DESCRIPTION

Title of the Invention: ETHYL 6-[(2-CHLORO-4-FLUOROPHENYL)

Technical Field METHANESULFONYL]-3-METHYLCYCLOHEX-1 -ENE-1 -

CARBOXYLATE AS TLR4 ANTAGONIST

[0001]

The present invention relates to a cyclohexene derivative, a pharmaceutical composition containing it and its use in

therapy, particularly for use in treating conditions mediated by Toll-like receptor 4 (TLR4) .

[0002]

(Background of the Invention)

The immune system is comprised of innate and acquired immunities, both of which work cooperatively to protect the host from infections. It has been shown that innate immunity can recognize conserved pathogen-associated molecular patterns or damage-associated molecular patterns in damaged/stressed tissues through Toll-like receptors (TLRs) expressed on the cell surface of immune cells. Thus, for example, recognition of invading pathogens triggers cytokine production (including interferon alpha (IFNc ) and upregulation of co-stimulatory molecules on phagocytes, leading to modulation of T cell

function. Thus, innate immunity is closely linked to acquired immunity and can influence the development and regulation of an acquired response.

[0003]

TLRs are a family of type I transmembrane receptors

characterized by an NH ₂ -terminal extracellular leucine-rich repeat domain (LRR) and a COOH-terminal intracellular tail containing a conserved region called the Toll/IL-1 receptor

(TIR) homology domain. The extracellular domain contains a.

varying number of LRR, which are thought to be involved in ligand binding. Ten human and twelve murine TLRs have been characterized, TLR1 to TLR10 in humans, and TLR1 to TLR9, TLRll, TLR12 and TLR13 in mice, the homolog of TLR10 being a

pseudogene. They differ from each other in ligand specificities, expression patterns, and in the target genes they can induce.

[0004]

It has been discovered that TLRs are not only expressed and functional in immune cells but are also expressed in non- immune cells such as neurons and glial cells. TLR4, in

particular, has been reported not only to initiate innate immunity in the brain but to cause central nervous system (CNS) neurodegeneration (see, for example, non-patent document 1 which demonstrated TLR4-dependent upregulation of cytokines in an Alzheimer's disease mouse model), suggesting that TLR4 activation needs to be tightly controlled to regulate the switch between its different roles in immune surveillance and neuroinflammatory propagation (see, for example, non-patent document 2) . Non-patent document 2 also comments that a well- known natural ligand of TLR4 is endotoxin/lipopolysaccharide (LPS) , one of the major cell wall components of Gram-negative bacteria. LPS has potent immunostimulatory effects, mainly exerted through TLR4 /MD2/CD14-bearing cells. The membrane anchor of LPS is a glucosamine-based phospholipid called lipid A, which represents the endotoxic principle of LPS and is responsible for its pathophysiological effects via TLR4. There is convincing evidence that LPS/TLR4 signaling is involved in various human and experimental CNS diseases (see, for example, non-patent document 3 and non-patent document 4).

[0005]

Consequently, there is a need for compounds having TLR4 antagonist activity that are capable of crossing the blood brain barrier for use in treating central nervous system diseases .

[0006]

Patent document 1 (Applicant: Takeda Pharmaceutical Company Limited) describes a certain class of cyclohexene derivatives of formula (1) and salts thereof, effective as inhibitors of nitric oxide and/or cytokine production for use in treating cardiac, autoimmune, inflammatory, CNS and infectious diseases but, in particular, sepsis

[0007]

[0008]

wherein

R ¹ is an optionally substituted aliphatic hydrocarbon group, an optionally substituted aromatic hydrocarbon group, an

optionally substituted heterocyclic group, a group of formula 0R ^la wherein R ^la is a hydrogen atom or an optionally substituted aliphatic hydrocarbon group, or a group of formula -NR ^lb R ^lc wherein R ^lb and R ^lc are the same or different and each is a hydrogen atom or an optionally substituted aliphatic

hydrocarbon group;

X is a methylene group, a nitrogen atom, a sulfur atom or an oxygen atom;

Y is an optionally substituted methylene group or an optionally substituted nitrogen atom;

ring A is a 5- to 8-membered ring optionally further

substituted by 1 to 4 substituent (s) selected from the

following (1) to (4):

(1) an optionally substituted aliphatic hydrocarbon group,

(2) an optionally substituted aromatic hydrocarbon group,

(3) a group of formula OR ² wherein R ² is a hydrogen atom or an optionally substituted aliphatic hydrocarbon group, and (4) a halogen atom;

Ar is an optionally substituted aromatic hydrocarbon group; a group of the formula

[0009]

[0012]

m is an integer of 0 to 2; and

n is an integer of 1 to 3 where the sum of m and n is not more than 4;

provided that when X is a methylene group, Y is an optionally substituted methylene group.

[0013]

One of the compounds encompassed in formula (1) of patent document 1 is ethyl ( 6R) -6- [N- (2-chloro-4- fluorophenyl) sulfamoyl] cyclohex-l-ene-l-carboxylate (also known as Resatorvid) which at one time was in clinical development for the treatment of sepsis.

Document List

Patent Document

[0014]

Patent Document 1: U.S. Patent No. 7,417,059

Non-Patent Document

[0015]

Non-Patent Document 1: Jin, J.J. et al. (2008). Toll-like receptor 4-dependent upregulation of cytokines in a transgenic mouse model of Alzheimer's disease. J Neuroinflammation, 5, 23- 32. Non-Patent Document 2: De Paola, M. et al. (2012).

Neuroprotective Effects of Toll-Like Receptor 4 Antagonism in Spinal Cord Cultures and in a Mouse Model of Motor Neuron Degeneration. Mol. Med., 18, 971-981.

Non-Patent Document 3: Walter, S. et al. (2007) Role of the toll-like receptor 4 in neuroinflammation in Alzheimer's disease. Cell Physiol. Biochem. , 20(6), 947-956.

Non-Patent Document 4: Panaro, M.A. et al. (2008) Expression of TLR4 and CD14 in the central nervous system (CNS) in a MPTP mouse model of Parkinson' s-like disease. Immunopharmacol .

Immunotoxicol. , 30(4), 729-740.

Summary of the Invention

Problems to be Solved by the Invention

[0016]

It has now been found that another compound encompassed in formula (1) of patent document 1 possesses surprising and unexpected properties.

Means of Solving the Problems

[0017]

Therefore, in accordance with the present invention, there is provided ethyl 6- [ (2-chloro-4- fluorophenyl) methanesulfonyl] -3-methylcyclohex-l-ene-l- carboxylate; and isomers and mixtures thereof.

Thus, the invention provides the followings.

[1] A compound of ethyl 6- [ (2-chloro-4- fluorophenyl) methanesulfonyl] -3-methylcyclohex-l-ene-l- carboxylate, as represented by formula (I) .

