Antimycotic Triazole Compound

  • Published: Jun 8, 2017
  • Earliest Priority: Dec 04 2015
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ANTIMYCOTIC TRIAZOLE COMPOUND

Field of the invention This invention relates to a compound useful in the treatment of mycoses, compositions containing it and its use in therapy.

Background of the invention The incidence of fungal infections has increased substantially over the past two decades and invasive forms are leading causes of morbidity and mortality, especially amongst immunocompromised or immunosuppressed patients. Disseminated candidiasis, pulmonary aspergillosis, and emerging opportunistic fungi are the most common agents producing these serious mycoses. It is a particular feature of fungi that they are able to generate an extracellular matrix (ECM) that binds them together and allows them to adhere to their in vitro or in vivo substrates. These biofilms serve to protect them against the hostile environments of the host immune system and to resist antimicrobial killing (Kaur and Singh, 2013).

Pulmonary aspergillosis can be segmented into those patients suffering with non-invasive disease versus those with an invasive condition. A further sub-division is used to characterise patients who exhibit an allergic component to aspergillosis (a condition known as ABPA; allergic bronchopulmonary aspergillosis) compared with those that do not. The factors precipitating pulmonary aspergillosis may be acute, such as exposure to high doses of immuno-suppressive medicines or to intubation in an intensive care unit. Alternatively, they may be chronic, such as a previous infection with tuberculosis (Denning et al., 201 1 a). Chronic lung infections with aspergillus can leave patients with extensive and permanent lung damage, requiring lifetime treatment with oral azole drugs (Limper et al., 201 1 ).

A growing body of research suggests that aspergillus infection may play an important role in clinical asthma (Chishimba et al., 2012; Pasqualotto et al., 2009). Furthermore, recently published work has correlated aspergillus infection with poorer clinical outcomes in patients with chronic obstructive pulmonary disease (COPD) (Bafadhel et al., 2013). Similarly cross- sectional studies have shown associations between the presence of Aspergillus spp. and Candida spp. in the sputum and worsened lung function (Chotirmall et al., 2010; Agbetile et al., 2012).

Invasive aspergillosis (IA) exhibits high mortality rates in immunocompromised patients, for example, those undergoing allogenic stem cell transplantation or solid organ transplants (such as lung transplants). The first case of IA reported in an immunocompromised patient occurred in 1953. This event was concurrent with the introduction of corticosteroids and cytotoxic chemotherapy into treatment regimens (Rankin, 1953). IA is a major concern in the treatment of leukaemia and other haematological malignancies given its high incidence and associated mortality. Death rates usually exceed 50% (Lin et al., 2001 ) and long term rates can reach 90% in allogeneic hematopoietic stem cell transplantation recipients, despite the availiability of oral triazole medicines (Salmeron et al., 2012). In patients undergoing solid organ transplantation (particularly of the lung), the use of high doses of steroids leaves patients vulnerable to infection (Thompson and Patterson, 2008) which is a serious problem. The disease has also appeared in less severely immunocompromised patient populations. These include those suffering with underlying COPD or cirrhosis, patients receiving high dose steroids, and individuals fitted with central venous catheters or supported by mechanical ventilation (Dimopoulos et al., 2012).

Existing anti-fungal medicines are predominantly dosed either orally or systemically. These commonly exploited routes of delivery are poor for treating lung airways infections, since drug concentrations achieved at the site of infection tend to be lower than those in unaffected organs. This is especially so for the liver, which is a site of toxicity: up to 15% of patients treated with voriconazole suffer raised transaminase levels (Levin et al., 2007; Lat and Thompson, 201 1 ). Exposure of the liver also results in significant drug interactions arising from the the inhibition of hepatic P450 enzymes (Jeong, et al., 2009; Wexler et ai, 2004).

Furthermore, the widespread use of triazoles, both in the clinic and in agriculture has led to a growing and problematic emergence of resistant mycoses in some locations (Denning et al., 201 1 b; Bowyer and Denning, 2014).

It is clearly evident that an urgent medical need exists for novel anti-fungal medicines that deliver improved efficacy and better systemic tolerability profiles.

Summary of the Invention

In a first aspect, the invention provides Compound (I),

Compound (I) which is: 4-(4-(4-(((3R,5R)-5-((1 H-1 ,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydro furan-3-yl)methoxy)phenyl)piperazin-1 -yl)-/V-(4-fluorophenyl)benzamide,

and pharmaceutically acceptable salts thereof (hereinafter sometimes referred to as the "compound of the invention").

Biological data disclosed herein below reveals that the compound of the invention, Compound (I), is a potent inhibitor of Aspergillus fumigatus growth in vitro assays. In immunosuppressed mice Compound (I) demonstrated potent inhibition of Aspergillus fumigatus infections. Brief description of the figures

Figure 1A shows the effects of Compound (I) on galactomannan levels in the serum of Aspergillus fumigatus infected, immunocompromised, neutropenic mice.

