g. triethylamine or

diisopropylethylamine at temperature ranging from 0°C to room temperature.

Scheme 2a

Y Y N N

O Y N O o N O NH2 HCI HCI N - N H = N O O NH + / 11 o N R4 R5 R4 O NH step (i) N: NH2 o R5 R5 step (ii) R4 O R4

(IX) (X) (VIII) (IV)

step (ii): Compounds of formula (IV) can be prepared by reaction of ureas of formula (VIII) and

a suitable base such as sodium methoxide in a suitable solvent such as methanol at

temperature ranging from 0°C to room temperature.

step (i): Ureas of formula (VIII) can be prepared by reaction of commercially available halo-

pyrazine derivative of formula (X), wherein typically Y=Cl, and amino esters (such as the

hydrochloride salt) of formula (IX) in a suitable solvent e.g. dichloromethane or ethyl acetate

with a carbonylating agent e.g. triphosgene preferentially prediluted in the same solvent in

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presence of a suitable base e.g. triethylamine or diisopropylethylamine at temperature ranging

from 0°C to room temperature.

Scheme 2b

Y Y NN NN // Y NN OH N

O NH2 .HCI HCI NN : NH N: N o NH + // o R4 R5 R4 step (i) o O NH NN : NH2 step (ii) R4 NH2 R4 R5 R5 NH (XII) (X) (XI) (IV)

step (ii): Compounds of formula (IV) can be prepared by cyclization of compounds of formula

(XI) in a suitable solvent e.g. dichloromethane with a carbonylating agent e.g. triphosgene

suitable base e.g. triethylamine.

step (i): Compounds of formula (XI) can be prepared from anilines of formula (X), wherein

typically Y=CI, and amino acids (as free base or hydrochloride salt) of formula (XII) by amidic

coupling in the presence of a coupling agent e.g. T3P in a suitable solvent such as ethyl

acetate, acetonitrile or a mixture of them.

Scheme 3

R1 R1 O o R1 O o R2 N R2 N o / O R3 R3 I " R3 N R3 N OH H N NH2 NH H (II) - X NH H o O N o N O R4 PG PG PG step (ii) PG PG R4 R5 R5 step (i) R4 R5 R4 R5 R4 R5 R4 R5

(XIII) (VII) (XIV)

step (ii): Compounds of formula (IV) can be prepared by metal catalysed cross coupling

reactions. In this reaction a halo-pyrazine derivative of formula (II) wherein typically X=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]phosphand (XPhos) and a suitable base such as cesium carbonate in

a suitable solvent, e.g. in 1,4-dioxane, with conventional heating or microwave heating.

Alternatively in this reaction a halo-pyrazine derivative of formula (II) wherein typically X=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

PCT/GB2020/050268

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dipotassium carbonate in a suitable solvent, e.g. in 1-butanol, with conventional heating or

microwave heating. A further alternative for the preparation of compounds of formula (IV) is to

react a halo-pyrazine derivative of formula (II) wherein typically X=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, e.g. in 1,4-

dioxane, with conventional heating or microwave heating.

step (i): Compounds of formula (XIII) can be prepared from N-protected (e.g. BOC) amino acids

of formula (XIV) and an amine such as hexamethyldisilazane by amidic coupling in the

presence of a base e.g. DIPEA and of a coupling agent e.g. HATU or TBTU in a solvent such as

N,N-dimethylformamide.

Scheme 4

Z R1 oO NN R1 R1 R2 N OH /

N + / R2 o R3 R3 /N > o step (i) XX R3 R3 - XX (II) (XV) (V)

step (i): Compounds of formula (II) wherein typically X=Br can be prepared by nucleophilic

aromatic substitution. In this reaction a halo-pyrazine derivative of formula (XV) wherein

typically X=Z=Br and a phenol of formula (V) are reacted in the presence of a base such as

potassium carbonate in a suitable solvent, e.g. in N,N-dimethylformamide, with conventional

heating or microwave heating.

Scheme 5

ZZ R1 OO N R1 R1 R2 R2 N OH OH // o O + R2 N R3 R3 NN = = NH2 o R3 step (i) NH2

(XVIII) (V) (XVI)

step (i): Anilines of formula (XVI) can be prepared by metal catalysed cross coupling reactions.

In this reaction a halo-pyrazine derivative of formula (XVIII) wherein typically Z=Br and a phenol

of formula (V) are reacted in the presence of a metal catalyst such as Copper(I)lodide, a

suitable ligand like picolinic acid, in a suitable solvent, e.g. in N,N-dimethylformamide or N,N-

dimethylacetamide, with conventional heating or microwave heating optionally a suitable base

such as potassium carbonate or caesium carbonate can be used.

PCT/GB2020/050268

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Scheme 6

(XXVI)

R2 R3 PG1 Br PG1 Br O, O O R2R3 R2 Rx R3 Rx OH R2 OH Zn Rx R3 Br O Br O O O OH step (i) step (ii) O step (iii)

OI R1 PG2 R1 R1 R1 PG2 (XXIII) (XXV) (XXII) (XXIV)

step (iv)

OH R2 R2 R3 PG1 PG1 PG1 PG1 R3 O R2 O R2 R3 R2R3 O R2 R3 R2 R2 R3 OH step (vii) step (vi) step (v) O O O OH O R1 R1 R1 R1 (V) (XIX) (XX) (XXI)

In Scheme 6 shown above, PG1 and PG represent suitable protecting groups. PG1 in steps (i)=-

(iii) may be different from PG1 in Steps (iv)-(vii). Suitable protecting groups include benzyl.

tetrahydropyranyl or methyloxymethyl. Suitably PG is the same as PG1, e.g. both are benzyl.

Description of the scheme wherein PG1 and PG are both benzyl

step (vii): Phenols of formula (V) can be prepared from the benzylated compounds of formula

(XIX), by deprotection such as using a metal catalyst such as palladium on carbon and a hydrogen

source such as hydrogen atmosphere or ammonium formate in a suitable solvent such as ethanol

or methanol at a temperature ranging from room temperature to reflux.

step (vi): 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 ranging from room temperature to reflux.

step (v): Diols of formula (XX) can be prepared from lactones of formula (XXI) using a reducing

agent such as lithium aluminium hydride in a suitable solvent such as THF at a temperature

ranging from 0°C to room temperature.

step (iv): Lactones of formula (XXI) can be prepared from phenols of formula (XXII) using a

benzylating agent such as benzyl bromide in presence of a base such as potassium carbonate in

a suitable solvent such as acetonitrile or THE or a mixture thereof at a temperature ranging from

room temperature to reflux.

step (iii): Phenols of formula (XXII) can be prepared from di-benzylated esters of formula (XXIII)

wherein Rx is a suitable alkylic group such as methyl or ethyl, using a metal catalyst such as

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palladium on carbon and a hydrogen source such as hydrogen atmosphere or ammonium formate

in a suitable solvent such as ethanol or methanol at a temperature ranging from room temperature

to reflux.

step (ii): Di-benzylated esters of formula (XXIII) wherein Rx is a suitable alkylic group such as

methyl or ethyl can be prepared from di-benzylated bromo derivatives of formula (XXIV) by using

pre-formed organozinc derivatives of formula (XXVI) wherein Rx is a suitable alkylic group such

as methyl or ethyl in presence of a metal catalyst 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 ranging from room temperature to reflux.

step (i): Di-benzylated bromo derivatives of formula (XXIV) can be prepared from commercially

available derivatives of formula (XXV) using a benzylating agent such as benzyl bromide in

presence of a base such as potassium carbonate in a suitable solvent such as acetonitrile or THF

or acetone or a mixture thereof at a temperature ranging from room temperature to reflux.

When PG and/or PG are protecting groups such as tetrahydropyranyl or methyloxymethyl, usual

protection/deprotection conditions apply:

Protection conditions of phenols with tetrahydropyranyl include the reaction of a phenol with

dihydro-2H-pyran in presence of a catalyst such C:Py *p-MePhSO3H in a suitable solvent

such us dichloromethane at a temperature ranging from 0°C to reflux.

Cleavage conditions for a tetrahydropyranyl protecting group from phenols include the

reaction of a THP protected phenol in presence of an acid such as sulphuric acid or p-

MePhSO3H or HCI in a suitable solvent such us methanol or ethanol at a temperature ranging

from 0oC to reflux.

Protection conditions of phenols with methyloxymethyl include the reaction of a phenol with

chloromethyl methyl ether in presence of a base such us potassium carbonate in a suitable

solvent such us tetrahydrofuran or acetonitrile at a temperature ranging from 0°C to reflux.

Cleavage conditions for a methyloxymethyl protecting group from phenols include the

reaction of a MOM protected phenol in presence of an acid such as sulphuric acid or p-

from 0°C to reflux.

Scheme 7

R2 R3 R2 R3 Br Br O. Br O Rx `Znl Rx Rx step (i) O (XXVII) (XXVI)

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Step (i): Organozinc derivatives of formula (XXVI) wherein Rx is a suitable alkylic group such as

methyl or ethyl can be prepared by adding commercially available bromo esters of formula (XXVII)

to a refluxing suspension of zinc (0) in presence of 1,2-dibromoethane and chlorotrimethylsilane

in a suitable solvent such as THF.

Processes of the invention

According to further aspects of the present invention are provided processes for the preparation

of compounds of formula (I) and derivatives thereof, as well as processes for preparing

intermediates in the synthesis of compounds of formula (I).

The processes of the invention are described above and include any individual step of a multi-

step scheme.

Intermediates

The present invention also relates to novel intermediates in the synthesis of compounds of

formula (I). Such novel intermediates include compounds of formulae (II), (IV), (VI), (VII), (VIII),

(XI), (XVI) and (XVII). Also of interest are intermediates of formulae (XIX) to (XXIV). Salts, such

as pharmaceutically acceptable salts, of such intermediates are also provided by the present

invention.

Intermediates of the invention therefore include:

- compounds of formula (II):

O N

R R2

o R N X R3

wherein R1, R2 and R3 are as defined previously, X is halo, such as Br;

- compounds of formula (IV):

Y N O N N N NH

o O R5 R4 R wherein R1, R2 and R3 are as defined previously, Y is halo, such as CI;

- compounds of formula (VI):

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R5 R4 NH2 NH HN N //

N R1 O

o R2 R3 (VI)

wherein R1, R2, R3, R4 and R5 are as defined previously;

- compounds of formula (VII):

R5 / PG PG R4 NH HN N O

N R1 O

O R2 R R3 (VII)

wherein R1, R2, R3, R4 and R5 are as defined previously, PG is a suitable protecting

group such as BOC;

- compounds of formula (XVI):

O N

R R2 NH2 o R N NH R3

wherein R1, R2 and R3 are as defined previously.

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

Compounds of formula (I) of the present invention are modulators of Kv3.1. Compounds of

formula (I) may also be modulators of Kv3.2 and/or Kv3.3. Compounds of the invention may be

tested in the assay of Biological Example 1 to determine their modulatory properties for Kv3.1

and/or Kv3.2 and/or Kv3.3 channels.

A 'modulator' as used herein refers to a compound which is capable of producing at least 10%

potentiation, and suitably at least 20% potentiation of whole-cell currents mediated by human

PCT/GB2020/050268

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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 be taken to 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 10% potentiation and suitably

at least 20% potentiation of whole-cell currents mediated by human Kv3.1 channels recombinantly expressed in mammalian cells. Suitably the pEC50 of the modulator is in the range

of 4-7 (such as 5-6.5).

at least 20% potentiation of whole-cell currents mediated by human Kv3.2 channels recombinantly expressed in mammalian cells. Suitably the pEC50 of the modulator is in the range

of 4-7 (such as 5-6.5).

at least 20% potentiation of whole-cell currents mediated by human Kv3.3 channels recombinantly expressed in mammalian cells. Suitably the pEC50 of the modulator is in the range

of 4-7 (such as 5-6.5).

In another embodiment the modulator is capable of producing at least 10% potentiation and

suitably at least 20% potentiation of whole-cell currents mediated by human Kv3.1 and Kv3.2

channels recombinantly expressed in mammalian cells.

suitably at least 20% potentiation of whole-cell currents mediated by human Kv3.1 and Kv3.3

channels recombinantly expressed in mammalian cells.

suitably at least 20% potentiation of whole-cell currents mediated by human Kv3.2 and Kv3.3

channels recombinantly expressed in mammalian cells.

In a further embodiment the modulator is capable of producing at least 10% potentiation and

suitably at least 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 may be of use for the treatment or prophylaxis of a disease or disorder where a

modulator of the 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 which 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 may be tested in

the assay of Biological Example 1 to determine their modulatory 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 modulation of Kv3.1

channels over modulation of Kv3.2 channels. By selective, is meant that compounds demonstrate, for example, at least a 2 fold, 5 fold or 10 fold activity for Kv3.1 channels than for

Kv3.2 channels. The activity of a compound is suitably quantified by its potency as indicated by

an 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 modulation of Kv3.2

channels over modulation of Kv3.1 channels. Once again, by selective is meant that compounds

demonstrate, for example at least a 2 fold, 5 fold or 10 fold activity for Kv3.2 channels than for

Kv3.1 channels.

In a particular embodiment of the invention the compounds of formula (I) or their pharmaceutically

acceptable salts and/or solvates and/or derivatives thereof demonstrate comparable activity

between modulation of Kv3.1 and Kv3.2 channels, for example the activity for one channel is less

than 2 fold that for the other channel, such as less than 1.5 fold or less than 1.2 fold.

In certain disorders it may be of benefit to utilise a modulator of Kv3.3 or Kv3.1, or Kv3.3 and

Kv3.1 which demonstrates a particular selectivity profile between the two channels. For example

a compound may be selective for modulation of Kv3.3 channels over modulation of Kv3.1

channels demonstrating, for example, at least a 2 fold, 5 fold or 10 fold activity for Kv3.3 channels

than for Kv3.1 channels.

channels over modulation of Kv3.3 channels. Once again, by selective is meant that compounds

PCT/GB2020/050268

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demonstrate, for example at least a 2 fold, 5 fold or 10 fold activity for Kv3.1 channels than for

Kv3.3 channels.

In a particular embodiment of the invention, a compound may demonstrate comparable activity

between modulation of Kv3.3 and Kv3.1 channels, for example the activity for each channel is

less than 2 fold that for the other channel, such as less than 1.5 fold or less than 1.2 fold.

In certain disorders it may be of benefit to utilise a modulator of Kv3.3 or Kv3.2, or Kv3.3 and

Kv3.2 which demonstrates a particular selectivity profile between the two channels. A compound

may be selective for modulation of Kv3.3 channels over modulation of Kv3.2 channels

demonstrating, for example, at least a 2 fold, 5 fold or 10 fold activity for Kv3.3 channels than for

Kv3.2 channels.

Kv3.3 channels.

In another particular embodiment a compound may demonstrate comparable activity between

modulation of Kv3.3 and Kv3.2 channels, for example the activity for each channel is less than 2

fold that for the other channel, such as less than 1.5 fold or less than 1.2 fold.

In a yet further particular embodiment of the invention a compound may demonstrate comparable

activity between modulation of Kv3.3, Kv3.2 and Kv3.1 channels, for example the activity for each

channel is less than 2 fold that for any other channel, such as less than 1.5 fold or less than 1.2

fold. The activity of a compound is suitably quantified by its potency as indicated by an EC50

value.

Therapeutic methods

The invention also provides a compound of formula (I), or a pharmaceutically acceptable salt

and/or solvate (e.g. salt) and/or derivative thereof, for use in the treatment or prophylaxis of a

disease or disorder where a modulator of Kv3.1, Kv3.2 and/or Kv3.3 is required, for example

those diseases and disorders mentioned herein below.

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The invention provides a method of treating or preventing a disease or disorder where a modulator

of Kv3.1 Kv3.2 and/or Kv3.3 is required, for example those diseases and disorders mentioned

herein below, which comprises 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. salt) and/or

derivative thereof.

The invention also provides the use of a compound of formula (I), or a pharmaceutically

acceptable salt and/or solvate thereof (e.g. salt) and/or derivative, in the manufacture of a

medicament for the treatment or prophylaxis of a disease or disorder where a modulator of Kv3. 1,

Kv3.2 and/or Kv3.3 is required, for example those diseases and disorders mentioned herein

below.

In one embodiment is provided a compound of formula (I) or a pharmaceutically acceptable salt

and/or solvate thereof and/or derivative thereof for use as a medicament.

The term "treatment" or "treating" as used herein includes the control, mitigation, reduction, or

modulation of the disease state or its symptoms.

The term "prophylaxis" is used herein to mean preventing symptoms of a disease or disorder in

a subject or preventing recurrence of symptoms of a disease or disorder in an afflicted subject

and is not limited to complete prevention of an affliction.

Suitably the subject is a human.

Diseases or disorders that may be mediated by modulation of Kv3.1 and/or Kv3.2 channels may

be selected from the list below. The numbers in brackets after the listed diseases below refer to

the classification code in Diagnostic and Statistical Manual of Mental Disorders, 4th Edition,

published by the American Psychiatric Association (DSM-IV) and/or the International

Classification of Diseases, 10th Edition (ICD-10).

In one embodiment of the invention, the compounds of formula (I) or their pharmaceutically

acceptable salts and/or solvates and/or derivatives may be of use for the treatment or prophylaxis

of a disease or disorder selected from the group consisting of hearing disorders, schizophrenia,

depression and mood disorders, bipolar disorder, substance abuse disorders, anxiety disorders,

sleep disorders, hyperacusis and disturbances of loudness perception, Ménière's disease,

disorders of balance, and disorders of the inner ear, impulse control disorder, personality

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disorders, attention-deficit/hyperactivity disorder, autism spectrum disorders, eating disorders,

cognition impairment, ataxia, pain such as neuropathic pain, inflammatory pain and miscellaneous pain, Lewy body dementia and Parkinson's disease.

of a disease or disorder selected from the group consisting of hearing disorders including hearing

loss and tinnitus, schizophrenia, substance abuse disorders, pain such as neuropathic pain,

inflammatory pain and miscellaneous pain, Lewy body dementia and Parkinson's disease.

of a disease or disorder selected from the group consisting of Fragile-X, Rett's Disorder and

Alzheimer's disease.