[0018]

[0019]

[2] The compound of the above-mentioned [1], which is ethyl (3S, 6R) -6- [ (2-chloro-4-fluorophenyl) methanesulfonyl] -3- methylcyclohex-l-ene-l-carboxylate, as represented by formula (II):

[0020]

[0021]

[3] A pharmaceutical composition comprising a compound of the above-mentioned [1] , in association with a pharmaceutically acceptable adjuvant, diluent or carrier, and optionally one or more other therapeutic agents.

[4] A pharmaceutical composition of the above-mentioned [3], wherein the optional therapeutic agent is an anti-inflammatory drug.

[5] The pharmaceutical composition of the above-mentioned [3] , which is a toll-like receptor 4 inhibitor.

[6] The pharmaceutical composition of the above-mentioned [3] , which is for prophylaxis and/or treatment of post-operative cognitive decline, epilepsy and/or schizophrenia.

[7] The pharmaceutical composition of the above-mentioned [3], which is for prophylaxis and/or treatment of chemotherapy- induced peripheral neuropathy (CIPN) , chemotherapy-induced neuropathic pain (CINP) , liver injury and/or ischemia- reperfusion injury (IRI) .

[0022]

[8] The compound of the above-mentioned [1] for use in prophylaxis and/or treating post-operative cognitive decline, epilepsy and/or schizophrenia.

[9] The compound of the above-mentioned [1] for use in

prophylaxis and/or treating chemotherapy-induced peripheral neuropathy (CIPN) , chemotherapy-induced neuropathic pain (CINP) , liver injury and/or ischemia-reperfusion injury (IRI) .

[0023]

[10] A method of inhibiting toll-like receptor 4 in a mammal, which comprises administering an effective amount of the compound of the above-mentioned [1] to the mammal.

[11] A method for the prophylaxis and/or treatment of postoperative cognitive decline, epilepsy and/or schizophrenia in a mammal, which comprises administering an effective amount of the compound of the above-mentioned [1] to the mammal.

[12] A method for the prophylaxis and/or treatment of

chemotherapy-induced peripheral neuropathy (CIPN) ,

chemotherapy-induced neuropathic pain (CINP) , liver injury and/or ischemia-reperfusion injury (IRI) in a mammal, which comprises administering an effective amount of the compound of the above-mentioned [1] to the mammal.

[0024]

[13] Use of the, compound of the above-mentioned [1] for the production of an agent for the prophylaxis and/or treatment of post-operative cognitive decline, epilepsy and/or schizophrenia.

[14] Use of the compound of the above-mentioned [1] for the production of an agent for the prophylaxis and/or treatment of chemotherapy-induced peripheral neuropathy (CIPN) ,

chemotherapy-induced neuropathic pain (CINP), liver injury and/or ischemia-reperfusion injury (IRI).

Effect of the Invention

[0025]

The compound of formula (I) possesses an advantageous combination of TLR4 antagonist properties and brain

permeability, and therefore, the compound would be expected to be useful in the treatment of central nervous system diseases mediated at least in part by TLR . [0026]

(Detailed Description of the Invention)

The present invention is explained in detail in the following .

[0027]

The compound according to the invention may be prepared by processes analogous to those known in the art, for example, as described in patent document 1.

[0028]

In one aspect of the invention, the compound of formula

(I) or (II) may be radiolabeled. Such radiolabeled versions may be produced by using radioisotope-containing reagents in the synthesis of the compound of formula (I) or (II), or may be produced by coupling the compound of formula (I) or (II) to chelating moieties capable of binding to a radioactive metal atom. Such radiolabeled versions of the compound of formula (I) or (II) may be used, for example, in diagnostic imaging studies.

[0029]

Unless stated otherwise, any atom specified herein may also be an isotope of said atom. For example, the term

"hydrogen" encompasses ¹ H, ² H and ³ H. Similarly carbon atoms are to be understood to include ¹² C, ¹³ C and ¹⁴ C, nitrogen atoms are to be understood to include ¹⁴ N and ¹⁵ N, and oxygen atoms are to be understood to include ¹⁶ 0, ¹⁷ 0 and ¹⁸ 0.

[0030]

In a further aspect, the compound of formula (I) or (II) may be isotopically labelled. As used herein, an "isotopically labelled" compound is one in which the abundance of a

particular nuclide at a particular atomic position within the molecule is increased above the level at which it occurs in nature.

[0031]

The compound of formula (I) or (II) may be in the form of hydrates or solvates which form an aspect of the present

invention. Such solvates may be formed with common organic solvents, including but not limited to, alcoholic solvents e.g. methanol, ethanol or isopropanol.

[0032]

The compound of formula (I) may exist in various

stereoisomeric forms and consequently it will be understood that the invention encompasses all geometric and optical isomers (including atropisomers) of the compound of formula (I) and mixtures thereof including racemates . Specifically, the compound of formula (I) has contains the following four isomers;

(1) ethyl (3S, 6R) -6- [ (2-chloro-4-fluorophenyl) methanesulfonyl] - 3-methylcyclohex-l-ene-l-carboxylate (i.e., the compound of formula (II) ) ,

(2) ethyl (3R, 6R) -6- [ (2-chloro-4-fluorophenyl) methanesulfonyl] - 3-methylcyclohex-l-ene-l-carboxylate,

(3) ethyl (3S, 6S) -6- [ (2-chloro-4-fluorophenyl) methanesulfonyl] - 3-methylcyclohex-l-ene-l-carboxylate, and

(4) ethyl (3R, 6S) -6- [ (2-chloro-4-fluorophenyl) methanesulfonyl] - 3-methylcyclohex-l-ene-l-carboxylate .

The present invention encompasses all four isomers, as well as mixture thereof in any ratio including racemate thereof, enantiomeric mixtures thereof and diastereomeric mixtures thereof.

[0033]

Enantiomerically pure forms are particularly desired.

"Enantiomerically pure" denotes the presence of at least 75%w, in particular at least 90%w and, more particularly at least 95%w of one of the two possible enantiomers of the compound. Techniques such as chiral supercritical fluid chromatography (chiral SFC) may be used for separating enantiomers. Both chiral SFC and other suitable techniques will be known to the person skilled in the art, for example, from "Chiral Separation Methods for Pharmaceutical and Biotechnological Products", edited by S. Ahuja, John Wiley & Sons, Inc. (2011) .

The compound of formula (I) or (II) may be amorphous or in a polymorphic form or a mixture of any of these, each of which forms an aspect of the present invention.

[0034]

The compound of formula (I) or (II) has activity as a pharmaceutical and may be used for treating central nervous system conditions mediated in full or in part by TLR4.

[0035]

Thus, the present invention provides a compound of formula (I) or (II) as hereinbefore defined, for the treatment of central nervous system conditions associated with TLR4.

[0036]

The present invention also provides the use of a compound of formula (I) or (II) as hereinbefore defined, for the preparation of a pharmaceutical composition for the treatment of central nervous system conditions associated with TLR4.

[0037]

The present invention still further provides a method of treating central nervous system conditions associated with TLR4 which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or (II) as hereinbefore defined.