Figure 1 B shows the effects of Compound (I) on fungal load in lung homogenates from Aspergillus fumigatus infected, immunocompromised, neutropenic mice.

Figures 2A-2C: show the effects of Compound (I) on neutrophil count (Figure 2A), on IL-17 concentration (Figure 2B) and on IFNy concentrations (Figure 2C) in bronchoalveolar lavage fluid of Aspergillus fumigatus infected, immunocompromised, neutropenic mice.

Detailed description of the invention

Alkyl as used herein refers to straight chain or branched chain alkyl, such as, without limitation, methyl, ethyl, n-propyl, /so-propyl, butyl, n-butyl and ie f-butyl. In one embodiment alkyl refers to straight chain alkyl.

The compound of the invention may be prepared from commercially available starting materials by the synthetic methodology (Route 1 ) depicted below (Scheme 1 ).

Scheme 1 : Route 1

The chemoselective /V-protection of 4-(piperazin-1 -yl)phenol, for example as a urethane, provides a phenol intermediate 1 '. A suitable, orthogonal protective group which may be employed for this purpose is a ie f-butyl carbamate (P = Boc). Reaction of the phenol with an electrophilic derivative of ((3R,5R)-5-((1 /-/-1 ,2,4-triazol-1 -yl)methyl)-5-(2,4-difluorophenyl) tetrahydrofuran-3-yl)methanol (2' X = OH) under basic conditions generates the ether 3'. An example of such a compound is the corresponding tosylate (2 X = OTs) which is readily available, in high enantiomeric purity, from commercial sources. Selective removal of the nitrogen protective group reveals the mono-substituted piperazine 4. Deprotection of a Boc derivative (R= Boc), for example, is typically undertaken by exposure to strong mineral acid or a strong organic acid, such as TFA.

Buchwald coupling of the amine 4 with an alkyl 4-bromobenzoate under basic conditions and the agency of a catalyst gives rise to the /V-arylated product 5' in which R1 represents alkyl such as Ci-5 alkyl e.g. methyl or ethyl. Those skilled in the art will appreciate that a wide variety of conditions may be used for affecting transformations of this kind. In particular, palladium catalysts and phosphine ligands such as RuPhosG3 and RuPhos are routinely employed in the presence of a base, for example, cesium carbonate or lithium hexamethyldisilazide. Saponification of the ester 5' is conveniently undertaken by treatment with a base, such an alkali metal hydroxide, in a mixture of water and a suitable miscible solvent. Reaction of the acid product 6, with 4-fluoroaniline under standard amide coupling conditions, widely available in the art, provides the compound of the invention, Compound (I).

The compound of the invention may be conveniently prepared, as depicted below (Scheme 2), using an alternative route (Route 2) which removes the need for protection (and subsequent deprotection) of the piperazine motif during the synthetic sequence and provides the final drug substance in three steps.

Scheme 2: Route 2

Acylation of 4-fluoroaniline with a suitable derivative of 4-bromobenzoic acid, such as 4- bromobenzoyl chloride (Y = CI), provides the known benzamide 7. It will be evident to those skilled in the art that this conversion can be undertaken on the corresponding acid (Y = OH) using standard amide coupling procedures. Subjecting this intermediate to a Buchwald coupling reaction with 4-(piperazin-1 -yl)phenol, under conditions selected from those routinely used for such transformations, results in the formation of the Λ/,Λ/'-bisaryl piperazine 8. Etherification of this phenol by reaction with an electrophilic alcohol derivative 2', such as the tosylate 2, (ex APIChem, Catalogue Number: AC-8330) as described above, generates the compound of the invention Compound (I) in an expedient manner.

Protective groups and the means for their removal are described in "Protective Groups in Organic Synthesis", by Theodora W. Greene and Peter G. M. Wuts, published by John Wiley & Sons Inc; 4th Rev Ed., 2006, ISBN-10: 0471697540. A review of methodologies for the preparation of amides is covered in: 'Amide bond formation and peptide coupling' Montalbetti, C.A.G.N. and Falque, V. Tetrahedron, 2005, 61 , 10827-10852.

Pharmaceutically acceptable salts of compounds of formula (I) include in particular pharmaceutically acceptable acid addition salts of said compounds. The pharmaceutically acceptable acid addition salts of compounds of formula (I) are meant to comprise the therapeutically active non-toxic acid addition salts that the compounds of formula (I) are able to form. These pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the free base form with such appropriate acids in a suitable solvent or mixture of solvents. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric acids and the like; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p- toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic acid and the like.

Conversely said salt forms can be converted by treatment with an appropriate base into the free base form.