The invention provides a method for the prophylaxis or treatment of a disease or disorder selected

from the group consisting of hearing disorders, schizophrenia, depression and mood disorders,

bipolar disorder, substance abuse disorders, anxiety disorders, sleep disorders, hyperacusis and

disturbances of loudness perception, Ménière's disease, disorders of balance, and disorders of

the inner ear, impulse control disorder, personality disorders, attention-deficit/hyperactivity

disorder, autism spectrum disorders, eating disorders, cognition impairment, ataxia, pain such as

neuropathic pain, inflammatory pain and miscellaneous pain, Lewy body dementia and Parkinson's disease, which comprises 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.

salt) and/or derivative thereof.

acceptable salt and/or solvate thereof (e.g. salt) and/or derivative thereof, in the manufacture of

a medicament for the treatment or prophylaxis of a disease or disorder selected from the group

consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder,

substance abuse disorders, anxiety disorders, sleep disorders, hyperacusis and disturbances of

loudness perception, Ménière's disease, disorders of balance, and disorders of the inner ear,

impulse control disorder, personality disorders, attention-deficit/hyperactivity disorder, autism

spectrum disorders, eating disorders, cognition impairment, ataxia, pain such as neuropathic

pain, inflammatory pain and miscellaneous pain, Lewy body dementia and Parkinson's disease.

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In a particular embodiment of the invention, there is provided a compound of formula (I) or their

pharmaceutically acceptable salts and/or solvates (e.g. salts) and/or derivatives thereof for use

in the treatment of prophylaxis 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 (presbycusis), and tinnitus.

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

salts) and/or derivatives thereof may be of use for the treatment or prophylaxis of Ménière's

disease, disorders of balance, and disorders of the inner ear.

in the treatment or prophylaxis of schizophrenia. Schizophrenia includes the subtypes Paranoid

Type (295.30), Disorganised 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 Type, Grandiose Type, Jealous Type, Persecutory

Type, Somatic Type, Mixed Type and Unspecified Type; Brief Psychotic Disorder (298.8); Shared

Psychotic Disorder (297.3); Psychotic Disorder Due to a General 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 Not Otherwise Specified (298.9).

salts) and/or derivatives thereof may be of use 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 Not Otherwise Specified (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 Not Otherwise Specified

(296.80); Other Mood Disorders including Mood Disorder Due to a General Medical Condition

(293.83) which includes 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 Not Otherwise Specified (296.90); Seasonal affective disorder.

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salts) and/or derivatives thereof may be of use for the treatment or prophylaxis of Epilepsy,

(including, but not limited to, localization-related epilepsies, generalized epilepsies, epilepsies

with both generalized and local 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's disease, Lundborg's progressive myoclonic

epilepsy, stroke, head trauma, stress, hormonal changes, drug use or withdrawal, alcohol use or

withdrawal, sleep deprivation, fever, infection, and the like), essential tremor, restless limb

syndrome, partial and generalised seizures (including tonic, clonic, tonic-clonic, atonic, myoclonic,

absence seizures), secondarily generalized seizures, temporal lobe epilepsy, absence epilepsies

(including childhood, juvenile, myoclonic, photo- and pattern-induced), severe epileptic

encephalopathies (including hypoxia-related and Rasmussen's syndrome), febrile convulsions,

epilepsy partialis continua, progressive myoclonus epilepsies (including Unverricht-Lundborg

disease and Lafora's disease), post-traumatic seizures/epilepsy including those related to head

injury, simple reflex epilepsies (including photosensive, somatosensory and proprioceptive,

audiogenic and vestibular), metabolic disorders commonly associated with epilepsy such as

pyridoxine-dependent epilepsy, Menkes' kinky hair disease, Krabbe's disease, epilepsy due to

alcohol and drug abuse (e.g. cocaine), cortical malformations associated with epilepsy (e.g.

double cortex syndrome or subcortical band heterotopia), chromosomal anomalies associated

with seizures or epilepsy such as Partial monosomy (15Q) / Angelman syndrome).

salts) and/or derivatives thereof may be of use for the treatment or prophylaxis of substance-

related disorders including Substance Use Disorders such as Substance Dependence, Substance Craving and Substance Abuse; Substance-Induced Disorders such as Substance

Intoxication, Substance Withdrawal, Substance-Induced Delirium, Substance-Induced Persisting

Dementia, Substance-Induced Persisting Amnestic Disorder, Substance-Induced Psychotic

Disorder, Substance-Induced Mood Disorder, Substance-Induced Anxiety Disorder, Substance-

Induced Sexual Dysfunction, Substance-Induced Sleep Disorder and Hallucinogen Persisting

Perception Disorder (Flashbacks); Alcohol-Related Disorders such as Alcohol Dependence

(303.90), Alcohol Abuse (305.00), Alcohol Intoxication (303.00), Alcohol Withdrawal (291.81),

Alcohol Intoxication Delirium, Alcohol Withdrawal Delirium, Alcohol-Induced Persisting Dementia,

Alcohol-Induced Persisting Amnestic Disorder, Alcohol-Induced Psychotic Disorder, Alcohol-

Induced Mood Disorder, Alcohol-Induced Anxiety Disorder, Alcohol-Induced Sexual Dysfunction,

Alcohol-Induced Sleep Disorder and Alcohol-Related Disorder Not Otherwise Specified (291.9);

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Amphetamine (or Amphetamine-Like)-Related Disorders such as Amphetamine Dependence (304.40), Amphetamine Abuse (305.70), Amphetamine Intoxication (292.89), Amphetamine

Withdrawal (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-Related Disorder Not Otherwise Specified (292.9); Caffeine Related Disorders

such as Caffeine Intoxication (305.90), Caffeine-Induced Anxiety Disorder, Caffeine-Induced

Sleep Disorder and Caffeine-Related Disorder Not Otherwise Specified (292.9); Cannabis-

Related Disorders such as Cannabis Dependence (304.30), Cannabis Abuse (305.20), Cannabis

Intoxication (292.89), Cannabis Intoxication Delirium, Cannabis-Induced Psychotic Disorder,

Cannabis-Induced Anxiety Disorder and Cannabis-Related Disorder Not Otherwise Specified

(292.9); Cocaine-Related Disorders such as Cocaine Dependence (304.20), Cocaine Abuse

(305.60), Cocaine Intoxication (292.89), Cocaine Withdrawal (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-Related Disorder Not Otherwise Specified (292.9); Hallucinogen-Related

Disorders such as Hallucinogen Dependence (304.50), Hallucinogen Abuse (305.30), Hallucinogen Intoxication (292.89), Hallucinogen Persisting Perception Disorder (Flashbacks)

(292.89), Hallucinogen Intoxication Delirium, Hallucinogen-Induced Psychotic Disorder,

Hallucinogen-Induced Mood Disorder, Hallucinogen-Induced Anxiety Disorder and Hallucinogen-

Related Disorder Not Otherwise Specified (292.9); Inhalant-Related Disorders such as Inhalant

Dependence (304.60), Inhalant Abuse (305.90), Inhalant Intoxication (292.89), Inhalant

Intoxication Delirium, Inhalant-Induced Persisting Dementia, Inhalant-Induced Psychotic

Disorder, Inhalant-Induced Mood Disorder, Inhalant-Induced Anxiety Disorder and Inhalant-

Related Disorder Not Otherwise Specified (292.9); Nicotine-Related Disorders such as Nicotine

Dependence (305.1), Nicotine Withdrawal (292.0) and Nicotine-Related Disorder Not Otherwise

Specified (292.9); Opioid-Related Disorders such as Opioid Dependence (304.00), Opioid Abuse

(305.50), Opioid Intoxication (292.89), Opioid Withdrawal (292.0), Opioid Intoxication Delirium,

Opioid-Induced Psychotic Disorder, Opioid-Induced Mood Disorder, Opioid-Induced Sexual

Dysfunction, Opioid-Induced Sleep Disorder and Opioid-Related Disorder Not Otherwise

Specified (292.9); Phencyclidine (or Phencyclidine-Like)-Related Disorders such as Phencyclidine Dependence (304.60), Phencyclidine Abuse (305.90), Phencyclidine Intoxication

(292.89), Phencyclidine Intoxication Delirium, Phencyclidine-Induced Psychotic Disorder,

Phencyclidine-Induced Mood Disorder, Phencyclidine-Induced Anxiety Disorder and Phencyclidine-Related Disorder Not Otherwise Specified (292.9); Sedative-, Hypnotic-, or

Anxiolytic-Related Disorders such as Sedative, Hypnotic, or Anxiolytic Dependence (304.10),

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Sedative, Hypnotic, or Anxiolytic Abuse (305.40), Sedative, Hypnotic, or Anxiolytic Intoxication

(292.89), Sedative, Hypnotic, or Anxiolytic Withdrawal (292.0), Sedative, Hypnotic, or Anxiolytic

Intoxication Delirium, Sedative, Hypnotic, or Anxiolytic Withdrawal Delirium, Sedative-, Hypnotic-

, or Anxiolytic-Persisting Dementia, Sedative-, Hypnotic-, or Anxiolytic- Persisting Amnestic

Disorder, Sedative-, Hypnotic-, or Anxiolytic-Induced Psychotic Disorder, Sedative-, Hypnotic-, or

Anxiolytic-Induced Mood Disorder, Sedative-, Hypnotic-, or Anxiolytic-Induced Anxiety Disorder

Sedative-, Hypnotic-, or Anxiolytic-Induced Sexual Dysfunction, Sedative-, Hypnotic-, or

Anxiolytic-Induced Sleep Disorder and Sedative-, Hypnotic-, or Anxiolytic-Related Disorder Not

Otherwise Specified (292.9); Polysubstance-Related Disorder such as Polysubstance Dependence (304.80); and Other (or Unknown) Substance-Related Disorders such as Anabolic

Steroids, Nitrate Inhalants and Nitrous Oxide.

salts) and/or derivatives thereof may be of use for the treatment or prophylaxis 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 Simple Phobia) including

the subtypes Animal Type, Natural Environment Type, Blood-Injection-Injury Type, Situational

Type and Other Type), 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 General Medical Condition

(293.84), Substance-Induced Anxiety Disorder, Separation Anxiety Disorder (309.21),

Adjustment Disorders with Anxiety (309.24) and Anxiety Disorder Not Otherwise Specified

(300.00).

salts) and/or derivatives thereof may be of use 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 Disorders (780.59),

Circadian Rhythm Sleep Disorder (307.45) and Dyssomnia Not Otherwise Specified (307.47);

primary sleep disorders such as Parasomnias such as Nightmare Disorder (307.47), Sleep Terror

Disorder (307.46), Sleepwalking Disorder (307.46) and Parasomnia Not Otherwise Specified

(307.47); Sleep Disorders Related to Another Mental Disorder such as Insomnia Related to

Another Mental Disorder (307.42) and Hypersomnia Related to Another Mental Disorder (307.44);

Sleep Disorder Due to a General Medical Condition, in particular sleep disturbances associated

with such diseases as neurological disorders, neuropathic pain, restless leg syndrome, heart and

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lung diseases; and Substance-Induced Sleep Disorder including the subtypes Insomnia Type,

Hypersomnia Type, Parasomnia Type and Mixed Type; sleep apnea and jet-lag syndrome.

salts) and/or derivatives thereof may be of use for the treatment or prophylaxis of hyperacusis

and disturbances of loudness perception, including Fragile-X syndrome and autism.

salts) and/or derivatives thereof may be of use for the treatment or prophylaxis of Impulse control

disorder including: Intermittent Explosive Disorder (312.34), Kleptomania (312.32), Pathological

Gambling (312.31), Pyromania (312.33), Trichotillomania (312.39), Impulse-Control Disorders

Not Otherwise Specified (312.3), Binge Eating, Compulsive Buying, Compulsive Sexual

Behaviour and Compulsive Hoarding.

salts) and/or derivatives thereof may be of use 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); sexual pain disorder such as Dyspareunia (302.76) and Vaginismus

(306.51); Sexual Dysfunction Not Otherwise Specified (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 Not Otherwise Specified (302.9); gender identity disorders such as Gender Identity

Disorder in Children (302.6) and Gender Identity Disorder in Adolescents or Adults (302.85); and

Sexual Disorder Not Otherwise Specified (302.9).

salts) and/or derivatives thereof may be of use for the treatment or prophylaxis of 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 Not Otherwise Specified (301.9).

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salts) and/or derivatives thereof may be of use 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 Predominantly Inattentive

Type (314.00), Attention-Deficit/Hyperactivity Disorder Hyperactive-Impulse Type (314.01) and

Attention-Deficit/Hyperactivity Disorder Not Otherwise Specified (314.9); Hyperkinetic Disorder;

Disruptive Behaviour Disorders such as Conduct Disorder including the subtypes childhood-onset

type (321.81), Adolescent-Onset Type (312.82) and Unspecified Onset (312.89), Oppositional

Defiant Disorder (313.81) and Disruptive Behaviour Disorder Not Otherwise Specified; and Tic

Disorders such as Tourette's Disorder (307.23).

salts) and/or derivatives thereof may be of use for the treatment or prophylaxis of Autism

Spectrum Disorders including Autistic Disorder (299.00), Asperger's Disorder (299.80), Rett's

Disorder (299.80), Childhood Disintegrative Disorder (299.10) and Pervasive Disorder Not

Otherwise Specified (299.80, including Atypical Autism).

salts) and/or derivatives thereof may be of use for the treatment or prophylaxis of eating disorders

such as Anorexia Nervosa (307.1) including the subtypes Restricting Type and Binge-

Eating/Purging Type; Bulimia Nervosa (307.51) including the subtypes Purging Type and

Nonpurging Type; Obesity; Compulsive Eating Disorder; Binge Eating Disorder; and Eating

Disorder Not Otherwise Specified (307.50).

salts) and/or derivatives thereof may be of use for the enhancement of cognition including the

treatment of cognition 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 may be of use for the prophylaxis of

cognition impairment, such as may be associated with in diseases such as schizophrenia, bipolar

disorder, depression, other psychiatric disorders and psychotic conditions associated with

cognitive impairment, e.g. Alzheimer's disease.

PCT/GB2020/050268

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salts) and/or derivatives thereof may be of use for the treatment or prophylaxis of ataxia including

ataxia, in particular spinocerebellar ataxia, especially ataxia associated with R420H, R423H or

F448L mutations.

salts) and/or derivatives thereof may be of use for the treatment or prophylaxis of pain including

nociceptive, neuropathic, inflammatory or miscellaneous pain.

Nociceptive pain represents the normal response to noxious insult or injury of tissues such as

skin, muscles, visceral organs, joints, tendons, or bones. Examples of nociceptive pain which

form part of the invention include somatic pain: musculoskeletal (joint pain, myofascial pain) or

cutaneous, which is often well localized; or visceral pain: hollow organs or smooth muscle.

Neuropathic pain is pain initiated or caused by a primary lesion or disease in the somatosensory

nervous system. Sensory abnormalities range from deficits perceived as paraesthesia

(numbness) to hypersensitivity (hyperalgesia or allodynia), and dysaesthesia (tingling and other

sensations). Examples of neuropathic pain which form part of the invention include, but are not

limited to, diabetic neuropathy, post-herpetic neuralgia, spinal cord injury pain, phantom limb

(post-amputation) pain, and post-stroke central pain. Other causes of neuropathic pain include

trauma, chemotherapy and heavy metal exposure.

Inflammatory pain occurs as a result of activation and sensitization of the nociceptive pain

pathway by a variety of mediators released at a site of tissue inflammation. Mediators that have

been implicated as key players in inflammatory pain are pro-inflammatory cytokines such IL-1- -

alpha, IL-1-beta, IL-6 and TNF-alpha, chemokines, reactive oxygen species, vasoactive amines,

lipids, ATP, acid, and other factors released by infiltrating leukocytes, vascular endothelial cells,

or tissue resident mast cells. Examples causes of inflammatory pain which form part of the

invention include appendicitis, rheumatoid arthritis, inflammatory bowel disease, and herpes

zoster.

Miscellaneous pain refers to pain conditions or disorders which are not easily classifiable. The

current understanding of their underlying mechanisms is still rudimentary though specific

therapies for those disorders are well known; they include cancer pain, migraine and other primary

headaches and wide-spread pain of the fibromyalgia type.

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Suitably, specific pain indications that may be mediated by a modulator of Kv3.1 and/or Kv3.2

and/or Kv3.3 channels are neuropathic pain and/or inflammatory pain.

Pain is a subjective condition and in a clinical setting tends to be measured by a patient's self-

assessment. Therefore it can be difficult to measure and quantify pain threshold. For chronic pain,

typically a subjective 11-point rating scale is used where 0 is no pain and 10 is the worst pain

imaginable. Subjects generally record their worst pain over a given period, usually a day. A

minimum mean baseline score is also recorded and response to the medication is measured

relative to the baseline, for example, a reduction of at least 10%, 20%, 30%, 40% or 50% in pain

from the baseline score may be observed.

Since individual responses to medicaments may vary, not all individuals may experience a

reduction in pain from the baseline score. Consequently, suitably a reduction is observed in at

least at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or all individuals tested.

Therefore, in one embodiment of the invention, a reduction of at least 10%, 20%, 30%, 40% or

50% in pain from the baseline score is observed upon administration 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 to a subject in need thereof.

Administration of a Kv3. 1/Kv3.2/Kv3.3 modulator can occur before an anticipated onset of pain or

after the onset of pain. In cases where it is anticipated that development of a disease or disorder

may lead to 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 can be administered. In cases where a 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 a subject in need

thereof.

Treatment of the subject in need thereof may continue for as long as treatment is required, for

example, 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

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.

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Reduction in pain in a subject can be measured by assessing the response to an external stimuli

such as mechanical or thermal (e.g. cold) stimuli (such as described in the Experimental section).

The reduction can either be considered as a percentage reversal (calculated by measuring the

pre- and post-dose thresholds of the affected pain site with a non-affected pain site, such as

described in more detail under Data Analysis in the Experimental Section) or by measuring

withdrawal thresholds of the affected pain site. Preferably, the percentage reversal calculation is

used.

Therefore, in one embodiment of the invention, the sensitivity to pain (such as neuropathic pain

or inflammatory pain) is reversed 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, the

sensitivity to pain is reversed by more than 80% or more than 90%

Subjects receiving the Kv3. 1/Kv3.2/Kv3.3 modulator may experience secondary benefits, such

as one or more of improved function, mood, sleep, quality of life, reduced time off work.

In a particular embodiment, the compounds of formula (I) or their pharmaceutically acceptable

salts and/or solvates (e.g. salts) and/or derivatives thereof may be of use for the treatment or

prophylaxis of neuropathic pain.

prophylaxis of inflammatory pain.

prophylaxis of miscellaneous pain.

In one embodiment is provided a compound of formula (I) for use in the prophylaxis of acute

noise-induced hearing loss.

In one embodiment is provided a method for the prophylaxis of acute noise-induced hearing loss,

comprising administering to a subject in need thereof a compound of formula (I).

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In one embodiment is provided the use of a compound of formula (I) in the manufacture of a

medicament for the prophylaxis of acute noise-induced hearing loss.

Acute noise-induced hearing loss may be caused by events such as exposure to loud noise or a

blast. In these cases, where it is anticipated that a future event may 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 before the event in order to prevent or reduce acute noise-

induced hearing loss. The 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 the acute noise-induced hearing loss or may mitigate other symptoms

arising from acute noise-induced hearing loss, such as tinnitus.