[0038]

Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of, the disorder or condition in question. Persons at risk of

developing a particular disorder or condition generally include those having a family history of the disorder or condition, or those who have been identified by genetic testing or by screening (e.g. for specific biomarkers or for displays of certain physiological or physical features which in either case are deemed to be risk factors) to be particularly susceptible to developing the disorder or condition or those in the prodromal phase of a disorder.

[0039] The terms "treat", "treatment" and "treating" include improvement of the conditions described herein. The terms

"treat", "treatment" and "treating" include all processes providing slowing, interrupting, arresting, controlling, or stopping of the state or progression of the conditions

described herein, but does not necessarily indicate a total elimination of all symptoms or a cure of the condition. The terms "treat", "treatment," and "treating" are intended to include therapeutic as well as prophylactic treatment of such conditions.

[0040]

As used herein, the terms "condition," "disorder," and "disease" relate to any unhealthy or abnormal state. The term "conditions associated with TLR4" includes conditions,

disorders and diseases in which the modulation of TLR4 may provide a therapeutic benefit, for example, Parkinson's disease, including dementia associated with Parkinson's disease;

Alzheimer's disease; Huntington's disease; amyotrophic lateral sclerosis; multiple sclerosis; bipolar disorder; and

psychiatric disorders such as schizophrenia, post-traumatic stress disorder, epilepsy (including treatment resistant epilepsy) , post-operative cognitive decline, post-operative cognitive delirium, anxiety disorders and depression (e.g.

major depressive disorder) . Preferred are post-operative cognitive decline, schizophrenia and epilepsy.

[0041]

Schizophrenia is a debilitating psychiatric disorder characterized by a combination of negative symptoms (such as social withdrawal, anhedonia, avolition and apathy) and

positive symptoms (including hallucinations, delusions and paranoia) as well as marked cognitive deficits (such as

impairment of executive function) . The executive function (EF) has been defined as "a set of abilities, which allows us to invoke voluntary control of our behavioral responses. These functions enable human beings to develop and carry out plans, make up analogies, obey social rules, solve problems, adapt to unexpected circumstances, do many tasks simultaneously, and locate episodes in time and place. EF includes divided

attention and sustained attention, working memory (WM) , set- shifting, flexibility, planning, and the regulation of goal directed behavior and can be defined as a brain function underlying the human faculty to act or think not only in reaction to external events but also in relation with internal goals and states" (Orellana G. and Slachevsky A., 2013.

Executive Functioning in Schizophrenia. Front. Psychiatry, 4, 35) .

[0042]

Accordingly, the present invention also provides a method of treating a negative symptom, a positive symptom and/or a cognitive deficit associated with a psychiatric disorder, in particular schizophrenia, which comprises administering to a patient in need thereof a compound of formula (I) or (II) as hereinbefore defined.

[0043]

For the above-mentioned therapeutic uses, the dosage administered will, of course, vary with the mode of

administration, the treatment desired and the disorder

indicated. For example, the daily dosage of a compound of formula (I) or (II) as hereinbefore defined, if inhaled, may be in the range from 0.05 micrograms per kilogram body weight ( g/kg) to 100 micrograms per kilogram body weight kg) . Alternatively, if a compound of formula (I) or (II) as

hereinbefore defined is administered orally, then the daily dosage may be in the range from 0.01 micrograms per kilogram body weight ^g/kg) to 100 milligrams per kilogram body weight (mg/kg) .

[0044]

A compound of formula (I) or (II) as hereinbefore defined may be used on its own but will generally be administered in the form of a pharmaceutical composition in which a compound of formula (I) or (II) (active ingredient) is in association with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0045]

Therefore, the present invention further provides a pharmaceutical composition comprising a compound of formula (I) or (II) as hereinbefore defined, in association with a

pharmaceutically acceptable adjuvant, diluent or carrier.

[0046]

The invention still further provides a process for the preparation of the pharmaceutical composition of the invention which comprises mixing a compound of formula (I) or (II) as hereinbefore defined with a pharmaceutically acceptable

adjuvant, diluent or carrier.

[0047]

Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, "Pharmaceutics - The Science of Dosage Form Design", M. E. Aulton, Churchill Livingstone, 1988.

[0048]

Pharmaceutically acceptable adjuvants, diluents or carriers that may be used in the pharmaceutical compositions of the invention are those conventionally employed in the field of pharmaceutical formulation, and include, but are not limited to, sugars, sugar alcohols, starches, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium

carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat. [0049]

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, rectally, nasally, buccally, vaginally or via an implanted reservoir. The pharmaceutical compositions of the invention may contain any conventional non-toxic pharmaceutically acceptable adjuvants, diluents or carriers. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0050]

The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. The suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol . The acceptable diluents and

solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides . Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil and castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant .

[0051]

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, powders, granules, and aqueous suspensions and solutions. These dosage forms are prepared according to techniques well-known in the art of pharmaceutical formulation. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

[0052]

The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal

administration. These compositions can be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active ingredient. Such materials include, but are not limited to, cocoa butter, beeswax .and polyethylene glycols.

[0053]

The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable

preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

[0054]

Depending on the mode of administration, the

pharmaceutical composition will preferably comprise from 0.05 to 99%w (per cent by weight), more preferably from 0.05 to 80%w, still more preferably from 0.10 to 70%w, and even more preferably from 0.10 to 50%w, of active ingredient, all

percentages by weight being based on total composition.

[0055]

A compound of formula (I) or (II) as hereinbefore defined may also be administered in conjunction with other compounds used for the treatment of the above conditions.

[0056]

The invention therefore further relates to combination therapies wherein a compound of formula (I) or (II) as

hereinbefore defined, or a pharmaceutical composition or formulation comprising a compound of formula (I) or (II) as hereinbefore defined, is administered with another therapeutic agent or agents for the treatment of one or more of the

conditions previously indicated. Such therapeutic agents may be selected from the following:

(i) antidepressants such as amitriptyline, amoxapine, bupropion, citalopram, clomipramine, desipramine, doxepin duloxetine, elzasonan, escitalopram, fluvoxamine, fluoxetine, gepirone, imipramine, ipsapirone, maprotiline, nortriptyline, nefazodone, paroxetine, phenelzine, protriptyline, reboxetine, robaizotan, sertraline, sibutramine, tianeptine, thionisoxetine,

tranylcypromaine, trazodone, trimipramine, venlafaxine. and vortioxetine, and equivalents and pharmaceutically active isomer (s) and/or metabolite (s) thereof;

(ii) antipsychotics including, for example, amisulpride, aripiprazole, asenapine, benzisoxidil, bifeprunox,

brexpiprazole, carbamazepine, cariprazine, clozapine,

chlorpromazine, debenzapine, divalproex, duloxetine,

eszopiclone, fluphenazine, haloperidol, iloperidone,

lamotrigine, loxapine, lurasidone, mesoridazine, olanzapine, paliperidone, perlapine, perphenazine, phenothiazine,

phenylbutlypiperidine, pimozide, prochlorperazine, quetiapine, risperidone, sertindole, sulpiride, suproclone, suriclone, thioridazine, trifluoperazine, trimetozine, valproate, valproic acid, zopiclone, zotepine, zicronapine and ziprasidone, and equivalents and pharmaceutically active isomer (s) and/or metabolite (s) thereof;