The definition of the compound of formula (I) is intended to include all tautomers of said compound. The definition of the compound of formula (I) is intended to include all solvates of said compound (including solvates of salts of said compound) unless the context specifically indicates otherwise. Examples of solvates include hydrates.

The compound of the disclosure includes embodiments wherein one or more atoms specified are naturally occurring or non-naturally occurring isotopes. In one embodiment the isotope is a stable isotope. Thus the compounds of the disclosure include, for example deuterium containing compounds and the like.

The disclosure also extends to all polymorphic forms of the compound herein defined. Novel intermediates as described herein such as compounds of formulae (5'), (6) and (8), and salts thereof, form a further aspect of the invention. In an embodiment there is provided a pharmaceutical composition comprising the compound of the invention optionally in combination with one or more pharmaceutically acceptable diluents or carriers.

Suitably the compound of the invention is administered topically to the lung or nose, particularly, topically to the lung. Thus, in an embodiment there is provided a pharmaceutical composition comprising the compound of the invention optionally in combination with one or more topically acceptable diluents or carriers.

Suitably compositions for pulmonary or intranasal administration include powders, liquid solutions, liquid suspensions, nasal drops comprising solutions or suspensions or pressurised or non-pressurised aerosols.

The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA., (1985). The compositions may also conveniently be administered in multiple unit dosage form.

Topical administration to the nose or lung may be achieved by use of a non-pressurised formulation such as an aqueous solution or suspension. Such formulations may be administered by means of a nebuliser e.g. one that can be hand-held and portable orfor home or hospital use (i.e. non-portable). An example device is a RESPIMAT inhaler. The formulation may comprise excipients such as water, buffers, tonicity adjusting agents, pH adjusting agents, viscosity modifiers, surfactants and co-solvents (such as ethanol). Suspension liquid and aerosol formulations (whether pressurised or unpressurised) will typically contain the compound of the invention in finely divided form, for example with a D5o of 0.5-10 μηη e.g. around 1 -5 μηι. Particle size distributions may be represented using Dio, D5o and D90 values. The D5o median value of particle size distributions is defined as the particle size in microns that divides the distribution in half. The measurement derived from laser diffraction is more accurately described as a volume distribution, and consequently the D5o value obtained using this procedure is more meaningfully referred to as a Dv5o value (median for a volume distribution). As used herein Dv values refer to particle size distributions measured using laser diffraction. Similarly, D10 and D90 values, used in the context of laser diffraction, are taken to mean Dvio and Dvgo values and refer to the particle size whereby 10% of the distribution lies below the D10 value, and 90% of the distribution lies below the D90 value, respectively.

According to one specific aspect of the invention there is provided a pharmaceutical composition comprising the compound of the invention in particulate form suspended in an aqueous medium. The aqueous medium typically comprises water and one or more excipients selected from buffers, tonicity adjusting agents, pH adjusting agents, viscosity modifiers and surfactants.

Topical administration to the nose or lung may also be achieved by use of an aerosol formulation. Aerosol formulations typically comprise the active ingredient suspended or dissolved in a suitable aerosol propellant, such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFC propellants include trichloromonofluoromethane (propellant 1 1 ), dichlorotetrafluoromethane (propellant 1 14), and dichlorodifluoromethane (propellant 12). Suitable HFC propellants include tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227). The propellant typically comprises 40%-99.5% e.g. 40%-90% by weight of the total inhalation composition. The formulation may comprise excipients including co-solvents (e.g. ethanol) and surfactants (e.g. lecithin, sorbitan trioleate and the like). Other possible excipients include polyethylene glycol, polyvinylpyrrolidone, glycerine and the like. Aerosol formulations are packaged in canisters and a suitable dose is delivered by means of a metering valve (e.g. as supplied by Bespak, Valois or 3M or alternatively by Aptar, Coster or Vari).

Topical administration to the lung may also be achieved by use of a dry-powder formulation. A dry powder formulation will contain the compound of the disclosure in finely divided form, typically with an MMD of 1 -10 μηη or a D5o of 0.5-10 μηη e.g. around 1 -5 μηη. Powders of the compound of the invention in finely divided form may be prepared by a micronization process or similar size reduction process. Micronization may be performed using a jet mill such as those manufactured by Hosokawa Alpine. The resultant particle size distribution may be measured using laser diffraction (e.g. with a Malvern Mastersizer 2000S instrument). The formulation will typically contain a topically acceptable diluent such as lactose, glucose or mannitol (preferably lactose), usually of comparatively large particle size e.g. an MMD of 50 μηη or more, e.g. 100 μηη or more or a D5o of 40-150 μηη. As used herein, the term "lactose" refers to a lactose-containing component, including olactose monohydrate, β-lactose monohydrate, a-lactose anhydrous, β-lactose anhydrous and amorphous lactose. Lactose components may be processed by micronization, sieving, milling, compression, agglomeration or spray drying. Commercially available forms of lactose in various forms are also encompassed, for example Lactohale® (inhalation grade lactose; DFE Pharma), lnhal_ac®70 (sieved lactose for dry powder inhaler; Meggle), Pharmatose® (DFE Pharma) and Respitose® (sieved inhalation grade lactose; DFE Pharma) products. In one embodiment, the lactose component is selected from the group consisting of a-lactose monohydrate, a-lactose anhydrous and amorphous lactose. Preferably, the lactose is α-lactose monohydrate.