"Acute hearing loss" is defined as hearing loss which occurs rapidly over a period of hours or

days. For example, hearing loss may occur over a period of minutes, hours or days (for example

over a period of up to 1 day, such as up to 2 days, 3 days, 4 days, 5 days, 6 days or 7 days).

Acute hearing loss will typically be caused by exposure to loud sound or blast. Hearing loss

caused by exposure to loud sound or blast is referred to herein as "noise-induced induced hearing

loss". "Acute noise induced hearing loss" is therefore hearing loss which occurs rapidly over a

period of hours or days caused by exposure to loud sound or blast.

Important symptoms of acute hearing loss include:

1. a shift in the auditory threshold, i.e. an increase in the minimum sound level of a pure tone

that can be heard with no other sound present;

2. tinnitus; and

3. degradation in central auditory processing, for example impaired auditory temporal

processing and/or speech understanding.

A "loud" noise or blast may be at least 90dB, for example, at least 100dB, at least 110dB, at least

120 dB or at least 130 dB.

In one embodiment, administration of the compound of formula (I) or a pharmaceutically

acceptable salt, solvate and/or derivative thereof is initiated before an event which is anticipated

to cause noise-induced acute hearing loss. For example, administration of the compound of

formula (I) or a pharmaceutically acceptable salt, solvate and/or derivative thereof may be initiated

up to 2 weeks in advance, such as up to 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 24 h, 12

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h, 6 h, 5 h, 4 h, 3 h, 2 h, 1 h, 30 minutes or up to 15 minutes in advance of an event which is

anticipated to cause noise-induced acute hearing loss. The compound of formula (I) or a

pharmaceutically acceptable salt, solvate and/or derivative thereof may be administered on

multiple occasions before event which is anticipated to cause noise-induced acute 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 which

is anticipated to cause acute noise-induced hearing loss, for preventing or reducing the

development of permanent tinnitus; for preventing or reducing the development of a permanent

shift in auditory thresholds; or for preventing or reducing the development of permanently

degraded central auditory processing, including for example auditory temporal processing and/or

speech understanding.

It will be appreciated that administration in advance may be in circumstances where the subject

is considered to be at risk of exposure to a noise or blast which is anticipated to cause acute

noise-induced hearing loss and is not limited to those circumstances where such exposure

ultimately occurs.

acceptable salt, solvate and/or derivative thereof is initiated during an event which is anticipated

to cause noise-induced acute hearing loss. The compound of formula (I) or a pharmaceutically

acceptable salt, solvate and/or derivative thereof may be administered on multiple occasions

during an event which is anticipated to cause noise-induced acute hearing loss.

and/or derivative thereof is initially administered during a noise or blast which is anticipated to

cause acute noise-induced hearing loss, for preventing or reducing the development of

permanent tinnitus; for preventing or reducing the development of a permanent shift in the

auditory threshold; or for preventing or reducing the development of permanently degraded

central auditory processing, including for example auditory temporal processing and/or speech

understanding.

acceptable salt, solvate and/or derivative thereof is initiated after an event which is anticipated to

cause acute noise-induced hearing loss.

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Thus, in one embodiment, a compound of formula (I) or a pharmaceutically acceptable salt,

solvate and/or derivative thereof is initially administered after a noise or blast which is anticipated

to cause acute noise-induced hearing loss, for preventing or reducing the development of

understanding.

When the compound of formula (I) is administered after an event which is anticipated to cause

acute noise-induced hearing loss, such administration is normally undertaken during the "acute

phase" i.e. before the hearing loss has become established.

acceptable salt, solvate and/or derivative thereof may be initiated up to 2 months after an event

which is anticipated to cause noise-induced acute hearing loss, such as up to 1 month, 2 weeks,

1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 24 h, 12 h, 6 h, 5 h, 4 h, 3 h, 2 h, 1 h, 30 minutes

or up to 15 minutes after an event which is anticipated 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 on multiple occasions after an event which is anticipated to cause

noise-induced acute hearing loss.

thereof may be administered over a period of up to 7 days (for example, 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), for 1-2 weeks (for example,

7-8 days, 7-9 days, 7-10 days, 7-11 days, 7-12 days, 7-13 days or 7-14 days), for 2-4 weeks (for

example, 2-3 weeks or 2-4 weeks) or for 1-2 months (for example, 4-6 weeks or 4-8 weeks).

thereof may initially be administered up to 1 day in advance, such as up to 2 days in advance, up

to 3 days in advance, up to 5 days in advance, up to 1 week in advance, up to 2 weeks in advance

or up to 1 month in advance of a noise or blast which is anticipated to cause acute noise-induced

hearing loss, administration which is initiated at any point in advance exposure to a noise or blast

which is anticipated to cause acute noise-induced hearing loss will typically continue for up to 2

months after exposure to the noise or blast which is anticipated to cause acute noise-induced

hearing loss, such as for 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.

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In one embodiment 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 shift in the auditory threshold, wherein the permanent shift in auditory threshold is

reduced by at least 10dB, such as at least 15dB, at least 20dB, at least 30dB, at least 40dB, or

completely.

Pharmaceutical compositions

For use in therapy the compounds of the invention are usually administered as a pharmaceutical

composition. The 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. salt) and/or derivative

thereof, for use in the treatment or prevention of a disease or disorder selected from the group

In a further embodiment, there is provided a method for the prophylaxis or treatment of a disease

or disorder selected from the group consisting of hearing disorders, schizophrenia, depression

and mood disorders, bipolar disorder, substance abuse disorders, anxiety disorders, sleep

disorders, hyperacusis and disturbances of loudness perception, Ménière's disease, disorders of

balance, and disorders of the inner ear, impulse control disorder, personality disorders, attention-

deficit/hyperactivity disorder, autism spectrum disorders, eating disorders, cognition impairment,

ataxia, pain such as neuropathic pain, inflammatory pain and miscellaneous pain, Lewy body

dementia and Parkinson's disease, which comprises 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.

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The invention also provides the use of a pharmaceutical composition comprising a compound of

formula (I), or a pharmaceutically acceptable salt and/or solvate thereof (e.g. salt) and/or

derivative thereof, in the manufacture of a medicament for the treatment or prophylaxis of a

disease or disorder selected from the group consisting of hearing disorders, schizophrenia,

cognition impairment, ataxia, pain such as neuropathic pain, inflammatory pain and

miscellaneous pain, Lewy body dementia and Parkinson's disease.

derivatives thereof may be administered by any convenient method, e.g. by oral, parenteral,

buccal, sublingual, nasal, rectal or transdermal administration, and the pharmaceutical

compositions adapted accordingly. Other possible routes of administration include intratympanic

and intracochlear.

derivatives thereof which are active when given orally can be formulated as liquids or solids, e.g.

as syrups, suspensions, emulsions, tablets, capsules or lozenges.

A liquid formulation will generally consist 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) e.g. an aqueous solvent such as water,

ethanol or glycerine, or a non-aqueous solvent, such as polyethylene glycol or an oil. The

formulation may also contain a suspending agent, preservative, flavouring and/or colouring agent.

A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s)

routinely used for preparing solid formulations, such as magnesium stearate, starch, lactose,

sucrose and cellulose.

A composition in the form of a capsule can be prepared using routine encapsulation procedures,

e.g. pellets 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) can be

prepared using standard carriers and then filled into a hard gelatin capsule; alternatively a

dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), e.g.

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aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft

gelatin capsule.

Typical parenteral compositions consist of a solution or suspension of the active ingredient (such

and/or derivative thereof) in a sterile aqueous carrier or parenterally acceptable oil, e.g.

polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the

solution can be lyophilised and then reconstituted with a suitable solvent just prior to

administration.

Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels

and powders. Aerosol formulations typically comprise a solution or fine suspension of the active

ingredient in a pharmaceutically acceptable aqueous or non-aqueous solvent and are usually

presented in single or multidose quantities in sterile form in a sealed container which can take the

form of a cartridge or refill for use with an atomising device. Alternatively, the sealed container

may be a disposable dispensing device such as a single dose nasal inhaler or an aerosol

dispenser fitted with a metering valve. Where the dosage form comprises an aerosol dispenser,

it will contain a propellant which can be a compressed gas e.g. air, or an organic propellant such

as a fluorochlorohydrocarbon or hydrofluorocarbon. Aerosol dosage forms can also take the form

of pump-atomisers.

Compositions suitable for buccal or sublingual administration include tablets, lozenges and

pastilles where the active ingredient is formulated with a carrier such as sugar and acacia,

tragacanth, or gelatin and glycerin.

Compositions for rectal administration are conveniently 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 unit dose form such as a tablet, capsule or ampoule.

The composition may contain from 0.1% to 100% by weight, for example from 10 to 60% by

weight, of the active material, depending on the method of administration. The composition may

contain from 0% to 99% by weight, for example 40% to 90% by weight, of the carrier, depending

on the method of administration. The composition may contain from 0.05 mg to 1000 mg, for

example from 1.0 mg to 500 mg, of the active material, depending on the method of

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administration. The composition may contain from 50 mg to 1000 mg, for example from 100 mg

to 400 mg of the carrier, depending on the method of administration. The dose of the compound

used in the treatment of the aforementioned disorders will vary in the usual way with the

seriousness of the disorders, the weight of the sufferer, and other similar factors. However, as a

general guide suitable unit doses may be 0.05 mg to 1000 mg, more suitably 1.0 mg to 500 mg,

and such unit doses may be administered more than once a day, for example two or three a day.

Such therapy may extend for a number of weeks or months.

The dose provided to a subject will typically be a safe and effective dose, i.e. an acceptable

balance of desired benefits and undesired side effects.

The invention provides, in a further aspect, a combination comprising a compound of formula (I)

or a pharmaceutically acceptable, salt, solvate and/or derivative thereof (e.g. a combination

comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof)

together with a further pharmaceutically acceptable active ingredient or ingredients.

The invention provides a compound of formula (I), for use in combination with a further

pharmaceutically acceptable active ingredient or ingredients.

When the compounds are used in combination with other therapeutic agents, the compounds

may be administered either sequentially or simultaneously by any convenient route. Alternatively,

the compounds may be administered separately.

The combinations referred to above may conveniently be presented for use in the form of a

pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as

defined above together with a pharmaceutically acceptable carrier or excipient comprise a further

aspect of the invention. The individual components of such combinations may be administered

either sequentially or simultaneously in separate or combined pharmaceutical formulations. The

individual components of combinations may also be administered separately, through the same

or 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 that when the compound is used alone. Appropriate doses will

be readily appreciated by those skilled in the art.

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Suitably, a compound of formula (I) or a pharmaceutically acceptable, salt, solvate and/or

derivative thereof is administered orally.

derivative thereof is administered at 2 to 400 mg per day, such as 2 to 300 mg per day, especially

5 to 250 mg per day.

derivative thereof is administered once or twice per day.

derivative thereof is administered for a period of at least three months.

Desirably, a compound of formula (I) or a pharmaceutically acceptable, salt, solvate and/or

derivative thereof is administered orally, once or twice per day, at 2 to 400 mg per day, such as 2

to 300 mg per day, especially 5 to 250 mg per day.

A human subject may be an adult, such as aged 18 to 65. Alternatively, a human subject may be

66 years old or older. A compound of formula (I) or a pharmaceutically acceptable, salt, solvate

and/or derivative thereof may be administered to a human subject of less than 18 years of age,

such as 4 to 17 years old. Administration to a human subject of less than 18 years of age may be

of particular relevance in the context of progressive myoclonic epilepsy and Fragile X syndrome.

For convenience and to assist with 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 a sustained period of time e.g. at least one week or at least

4 weeks.

Experimental

The invention is illustrated by the compounds described below. The following examples describe

the laboratory synthesis of specific compounds of the invention and are not meant to limit the

scope of the invention in any way with respect to compounds or processes. It is understood that,

although specific reagents, solvents, temperatures and time periods are used, there are many

possible equivalent alternatives that can be used to produce similar results. This invention is

meant to include such equivalents.

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Analytical Equipment

Starting materials, reagents and solvents were obtained from commercial suppliers and used

without further purification unless otherwise stated. Unless otherwise stated, all compounds with

chiral centres are racemic. Where reactions are described as having been carried out in a similar

manner to earlier, more completely described reactions, the general reaction conditions used

were essentially the same. Work up conditions used were of the types standard in the art, but

may have been adapted from one reaction to another. The starting material may not necessarily

have been prepared from the batch referred to. Compounds synthesised may have various

purities, ranging from for example 85% to 99%. Calculations of number of moles and yield are in

some cases adjusted for this.

HPLC-Mass spectra (HPLC-MS) were taken on an Agilent 1100 Series LC/MSD Mass Spectrometer coupled with HPLC instrument Agilent 1100 Series, operating in positive

electrospray ionization mode and in acidic gradient conditions.

Quality Control (3 minutes method): LC/MS-ES+ under acidic conditions was performed on a

Zorbax SB C18 column (1.8 um 3 X 50 mm). Mobile phase: A: (H2O + 0.05% TFA by vol.) / B:

(CH3CN + 0.05% TFA by vol). Gradient: t = 0 min 0% (B), from 0 to 95% (B) in 2.5 min, 95%

(B)for 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. Stop time 4 min. Column T = 60°C. Flow rate: 1.5 ml/min. Mass range ES+: (100-1000 amu,

F=60). UV detection wavelengths: DAD 1A = 220.8, DAD 1B = 254.8. The use of this methodology is indicated by "QC_3_MIN" in the analytic characterization of the described

compounds.

Chiral control: LC/MS-ES+ under acidic conditions was performed on a CHIRALCEL® OD-H (250

X 4,6 mm - 5 um). Mobile phase: A: (H2O + 0.05% TFA by vol.) / B: (CH3CN + 0.05% TFA by

vol). 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). Stop 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 either on Varian instruments at 300,

400, 500 or 600 MHz, or on Bruker instruments at 400 MHz. Chemical shifts are reported in ppm

() using the residual solvent line as internal standard. Splitting patterns are designed as S

(singlet), br.s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet wo 2021/156584 WO PCT/GB2020/050268

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of triplets) and m (multiplet). The NMR spectra were recorded at temperatures ranging from 25 to

60°C.

2D NMR NOESY experiments were acquired with a mixing time of 500 ms using a spectral width

of 3355 Hz in both f1 and f2. A total of 256 increments were collected, processed to 1 K with linear

prediction, 8 scans each. Data were processed with sine bell shift in both dimensions and with

lb=0.3 Hz in f1. In a number of preparations, purification was performed using Biotage automatic

flash chromatography (SP1 and SP4) or Flash Master Personal systems.

Flash chromatographies were carried out on silica gel 230-400 mesh (supplied by Merck AG

Darmstadt, Germany) or on silica gel 300-400 mesh (supplied by Sinopharm Chemical Reagent

Co., Ltd.), Varian Mega Be-Si pre-packed cartridges, pre-packed Biotage silica cartridges (e.g.

Biotage SNAP cartridge).

Abbreviations

AIBN AIBN azobisisobutyronitrile

BuLi BuLi butyllithium

CDCl3 CDCl deuterated chloroform

CCI4 carbon tetrachloride

D2O deuterated water

dichloromethane DCM DIPEA N,N-diisopropylethylamine

DMAP 4-dimethylaminopyridine DMAP DMF N,N-dimethylformamide

dimethylsulfoxide DMSO DMSO-d6 deutrated dimethylsulfoxide

Et2O diethyl ether

EtOAc EtOAc ethyl acetate

h hours

(O-7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumhexafluoro HATU phosphate)

HCI hydrogen chloride

K2CO3 potassium carbonate

acetonitrile MeCN /CH3CN methanol MeOH methyloxymethyl MOM wo 2021/156584 WO PCT/GB2020/050268 PCT/GB2020/050268

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NaH sodium hydride

NaSO4 sodium sulphate

sodium carbonate NaCO NaCO NaOH sodium hydroxide

NaOMe sodium methoxide

Nuclear Magnetic Resonance NMR r.t. room temperature

T3P propylphosphonic anhydride

Methyl tert-butyl ether MTBE Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate TBTU triethylamine TEA TFA trifluoroacetic acid

THF tetrahydrofuran THF THP tetrahydropyran

wt. weight

Compound Examples

Intermediate 1

2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxy-pyrazine

o O N 11

N Br Br

A mixture of (-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-c (Intermediate 156

WO2012076877, 1.11g, 6,30mmol), 2,5-dibromopyrazine (1.5g, 6,30mmol) and dipotassium

carbonate (1.31g, 9.46mmol) in N,N-dimethylformamide (14mL) was stirred at 120°C for 3

hours. After cooling, the reaction mixture was diluted with MTBE (100 ml) and washed with

brine (50 ml). Phases were separated and the aqueous layer was washed with MTBE (100ml)

and EtOAc (100ml). All organic phases are collected, dried over Na2SO4, filtered and

evaporated. The residue was purified by flash chromatography (Biotage System) on silica gel

using a SNAP 100g as column and Cyclohexane: Ethyl acetate from 100:0 to 90:10 as eluent

affording2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxy-pyrazine(1.8g)

as white solid.

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

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The following compounds were prepared using the foregoing methodology, replacing 7-

methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-ol with the appropriate phenol. Final products

were purified by flash-chromatography (Silica cartridge; Cyclohexane/EtOAc or other

appropriate solvent system).

Int. Structure Name Phenol LCMS 2 2-bromo-5- spiro[2H-benzofuran- LC/MS: oO spiro[2H- 3,1'-cyclopropane]-4- QC_3_MIN: Rt = N 11 benzofuran-3,1'- ol (Intermediate 85 2.575 min; m/z NN Br cyclopropane]-4- WO2012076877) 319 & 321

yloxy-pyrazine [M+H]+.

3 2-bromo-5-[(3,3,7- 3,3,7-trimethyl-2H- LC/MS: O N N trimethyl-2H- benzofuran-4-ol QC_3_MIN: Rt = o O benzofuran-4- (Intermediate 184 2.365 min; m/z N Br Br yl)oxy]pyrazine WO2012076877) 335 & 337

[M+H]+.

4 2-bromo-5-[(3,3- 3,3-dimethyl-2H- LC/MS: O O dimethyl-2H- benzofuran-4-ol QC_3_MIN: Rt = N N 11 o O benzofuran-4- (Intermediate 50 2.632 min; m/z NN yl)oxy]pyrazine WO2012076877) 321 & 323 Br

[M+H]+.