(iii) anxiolytics including, for example, alnespirone,

azapirones, benzodiazepines and barbiturates, and equivalents and pharmaceutically active isomer (s) and/or metabolite (s) thereof. Example anxiolytics include adinazolam, alprazolam, balezepam, bentazepam, bromazepam, brotizolam, buspirone, clonazepam, clorazepate, chlordiazepoxide, cyprazepam, diazepam, diphenhydramine, estazolam, fenobam, flunitrazepam, flurazepam, fosazepam, lorazepam, lormetazepam, meprobamate, midazolam, nitrazepam, oxazepam, prazepam, prazosin, quazepam, reclazepam, tracazolate, trepipam, temazepam, triazolam, uldazepam and zolazepam, and equivalents and pharmaceutically active

isomer (s) and/or metabolite (s) thereof;

(iv) anticonvulsants including, for example, carbamazepine, valproate, lamotrigine, levetiracetam, oxcarbazepine and gabapentin, and equivalents and pharmaceutically active

isomer (s) and/or metabolite (s) thereof;

(v) Alzheimer's therapies including, for example, donepezil, galantamine, memantine, rivastigmine and tacrine, and

equivalents and pharmaceutically active isomer (s) and/or metabolite (s) thereof;

(vi) Parkinson's therapies including, for example, L-dopa, ropinirole, pramipexole, monoamine oxidase type B (MAO-B) inhibitors such as deprenyl, selegiline and rasagiline,

catechol-O-methyl transferase (COMT) inhibitors such as

entacapone and tolcapone, adenosine A-2 inhibitors, dopamine re-uptake inhibitors, NMDA antagonists, Nicotine agonists, dopamine agonists and inhibitors of neuronal nitric oxide synthase, and equivalents and pharmaceutically active isomer (s) and/or metabolite (s) thereof;

(vii) insomnia therapies including, for example, allobarbital, alonimid, amobarbital, benzoctamine, butabarbital, capuride, chloral, cloperidone, clorethate, dexclamol, ethchlorvynol, eszopiclone, etomidate, glutethimide, halazepam, hydroxyzine, lorediplon, mecloqualone, melatonin, mephobarbital,

methaqualone, midaflur, nisobamate, pentobarbital,

phenobarbital, propofol, ralmeteon, roletamide, suvorexant, triclofos, secobarbital, zaleplon, Zolpidem and zopiclone, and equivalents and pharmaceutically active isomer (s) and/or metabolite (s) thereof;

(viii) mood stabilizers including, for example, carbamazepine, divalproex, gabapentin, lamotrigine, lithium carbonate

(generally referred to as lithium' ) , olanzapine, quetiapine, valproate, valproic acid and verapamil, and equivalents and pharmaceutically active isomer (s) and/or metabolite (s) thereof; and

(ix) anti-inflammatories including, for example, non-steroidal anti-inflammatory drugs (NSAIDs, such as ibuprofen,

dexibuprofen, naproxen, fenoprofen, flurbiprofen, diclofenac and aceclofenac) and belnacasan (a Caspase-I inhibitor) .

[0057]

Such combination products employ the compound of formula (I) or (II) of this invention within the dosage ranges

described herein and the other pharmaceutically active agent within approved dosage ranges.

[0058]

In a further aspect the present invention provides a fixed dose combination (for example, for the treatment of a psychiatric disorder) of a compound of formula (I) or (II) as hereinbefore defined and one or more agents selected from carbamazepine, olanzapine, quetiapine, verapamil, lamotrigine, oxcarbazepine, risperidone, aripiprazole, ziprasidone and lithium.

[0059]

The invention also provides a pharmaceutical product comprising, in combination, a preparation of a first active ingredient which is a compound of formula (I) or (II) as hereinbefore defined, and a preparation of a second active ingredient which is carbamazepine, olanzapine, quetiapine, verapamil, lamotrigine, oxcarbazepine, risperidone, aripiprazole, ziprasidone or lithium, for simultaneous,

sequential or separate use in therapy.

[0060]

In another aspect, the invention provides a kit

comprising a preparation of a first active ingredient which is a compound of formula (I) or (II) as hereinbefore defined, and a preparation of a second active ingredient which is

carbamazepine, olanzapine, quetiapine, verapamil, lamotrigine, oxcarbazepine, risperidone, aripiprazole, ziprasidone or lithium, and instructions for the simultaneous, sequential or separate administration of the preparations to a patient in need thereof.

Example

[0061]

The present invention will now be further explained by reference to the following illustrative examples, in which the starting materials and reagents used are available from

commercial suppliers or prepared via literature procedures.

[0062]

In addition, a compound of formula (I) or (II) has excellent TLR4 signaling inhibitory action, and so said

compounds are useful as safe drugs based on the said action.

Accordingly, a compound of formula (I) or (II) in the present invention can be used with respect to mammalian

organisms (for example, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, human, or the like) as preventive and/or therapeutic agents of, for example, autoimmune disease and/or inflammatory disease, and diseases such as infectious disease, cardiac disease, central nervous system disease, hypoimmunity and the like; for example, sepsis including serious sepsis, septic shock, septicemia, endotoxic shock, exotoxic shock, systemic inflammatory response syndrome (SIRS) , compensatory antiinflammatory reaction syndrome (CARS) , burn injury, trauma, postoperative complication, cardiac _. failure, shock, hypotension, rheumatoid arthritis, osteoarthritis, gastritis, ulcerative colitis, peptic ulcer, stress-induced peptic ulcer, Crohn's disease, autoimmune disease, graft rejection after organ transplantation, ischemia-reperfusion injury (IRI), liver injury (acute liver injury (ALI) , ACLF) , acute coronary microvascular embolism, shock-induced vascular embolism

(disseminated intravascular coagulation (DIC) or the like) , ischemic encephalopathy, arteriosclerosis, pernicious anemia, Fanconi anemia, sickle cell anemia, pancreatitis, nephrotic syndrome, acute and chronic nephropathy, nephritis, renal failure, insulin dependent diabetes mellitus, non-insulin dependent diabetes mellitus, hepatic porphyria, alcohol poisoning, Parkinson's disease, chronic leukemia, acute leukemia, tumor, myeloma, infant and adult respiratory distress syndrome, chronic obstructive pulmonary disease, dementia,