Intermediate 5 route 1

3-(5-chloropyrazin-2-yl)-5,5-dimethyl-imidazolidine-2,4-dion CI

NN 11

N: o N

o O NH

To a solution of bis(trichloromethyl) carbonate (950mg, 3.20mmol) in ethyl acetate (30mL) at

0°C a solution of 5-chloropyrazin-2-amine (0.75g, 5.79mmol)/N,N-diisopropylethylamine

(6.05ml, 34.74mmol) in ethyl acetate (12mL) was added dropwise and the reaction mixture was

stirred for 15 minutes at the same temperature. Maintaining the reaction mixture at 0°C, vacuum

was applied (5 minutes) in order to remove the excess of phosgene. A solution of 4-

(dimethylamino)pyridine (710mg, 5.81mmol) in ethyl acetate (8mL)/ dichloromethane (2mL) was

added and the reaction mixture was stirred for 5 minutes at the same temperature. Then, methyl wo 2021/156584 WO PCT/GB2020/050268 PCT/GB2020/050268

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2-amino-2-methyl-propanoate hydrochloride (1.4g, 9.1mmol) was added at 0°C and the reaction

mixture was stirred for 30 minutes at the same temperature. The reaction was quenched with a

solution 0.2 N of HCI (100 ml) and the two phases were separated. The organic layer was

washed with brine (100 ml), dried over Na2SO4, filtered and evaporated affording the urea

intermediate.

The urea was dissolved in dichloromethane (20mL) and at 0°C sodium methoxide (315mg,

5.83mmol) was added. The reaction mixture was stirred 15 minutes at the same temperature;

the reaction was quenched with a saturated solution of NH4CI to allow the pH to reach 3-4. The

mixture was extracted with ethyl acetate (50 ml); phases were separated, and the organic layer

was washed with brine (50 ml), dried over Na2SO4, filtered and evaporated. The residue were

purified by reverse phase flash chromatography (Biotage System) on C-18 phase using a SNAP

30g as column and Water:Acetonitrile from 95:5 to 40:60 as eluent. The appropriate fractions

were combined and evaporated to dryness affording 3-(5-chloropyrazin-2-yl)-5,5-dimethyl-

imidazolidine-2,4-dione (220mg) as a pale brown solid.

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

The following compounds were prepared using the foregoing methodology, replacing 2,2-

dimethylglycine methyl ester hydrochloride with the appropriate amino ester hydrochloride. Final

products were purified by flash-chromatography (Silica cartridge; Cyclohexane/EtOAc or other

appropriate solvent system) or triturated in an appropriate solvent or crystallised from an

appropriate solvent.

Int. Structure Name Amino ester LCMS hydrochloride

CI 6 5R)-3-(5- methyl (2R)-2- LC/MS: NN // 11 chloropyrazin-2- amino-2-methyl- QC_3_MIN: Rt = N - O yl)-5-ethyl-5- butanoate 1.546 min; m/z 255 NN methyl- hydrochloride & 257 [M+H]+. O NH E imidazolidine-

2,4-dione

Intermediate 5 route 2

3-(5-chloropyrazin-2-yl)-5,5-dimethyl-imidazolidine-2,4-dione

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CI N

N o - N o NH

To a solution of 5-chloropyrazin-2-amine (500mg, 3.86mmol) and 2-amino-2-methyl-propanoic

acid hydrochloride (646mg, 4.63mmol) in acetonitrile (10mL), Propylphosphonic anhydride

solution 50 wt. % in ethyl acetate (3.68g, 5.78mmol) was slowly added at RT. The reaction

mixture was stirred at 80°C for 6h. The reaction mixture was diluted with Ethyl Acetate (10ml)

and an aqueous solution of NaOH 1 N was added, while the ph was allowed to reach ~8. The

two phases were separated and the organic one was washed with brine (10ml), dried with

Na2SO4, concentrated under vacuum and the crude was purified by Flash Chromatography on

silica gel (BIOTAGE SYSTEM), using a SNAP 25g as column and DCM:MEOH from 99/1 to

90/10 as eluent, affording 2-amino-N-(5-chloropyrazin-2-yl)-2-methyl-propanamide (190mg) as

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-methyl-propanamide (190mg,

0.88mmol) and triethylamine (268mg, 2,6555mmol) in dichloromethane (5mL), at 0°C a solution

of bis(trichloromethyl) carbonate (105,07mg,0,3541mmol) in dichloromethane (4mL) was slowly

added. and the reaction mixture was stirred for 30 minutes at the same temperature. The

reaction mixture was diluted in DCM (10mL), washed with an aqueous solution 0.2N of HCI

(10mL) and Brine (10mL). The organic phases were concentrated under vacuum and the crude

was purified by flash chromatography on silica gel (Biotage system) using a SNAP 25g as

column and Chexane/EtOAc from 80/20 to 0/100 as eluent affording 3-(5-chloropyrazin-2-yl)-

5,5-dimethyl-imidazolidine-2,4-dione (130mg) as white solid.

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

Intermediate 7

tert-butyl N-[(1R)-1-carbamoylpropyl]carbamate

NH2 H o N o

A mixture of[dimethylamino-(3-oxidotriazolo[4,5-b]pyridin-3-ium-1-yl)methylene]-dimethyl

ammonium tetrafluoroborate (1,1084g,3,4415mmol), N,N-diisopropylethylamine

(0,7939g,6,1431mmol) and (2R)-2-(tert-butoxycarbonylamino)butanoic acid

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(0,5000g,2,4601mmol) in dry N,N-dimethylformamide (8mL) was stirred at room temperature for

10 minutes. Hexamethyldisilazane (0,5960g,3,6928mmol) was added and the mixture stirred for

18h.

Reaction mixture was separated in MTBE (30 mL) and Brine (20 mL). The organic layer was

dried with sodium sulphate, filtered and the solvent removed. The resulting oil triturated in

MTBE (3 mL) and the resulting precipitate was washed with MTBE and dried via vacuum to give

tert-butyl N-[(1R)-1-carbamoylpropyl]carbamate (0,3000g,1,4833mmol, 60,294 %) as a white

solid.

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

The following compounds were prepared using the foregoing methodology, replacing (2R)-2-

(tert-butoxycarbonilamino) butanoic acid with the appropriate protected amino-acid.

Int. Structure Amino-acid Name LCMS 8 NH2 tert-butyl N-[(1R)-1- (2R)-2-(tert- LC/MS: NH HH o N o carbamoyl-1-methyl- butoxycarbonylami QC_3_MIN: = O - propyl]carbamate no)-2-methyl- m/z 455

butanoic acid [2M+Na]+.

Intermediate 9 (route 1)

tert-butylN-(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazir

2-yl]carbamoyl]propyl]carbamate

O N O N NH H O N o : n O

A mixture of2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxy-pyrazine

(Intermediate 1, 50mg, 0.15mmol), tert-butyl N-[(1R)-1-carbamoylpropyl]carbamate

(Intermediate 7, 46mg, 0.23mmol), Tris(dibenzylideneacetone)dipalladium(0) (10.3mg,

0.011mmol), dicyclohexyl-[2-(2,4,6-triisopropylphenyl)phenyl]phosphane (XPhos) (5.4mg,

0.011mmol) and cesium carbonate (73mg, 0.22mmol) in 1,4-dioxane (2mL) was stirred under

an atmosphere of nitrogen at 80°C for 3h.

The reaction was partitioned between ethyl acetate and brine, The organic layer was separated,

dried with sodium sulphate, filtered and evaporated to dryness. The residue was purified by

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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, affording tert-butyl N-[(1R)-1-[5-(7-methylspiro[2H-

nzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]carbamoyl]propyl]carbamate(10mg).

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

Intermediate 9 (route 2)

tert-butyl IN-[(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-

2-yl]carbamoyl]propyl]carbamate

O NN

N N NH H O N o : n O

To a ixture of 2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxy-pyrazine

(Intermediate 1, 16g, 48.0mmol), tert-butyl IN-[(1R)-1-carbamoylpropyl]carbamate (Intermediate

7, 10g, 49.4mmol), cesium carbonate (24.16g, 74.17mmol) in 1,4-dioxane (150mL), after

flushing with argon, diacetoxypalladium (0.555g, 2.47mmol) and (5-diphenylphosphanyl-9,9-

dimethyl-xanthen-4-yl)-diphenyl-phosphane (2.15g, 3.71mmol) were added. For three times

cycle vacuum-argon was applied and the reaction mixture was stirred at 95°C for 1.5h. 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 (150ml) and washed

with an aqueous saturated solution of NH4CI (100ml) and then with a n aqueous saturated

solution of NaCI (100ml), dried with sodium sulphate, filtered and evaporated to dryness.

The residue was purified by flash chromatography on silica gel (Biotage system) using 2x SNAP

100g column (200g silica) and cyclohexane/EtOAc from 0 to 40% as eluent affording tert-butyl

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

yl]carbamoyl]propyl]carbamate (16.8g) as yellow solid.

The following compounds were prepared using the foregoing methodology (either route 1 or

route 2), replacing2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxy-

pyrazine (Intermediate 1) with the appropriate bromopyrazine. Final products were purified by

flash-chromatography (Silica cartridge; Cyclohexane/EtOAc or other appropriate solvent

system).

Int. Structure Name bromopyrazine LCMS wo 2021/156584 WO PCT/GB2020/050268

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10 tert-butyl N-[(1R)-1- 2-bromo-5- LC/MS: o

[(5-spiro[2H- spiro[2H- N QC_3_MIN: benzofuran-3,1'- benzofuran-3,1'- Rt = 2.246 o cyclopropane]-4- cyclopropane]-4- min; m/z 441 N o H N yloxypyrazin-2- yloxy-pyrazine [M+H]+. e H

yl)carbamoy/]propyl]c (Intermediate 2)

arbamate 11 tert-butyl N-[(1R)-1- 2-bromo-5-[(3,3,7- LC/MS: o O

[[5-[(3,3,7-trimethyl- trimethyl-2H- N QC_3_MIN: O o 2H-benzofuran-4- benzofuran-4- - Rt = 2.309 N O O N o yl)oxy]pyrazin-2- yl)oxy]pyrazine min; m/z 457 H N H yljcarbamoyl]propyl]c (Intermediate 3) [M+H]+.

arbamate

12 tert-butyl N-[(1R)-1- 2-bromo-5-[(3,3- LC/MS: O [[5-[(3,3-dimethyl-2H- dimethyl-2H- QC_3_MIN: N - benzofuran-4- benzofuran-4- Rt =2.366 N N yl)oxy]pyrazin-2- yl)oxy]pyrazine min; m/z 443 N H N yl]carbamoyl]propyl]c (Intermediate 4) [M+H]+. : H

arbamate

Intermediate 13

2R)-2-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1-cyclopropane]-4-yl)oxypyrazin-

yl]butanamide

O N O O N N: NH O NH2 :

1 A mixture of tert-butyl IN-[(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-

yl) loxypyrazin-2-yl]carbamoyl]propyl]carbamate (Intermediate 9, 16mg, 0.035mmol) and 2,2,2-

trifluoroacetic acid (0.50mL, 6.53mmol) in dichloromethane (2mL) was stirred at room

temperature for 2h.

The reaction mixture was diluted with dichloromethane (20 ml) and a saturated solution of

NaHCO3 (aq) was added while the pH was allowed to reach 8. The phases were separated and

the organic layer was washed with brine (20 ml), dried over Na2SO4, filtered and evaporated

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affording(2R)-2-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-

2-yl]butanamide (13mg) that 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 foregoing methodology, replacing tert-butyl

yl]carbamoyl]propyl]carbamate (Intermediate 9) with the appropriate Boc amine.

Int. Structure Name Boc amine LCMS 14 (2R)-2-amino-N-(5- tert-butyl N-[(1R)-1-[(5- LC/MS: O o spiro[2H- spiro[2H-benzofuran-3,1'- QC_3_MIN: Rt N O benzofuran-3,1'- cyclopropane]-4- 1.675min N o - N cyclopropane]-4- yloxypyrazin-2- m/z 342 H NH2 NH2 yloxypyrazin-2- yl)carbamoyl]propyl]carba [M+H]+.

yl)butanamide mate (Intermediate 10

15 (2R)-2-amino-N-[5- tert-butyl N-[(1R)-1-[[5- LC/MS: O

[(3,3,7-trimethyl- [(3,3,7-trimethyl-2H- QC_3_MIN: Rt N QC_3_MIN:R O 2H-benzofuran-4- benzofuran-4- =1.756 min; N o N yl)oxy]pyrazin-2- yl)oxy]pyrazin-2- m/z 357 H N H2 yl]butanamide yl]carbamoyl]propyl]carba [M+H]+.

mate (Intermediate 11

16 (2R)-2-amino-N-[5- tert-butyl N-[(1R)-1-[[5- LC/MS: OO

[(3,3-dimethyl-2H- [(3,3-dimethyl-2H- QC_3_MIN: Rt N o benzofuran-4- benzofuran-4- =1.673 min; N O yl)oxy]pyrazin-2- yl)oxy]pyrazin-2- m/z m/z 343 343 N H NH2 NH yl]butanamide yl]carbamoyl]propyl]carba [M+H]+.

mate (Intermediate 12)

Intermediate 17

5R)-5-ethyl-5-methyl-imidazolidine-2,4-dione

H O N

NH =

A mixture of tert-butyl N-[(1R)-1-carbamoyl-1-methyl-propyl]carbamate (Intermediate 8,

100mg,0,4624mmol) and potassium carbonate (191,71mg,1,3871mmol) in 1-butanol (5mL) was

stirred under an atmosphere of 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

an aqueous 0.1N HCI solution (30 ml) and then with brine (30 ml). Phases were separated and wo 2021/156584 WO PCT/GB2020/050268

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the organic layer was collected, dried over Na2SO4, filtered and evaporated affording (5R)-5-

ethyl-5-methyl-imidazolidine-2,4-dione (60mg,0,4221mmol, 91,283%).

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

Intermediate 18

tert-butyl N-(1-carbamoylcyclobutyl)carbamate

NH2 H o O N O

Intermediate 18 was prepared using the methodology described for Intermediate 7, replacing

(2R)-2-(tert-butoxycarbonylamino)butanoi acid with 1-(tert-

sutoxycarbonylamino)cyclobutanecarboxylic acid.

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

Intermediate 19

tert-butylN-[1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-

yl]carbamoyl]cyclobutyl]carbamate

O N // 11

NN NH H O N o

O

(Intermediate 1, 50mg, 0.1501mmol), tert-butyl N-(1-carbamoylcyclobutyl)carbamate

(Intermediate 18, 64mg, 0.2987mmol), dipotassium carbonate (62mg,0,4486mmol), copper(I)

iodide (2.9mg.0.0152mmol) and N,N'-dimethylethane-1,2-diamine (0.0065mL,0.0601mmol) in 1-

butanol (1mL) was stirred under an atmosphere of nitrogen at 95°C for 4h. After cooling, the

reaction mixture was diluted with ethyl acetate (30 ml) and washed with an aqueous 0.1M HCI

solution (30 ml) and then with brine (30 ml). Phases were separated, and the organic layer was

collected, dried over Na2SO4, filtered and evaporated. The residue was purified by flash

chromatography (Biotage System) on silica gel using a SNAP 10g as column and Cyclohexane:

Ethyl acetate from 100:0 to 30:70 as eluent affording tert-butyl N-[1-[[5-(7-methylspiro[2H-

benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]carbamoyl]cyclobutyl]carbamate (18mg).

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

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Intermediate 20

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

yl]cyclobutanecarboxamide

O NN 11 o < N : NH o NH2

Intermediate 20 was prepared using the methodology described for Intermediate 13, replacing

tert-butylN-[(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-

yl]carbamoyl]propyl]carbamate (Intermediate 9) with tert-butyl N-[1-[[5-(7-methylspiro[2H-

an-3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]carbamoyl]cyclobutyl]carbamate

(Intermediate 19).

LC/MS: QC_3_MIN: LC/MS: Rt = 1.979 min; m/z367 QC_3_MIN:Rt=1.979min;m/z 367 [M+H]+.

[M+H]+.

Intermediate 21

tert-butyl N-(1-carbamoylcyclopropyl)carbamate

NH2 NH H :

O N N O O

Intermediate 21 was prepared using the methodology described for Intermediate 7, replacing

2R)-2-(tert-butoxycarbonylamino)butanoi acid with 1-(tert-

butoxycarbonylamino)cyclopropanecarboxylic acid.

Intermediate 22

tert-butylN-[1-r[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2

yl]carbamoyl]cyclopropyl]carbamate

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A mixture of fdicyclohexyl-[2-(2,4,6-triisopropylphenyl)phenyl]phosphane(12mg, 0.0252mmol),

tert-butyl N-(1-carbamoylcyclopropyl)carbamate (Intermediate 21, 67mg, 0.3346mmol),

Tris(dibenzylideneacetone)dipalladium(0) (22mg, 0.0240mmol), 2-bromo-5-(7-methylspiro[2H-

benzofuran-3,1'-cyclopropane]-4-yl)oxy-pyrazine, (Intermediate 1, 79.518mg, 0.2387mmol) and

caesium carbonate (116mg, 0.3560mmol) in 1,4-dioxane (1mL) were stirred under an

atmosphere of nitrogen at 95°C for 2h. Additional tert-butyl N-(1-

carbamoylcyclopropyl)carbamate (Intermediate 21, 67mg, 0.3346mmol) and

Tris(dibenzylideneacetone)dipalladium(0 (22mg, 0.0240mmol) was added and the reaction

mixture was stirred at 95°C under nitrogen for a further 2h, followed by the addition of a further

dicyclohexyl-[2-(2,4,6-triisopropylphenyl)phenyl]phosphane (12mg, 0.0252mmol),

Tris(dibenzylideneacetone)dipalladium(0 (22mg, 0.0240mmol) and caesium carbonate (58mg)

and the mixture was stirred under nitrogen for a further 2h. The reaction mixture was then

quenched with water (10mL), NH4CI (10mL) and extracted with ethyl acetate (20mL). The

organic layer was then washed with brine (15mL), dried over Na2SO4, filtered, then

concentrated in vacuo. The crude was purified by flash chromatography (Biotage System) on

silica gel using a SNAP 10g as column and Cyclohexane:Ethyl acetate 90:10 to 70:30 as eluent

to afford tert-butyl N-[1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2

yl]carbamoyl]cyclopropyl]carbamate (55mg) as a yellow solid.

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

Intermediate 23

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

yl]cyclopropanecarboxamide

O NN O

NH NH2 o

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

yl]carbamoyl]cyclopropyl]carbamate (Intermediate 22, 55mg, 0.1215mmol) was dissolved in

dichloromethane (4mL) and cooled to 0 °C. 2,2,2-trifluoroacetic acid (1154.7mg, 10.026mmol)

(0.8mL) was added dropwise and the reaction was stirred at room temperature for 1 hour. The

reaction mixture was then cooled to 0 1°C and NaHCO was added until the pH reached 8. The

mixture was then allowed to warm to room temperature and extracted with DCM (10mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuum to afford1-amino-N-

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

yl]cyclopropanecarboxamide (40mg) as a yellow oil.

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

Intermediate 24

1,3-dibenzyloxy-2-bromo-benzene

O Br Br

O

To a solution of 2-bromobenzene-1,3-diol (20g,105.8mmol) in acetone (200mL), potassium

carbonate (43.87g, 317.4mmol) was added followed by the addition of benzyl bromide (40.72g,

238. 1mmol) (28ml) and the reaction mixture was refluxed for 1.5 hours. 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 with

brine (100 ml). Phases were separated and the organic layer was dried over Na2SO4, filtered

and concentrated. The residue was suspended in isopropanol (8 volumes) and the mixture

heated at 80°C and stirred for 1 hour at this temperature (to obtain a clear solution). Then, the

mixture was allowed to reach room temperature (in 1h) and the obtained suspension was

filtered. The solid was washed with ice cold isopropanol and then dried affording the title

compound 1,3-dibenzyloxy-2-bromo-benzene, (34g) as pale pink solid.

LC/MS: QC_3_MIN: Rt = 2.688 min.

Intermediate 25

bromo-(1-methoxycarbonylcyclopropyl)zino

Br Zn o Zn

In a two-neck round-bottom flask activated zinc powder (6.84g,104.6mmol) was added and the

powder was heated under vacuum. The system was put under argon and dry tetrahydrofuran

(58mL) was added. Then, 1,2-dibromoethane (2.18g, 11.62mmol) was added and the mixture

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was heated to reflux. Chlorotrimethylsilane (505mg, 4.65mmol) was added in a single portion

and the mixture kept stirring at reflux temperature. A solution of methyl 1-

bromocyclopropylcarboxylate (10.4g, 58.1mmol) in dry tetrahydrofuran (12mL) was slowly

added at the same temperature and the reaction mixture was refluxed for 1.5h. The reaction

mixture was cooled down to room temperature and the zinc was allowed to settle affording 70ml

of a 0.83M (theoretical) solution of bromo-(1-methoxycarbonylcyclopropyl)zino in THE which

was used in the next step without further work up.

Intermediate 26

methyl 1-(2,6-dibenzyloxyphenyl)cyclopropanecarboxylate

To a solution of 1,3-dibenzyloxy-2-bromo-benzene (Intermediate 24, 16g, 43.33mmol) and

Bis(tri-tert-butylphosphine)palladium( (221mg, 0.43mmol) in N,N-dimethylformamide (150mL)

pre-heated at 70 °C, a 0.83M (theoretical) solution of bromo-(1-

methoxycarbonylcyclopropyl)zino in THF (Intermediate 25, 60ml) was added (via cannulation)

and the reaction mixture was stirred at the same temperature for 40 minutes. After cooling, the

reaction mixture was concentrated under vacuum up to ~30 ml and the residue was diluted with

ethyl acetate (450 ml) and washed twice with a 1N aqueous solution of HCI (2x100 ml) and then

three times with ice cold brine (3x100ml). Phases were separated and the organic layer was

filtered under vacuum on a Gooch filter assembled with filter paper and cellulose and washing

with ethyl acetate. The filtrate was dried over Na2SO4, filtered and evaporated affording the title

compound methyl 1-(2,6-dibenzyloxyphenyl)cyclopropanecarboxylate (15.5g) that was in the

next step without further purification.

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

Intermediate 27

4-hydroxyspiro[benzofuran-3,1'-cyclopropane]-2-on

OH O O

The reaction was performed in three different runs using about 20g of starting material each.

General procedure: to a mixture of methyl I-(2,6-dibenzyloxyohenyl)cyclopropanecarboxylate

(Intermediate 26, 20.4g, 52.52mmol) and palladium 5% wt. on carbon (1.02g) in ethanol

(200ml), ammonium formate (16.56g, 262.6mmol) was added and the reaction mixture was

stirred at 80°C for 1 hour. After cooling, the catalyst was filtered off on a cellulose pad and the

filtrate was concentrated under vacuum up to ~20ml.

The residues coming from the 3 runs were put together and diluted with ethyl acetate (400ml)

and washed twice with water (2x300 ml). The two phases were separated and the organic one

was washed with brine (300 ml), dried with Na2SO4 and concentrated under vacuum affording 4-

hydroxyspiro[benzofuran-3, l'-cyclopropane]-2-one (27.55g) (containing ~10-15% of the

uncyclized methyl 1-(2,6-dihydroxyphenyl)cyclopropanecarboxylate intermediate) that was used

in the next step without further purification.

LC/MS: QC_3_MIN: Rt = 1.707 min.

Intermediate Intermediate2828

4-benzyloxyspiro[benzofuran-3,1'-cyclopropane]-2-one

O

To a solution of f4-hydroxyspiro[benzofuran-3,1'-cyclopropane]-2-one, (Intermediate 27,

28.5g,161.8mmol) (containing ~10-15% of the uncyclized methyl 1-(2,6-

dihydroxyphenyl)cyclopropanecarboxylate intermediate) in acetonitrile (200mL)/tetrahydrofuran

(50mL), potassium carbonate (33.54g, 242,7mmol) was added and the reaction mixture was

stirred at 70°C for 1.5 hours. The reaction mixture was then cooled to room temperature and

benzyl bromide (27.67g,161.8mmol) was slowly added. The reaction mixture was stirred at 60°C

for 5 hours. After cooling, the reaction mixture was filtered under vacuum and the solid

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discarded, the filtrate was concentrated up to 50 ml, diluted with ethyl acetate (250 ml) and

washed twice with brine (2x100 ml). Phases were separated and the organic layer was dried

over Na2SO4, filtered and evaporated affording the title compound 4-benzyloxyspiro[benzofuran-

3,1 1'-cyclopropane]-2-one (42,4g) that was used in the next step without further purification.

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

Intermediate 29

3-benzyloxy-2-[1-(hydroxymethyl)cyclopropyl]pheno

OH OH

To a solution of4-benzyloxyspiro[benzofuran-3,1'-cyclopropane]-2-one (Intermediate 28,

42.4g,159.2mmol) in dry tetrahydrofuran (300mL), a 1M solution of lithium aluminium hydride in

THF (79.6ml, 79,6mmol) was slowly added at 0 °C and the reaction mixture was stirred at the

same temperature for 30 minutes. The reaction was quenched with ice, water (400 ml) and an

aqueous 1M solution of HCI (160 ml) and then diluted with ethyl acetate (700 ml). Phases were

separated and the aqueous layer was back extracted with ethyl acetate (500 ml). The combined

organic phases were washed with brine (600 ml), dried over Na2SO4, filtered and evaporated

affording the title compound 3-benzyloxy-2-[1-(hydroxymethyl)cyclopropyl]phenol (43g) 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 30

4-benzyloxyspiro[2H-benzofuran-3,1'-cyclopropane)

O

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To a solution of 3-benzyloxy-2-[1-(hydroxymethyl)cyclopropyl]phenol, (Intermediate 29, 43g,

159. 1mmol) in dimethyl carbonate (430 mL), potassium tert-butoxide (35.7g, 318. 1mmol) was

slowly added and the reaction mixture was stirred at 85°C for 3.5 hours. The reaction mixture

was cooled to room temperature, concentrated under vacuum up to 150mL, diluted with MTBE

(400 ml) and washed with water (400 ml). Phases were separated and the aqueous layer was

back extracted with MTBE (250 ml). The combined organic layers were washed with brine (350

ml), dried over Na2SO4, filtered and concentrated affording the title compound 4-

benzyloxyspiro[2H-benzofuran-3,1'-cyclopropane](40g) that 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 WO2012/076877)

1 spiro[2H-benzofuran-3,1'-cyclopropane]-4-6

OH

The reaction was done in two runs using 20g of starting material each.

To a mixture of f4-benzyloxyspiro[2H-benzofuran-3,1'-cyclopropane] (Intermediate 30, 20g,

79.27mmol) and ammonium formate (24.99g, 396.34mmol) in ethanol (160ml), palladium 5%

wt. on carbon (2.0g) was added and the reaction mixture was stirred at 80°C for 10 minutes.

After cooling, the catalyst was filtered off through a cellulose pad and the filtrate was

concentrated under vacuum up to ~20 ml. The residues coming from the two reactions were

combined and the mixture was diluted with ethyl acetate (300 ml) and washed three times with

water (3x200 ml) and then with brine (200 ml). The two phases were separated and the organic

one was dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash

chromatography (Biotage System) on silica gel using Cyclohexane: Ethyl acetate from 99:1 to

85:15 as eluent affording piro[2H-benzofuran-3,1'-cyclopropane]-4-ol (17,75g) as white solid.

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

Intermediate 32

tert-butyl N-[(1S)-1-carbamoylpropyl]carbamate

NH2 NH H o O N

O

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The title compound was synthesized following 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 33

tert-butylN-[(1S)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-

2-yl]carbamoyl]propyl]carbamate

O o NN 11 o o / N NH NH NN o: o II o

The title compound was synthesized following the "route 1" methodology used for the synthesis

of Intermediate 9 replacing tert-butyl N-[(1R)-1-carbamoylpropyl]carbamate (Intermediate 7) with

tert-butyl N-[(1S)-1-carbamoylpropyl]carbamate (Intermediate 32).

LC/MS: QC_3_MIN:Rt=2.65min; m/z455 [M+H]+.

Intermediate 34

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

yl]butanamide

o N // 11

N N - NH

O NH2

Intermediate 13 replacing tert-butyl IN-[(1R)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-

cyclopropane]-4-yl)oxypyrazin-2-yl]carbamoyl]propyl]carbamate (Intermediate 9 with tert-butyl

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N-[(1S)-1-[[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-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 route 1

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

yl]imidazolidine-2,4-dione

O NN // o N O N O NH

To a solution of2-bromo-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxy-pyrazine

(Intermediate 1, 30mg, 0.069mmol) in N,N-dimethylacetamide (1mL) 5,5-dimethylimidazolidine-

2,4-dione (44.4mg, 0.345mmol) and copper (I) oxide (5mg, 0.035mmol) were added. The flask

was flushed with nitrogen gas and left stirring overnight at 135 °C. The reaction was diluted with

EtOAc (10 mL) and first washed with an aqueous saturated solution of ammonium chloride (20

mL) and then brine (20 mL). The organic layer was collected, dried with sodium sulphate and

evaporated to dryness. The residue was then purified using flash column chromatography using

cyclohexane:ethyl acetate from 80:20 to 40:60 as eluent to afford 5,5-dimethyl-3-[5-(7-

ethylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione

(17mg) as a white solid.

1H-NMR (400 MHz; DMSO-d6): 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 foregoing methodology, replacing 2-bromo-

5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxy-pyrazine(Intermediate 1) with the

appropriate bromopyrazine and 5,5-dimethylimidazolidine-2,4-dione with the appropriate

hydantoin. Final products were purified by flash-chromatography (Silica cartridge;

Cyclohexane/EtOAc or other appropriate solvent system) and/or reverse chromatography (C-18

cartridge; water/acetonitrile or other appropriate solvent system).

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Ex. Structure Name Bromopyrazine Hydantoin LCMS/NMR 2 3-[5-[(3,3- 2-bromo-5-[(3,3- 5,5- LC/MS: O N dimethyl-2H- dimethyl-2H- dimethylimidaz QC_3_MIN: Rt o benzofuran-4- benzofuran-4- olidine-2,4- = 2.288 min; =2.288 min; N O N yl)oxy]pyrazin-2- yl)oxy]pyrazine dione m/z 369 O NH yl]-5,5-dimethyl- (Intermediate 4 [M+H]+.

imidazolidine-2,4- 1H-NMR (500

dione MHz; DMSO- d6): ppm d6): 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 (5R)-5-ethyl-5- 2-bromo-5- (5R)-5-ethyl-5- LC/MS: O N methyl-3-(5- spiro[2H- methyl- QC_3_MIN: Rt O o spiro[2H- benzofuran-3,1'- imidazolidine- = 2.228 min; min; =2.228 N - N N O benzofuran-3,1'- cyclopropane]- 2,4-dione m/z 381 o O NH cyclopropane]-4- 4-yloxy-pyrazine (Intermediate [M+H]+.

[M+H]+. = yloxypyrazin-2- (Intermediate 2) 17)

yl)imidazolidine-

2,4-dione

Example 1 route 2

5,5-dimethyl-3-5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropanel-4-yl)oxypyrazin-2-

yllimidazolidine-2,4-dione

o NN // o NN o = N

o O NH

To a solution of 3-(5-chloropyrazin-2-yl)-5,5-dimethyl-imidazolidine-2,4-dione, (Intermediate 5,

20mg, 0.083mmol) and 17-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-ol( (Intermediate 156

WO2012076877, 22mg, 0.125mmol) in acetonitrile (1mL), dipotassium carbonate (17.2mg, wo 2021/156584 WO PCT/GB2020/050268

67 67

0.12mmol) was added. The reaction mixture was stirred overnight at 60°C and then for 3h at

80°C. The reaction mixture was concentrated under vacuum and the crude was purified by flash

chromatography on silica gel (BIOTAGE SYSTEM) using a SNAP 10g as column and

Chexane/EtOAc from 80/20 to 20/80 as eluent. The fraction were still impure and they were

purified by reverse chromatography using a SNAP C-18 as column and H2O/ACN from 95/5 to

5/95 as eluent affording 5,5-dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4

yl)oxypyrazin-2-yl]imidazolidine-2,4-dione (9.4mg) as a white solid.

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

methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-ol with the appropriate phenol and use 3-(5-

chloropyrazin-2-yl)-5,5-dimethyl-imidazolidine-2,4-dione (Intermediate 5) or replace it with the

appropriate chloropyrazine intermediate. Final products were purified by flash-chromatography

(Silica cartridge; Cyclohexane/EtOAc or other appropriate solvent system) and/or reverse

chromatography (C-18 cartridge; water/acetonitrile or other appropriate solvent system).

Ex. Structure Name Phenol Chloro- LCMS/NMR pyrazine

intermediate

4 5,5-dimethyl-3- spiro[2H- 3-(5- LC/MS: OO (5-spiro[2H- benzofuran- chloropyrazin- QC_3_MIN: N // benzofuran-3,1'- 3,1'- 2-yl)-5,5- Rt = 2.085 N O N cyclopropane]- cyclopropane]- dimethyl- min; m/z 367 O NH 4-ol imidazolidine- 4-yloxypyrazin- [M+H]+.

2- (Intermediate 2,4-dione 1-H-NMR (500

yl)imidazolidine- 85, (Intermediate MHz; DMSO- 2,4-dione 5) d6): ppm d6): ppm WO2012/0768 77) 8.73 (bs,

1H), 8.54 (d,

1H), 8.32 (d,

1H), 7.11

(dd, 1H),

6.71 (d, 1H),

6.62 (d, 1H),

4.46 (s, 2H),

1.42 (s, 6H),

1.12-1.16 (m,

2H), 0.92-

0.97 (m, 5H).

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(5R)-5-ethyl-5- 7- 5R)-3-(5- LC/MS: o N methyl-3-[5-(7- methylspiro[2H chloropyrazin- QC_3_MIN: - - methylspiro[2H- -benzofuran- 2-yl)-5-ethyl-5- Rt = 2.361 N o N N benzofuran-3,1'- 3,1'- methyl- min; m/z 395 o NH imidazolidine- cyclopropane]- cyclopropane]- [M+H]+.

4-yl)oxypyrazin- 4-ol 2,4-dione 1H-NMR (500 2- (Intermediate (Intermediate MHz; DMSO- yl]imidazolidine- 156 6) d6): d6): ppm ppm 2,4-dione 8.64 (bs, WO2012/0768 77) 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 (m, 1H),

1.38 (s, 3H),

1.02-1.09 (m,

2H), 0.82-

0.92 (m, 5H).

(5R)-3-[5-[(3,3- 3,3-dimethyl- 5R)-3-(5- LC /I MS: 6 o O NN dimethyl-2H- 2H- chloropyrazin- QC 3 MIN: - - benzofuran-4- benzofuran-4- 2-yl)-5-ethyl-5- Rt = 2.008 N N N yl)oxy]pyrazin-2- ol methyl- min; m/z 383 oO NH yl]-5-ethyl-5- imidazolidine- (Intermediate [M+H]+.

methyl- 50 2,4-dione

imidazolidine- (Intermediate WO2012/0768 2,4-dione 77) 6)

7 5,5-dimethyl-3- 3,3,7-trimethyl- 3-(5- LC/MS: O

[5-[(3,3,7- 2H- chloropyrazin- QC_3_MIN: NN o - - trimethyl-2H- benzofuran-4- 2-yl)-5,5- Rt = 2.025 NN O N N benzofuran-4- ol dimethyl- min; m/z 383

O NH yl)oxy]pyrazin-2- imidazolidine- (Intermediate [M+H]+.

yl]imidazolidine- 184 2,4-dione

2,4-dione WO2012/0768 (Intermediate

77) 5) wo 2021/156584 WO PCT/GB2020/050268

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8 (5R)-5-ethyl-5- 3,3,7-trimethyl- (5R)-3-(5- LC/MS: o methyl-3-[5- 2H- chloropyrazin- N QC_3_MIN:

[(3,3,7-trimethyl- 2-yl)-5-ethyl-5- - benzofuran-4- Rt=2.111 NN o N 2H-benzofuran- ol methyl- min; m/z 397 o NH NH 4-yl)oxy]pyrazin- (Intermediate imidazolidine- [M+H]+. = 2- 184 2,4-dione

yl]imidazolidine- (Intermediate WO2012/0768 2,4-dione 77) 6)

Example 9 (route 1)

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

yl]imidazolidine-2,4-dione

O NN // O NN o NN

o NH :

A mixture of 2R)-2-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-

yl)oxypyrazin-2-yl]butanamide (Intermediate 13, 13mg, 0.037mmol) and N,N-diethylethanamine

(11mg, 0.11mmol) in dichloromethane (2mL) was cooled to 0°C. A solution of

bis(trichloromethyl) carbonate (4,5mg, 0.015mmol) in dichloromethane (0.5mL) was added

dropwise and the reaction mixture was stirred for 1 hour at the same temperature. Additional

bis(trichloromethyl) carbonate (1.5mg) in dichloromethane (0.5mL) was added and stirring

continued for 30 minutes. 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 an

aqueous solution 0. 1N HCI (20 ml) and then with brine (20 ml). Phases were separated and the

organic layer was dried over Na2SO4, filtered and evaporated. The residue was purified by

reverse phase chromatography using a SNAP C-18 column, eluting with water:acetonitrile from

90:10 to 0:100. The appropriate fractions were combined and evaporated to dryness,

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

yl]imidazolidine-2,4-dione (7.5mg) as a white solid.

LC/MS: QC_3_MIN: Rt = 2.305 min; m/z 381 [M+H]+. Enantiomeric purity was confirmed as

>95% using Chiral Control method.