Alzheimer's disease, multiple sclerosis, optic nerve myelitis, vitamin E deficiency, ageing, sunburn, muscular dystrophy, myocarditis, cardiomyopathy, myocardial infarction, myocardial infarction sequellae, osteoporosis, pneumonia, hepatitis, psoriasis, pain, cataract, influenza infection, malaria, human immunodeficiency virus (HIV) infection, radiation damage, burn, hypercalcemia, ankylosing spondylitis, osteopenia, Paget ' s disease, osteomalacia, bone fracture, acute bacterial

meningitis, Helicobacter pylori infection, invasive

Staphylococcus infection, tuberculosis, systemic fungal infection, herpes simplex viral infection, varicella-zoster viral infection, human papilloma virus infection, acute viral encephalitis, encephalitis, meningitis, hypoimmunity

accompanying infection, bronchial asthma, atopic dermatitis, allergic rhinitis, reflux esophagitis, fever,

hypercholesterolemia, hyperglyceridemia, hyperlipidemia, diabetic complications, diabetic nephropathy, diabetic

neuropathy, diabetic retinopathy, gout, gastric atony,

hemorrhoids, systemic lupus erythematosus, spinal cord injury, insomnia, schizophrenia, epilepsy, cirrhosis, hepatic insufficiency, unstable angina, cardiac valvular disease, thrombocytopenia or hypotension due to dialysis, acute ischemic cerebral apoplexy, acute cerebral thrombosis, cancer metastasis, urinary bladder cancer, breast cancer, uterine cervical cancer, colorectal cancer, gastric cancer, ovarian cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, malignant melanoma, Hodgkin's disease, non-Hodgkin ' s lymphoma, side effects due to anticancer agent and immunosuppressant drug administration, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary fibrosis, autoimmune hemolytic anemia, meningitis, inflammatory pulmonary disease (for example, silicosis, pulmonary sarcoidosis, tuberculosis), endometriosis, cachexia (for example, cachexia due to infection, cancerous cachexia, cachexia due to acquired immunodeficiency syndrome) , cancer pain, Addison's disease, acute pain due to inflammation, pain due to chronic inflammation, post-operative pain (incision wound pain, deep pain, visceral pain, chronic post-operative pain, or the like) , myalgia (myalgia accompanying chronic pain, stiff neck, or the like) , arthralgia, toothache,

temporomandibular arthralgia, headache (migraine headache, tension headache, headache accompanying pyrexia, headache accompanying hypertension) , visceral pain (cardialgia, anginal pain, abdominal pain, kidney pain, urinary tract pain, bladder pain) , pain of the obstetric and gynecological area

(intermenstrual pain, dysmenorrheal and labour pain) ,

neurogenic pain (spinal disc herniation, nerve root pain, post- herpes zoster neuralgia, trigeminal neuralgia, lumbago, or the like) , chemotherapys ( (taxane anticancer drugs (for example, paclitaxel (taxol) , docetaxel) , vinca alkaloid anticancer drugs (for example, vincristine, vinblastine) , platinum preparations (for example, cisplatin, carboplatin, oxaliplatin) , molecular target drug (for example, bortezomib) or the like) ) -induced peripheral neuropathy (CIPN) and associated neurological symptoms (chemotherapy-induced neuropathic pain (CINP)

(dysesthesia such as numbness and/or pain (for example, muscle pain, nerve pain) ) ) , reflex sympathetic atrophy, complex local pain syndrome, pituitary gland abscess, thyroiditis,

peritonitis, erythema nodosum) , allergic conjunctivitis, pollinosis, metal allergy, exudative otitis media, Meniere's disease, contact dermatitis, anaphylaxis, urticaria, myasthenia gravis, Sjogren's syndrome, Basedow's disease, leukocyte abnormality, renal tubulointerstitial disorder (including fibrillary pathology) , acute coronary artery syndrome,

atherosclerotic aortic aneurysm, cardiac anaphylaxis, deep vein thrombosis, ophthalmologic diseases (for example, pterygium, spring catarrh, dry eye, or the like) , food allergy, NUD (Non Ulcer Dyspepsia) , gastric MALT lymphoma, ulcer due to nonsteroid anti-inflammatory drug, gastric hyperacidity, gastric hyperacidity and ulcer due to postoperative stress, obesity, edema, granuloma, atopic myelitis, neurofibroma, nasal mucosal hypersensitivity, osteoarthritis, scleroderma, or the like, preferably chemotherapy-induced peripheral neuropathy (CIPN) , chemotherapy-induced neuropathic pain (CINP) , liver injury and/or ischemia-reperfusion injury (IRI) . Moreover, the TLR4 signaling inhibiting substance of the present invention can also be used for increasing efficiency of in vitro

fertilization .

[0063]

Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz or 300 MHz as stated and at 300.3K, 298.2K or 293K unless otherwise stated; the chemical shifts (δ) were reported in parts per million. Spectra were recorded using a Bruker 400 AVANCE instrument fitted with a 5mm BBFO probe with instrument controlled by Bruker TopSpin 2.1 software, or by a Bruker 400 AVANCE-III HD instrument fitted with a 5mm BBFO smart probe or a 5mm BBFO probe with instrument controlled by Bruker TopSpin 3.2 software, or by a Bruker 400 AVANCE-III instrument fitted with a 5mm BBFO probe with instrument controlled by Bruker Topspin 3.0 software or by a Bruker 300MHz AVANCE II

instrument fitted with a 5mm DUL probe with instrument controlled by Bruker TopSpin 1.3 software, or 5mm BBFO probe controlled by Bruker Topspin 3.2 software.

[0064]

Purity was assessed using one or more of the following: (1) UPLC with UV (photodiode array) detection over a wide range of wavelengths, normally 220-450 nm, using a Waters Acquity UPLC system equipped with Acquity UPLC BEH, HSS or HSS T3 C18 columns (2.1mm id x 50mm long) operated at 50 or 60°C. Mobile phases typically consisted of acetonitrile mixed with water containing either 0.1% formic acid, 0.1% TFA or 0.025% ammonia. Mass spectra were recorded with a Waters SQD single quadrupole mass spectrometer using atmospheric pressure ionization.

(2) UPLC with UV (photodiode array) detection over a wide range of wavelengths, normally 220-450 nm, using Shimadzu Nexera X2 UPLC controlled by Lab Solution software equipped with Acquity UPLC BEH, HSS or HSS T3 C18 columns (2.1mm id x 50mm long) operated at 50°C. Mobile phases typically consisted of

acetonitrile mixed with water containing either 0.1% formic acid, 0.1% TFA or 0.025% ammonia. Mass spectra were recorded with a Shimadzu single quadrupole mass spectrometer using DUIS ionization .

[0065]

Compounds were purified using normal phase chromatography on silica, using Biotage KP-Sil cartridges, Interchim PuriFlash cartridges or Kinesis Telos silica cartridges, or on basic silica using Biotage KP-NH cartridges, or by reverse phase chromatographic methods using Biotage KP-C18-HS cartridges or by Biotage Isolute SCX-2 or Phenomenex Strata ABW catch-release cartridges, or by preparative HPLC.

[0066]

Preparative HPLC was performed using Agilent Technologies 1100 Series system or a Waters autopurification LC/MS system typically using Waters 19 mm id x 250 mm long C18 columns such as XBridge or SunFire 5 μιη materials at rt . Mobile phases typically consisted of acetonitrile mixed with water containing either 0.1% formic acid or 0.1% ammonia, unless stated otherwise .