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Example 9 (route 2)

yl]imidazolidine-2,4-dione

O NN O N o N

O NH =

To a solution of f(2R)-2-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1-cyclopropane]-4-

yl)oxypyrazin-2-yl]butanamide (Intermediate 13, 21g, 59.26mmol) in ethyl acetate (500mL) 1-1'-

carbonyldiimidazole (10.57g, 65.18mmol) was added in 5 portions of about 2g each, and stirred

at room temperature for 4h. The reaction was quenched with ice and an aqueous 0.2N solution

of HCI (250ml) was added. The two phases were separated and the organic layer was washed

with an aqueous 0.2N solution of HCI (250ml) and with brine (200ml), then dried with sodium

sulphate, filtered and evaporated to dryness. The crude was split into 4 aliquots of ~4.2g each

and each aliquot was purified by flash chromatography on silica gel using a SNAP (100G) as

column and Cyclohexane/Ethyl acetate from 80/20 to 20/80 as eluent. The desired fractions

from each run were collected and the solvent evaporated to dryness. The obtained light-yellow

solid was suspended in a solution of Cyclohexane/Ethyl acetate (1/1, 3volumes) (90ml) and

stirred for 2h 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 (15ml), the solid was

collected and dried to afford the title compound (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-

3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione (13.6g) as a white solid.

1H-NMR (500 MHz; DMSO-d6): 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).

route 2), replacing (2R)-2-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-

yl)oxypyrazin-2-yl]butanamide (Intermediate 13) with the appropriate butanamide. Final

appropriate solvent system) and/or reverse chromatography (C-18 cartridge; water/acetonitrile

or other appropriate solvent system).

Ex. Structure Name Butanamide LCMS/NMR

(5R)-5-ethyl-3-(5- (2R)-2-amino-N- LC/MS: :QC_3_MIN:Rt= O (5-spiro[2H- - - NN spiro[2H- 2.081 min; m/z 367 [M+H]+.

o benzofuran-3,1'- benzofuran-3,1'- Enantiomeric purity was N O N cyclopropane]-4- cyclopropane]-4- confirmed as >95% using O NH yloxypyrazin-2- yloxypyrazin-2- Chiral Control method.

yl)imidazolidine- yl)butanamide 1H-NMR (500 MHz; DMSO-

2,4-dione (Intermediate 14) d6): 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 (m, 1H),

1.76-1.87 (m, 1H), 1.65-

1.76 (m, 1H), 1.11-1.17 (m,

2H), 0.92-0.98 (m, 5H).

11 (5R)-3-[5-[(3,3- (2R)-2-amino-N- LC/MS: QC_3_MIN: Rt = o O dimethyl-2H- [5-[(3,3-dimethyl- 2.142 min; m/z 369 [M+H]+. N o benzofuran-4- 2H-benzofuran-4- N o N yl)oxy]pyrazin-2- yl)oxy]pyrazin-2-

O o NH yl]-5-ethyl- yl]butanamide

imidazolidine-2,4- (Intermediate 1

dione

12 (5R)-5-ethyl-3-[5- (2R)-2-amino-N- LC/MS: QC_3_MIN: = o

[(3,3,7-trimethyl- [5-[(3,3,7- 2.111 min; m/z 383 [M+H]+. N

2H-benzofuran-4- trimethyl-2H- NN O N yl)oxy]pyrazin-2- benzofuran-4- o O NH yl]imidazolidine- yl)oxy]pyrazin-2-

2,4-dione yl]butanamide

(Intermediate 15)

13 7-[5-(7- 1-amino-N-[5-(7- LC/MS: QC_3_MIN: Rt = o N methylspiro[2H- methylspiro[2H- 2.309 min; 393 m/z

benzofuran-3,1'- benzofuran-3,1'- [M+H]+. NN O N cyclopropane]-4- cyclopropane]-4- o NH yl)oxypyrazin-2- yl)oxypyrazin-2-

yl]-5,7- yl]cyclobutanecar

diazaspiro[3.4]oct boxamide ane-6,8-dione (Intermediate : 20)

WO wo 2021/156584 PCT/GB2020/050268

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14 6-[5-(7- 1-amino-N-[5-(7- LC/MS: QC_3_MIN: Rt = O methylspiro[2H- methylspiro[2H- 2.236 min; 379 m/z N // O benzofuran-3,1'- benzofuran-3,1'- [M+H]+. N = N O cyclopropane]-4- cyclopropane]-4-

yl)oxypyrazin-2- yl)oxypyrazin-2- O NH yl]-4,6- yl]cyclopropaneca

diazaspiro[2.4]he rboxamide ptane-5,7-dione

Example 15 5S)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-

yl]imidazolidine-2,4-dione

O N // 11 o O N = O

O NH

of Intermediate 9 replacing (2R)-2-amino-N-[5-(7-methylspiro[2H-benzofuran-3,1'-

yclopropane]-4-yl)oxypyrazin-2-yl]butanamide(Intermediate 13) with (2S)-2-amino-N-[5-(7-

methylspiro[2H-benzofuran-3,1'-cyclopropane]-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: Measurement of Kv3.1, Kv3.2 and Kv3.3 channel modulation

The ability of the compounds of the invention to modulate the voltage-gated potassium channel

subtypes Kv3.3/Kv3.2/Kv3.1 may be determined using the following assay. Analogous methods

may be used to investigate the ability of the compounds of the invention to modulate other channel

subtypes.

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Cell biology

To assess compound effects on human Kv3.3 channels (hKv3.3), a stable cell line expressing

human Kv3.3 channels is created by transfecting Chinese Hamster Ovary (CHO)-K1 cells with a

pBacMire_KCNC-3 vector. Cells are cultured in DMEM/F12 (Gibco) supplemented with 10%

Foetal Bovine Serum (Gibco), 1X non-essential amino acids (Invitrogen) and geneticin (G418)

400 microg/mL. Cells are grown and maintained at 37 °C in a humidified environment containing

5% CO2 in air.

To assess compound effects on human Kv3.2 channels (hKv3.2), a stable cell line expressing

human Kv3.2 channels (hKv3.2) is created by transfecting CHO-K1 cells with a pCIH5-hKv3.2

vector. Cells are cultured in DMEM/F12 medium supplemented by 10% Foetal Bovine Serum, 1X

non-essential amino acids (Invitrogen) and 500ug/ml of Hygromycin-B (Invitrogen). Cells are

grown and maintained at 37 °C in a humidified environment containing 5% CO2 in air.

To assess compound effects on human Kv3.1 channels (hKv3.1):

Human embryonic kidney (HEK)-hKv3.1 cell line is generated by transfecting HEK-293 cells with

an expression vector with human Kv3.1 (NM_004976.4). Cells are cultured with MEM supplemented with 10% Heat-Inactivated FBS, 2 mM L-glutamine, 1% Penicillin-Streptomycin,

and 0.6 mg/ml of Geneticin (G418). HEK-hKv3.1b cells were amplified in T175 cm2 flask at 37°C

with 5% CO2, using MEM amplification medium, containing the G418 selection antibiotic

(0.6mg/ml). Cells were detached every 3-4 days, using DPBS to wash twice the flask, then TrypLE

to dislodge the cells, and re-plated at a density of 2-4x106 cells/flask.

Cell preparation for IonWorks Quattro experiments

The day of the experiment, cells are removed from the incubator and the culture medium

removed. Cells are washed with 5 ml of Dulbecco's PBS (DPBS) calcium and magnesium free

and detached by the addition of 3 ml Versene (Invitrogen, Italy) followed by a brief incubation at

37 °C for 5 minutes. The flask is tapped to dislodge cells and 10 ml of DPBS containing calcium

and magnesium is added to prepare a cell suspension. The cell suspension is then placed into a

15 ml centrifuge tube and centrifuged for 2 min at 1200 rpm. After centrifugation, the supernatant

is removed and the cell pellet re-suspended in 4 ml of DPBS containing calcium and magnesium

using a 5 ml pipette to break up the pellet. Cell suspension volume is then corrected to give a cell

concentration for the assay of approximately 3 million cells per ml.

All the solutions added to the cells are pre-warmed to 37 °C.

PCT/GB2020/050268

74

Electrophysiology

lonworks

Experiments are conducted at r.t. using IonWorks Quattro planar array electrophysiology

technology (Molecular Devices Corp.) with PatchPlateTM PPC. Stimulation protocols and data

acquisition are carried out using a microcomputer (Dell Pentium 4). Planar electrode hole

resistances (Rp) are determined by applying a 10 mV voltage step across each well. These

measurements are performed before cell addition. After cell addition and seal formation, a seal

test is performed by applying a voltage step from -80 mV to -70 mV for 160 ms. Following this,

amphotericin-B solution is added to the intracellular face of the electrode to achieve intracellular

access. Cells are held at -70 mV. Leak subtraction is conducted in all experiments by applying

50 ms hyperpolarizing (10 mV) prepulses to evoke leak currents followed by a 20 ms period at

the holding potential before test pulses.

For hKv3.2 and hKv3. 1, assays from the holding potential of -70 mV, a first test pulse at -15 mV

was applied for 100 ms and after 100 ms at -70 mV a second pulse at +40 mV was applied for 50

ms. Cells were then maintained for 100 ms at -100 mV and another pulse from -70mV to +40 mV

(duration 50 ms) was applied to clamp later the voltage at -40 mV during 200ms

For hKv3.3 assays, from the holding potential of -70 mV, a first test pulse to 0 mV is applied for

500 ms and following a further 100 ms at -70 mV, a second pulse to 40 mV is applied for 200 ms.

These longer test pulses are used to study inactivation of hKv3.3 channels. Test pulses protocol

may be performed in the absence (pre-read) and presence (post-read) of the test compound. Pre-

and post-reads may be separated by the compound addition followed by a 3 minute incubation.

Solutions and drugs

The intracellular solution contains the following (in mM): K-gluconate 100, KCI 54, MgCl2 3.2,

HEPES 5, adjusted to pH 7.3 with KOH. Amphotericin-B solution is prepared as 50mg/ml stock

solution in DMSO and diluted to a final working concentration of 0.1 mg/ml in intracellular solution.

The external solution is Dulbecco's Phosphate Buffered Saline (DPBS) and contained the

following (in mM): CaCl2 0.90, KCI 2.67, KH2PO4 1.47, MgCl.6H2O 0.493, NaCI 136.9, NaPO4

8.06, with a pH of 7.4.

Compounds of use in the invention (or reference compounds such as N-cyclohexyl-N-[(7,8-

dimethyl-2-oxo-1,2-dihydro-3-quinolinyl)methyl]-N'-phenylurea) dissolved in are dimethylsulfoxide (DMSO) at a stock concentration of 10 mM. These solutions are further diluted wo 2021/156584 WO PCT/GB2020/050268 PCT/GB2020/050268

75

with DMSO using a Biomek FX (Beckman Coulter) in a 384 compound plate. Each dilution (1 uL)

is transferred to another compound plate and external solution containing 0.05% pluronic acid (66

uL) is added. 3.5 uL from each plate containing a compound of the invention is added and

incubated with the cells during the IonWorks Quattro experiment. The final assay dilution is 200

and the final compound concentrations are in the range 50 uM to 50 nM.

Data analysis

The recordings are analysed and filtered using both seal resistance (>20 MO) and peak current

amplitude (>500 pA at the voltage step of 40 mV) in the absence of compound to eliminate

unsuitable cells from further analysis. For hKv3.2 and hKv3.1 assays, paired comparisons of

evoked currents between pre- and post-drug additions measured for the -15 mV voltage step are

used to determine the positive modulation effect of each compound. Kv3 channel-mediated

outward currents are measured determined from the mean amplitude of the current over the final

10 ms of the -15 mV voltage pulse minus the mean baseline current at -70 mV over a 10 ms

period just prior to the -15 mV step. These Kv3 channel currents following addition of the test

compound are then compared with the currents recorded prior to compound addition. Data are

normalised to the maximum effect of the reference compound (50microM of N-cyclohexyl-N-[(7,8-

limethyl-2-oxo-1,2-dihydro-3-quinolinyl)methyl]-N'-phenylurea) and to the effect of a vehicle

control (0.5% DMSO). The normalised data are analysed using ActivityBase or Excel software.

The concentration of compound required to increase currents by 50% of the maximum increase

produced by the reference compound (EC50) is determined by fitting of the concentration-

response data using a four parameter logistic function in ActivityBase. For hKv3.3 assays, paired

comparisons of evoked currents between pre- and post-drug additions are measured for the 0mV

step, considering the peak current and the decay (inactivation) of the current over the duration of

the Omv test pulse (500 ms).

I-cyclohexyl-N-[(7,8-dimethyl-2-oxo-1,2-dihydro-3-quinolinyl)methyl]-N'-phenylurea is obtained

from ASINEX (Registry Number: 552311-06-5).

Ex. Compound Kv3.1 pEC50 Kv3.1 max R% Reference / LCMS

RE1 O H 4.78 Ex57 WO2011/069951

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Ex. Compound Kv3.1 pEC50 Kv3.1 max R% Reference / LCMS

RE2 O H 5.25 N 118 Ex45 WO2011/069951

O N o -F

FF

o HH 4.89 RE3 N 79 LC/MS: QC_3_MIN: Rt =

N 2.376 min; m/z 396

N [M+H]+. O O

F F

O H <4.3 LC/MS: QC_3_MIN: Rt = RE4 N 24 N 2.346 min; m/z 397

O

[M+H]+. O

F F

As shown by testing of RE1-RE4, the incorporation of a pyrazine ring can detrimentally impact

the pEC50 and maxR of Kv3.1 modulators.

Ex. Compound Kv3.1 Kv3.1 max Reference / LCMS

pEC50 R% R% O o H 5.14 RE5 N 158 Ex58 WO2012/076877 N N

O

RE6 O H N 5.58 144 Ex70 WO2012/076877 N

O N

RE7 O H N 5.56 130 Ex3 WO2017/103604 N

oo

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Ex. Compound Kv3.1 Kv3.1 max Reference / LCMS

pEC50 R% RE8 o IZ 4.98 LC/MS: QC_3_MIN: Rt 42 N, N : 2.224 min; m/z 381

NN O [M+H]+.

RE9 o O H <4.3 LC/MS: QC_3_MIN: Rt 16 = 2.043 min; m/z 381

[M+H]+.

RE10 <4.3 LC/MS: QC_3_MIN: Rt 22 = 2.29 min; m/z 381

the [M+H]+. My

1+ o O 5.47 Example 1 164

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

As shown by testing of RE5-RE9 as compared to Example 1, the incorporation of a para-pyrazine

ring in Example 1 unexpectedly results in high pEC50 and high maxR in the Kv3.1 assay. RE10

shows that a meta-pyrazine central ring has greatly reduced pEC50 and maxR as compared to

the para-pyrazine of Example 1.

Kv3.1 Kv3.1 max Kv3.1 Kv3.1 max Example Example pEC50 R% pEC50 R% 1+ 5.47 164 8 5.29 119

2 4.68 149 9* 5.88 5,88 172

3 5.15 205 10$ 5.45 153

4 5.17 170 11 4.89 165

5 5.69 149 12 5.56 5,56 118

6 4.75 165 13 5.09 165

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Kv3.1 Kv3.1 max Kv3.1 Kv3.1 max Example Example pEC50 R% pEC50 R% 7 5.12 134 14 14 5.51 145

15 5.10 136 + n=10. For n=18, pEC50 was 5.56 and maxR% 152 * n=4. For n=22, pEC50 was 5.90 and maxR% 146

n=2. For n=26, pEC50 was 5.63 and maxR% 147

Ex. Compound Kv3.1 Kv3.1 max Reference / LCMS

pEC50 R% R% 6.1 RE11 152 Ex62 WO2012/076877

5.6 5.6 RE12 149 Ex4 WO2017/102604

9 9 5.90 146 Example 9

Ex. Compound Kv3.1 Kv3.1 max Reference / LCMS

pEC50 R% 6.1 RE13 149 Ex15 WO2012/076877

RE14 5.2 150 Ex6 WO2017/102604

10 10 o N 5.63 147 Example 10

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All tested examples of the compounds of formula (I) are shown above and demonstrate good

pEC50 and maxR properties in the Kv3.1 assay. Previous disclosures of Kv3.1 data for

comparator compounds may differ slightly due to a lower number of measurements.

A secondary analysis of the data from the hKv3.1, hKv3.2 and hKv3.3 assays described in may

be used to investigate the effect of the compounds on rate of rise of the current from the start of

the depolarising voltage pulses. The magnitude of the effect of a compound can be determined

from the time constant (Tauact) obtained from a non-linear fit, using the equation given below, of

the rise in Kv3.1, Kv3.2 and Kv3.3 currents following the start of the - 15mV depolarising voltage

pulse.

Y = (YO - Ymax) * exp(-K*X) + Ymax

where:

YO is the current value at the start of the depolarising voltage pulse;

Ymax is the plateau current;

K is the rate constant, and Tauact is the activation time constant, which is the reciprocal of

K.

Similarly, the effect of the compounds on the time taken for Kv3.1, Kv3.2 or Kv3.3 currents to

decay on closing of the channels at the end of the -15mV depolarising voltage pulses can also be

investigated. In this latter case, the magnitude of the effect of a compound on channel closing

can be determined from the time constant (Taudeact) of a non-linear fit of the decay of the current

("tail current") immediately following the end of the depolarising voltage pulse.

Kv3.1, Kv3.2 and Kv3.3 channels must activate and deactivate very rapidly in order to allow

neurons to fire actions potentials at high frequency (Rudy et al., 2001). Slowing of activation is

likely to delay the onset of action potential repolarisation; slowing of deactivation could lead to

hyperpolarising currents that reduce the excitability of the neuron and delay the time before the

neuron can fire a further action potential. Together these two slowing effects on channel

activation and deactivation are likely to lead to a reduction rather than a facilitation of the neurons

ability to fire at high frequencies. Thus compounds that have this slowing effect on the Kv3.

and/or Kv3.2, and/or Kv3.3 channels will effectively behave as negative modulators of the

channels, leading to a slowing of neuronal firing. This latter effect has been shown for certain of

the compounds disclosed in WO2011/069951, where marked increases in Tauact can be observed

from recordings made from "fast-firing" interneurons in the cortex of rat brain, using

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electrophysiological techniques, in vitro. The addition of the relevant compounds reduces the

ability of the neurons to fire in response to trains of depolarising pulses at 300Hz.

Therefore, although certain compounds may be identified act as positive modulators in the

recombinant cell assay, those compounds which markedly increase the value of Tauact can reduce

the ability of neurons in native tissues to fire at high frequency.

Biological Example 2: Determination of blood and brain tissue binding

Materials and Methods

Sprague Dawley rat whole blood, collected on the week of the experiment using K3-EDTA as an

anti-coagulant, is diluted with isotonic phosphate buffer 1:1 (v/v). Sprague Dawley rat whole brain,

stored frozen at -20 °C, is thawed and homogenised in artificial cerebrospinal fluid (CSF) 1:2

(w/v).