[0067]

SFC chiral separations were performed on a Waters prep30/MS system, using a flow rate of 30 mL/min, temperature of 40°C and a pressure of 100 bar. Mobile phases typically consisted of supercritical C0 ₂ and a polar solvent such as methanol, ethanol or isopropanol. Column type and eluent are detailed for individual examples.

[0068]

^oom temperature' as used herein means a temperature in the range from about 18°C to about 25°C.

[0069]

Abbreviations

DBU: 1, 8-Diazabicycloundec-7-ene

DCM: Dichloromethane

DIPEA: N, IV-Diisopropylethylamine

DMSO: Dimethylsulfoxide

EtOAc: Ethyl acetate

EtOH: Ethanol

h: hour

KO ^fc Bu: Potassium tert-butoxide

mCPBA: 3-Chloroperbenzoic acid

MeCN: Acetonitrile

min: minute

SFC: Super Critical Fluid Chromatography

TEA: Triethylamine

TFA: Trifluoroacetic acid

THF: Tetrahydrofuran

[0070]

1. Preparation of Intermediates

Intermediate 1 : Ethyl 8- (trifluoromethanesulfonyloxy) -1,4- dioxaspiro [4.5] dec-7-ene-7-carboxylate

[0071]

[0072]

Step (i) : Ethyl 8-hydroxy-l , 4-dioxaspiro [4.5] dec-7-ene-7- carboxylate

A THF (200 mL) solution of 1, 4-dioxaspiro [4.5] decan-8-one

(35 g, 224 mmol) was added while heating under reflux to a mixture of diethyl carbonate (67.9 mL, 560 mmol) and KO ^t Bu (1M in THF) (672 mL, 672 mmol), and the mixture was stirred at the same temperature for 5h. The mixture was allowed to cool to room temperature for 17h. The solids recovered by filtration were washed with EtOAc and dissolved in water. Water (50 mL) and acetic acid (50 mL) were added with ice cooling. The mixture was extracted three times with EtOAc. The organics were combined, washed twice with water, saturated aqueous sodium bicarbonate and saturated aqueous sodium chloride solution, dried (MgS0 ₄ ) and filtered through a pad of silica gel. The filtrate was concentrated in vacuo to afford the title compound. ¾ NMR (400 MHz, DMSO-ds) 5ppm 1.21 - 1.28 (m, 3 H) , 1.72 - 1.80 (m, 2 H) , 2.36 -2.43 (m, 4 H) , 3.87 - 3.93 (m, 4 H) , 4.13 - 4.22 (m, 2 H) , 12.07 - 12.20 (m, 1 H)

MS ES+: 229

[0073]

Step (ii) : Ethyl 8- (trifluoromethanesulfonyloxy) -1 , 4- dioxaspiro [4.5] dec-7-ene-7-carboxylate

To a stirred solution of ethyl 8-hydroxy-l, 4- dioxaspiro [4.5] dec-7-ene-7-carboxylate (33.0 g, 145 mmol) and

DIPEA (126 mL, 723 mmol) in toluene (500 mL) at -78°C was added triflie anhydride (35.6 mL, 217 mmol) drop wise. The mixture was stirred at -78°C for 2h and then allowed to warm to room temperature. A saturated solution of sodium bicarbonate was added and the mixture was stirred at room temperature for 10 min. The organics were washed with 5% aqueous citric acid solution, dried (phase separator) and concentrated in vacuo. The product was taken up into EtOAc and washed three times with aqueous citric acid solution, dried (phase separator) and concentrated in vacuo to afford the title compound.

XH NMR (300 MHz, DMSO-d<j) 5ppm 1.21 - 1.27 (m, 3 H) , 2.53 - 2.65 (m, 6 H) , 3.90 -3.96 (m, 4 H) , 4.11 - 4.23 (m, 2 H)

[0074]

Intermediate 2 : (2-Chloro-4-fluorophenyl)methanethiol

[0075]

[0076]

To a solution of 1- (bromomethyl) -2-chloro-4-fluorobenzene (7.7 g, 34.5 mmol) in dry methanol (115 mL) at room temperature was added K ₂ C0 ₃ (5.71 g, 41.3 mmol) followed by ethanethioic S- acid (2.95 mL, 41.3 mmol). The mixture was stirred at room temperature under an atmosphere of nitrogen for 30 min. To this was then added K ₂ C0 ₃ (5.71 g, 41.3 mmol) and the mixture was stirred at room temperature for lh. The mixture was acidified by the addition of 2M HC1 and water. The organics were

extracted with DCM, dried (phase separator) and concentrated in vacuo to afford the title compound.

^X H NMR (300 MHz, DMSO-d ₆ ) 5ppm 2.84 - 2.96 (m, 1 H) , 3.76 - 3.84 (m, 2 H), 7.13 -7.26 (m, 1 H) , 7.37 - 7.46 (m, 1 H) , 7.50 - 7.59 (m, 1 H)

[0077]

2. Preparation of Compound of the Invention : Ethyl (3S,6R)-6- [ (2-chloro-4-fluorophenyl)methanesulfonyl] -3-methylcyclohex-l- ene-l-carboxylate (Compound 1)

[0078]

[0079]

Step (i) : Ethyl 8- {[ (2-chloro-4-fluorophenyl)methyl] sulfanyl} - 1 , -dioxaspiro [ .5] dec-7-ene-7-carboxylate

To a solution of ethyl 8- (trifluoromethanesulfonyloxy) -

1, 4-dioxaspiro [4.5] dec-7-ene-7-carboxylate (Intermediate 1, 8.42 g, 23.37 mmol) in ethanol (78 mL) were added TEA (3.58 mL, 25.7 mmol) and (2-chloro-4-fluorophenyl) methanethiol

(Intermediate 2, 4.95 g, 28.0 mmol). The mixture was stirred at room temperature for 72h. The solid was collected by filtration and washed with a minimum amount of EtOH to afford the title compound.

¾ NMR (300 MHz, DMSO-d ₆ ) 5ppm 1.18 (t, J = 7.08 Hz, 3 H) , 1.72 - 1.83 (m, 2 H) , 2.45 (s, 2 H) , 2.68 - 2.80 (m, 2 H) , 3.91 (s, 4 H) , 4.06 (q, J= 7.15 Hz, 2 H) , 4.19 (s, 2 H) , 7.17 - 7.30 (m, 1 H) , 7.43 - 7.51 (m, 1 H) , 7.51 - 7.61 (m, 1 H)

MS ES+: 409 (M+Na)

[0080]

Step (ii) : Ethyl 2-{ [ (2-chloro-4-fluorophenyl)methyl] sulfanyl}- 5-oxocyclohex-l-ene-l-carboxylate

To a solution of ethyl 8- { [ (2-chloro-4- fluorophenyl) methyl] sulfanyl}-l, 4-dioxaspiro [4.5] dec-7-ene-7- carboxylate (6.97 g, 18.02 mmol) in THF (60 mL) at 0°C was added HC1 (aq) (6M, 60.1 mL, 360 mmol) . The mixture was then allowed to warm to room temperature for 17h. Water was added and the precipitate was collected by filtration and purified by flash chromatography (20-40% EtOAc in petroleum ether on silica) to afford the title compound.