An appropriate amount of test compound is dissolved in DMSO to give a 10 millimolar solution.

Further dilutions, to obtain a 166.7 micromolar working solution are then prepared using 50%

acetonitrile in MilliQ water. This working solution is used to spike the blood to obtain a final

concentration of 0.5 micromolar in whole blood. Similarly, the working solution is used to spike

brain samples to obtain a final concentration of 5 micromolar in whole brain. From these spiked

blood and brain preparations, control samples (n=3), are immediately extracted and used to

calculate the initial recovery of the test items.

150 microl of compound-free buffer (isotonic phosphate buffer for blood or artificial CSF buffer

for brain) is dispensed in one half-well and 150 microl of spiked matrix (blood or brain) is loaded

in the other half-well, with the two halves separated by a semi-permeable membrane. After an

equilibration period of 5 h at 37°C, 50 microl of dialysed matrix (blood or brain) is added to 50

microl of corresponding compound-free buffer, and vice-versa for buffer, such that the volume of

buffer to matrix (blood or brain) remains the same. Samples are then extracted by protein

precipitation with 300 microl of acetonitrile containing rolipram (control for positive ionization

mode) or diclofenac (control for negative ionization mode) as internal standards and centrifuged

for 10min at 3000rpm. Supernatants are collected (100 microL), diluted with 27% AcN in MilliQ

water (200 microL) and then injected into an HPLC-MS/MS or UPLC-MS/MS system to determine

the concentration of test compound present.

Analysis

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Blood and brain tissue binding are then determined using the following formulas:

Afu=Buffer/Blood or Afu=CSF/Brain

Where Afu = apparent fraction unbound; Buffer= analyte/intemal standard ratio determined in the

buffer compartment; Blood= analyte/intemal standard ratio determined in the blood compartment;

Brain= analyte/intemal standard ratio determined in the brain compartment.

Fucr = 1/D Fucr = 1/D

[(1/Afu - 1)+1/D]

where: fucr = Fraction unbound corrected; D =matrix dilution factor (D=2 for blood and D=3 for

brain).

Then:

%Binding =(1-fucr) x 100

%Unbound = 100 -%Bound

Brain/Blood partition ratio (Kbb) Determination

For compounds freely permeable across the blood/brain barrier (BBB), the unbound concentrations in blood and brain would be equivalent under steady-state distribution conditions.

Therefore, the Kbb value could be calculated as:

20 Fu(blood)/Fu(brain)

which is expected to be equivalent to the brain-to-blood concentration ratio (Ct(brain)/Ct(blood))

if efflux pump transporters are not involved.

Results

Examples 1, 9 and 10, and certain comparator compounds, were tested in the above described

methodology to determine the brain fraction unbound. The results were as follows:

Ex. Compound Brain fraction

unbound (%)

RE5 H 5.1 RE5 N

0

H :

RE6 RE6 O N 2.8

N

Ex. Compound Brain fraction

unbound (%)

RE7 O HH 2.3* N

O O

1 H 4.3 O N

O

RE11 2.1 N

N N

RE12 1.9

will

9 3.0 N

N

RE13 O N 6.2

RE14 5.8 5.8 ..............

10 8.7 N

* Supernatant diluted with 18% AcN in water

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Pyrazine compounds of the invention, demonstrated an increased brain fraction unbound as

compared to their pyridine comparator compounds.

Biological Example 3: Determination of in vivo pharmacokinetic parameters

Materials and Methods

Adult male rats (Charles River, Italy) are dosed with test compound orally at 1mg/kg (5 ml/kg, in

5% v/v DMSO, 0.5% w/v HPMC in water) and intravenously at 0.5mg/kg (2ml/kg, in 5% v/v DMSO

40% w/v PEG400 in saline). After oral administration, blood samples are 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 are collected from the lateral tail vein of each

rat. A further group of rats (n=1 per test compound) receive a single intravenous administration

of the PgP transport inhibitor, Elacridar (3 mg/kg) shortly before the oral administration of the test

compound at 1 mg/kg, as above. Blood and brain samples ar collected at a single timepoint of

0.5 h after dose administration for these animals. In all cases, blood samples are collected into

potassium EDTA tubes.

Blood and brain samples can be assayed for test compound concentration using a method based

on protein precipitation with acetonitrile followed by HPLC/MS-MS analysis with an optimized

analytical method.

Analysis

The concentrations of test compound in blood (expressed as ng/ml) and brain (expressed as ng/g)

at the different time points following either oral or intravenous dosing are analysed using a non-

compartmental pharmacokinetic model using WinNonLin Professional version 4.1. The following

parameters are derived:

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 of maximum concentration (Tmax), AUC, bioavailability (F%), fraction

absorbed (Fa%), blood to brain ratio (AUC BB), and Fold-change in AUC BB in the presence of

Elacridar.

Compounds of the invention may be expected to demonstrate good availability in brain tissue.

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Biological Example 4: In Vitro Metabolic Stability Study in Human Hepatocytes

Methodology The objective of this study was to determine metabolic stability in mixed gender human

cryopreserved hepatocytes. Testosterone and 7-Hydroxycoumarin were used as positive

controls for Phase I and Phase II metabolism, respectively.

Incubation medium was prepared by combining William's medium E, HEPES buffer 1 M and L-

glutamine 200 mM in the following proportions: 88%, 10% and 2%, respectively (440 mL, 50 mL

and 10 mL, respectively). The medium obtained was bubbled with carbogen (5% CO2, 95% O2)

for 30 minutes prior to use. Cryopreserved hepatocytes were thawed and suspended in

incubation medium pre-warmed at 37°C. Cells were centrifuged, re-suspended in medium and

counted by means of a haemocytometer (Burker's chamber). Cell viability was measured using

the Trypan Blue exclusion test.

Test compounds were separately dissolved in DMF to obtain 50 mM stock solutions that were

further diluted in water/acetonitrile 50/50 (v/v) to obtain the corresponding 50 uM working

solutions. Testosterone and 7-Hydroxy-Coumarin were dissolved in DMF in order to obtain a 50

mM Testosterone solution and 5 mM 7-Hydroxy-Coumarin solution. These solutions were then

diluted in the incubation medium in order to obtain a 1 mM Testosterone working solution and a

500 uM 7-Hydroxy-Coumarin working solution.

10 uL of each working solution, i.e. 50 uM test compound, 1 mM Testosterone and 500 uM of

7-Hydroxy-Coumarin were added to 990 uL of 0.5x106 cell suspensions in order to obtain the

final concentrations of 0.5 uM, 10 uM and 5 uM, respectively. The concentration of the organic

solvent in each incubation was constant and < 1% (v/v).

Test compounds were separately incubated at 0.5 uM for 0, 5, 10, 15, 20, 30, 45, 60, 90, 120,

150 and 180 min (12 time points) with mixed gender human cryopreserved hepatocytes at 37°C

in a 24 well plate. At each time point a robotic handling processor aspirated 50 uL of incubation

mixture from each well and dispensed it into a refrigerated 96 well plate, containing 100 uL of

acetonitrile with the corresponding internal standard 150 ng/mL to stop the reaction. Then an

aliquot of water (120 uL) was added to equilibrate the organic solvent content at 37%. Samples

were centrifuged (ca. 3500 g for 10 minutes) prior to LC MS/MS analysis.

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Positive controls, Testosterone and 7-Hydroxy-Coumarin, were incubated in single (n=1) at 10

and 5 uM, respectively, for 0, 5, 10, 15, 20, 30, 45, 60, 90, 120, 150 and 180 min (12 time points)

with mixed gender human cryopreserved hepatocytes at the same conditions reported above for

the test items, to demonstrate Phase I and Phase Il metabolism in the hepatocytes systems. At

each time point a robotic handling processor aspirated 50 uL of incubation mixture from each well

and dispensed it into a refrigerated 96 well plate, containing 100 ul of acetonitrile with Rolipram

as internal standard to stop the reaction. Then an aliquot of water (120 uL) was added to

equilibrate the organic solvent content at 37%. Samples were centrifuged (ca. 3500 g for 10

minutes) prior to LC MS/MS analysis.

Metabolic stability was calculated from the peak area ratio of the remaining test compound with

internal standard versus time.

The intrinsic clearance (CLint) was determined from the first order elimination constant k (min-1)

(obtained from GraphPad by plotting the natural logarithm of the peak area ratio of the remaining

test item with internal standard versus time), using the actual volume of the incubation V (mL),

the amount of hepatocytes in the incubation M (million cells) and the hepatocellularity number per

g liver Hn (120 for human).

CLint

Values for CLint were expressed as mL/min/g liver.

Ex. Rate constant k In vitro Clint Compound (min-1 (mL/min/g liver)

RE5 H 0.002 0.31

RE11 0.02 3.58

N

9 0.004 1.03

N

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Ex. Rate constant k In vitro Clint Compound (min-1) (mL/min/g liver)

RE13 0.009 2.16 N

10 0.003 0.70 N

N

Examples 9 and 10 demonstrate low clearance compared to pyridine comparator compounds

RE11 and RE13.

Biological Example 5: Ames testing

Methodology The objective of this in vitro study was to assess the potential of test articles to induce gene

mutations in vitro in bacterial strains of Salmonella typhimurium (TA1535, TA1537, TA98 and

TA100) and Escherichia coli WP2 uvrA (pKM101); test methodology was based on established

procedures for bacterial mutagenicity testing, and assays were performed in the presence and

absence of an exogenous mammalian oxidative metabolizing system (S9-mix).

The study was designed in accordance with national and international guidelines, to fulfil the

requirements of regulatory authorities, for the toxicity testing of new drugs. The study design is

in agreement with the following test guidelines:

ICH guideline M3(R2) on non-clinical safety studies for the conduct of human clinical trials

and marketing authorisation for pharmaceuticals (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:

Species Strain Genotype S. typhimurium TA1535 hisG46 rfa A uvrB

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Species Strain Genotype S. typhimurium TA1537 hisC3076 rfa A uvrB

S. typhimurium TA98 hisD3052 rfa A uvrB (pKM101)

S. typhimurium TA100 hisG46 rfa A uvrB (pKM101)

E. coli WP2 uvrA (pKM101) TrpE

Ochre uvrA (pKM101)

Source Molecular Toxicology Incorporated, Boone, NC, USA (MolToxTM)

Growth Phase Late log phase

The strains TA1535, TA100, and WP2 uvrA pKM101 detect base change mutations. The strains

TA1537 and TA98 detect frameshift mutations.

Bacteria inocula were used to prepare fresh cultures in 10 mL of nutrient broth (NB2, containing

ampicillin for the pKM101 plasmid containing strains S. typhimurium strains TA98 and TA100 and

E. coli WP2 uvrA (pKM101) to maintain the plasmid copy number). Bacteria were cultured for

10-12 hours in a shaking incubator at 37+2°C to yield 1-2x109 cells/mL.

The bacteria suspension was added to the Top Agar (containing trace amounts of the amino acids

required for auxotrophy) at a volume of 100 uL.

Mammalian Oxidative Metabolizing System

Phenobarbital, 5 6 Benzoflavone induced rat liver post mitochondrial fraction (S9) from Molecular

Toxicology Incorporated, USA (MolToxTM) was used as an exogenous oxidative metabolizing

system. Batches of S9 fraction stored as frozen aliquots at approximately -80°C were thawed

immediately prior to use. S9 mix was prepared by the addition of S9 (10% v/v) to a NADPH

generating system, which included NADP (3.15 mg/mL), glucose 6 phosphate (1.5 mg/mL), and

2% v/v of a saline solution containing MgCl2 (81.3 mg/mL) and KCI (123 mg/mL) in phosphate

buffer pH 7.4. For treatment in the presence of S9 mix, S9 mix was used at a final volume of 500

uL/plate. For treatment in the absence of S9 mix, an equivalent volume of sterile phosphate buffer

pH 7.4 was added in place of the S9 mix.

Positive Control Formulations

The following positive controls (supplied by MolToxTM through Trinova Biochem GmbH, Giessen,

Germany and Sigma Aldrich, Milano, Italy) were used and formulated as follows:

Bacterial Positive Control Conc. Vehicle S9-mix Strain (ug/plate) (Solvent)

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TA98 2-Nitrofluorene (2NF) 2 Dimethyl Sulfoxide No (DMSO) TA1535, TA100 Sodium Azide (NaAz) 2 H2O No TA1537 ICR-191 1 DMSO No 4-Nitroquinoline-1-oxide 1 WP2 uvrA (pKM101) DMSO No (4NQO) TA98 Benzo[a]pyrene (B[a]P) 1.25 Yes DMSO TA1535, TA1537, TA100, 2-Aminoanthracene (2AAN) 5 DMSO Yes Yes WP2 uvrA (pKM101)

Positive controls were prepared from frozen (approximately -20°C) stock solutions and stored at

ambient temperature during the use.

Test Articles

The test consisted of 4 replicate plates for vehicle (DMSO) controls and 2 replicate plates for the

test article and positive controls, treated in the absence and in the presence of S9-mix. A range

of test article concentrations starting from 5 ug/plate to 5000 ug/plate was tested, as follows:

Species Strain Test Item S9-mix

Concentrations (ug/plate)

S. typhimurium TA1535, TA1537, TA98 and TA100 5, 15, 50, 150, 500, No E. coli WP2 uvrA (pKM101) 1500 and 5000

S. typhimurium TA1535, TA1537, TA98 and TA100 Yes E. coli WP2 uvrA (pKM101)

The vehicle, test article and positive control formulations were added to plates at a volume of

100 uL/plate.

Plate Treatment and Incubation

Top agar was supplemented with trace amounts of histidine and biotin, or tryptophan, aliquoted

(2 mL/plate), and maintained at 46+2°C. The appropriate bacterial suspension was added to 2 ml

of top agar followed by the test article, or vehicle/positive control solutions, and sterile phosphate

buffer pH 7.4 or S9-mix. This final treatment mixture was poured over minimal agar plates (Vögel

Bonner plates) and incubated in the dark for approximately 64 hours at 37+2°C.

Plate Scoring and Analysis

At the end of the incubation period, plates were evaluated (by visual examination) for test article

precipitation. Plates were scored electronically for bacterial colony formation using the colony

counter ProtoCOL3 Synbiosis. Where test article precipitation occurred, the bacterial colony count for each strain was performed manually and halted at the lowest treatment concentration that did not interfere with the manual scoring.

The scoring was followed by the inspection of the plates for signs of toxicity (i.e. reduced

growth/diminution of background lawn, the presence of pin dot/pseudorevertant colonies, and/or

a reduction in colony numbers).

If the data for any treatment concentration show a response 2 times the concurrent vehicle

control value for TA98, TA100, and WP2 uvrA (pKM101), or 3 times the concurrent vehicle

control value for TA1535 and TA1537, in conjunction with a dose related response, the result

should be considered positive. Results that only partially satisfy these criteria or where the data

for any strain show a dose related response, but do not exceed the 2 or 3 fold threshold as

detailed, are considered equivocal.

The following acceptance criteria were applied:

1. The highest concentration tested should be 5000 ug/plate, or limited by solubility of test item

in the vehicle.

2. If the test item solubility is a limiting factor, the maximum concentration chosen for plate

scoring would be the lowest concentration at which the test item precipitation is observed

on treatment plates at the end of the incubation period and that does not interfere with

the scoring.

If toxicity is a limiting factor, the maximum concentration evaluable for gene mutation would be

the lowest concentration at which signs of significant bacterial toxicity are observed during plate

scoring.

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Results

Ex. Ames Aniline Compound Ames Result Result

RE5 Non- Not tested Not tested RE5 mutagenic

O N Non- RE6 mutagenic NH2 Mutagenic for TA1535 in the N presence of metabolic activation at

150 ug/plate Non- RE11 mutagenic

o

Non- 1 mutagenic NH2

o N Non-mutagenic

Non- 9 mutagenic

Non- 10 Non-mutagenic mutagenic

The aniline associated with RE6/RE11, which has been shown to be a degradant under certain

conditions, was found to be mutagenic. This finding presents a risk in the future development of

RE6/RE11 and also for compounds which could 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 which may be distinguished on the basis of their associated anilines are advantageous.

Anilines for Examples 1, 9 and 10 are non-mutagenic, which may be expected to apply to other

compounds of the invention which could produce related anilines.

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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 the testing of

compounds) demonstrate the activity of compounds which are modulators of Kv3.1 and Kv3.2 in

animal models of seizure, hyperactivity, sleep disorders, psychosis, hearing disorders 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 the testing of compounds)

demonstrates the efficacy of a compound which is a modulator of Kv3.1 and Kv3.2 in a model of

acute noise-induced hearing loss in the chinchilla, 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 demonstrate the efficacy of a

modulator of Kv3.1 and Kv3.2 in hearing associated models.

Patent application WO2017/098254 (incorporated by reference for the purpose of illustrating the

demonstrates the efficacy of a compound which is a modulator of Kv3.1 and Kv3.2 in models of

neuropathic and inflammatory pain.

Throughout the specification and the claims which follow, unless the context requires otherwise,

the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to

imply the inclusion of a stated integer, step, group of integers or group of steps but not to the

exclusion of any other integer, step, group of integers or group of steps.

The application of which this description and claims forms part may be used as a basis for priority

in respect of any subsequent application. The claims of such subsequent application may be

directed to any feature or combination of features described herein. They may take the form of

product, composition, process, or use claims and may include, by way of example and without

limitation, the claims which follow.

Clauses of the invention:

Clause 1 - A compound of formula (I):

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O N O R-

R2

O R N N NH R3

O O R4 R5 (I)

wherein:

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, are a spirocyclopropyl ring;

R4 is methyl or ethyl;

R5 is H or methyl;

or R4 and R5, together with the carbon atom to which they are attached,

form a C3-C4 spiro carbocyclyl;

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

Clause 2 - The compound according to clause 1 wherein R1 is H.

Clause 3 - The compound according to clause 1 wherein R1 is methyl.

Clause 4 - The compound according to any one of clauses 1 to 3, wherein R2 and R3 are a

spiro cyclopropyl ring.

Clause 5 - The compound according to any one of clauses 1 to 3, wherein R2 is methyl and

R3 is methyl

Clause 6 - The compound according to any one of clauses 1 to 5, wherein R4 is methyl.

Clause 7 - The compound according to any one of clauses 1 to 5, wherein R4 is ethyl.

Clause 8 - The compound according to any one of clauses 1 to 7, wherein R5 is H.

Clause 9 - The compound according to any one of clauses 1 to 7, wherein R5 is methyl.

Clause 10 - The compound according to any one of clauses 1 to 9 wherein when R4 and R5

are different and they have the following stereochemical arrangement: wo 2021/156584 WO PCT/GB2020/050268

93

o NH R4 : N N Il R 11/R5

O Clause 11 - The compound according to any one of clauses 1 to 9 wherein when R4 and R5

are different and they have the following stereochemical arrangement:

O / NH R5

I N R 11/R4

Clause 12 - The compound according to any one of clauses 1 to 5, wherein R4 and R5,

together with the carbon atom to which they are attached, form a

spirocyclopropyl.

Clause 13 - The compound according to any one of clauses 1 to 5, wherein R4 and R5,

together with the carbon atom to which they are attached, form a spirocyclobutyl.

Clause 14 - The compound according to clause 1 selected from the group consisting of:

5,5-dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopro

yl)oxypyrazin-2-yl]imidazolidine-2,4-dione;

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

imidazolidine-2,4-dione;

5R)-5-ethyl-5-methyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-

loxypyrazin-2-yl)imidazolidine-2,4-dione;

5,5-dimethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-

yl)imidazolidine-2,4-dione;

(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-

limidazolidine-2,4-dione;

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

yl]imidazolidine-2,4-dione;

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

yl]imidazolidine-2,4-dione;

(5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-

yl)oxypyrazin-2-yl]imidazolidine-2,4-dione; wo 2021/156584 WO PCT/GB2020/050268

94

(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]imidazolidine-2,4-dione;

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

diazaspiro[3.4]octane-6,8-dione;

6-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]-4,6-

diazaspiro[2.4]heptane-5,7-dione

(5S)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-

yl)oxypyrazin-2-yl]imidazolidine-2,4-dione;

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

Clause 15 - The compound according to clause 1 which is:

5,5-dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cycopropane]-4

yl)oxypyrazin-2-yl]imidazolidine-2,4-dione.

Clause 16 - The compound according to clause 1 which is:

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

imidazolidine-2,4-dione.

Clause 17 - The compound according to clause 1 which is:

(5R)-5-ethyl-5-methyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-

yloxypyrazin-2-yl)imidazolidine-2,4-dione.

Clause 18 - The compound according to clause 1 which is:

5,5-dimethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-

yl)imidazolidine-2,4-dione.

Clause 19 - The compound according to clause 1 which is:

(5R)-5-ethyl-5-methyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-

I)oxypyrazin-2-yl]imidazolidine-2,4-dione.

Clause 20 - The compound according to clause 1 which is: wo 2021/156584 WO PCT/GB2020/050268

95

(5R)-3-[5-[(3,3-dimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5-ethyl-5-methyl-

imidazolidine-2,4-dione,

Clause 21 - The compound according to clause 1 which is:

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

yl]imidazolidine-2,4-dione.

Clause 22 - The compound according to clause 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.

Clause 23 - The compound according to clause 1 which is:

(5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-

yl)oxypyrazin-2-yl]imidazolidine-2,4-dione.

Clause 24 - The compound according to clause 1 which is:

(5R)-5-ethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-

yl)imidazolidine-2,4-dione.

Clause 25 - The compound according to clause 1 which is:

(5R)-3-[5-[(3,3-dimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5-ethyl-

limidazolidine-2,4-dione.

Clause 26 - The compound according to clause 1 which is:

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

yl]imidazolidine-2,4-dione.

Clause 27 - The compound according to clause 1 which is:

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

diazaspiro[3.4]octane-6,8-dione,

Clause 28 - The compound according to clause 1 which is:

6-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]-4,6-

diazaspiro[2.4]heptane-5,7-dione,

Clause 29 - The compound according to clause 1 which is:

PCT/GB2020/050268

96

(5S)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-

Vl)oxypyrazin-2-yl]imidazolidine-2,4-dione.

Clause 30 - The compound of formula (I) according to any one of clauses 1 to 29, or a

pharmaceutically acceptable salt and/or solvate thereof.

Clause 31 - The compound according to any one of clauses 1 to 30 for use as a medicament.

Clause 32 - The compound according to clause 31 for use in the prophylaxis or treatment of a

disease or disorder selected from the group consisting of hearing disorders,

schizophrenia, depression and mood disorders, bipolar disorder, substance

abuse disorders, anxiety disorders, sleep disorders, hyperacusis and

disturbances of loudness perception, Ménière's disease, disorders of balance,

and disorders of the inner ear, impulse control disorder, personality disorders,

attention-deficit/hyperactivity disorder, autism spectrum disorders, eating

disorders, cognition impairment, ataxia, pain such as neuropathic pain,

inflammatory pain and miscellaneous pain, Lewy body dementia and Parkinson's

disease.

Clause 33 - The compound according to clause 31 for use in the prophylaxis or treatment of

schizophrenia.

Clause 34 - The compound according to clause 31 for use in the prophylaxis or treatment of

hearing disorders.

Clause 35 - The compound according to clause 31 for use in the prophylaxis or treatment of

pain.

Clause 36 - The compound according to clause 31 for use in the treatment of Fragile X.

Clause 37 - A method for the prophylaxis or treatment of a disease or disorder selected from

the group consisting of hearing disorders, schizophrenia, depression and mood

disorders, bipolar disorder, substance abuse disorders, anxiety disorders, sleep

disorders, hyperacusis and disturbances of loudness perception, Ménière's

disease, disorders of balance, and disorders of the inner ear, impulse control

disorder, personality disorders, attention-deficit/hyperactivity disorder, autism

WO wo 2021/156584 PCT/GB2020/050268 PCT/GB2020/050268

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spectrum disorders, eating disorders, cognition impairment, ataxia, pain such as

neuropathic pain, inflammatory pain and miscellaneous pain, Lewy body

dementia and Parkinson's disease which comprises administering to a subject in

need thereof an effective amount of a compound according to any one of clauses

1 to 30.

Clause 38 - A method for the prophylaxis or treatment of schizophrenia, comprising

administering to a subject in need thereof a compound according to any one of

clauses 1 to 30.

Clause 39 - A method for the prophylaxis or treatment of hearing disorders, comprising

administering to a subject in need thereof a compound according to any one of

clauses 1 to 30.

Clause 40 - A method for the prophylaxis or treatment of pain, comprising administering to a

subject in need thereof a compound according to any one of clauses 1 to 30.

Clause 41 - A method for the treatment of Fragile X, comprising administering to a subject in

need thereof a compound according to any one of clauses 1 to 30.

Clause 42 - Use of a compound according to any one of clauses 1 to 30 in the manufacture

of a medicament for the prophylaxis or treatment of a disease or disorder

selected from the group consisting of hearing disorders, schizophrenia,

depression and mood disorders, bipolar disorder, substance abuse disorders,

anxiety disorders, sleep disorders, hyperacusis and disturbances of loudness

perception, Ménière's disease, disorders of balance, and disorders of the inner

ear, impulse control disorder, personality disorders, attention-deficit/hyperactivity

disorder, autism spectrum disorders, eating disorders, cognition impairment,

ataxia, pain such as neuropathic pain, inflammatory pain and miscellaneous pain,

Lewy body dementia and Parkinson's disease.

Clause 43 - Use of a compound according to any one of clauses 1 to 30 in the manufacture

of a medicament for the prophylaxis or treatment of schizophrenia.

Clause 44 - Use of a compound according to any one of clauses 1 to 30 in the manufacture

of a medicament for the prophylaxis or treatment of hearing disorders.

WO wo 2021/156584 PCT/GB2020/050268 PCT/GB2020/050268

98

Clause 45 - Use of a compound according to any one of clauses 1 to 30 in the manufacture

of a medicament for the prophylaxis or treatment of pain.

Clause 46 - Use of a compound according to any one of clauses 1 to 30 in the manufacture

of a medicament for the treatment of Fragile X.

Clause 47 - A pharmaceutical composition comprising a compound of any one of clauses 1 to

30 and a pharmaceutically acceptable carrier or excipient.

Clause 48 - The compound according to any one of clauses 1 to 30 for use in combination

with a further pharmaceutically acceptable active ingredient.

Clause 49 - A compound of formula (II) or (XVI):

0 N R1 R2

O R3 R N X (II),

wherein R1, R2 and R3 are as defined in clause 1, X is halo, such as Br.

Clause 50 - A compound of formula (XVI):

o O N

R1 R2 R N N NH2 NH R3 (XVI), R wherein R1, R2 and R3 are as defined in clause 1.

Clause 51 - A compound of formula (IV): Y, N Y O

N N NH NH

R4 R5 (IV)

wherein R4 and R5 are as defined in clause 1, Y is halo, such as CI.

Clause 52 - A derivative of a compound of formula (I), or salt and/or solvate thereof,

according to any one of clauses 1 to 30 functionalised via the secondary nitrogen

WO wo 2021/156584 PCT/GB2020/050268 PCT/GB2020/050268

99

of the hydantoin or via the secondary nitrogen of the triazolone with a group L,

wherein L is selected from the groups consisting of:

a) -PO(OH)O .M+, wherein M+ is a pharmaceutically acceptable monovalent

counterion,

-PO(O)2 .2M+, c) -PO(O) 2.D2 wherein D2+ is a pharmaceutically acceptable divalent

counterion,

d) -CH(RX)-PO(OH)OM,wherein RX is hydrogen or C1-3 alkyl,

e) -CH(R*)-PO(O')2 +2M*, f)

g) -SO, h) -CH(R*)-SO*M*, and i) -CO-CH2CH2-COM+.

Clause 53 - The compound according to any one of clauses 1 to 36, which is in natural isotopic

form.

Clause 54 - The compound, method, use, composition or derivative according to any one of

clauses 1 to 48, 52 or 53, for oral administration.

Clause 55 - The compound, method, use, composition or derivative according to any one of

clauses 1 to 48 or 52 to 54 for administration at 2 to 400 mg per day, such as 2 to 300 mg per

day, especially 5 to 250 mg per day.

Clause 56 - The compound, method, use, composition or derivative according to any one of

clauses 1 to 48 or 52 to 55 for administration once or twice per day.

Clause 57 - The compound according to clause 56 for administration once per day.

Clause 58 - The compound according to clause 56 for administration twice per day.

Clause 59 - The compound, method, use, composition or derivative according to any one of

clauses 1 to 48 or 52 to 58 for administration for a period of at least three months.

Clause 60 - The compound, method, use, composition or derivative according to any one of

clauses 1 to 48 or 52 to 58 for administration to a human subject.

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Clause 61 - The compound, method, use, composition or derivative according to clause 60 for

administration to a human adult, such as aged 18 to 65.

Clause 62 - The compound, method, use, composition or derivative according to clause 60 for

administration to a human aged 66 years old or older.

Clause 63 - The compound, method, use, composition or derivative according to clause 60 to

a human subject of less than 18 years of age, such as 4 to 17 years old.

Clause 64 - The compound, method, use, composition or derivative according to according to

any one of clauses 1 to 48, 52, 53 or 59 to 63 wherein a compound of formula (I) or a

pharmaceutically acceptable, salt, solvate and/or derivative thereof is delivered by a patch or

implant.

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specifically and individually indicated to be incorporated by reference herein as though fully set

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Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 21 Nov 2025

1. Use of a compound of formula (I):

(I) 2020427632

wherein: 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, are a spirocyclopropyl ring; R4 is methyl or ethyl; R5 is H or methyl; or R4 and R5, together with the carbon atom to which they are attached, form a C3-C4 spiro carbocyclyl; or a pharmaceutically acceptable salt and/or solvate thereof, in the manufacture of a medicament wherein: i) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered at 2 to 400 mg per day; or ii) wherein the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered for a period of at least three months; or iii) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered orally at 1 to 500 mg per day once or twice per day; or iv) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered to an adult human subject.

2. The use according to claim 1, wherein the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered at 2 to 400 mg per day.

3. The use according to claim 1, wherein the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered for a period of at least three months.

4. The use according to claim 1, wherein the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered orally at 1 to 500 mg per day once or twice per day.

5. The use according to claim 1, wherein the compound of formula (I), or a pharmaceutically 21 Nov 2025

acceptable salt and/or solvate thereof is administered to an adult human subject.

6. Use of a compound of formula (I): 2020427632

(I) wherein: 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, are a spirocyclopropyl ring; R4 is methyl or ethyl; R5 is H or methyl; or R4 and R5, together with the carbon atom to which they are attached, form a C3-C4 spiro carbocyclyl; or a pharmaceutically acceptable salt and/or solvate thereof, in the manufacture of a medicament wherein the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered to a human subject of less than 18 years of age.

7. The use according to any one of claims 1 to 6 wherein R1 is H.

8. The use according to any one of claims 1 to 6 wherein R1 is methyl.

9. The use according to any one of claims 1 to 8, wherein R2 and R3 are a spiro cyclopropyl.

10. The use according to any one of claims 1 to 8, wherein R2 is methyl and R3 is methyl.

11. The use according to any one of claims 1 to 10, wherein R4 is methyl.

12. The use according to any one of claims 1 to 10, wherein R4 is ethyl.

13. The use according to any one of claims 1 to 12, wherein R5 is H.

14. The use according to any one of claims 1 to 12, wherein R5 is methyl.

15. The use according to any of claims 1 to 6, wherein the compound, or pharmaceutically acceptable salt and/or solvate thereof is a compound selected from the group consisting of: 5,5-dimethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4- yl)oxypyrazin-2-yl]imidazolidine-2,4-dione; 3-[5-[(3,3-dimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2-yl]-5,5-dimethyl- imidazolidine-2,4-dione; (5R)-5-ethyl-5-methyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4- 2020427632

yloxypyrazin-2-yl)imidazolidine-2,4-dione; 5,5-dimethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2- yl)imidazolidine-2,4-dione; (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-2,4-dione; 5,5-dimethyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2- yl]imidazolidine-2,4-dione; (5R)-5-ethyl-5-methyl-3-[5-[(3,3,7-trimethyl-2H-benzofuran-4-yl)oxy]pyrazin-2- yl]imidazolidine-2,4-dione; (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4- yl)oxypyrazin-2-yl]imidazolidine-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]imidazolidine-2,4-dione; 7-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]-5,7- diazaspiro[3.4]octane-6,8-dione; 6-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-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'-cyclopropane]-4- yl)oxypyrazin-2-yl]imidazolidine-2,4-dione; or a pharmaceutically acceptable salt and/or solvate thereof.

16. The use according to any one of claims 1 to 6, wherein the compound, or pharmaceutically acceptable salt and/or solvate thereof is a compound which is (5R)-5-ethyl-3-

[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4- 21 Nov 2025

dione:

. 2020427632

17. The use according to any one of claims 1 to 6, wherein the compound, or pharmaceutically acceptable salt and/or solvate thereof is a pharmaceutically acceptable salt of (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2- yl]imidazolidine-2,4-dione:

.

18. The use according to any one of claims 1 to 6, wherein the compound, or pharmaceutically acceptable salt and/or solvate thereof is a compound which is (5R)-5-ethyl-3- (5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-yl)imidazolidine-2,4-dione:

.

19. The use according to any one of claims 1 to 6, wherein the compound, or pharmaceutically acceptable salt and/or solvate thereof is a pharmaceutically acceptable salt of (5R)-5-ethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-yl)imidazolidine-2,4- dione:

.

20. The use according to claims 16 or 17 wherein the compound, or pharmaceutically acceptable salt and/or solvate thereof is administered to an adult human subject. 2020427632

21. The use according to claims 16 or 17 wherein the compound, or pharmaceutically acceptable salt and/or solvate thereof is administered to a human subject of less than 18 years of age.

22. The use according to claims 18 or 19 wherein the compound, or pharmaceutically acceptable salt and/or solvate thereof is administered to an adult human subject.

23. The use according to claims 18 or 19 wherein the compound, or pharmaceutically acceptable salt and/or solvate thereof is administered to a human subject of less than 18 years of age.

24. Use of the compound, pharmaceutically acceptable salt and/or solvate thereof according to any one of claims 1 to 23 in the manufacture of a medicament for the prophylaxis or treatment of a disease or disorder selected from the group consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse disorders, anxiety disorders, sleep disorders, hyperacusis and disturbances of loudness perception, Ménière's disease, disorders of balance, and disorders of the inner ear, impulse control disorder, personality disorders, attention-deficit/hyperactivity disorder, autism spectrum disorders, eating disorders, cognition impairment, ataxia, pain such as neuropathic pain, inflammatory pain and miscellaneous pain, Lewy body dementia and Parkinson’s disease.

25. A method for the prophylaxis or treatment of a disease or disorder selected from the group consisting of hearing disorders, schizophrenia, depression and mood disorders, bipolar disorder, substance abuse disorders, anxiety disorders, sleep disorders, hyperacusis and disturbances of loudness perception, Ménière's disease, disorders of balance, and disorders of the inner ear, impulse control disorder, personality disorders, attention-deficit/hyperactivity disorder, autism spectrum disorders, eating disorders, cognition impairment, ataxia, pain such as neuropathic pain, inflammatory pain and miscellaneous pain, Lewy body dementia and

Parkinson’s disease which comprises administering to a subject in need thereof an effective 21 Nov 2025

amount of a compound of formula (I):

(I) wherein: 2020427632

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, are a spirocyclopropyl ring; R4 is methyl or ethyl; R5 is H or methyl; or R4 and R5, together with the carbon atom to which they are attached, form a C3-C4 spiro carbocyclyl; or a pharmaceutically acceptable salt and/or solvate thereof wherein: i) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered at 2 to 400 mg per day; or ii) wherein the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered for a period of at least three months; or iii) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered orally at 1 to 500 mg per day once or twice per day; or iv) the compound of formula (I), or a pharmaceutically acceptable salt and/or solvate thereof is administered to an adult human subject.

26. A compound of formula (IV):

(IV) wherein R4 and R5 are as defined in claim 1, Y is halo, such as Cl; or a salt thereof.

27. A compound which is selected from: (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4-yl)oxypyrazin-2- yl]imidazolidine-2,4-dione:

; and (5R)-5-ethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2-yl)imidazolidine-2,4- dione: 2020427632

.

28. A salt of a compound, the salt being selected from: a salt of (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'-cyclopropane]-4- yl)oxypyrazin-2-yl]imidazolidine-2,4-dione:

; and a salt of (5R)-5-ethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]-4-yloxypyrazin-2- yl)imidazolidine-2,4-dione:

.

29. A pharmaceutically acceptable salt of a compound, the pharmaceutically acceptable salt being selected from: a pharmaceutically acceptable salt of (5R)-5-ethyl-3-[5-(7-methylspiro[2H-benzofuran-3,1'- cyclopropane]-4-yl)oxypyrazin-2-yl]imidazolidine-2,4-dione:

; and a pharmaceutically acceptable salt of (5R)-5-ethyl-3-(5-spiro[2H-benzofuran-3,1'-cyclopropane]- 4-yloxypyrazin-2-yl)imidazolidine-2,4-dione: 2020427632

.