¾ N R (300 MHz, CDC1 ₃ ) 5ppm 1.30 (t, J = 7.12 Hz, 3 H) , 2.45 - 2.56 (m, 2 H), 2.82 -2.93 (m, 2 H) , 3.26 (s, 2 H) , 4.17 (s, 2 H) , 4.23 (q, J = 7.22 Hz, 2 H) , 6.93 - 7.04 (m, 1 H) , 7.10 - 7.19 (m, 1 H) , 7.41 - 7.52 (m, 1 H)

MS ES+: 297 (M-OEt)

[0081]

Step (iii) : Ethyl 6-{ [ (2-chloro-4- fluorophenyl)methyl] sulfanyl} -3-oxocyclohex-l-ene-l-carboxylate

To a solution of ethyl 2- { [ (2-chloro-4- fluorophenyl ) methyl ] sulfanyl } -5-oxocyclohex-l-ene-l-carboxylate

(5.76 g, 16.80 mmol) in dry toluene (96 mL) at 0°C was added DBU (0.253 mL, 1.680 mmol) in a drop wise fashion. The mixture was stirred at 0°C for 2h. The mixture was quenched by the addition of water and extracted with EtOAc. The organics were washed with water and brine, dried over MgSC , filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (0-40% EtOAc in petroleum ether on silica) to afford the title compound.

¾ NMR (300 MHz, CDC1 ₃ ) δρρπι 1.32 (t, J = 7.12 Hz, 3 H), 2.08 - 2.51 (m, 3 H), 2.79 -3.00 (m, 1 H) , 3.94 - 4.07 (m, 3 H) , 4.28

(q, J = 7.13 Hz, 2 H) , 6.61 (s, 1 H) , 6.93 - 7.06 (m, 1 H) , 7.10 - 7.21 (m, 1 H) , 7.36 - 7.49 (m, 1 H)

MS ES+: 341

[0082]

Step (iv) : Ethyl 6- {[ (2-chloro- -fluorophenyl) methyl] sulfanyl} - 3-hydroxy-3-methylcyclohex-1-ene-1-carboxylate

To a solution of ethyl 6- { [ (2-chloro-4- fluorophenyl ) methyl] sulfanyl } -3-oxocyclohex-l-ene-l-carboxylate (4.15 g, 12.11 mmol) in dry THF (40.4 mL) at 0°C under nitrogen was added methylmagnesium bromide (3M in Et ₂ 0, 4.84 mL, 14.53 mmol) in a drop wise fashion. The mixture was stirred at 0°C for 4h. The mixture was quenched with sat. NH ₄ C1 at room

temperature and extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgS0 ₄ and concentrated in vacuo. The residue was purified by flash chromatography (5- 50% EtOAc in hexane on silica) to afford the title compound. ^X H NMR (300 MHz, CDC1 ₃ ) 5ppm 1.23 - 1.33 (m, 7 H) , 1.75 - 1.95 (m, 3 H) , 1.98 - 2.14 (m, 1 H) , 3.69 - 3.82 (m, 1 H) , 3.95 (s, 2 H) , 4.14 - 4.28 (m, 2 H) , 6.67 (s, 1 H) , 6.90 - 7.03 (m, 1 H) , 7.09 - 7.20 (m, 1 H) , 7.39 - 7.49 (m, 1 H)

MS ES+: 381 (M+Na)

[0083]

Step (v) : Ethyl (3S , 6R) -6- [ (2-chloro-4- fluorophenyl)methanesulfonyl] -3-methylcyclohex-l-ene-l- carboxylate

To a solution of ethyl 6- { [ (2-chloro-4- fluorophenyl) methyl] sulfanyl } -3-hydroxy-3-methylcyclohex-l-ene- 1-carboxylate (2.29 g, 6.38 mmol) in dry DCM (21 mL) at 0°C under an atmosphere of nitrogen was added triethylsilane (5.10 mL, 31.9 mmol) in a drop wise fashion followed by TFA (3.44 mL,

44.7 mmol). The mixture was stirred at 0°C for 2 hours and allowed to warm to room temperature for 17 hours. The mixture was quenched by the addition of a saturated aqueous solution of sodium bicarbonate at 0°C and then extracted with EtOAc. The organics were washed with water and brine, dried (MgS0 ₄ ) , filtered and concentrated in vacuo. The resulting residue was purified by reverse phase chromatography with 0.01% formic acid to afford ethyl 6- [ (2-chloro-4-fluorophenyl) methanesulfanyl] -3- methylcyclohex-l-ene-l-carboxylate . To a solution of ethyl 6- [ (2-chloro-4-fluorophenyl) methanesulfanyl] -3-methylcyclohex-l- ene-l-carboxylate (1.72 g, 5.02 mmol) in dry MeCN (20 mL) at 0°C under an atmosphere of nitrogen was added mCPBA (3.01 g, 12.5 mmol) . The mixture was allowed to warm to room temperature and stirred for 1 hour. The mixture was partitioned between EtOAc and a saturated aqueous solution of potassium carbonate. The organics were washed with brine, dried (phase separator) and concentrated in vacuo. The resulting residue was purified by flash chromatography (0-40% EtOAc in petroleum ether on silica) . The residue was purified further by reverse phase chromatography with 0.01% formic acid. The resulting residue was further purified by chiral SFC (IC Daicel CHIRALPAK, 40% IPA) . The residue was recrystallized from EtOAc/pentane to afford the title compound.

¾ NMR (300 MHz, CDC1 ₃ ) 5ppm 1.12 - 1.22 (m, 3 H) , 1.34 (t, J = 7.08 Hz, 3 H) , 1.58 -1.93 (m, 3 H) , 2.29 - 2.56 (m, 2 H) , 4.27 (q, J = 7.18 Hz, 2 H) , 4.36 - 4.43 (m, 1 H) , 4.60 (s, 2 H) , 6.98 - 7.12 (m, 1 H) , 7.17 - 7.25 (m, 2 H) , 7.54 - 7.68 (m, 1

H)

MS ES+: 397 (M+Na)

[0084]

3. Biological efficacy

(a) TLR4 in vitro assay

Compound inhibition at TLR4 was determined by measuring inhibition of lipopolysaccharide (LPS) and mouse interferon gamma (mlFNy) stimulated nitrite production in the mouse macrophage cell line RAW 264.7 (ATCC) . Complete culture media consisted of RPMI-1640 medium supplemented with 10% heat inactivated fetal bovine serum and 10 mM 4- (2- hydroxyethyl) iperazine-l-ethanesulfonic acid (HEPES) and the cell cultures were maintained in a humidified environment with 5% C0 ₂ , at 37°C. For assays, cells were seeded overnight into 384 well plates at 60,000 cells/well, in 30 of assay medium (phenol red free culture media) . On the first day of the assay, Compound 1 (dissolved in DMSO) was diluted in assay medium to a 5x final concentration in 2.5% DMSO, 10 μL of which was

transferred to the cell plate for a 60 minute incubation (37°C) . The stimulants LPS (Sigma) and mlFNy (R&D Systems) were diluted in assay medium (final concentrations of 1.25 and 0.2 ng/mL respectively) and 10 μL added to the cell plate, after which the plate was incubated overnight (37°C; 50 L assay volume) . To measure the nitrite production, 20 L of cell supernatant was transferred to a 384 well plate and 10 μ∑ of a 20 μς/ηχΐ 2, 3-diaminonaphthalene solution added (Sigma; dissolved in 0.2M HC1) . After a 10 minute incubation at room temperature, 10 μL of 0.5M NaOH was added to each well and the fluorescence measured on the SpectraMax i3 plate reader (Molecular Devices; excitation/emission wavelengths: 355/460 nm) . The activity of Compound 1 was determined as ^x % response', for which

fluorescent responses were normalized to the assay window obtained between the response to LPS/mlFNy alone (0%

inhibition) and the response to LPS/mlFNy in the presence of an ECIOO concentration of a standard compound (100% inhibition) .

The mean IC50 for Compound 1 was determined to be 21 nanomolar (nM) .

[0085]

(b) in vivo Pharmacodynamical models

(i) Peripheral LPS challenge model

C57Bl6/j male mice were used and were 2-4 months old at the time of testing. Mice were orally administered with vehicle (an aqueous solution of 1% polyoxyethylenesorbitan monooleate (commercially sold under the trade mark "Tween 80") /0.5% methyl cellulose at a dose volume of 10 mL/kg) or with Compound 1 (at a dosage of 1, 3.0, 5.0, 10.0 or 30.0 mg/kg suspended in the above vehicle and at a dose volume of 10 mL/kg) and returned to their home cages. One hour later mice were dosed with

lipopolysaccharide (LPS) (Sigma; E.coli 0111. B4),

intraperitoneally, (at a dosage of 0.01 mL/kg at a dose volume of 10 mL/kg) suspended in phosphate buffered saline (PBS) .

Animals were sacrificed, 1 hour post LPS dose, by a rising concentration of C0 ₂ followed by a cervical dislocation to confirm death. Blood was immediately removed from the heart using a 25G needle attached to a 1 mL syringe. Blood was transferred to 1.3 mL tubes containing 1.6 mg EDTA (Sarstedt) for centrifugation ( 1500g/10min/4°C) to separate the plasma. The resulting plasma was used for TNF quantification. [0086]

(ii) CNS Intra-Cerebroventricular (icv) LPS Challenge Model

C57B16/j male mice (2.5 month old) were orally

administered with vehicle (an aqueous solution of 1%

polyoxyethylenesorbitan monooleate (commercially sold under the trade mark "Tween 80") /0.5% methyl cellulose at a dose volume of 10 mL/kg) or with Compound 1 (at a dosage of 30.0 mg/kg suspended in the above vehicle and at a dose volume of 10 mL/kg) and returned to their home cages. Fifty minutes later mice were administered with l g lipopolysaccharide (Sigma

L2630; E.coli 0111. B4) or vehicle (PBS, pH 7.4) by unilateral intra-cerebroventricular (icv) infusions (2 μL over 10 minutes) delivered stereotactically to the right lateral ventricle (AP - 0.3, ML -1, DV -2.2mm vs. bregma) under isoflurane anesthesia. Mice were allowed to recover from anesthesia, and were

euthanized by cervical dislocation 4 hours after the LPS infusions. Whole brains were dissected out and 3mm coronal hemi-brain slices were produced using a brain matrix. Brain slices with the LPS infusion site (anterior posterior

coordinates of 0 to -3mm relative to bregma) were collected and flash frozen on dry ice. The frozen section was used for TNF quantification.

[0087],

Brain tissue processing

Frozen sections were homogenized in 200 mL PBS

supplemented with complete protease inhibitors (Roche) ; using the LT Tissue lyser (Qiagen) (20Hz for 2.5 minutes). Protein levels were quantified with a bicinchoninic acid (BCA) assay (Pierce) according to manufacturer's instructions. Samples were normalized to 5.5 mg/mL with PBS and 1% of a 10% N-dodecyl-b-D- maltoside solution was added to each homogenate. Samples were briefly mixed and left on ice for 30 minutes. Samples were then centrifuged at 16000g/20minutes/4°C . Supernatants were removed for TNFa quantification.

[0088] TNFa quantification

Twenty five microliters of plasma or 5 μL of brain homogenate was added to a 96 well TNFa U-Plex Mesoscale

Discovery (MSD, K152UCK) ELISA plate. Manufacturer's protocols were followed.

[0089]

Results

Peripheral LPS challenge model :

Compound 1 reduced plasma LPS induced TNFa production in a dose dependent manner. Compound 1 significantly reduced LPS stimulated TNFa production by 95.2% when dosed at 30 mg/kg (p<0.0001), 83.6% when dosed at 10 mg/kg (p<0.0001), 70.1% when dosed at 5 mg/kg (p<0.001) or 50% when dosed at 3 mg/kg

(p<0.01). A non-significant reduction of LPS stimulated TNFa production was observed with a 1 mg/kg dose of Compound 1.

[0090]

C S Intra-Cerebroventricular (icv) LPS Challenge Model:

Compound 1 significantly reduced LPS stimulated TNFa production by 74.1% when dosed at 30 mg/kg (p<0.0001).

[0091]

Pharmaceutical Preparation Example 1 (Production of capsule)

1) Compound of Example 1 30 mg

2) Finely powdered cellulose 10 mg

3) Lactose 19 mg

4) Magnesium stearate 1 mg

Total 60 mg

1), 2), 3) and 4) are mixed, and packed into a gelatin capsule.

[0092]

Pharmaceutical Preparation Example 2 (Production of tablets)

1) Compound of Example 1 30 g.

2) Lactose 50 g

3) Corn starch 15 g

4) Carboxymethylcellulose calcium 44 g

5) Magnesium stearate 1 g 1000 tablets, total 140 g

The total quantities of 1), 2) and 3) and 30 g of 4) are kneaded with water, and the kneaded mixture is then subjected to vacuum drying and granulation. To said granular powder is added 14 g of 4) and lg of 5) and the mixture is subjected to tableting using a tableting machine. In this way, 1000 tablets containing 30 mg of compound of Example 1 per tablet are obtained.

Industrial Applicability

[0093]

The compound of formula (I) possesses an advantageous combination of TLR4 antagonist properties and brain

permeability, and therefore, the compound would be expected to be useful in the treatment of central nervous system diseases mediated at least in part by TLR .

[0094]

This application is based on patent application No.

GB1612652.6 filed on July 21, 2016 in United Kingdom, the contents of which are encompassed in full herein.