Dry powder formulations may also contain other excipients such as sodium stearate, calcium stearate or magnesium stearate.

A dry powder formulation is typically delivered using a dry powder inhaler (DPI) device. Example dry powder delivery systems include SPINHALER, DISKHALER, TURBOHALER, DISKUS, SKYEHALER, ACCUHALER and CLICKHALER. Further examples of dry powder delivery systems include ECLIPSE, NEXT, ROTAHALER, HANDIHALER, AEROLISER, CYCLOHALER, BREEZHALER/NEOHALER, MONODOSE, FLOWCAPS, TWINCAPS, X- CAPS, TURBOSPIN, ELPENHALER, MIATHALER, TWISTHALER, NOVOLIZER, PRESSAIR, ELLIPTA, ORIEL dry powder inhaler, MICRODOSE, PULVINAL, EASYHALER, ULTRAHALER, TAIFUN, PULMOJET, OMNIHALER, GYROHALER, TAPER, CONIX, XCELOVAIR and PROHALER.

The compound of the invention is useful in the treatment of mycoses and for the prevention or treatment of disease associated with mycoses. In an aspect of the invention there is provided use of the compound of the invention in the manufacture of a medicament for the treatment of mycoses and for the prevention or treatment of disease associated with mycoses.

In another aspect of the invention there is provided a method of treatment of a subject with a mycosis which comprises administering to said subject an effective amount of the compound of the invention.

In another aspect of the invention there is provided a method of prevention or treatment of disease associated with a mycosis in a subject which comprises administering to said subject an effective amount of the compound of the invention.

Mycoses may, in particular, be caused by Aspergillus spp. such as Aspergillus fumigates.

A disease associated with a mycosis is, for example, pulmonary aspergillosis.

The compound of the invention may be used in a prophylactic setting by administering the said compound prior to onset of the mycosis.

In another aspect of the invention there is provided a method of treatment of a subject with an aspergilloma which comprises administering to said subject an effective amount of the compound of the invention. There is also provided a method of preventing recurrence of an aspergilloma in a subject which comprises administering to said subject an effective amount of the compound of the invention. There is also provided the compound of the invention for use in the treatment of a subject with an aspergilloma or for preventing recurrence of an aspergilloma in a subject.

Subjects include human and animal subjects, especially human subjects.

The compound of the invention is especially useful for the treatment of mycoses such as Aspergillus fumigatus infection and for the prevention or treatment of disease associated with mycoses such as Aspergillus fumigatus infection in at risk subjects. At risk subjects include premature infants, children with congenital defects of the lung or heart, immunocompromised subjects (e.g. those suffering from HIV infection), asthmatics, subjects with cystic fibrosis, elderly subjects and subjects suffering from a chronic health condition affecting the heart or lung (e.g. congestive heart failure or chronic obstructive pulmonary disease).

The compound of the invention may be administered in combination with a second or further active ingredient. Second or further active ingredients may, for example, be selected from other anti-fungal agents (such as voriconazole or posaconazole), amphotericin B, an echnocandin (such as caspofungin) and an inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (such as lovastatin, pravastatin or fluvastatin). Second or further active ingredients include active ingredients suitable for the treatment or prevention of a mycosis such as Aspergillus fumigatus infection or disease associated with a mycosis such as Aspergillus fumigatus infection or conditions co-morbid with a mycosis such as Aspergillus fumigatus infection. The compound of the invention may be co-formulated with a second or further active ingredient or the second or further active ingredient may be formulated to be administered separately by the same or a different route.

For example, the compound of the invention may be administered to patients already being treated systemically with an anti-fungal, such as voriconazole or posaconazole.

For example, the compound of the invention may be co-formulated with one or more agents selected from amphotericin B, an echnocandin, such as caspofungin, and an inhibitor of 3- hydroxy-3-methyl-glutaryl-CoA reductase, such as lovastatin, pravastatin or fluvastatin.

According to an aspect of the invention there is provided a kit of parts comprising (a) a pharmaceutical composition comprising the compound of the invention optionally in combination with one or more diluents or carriers; (b) a pharmaceutical composition comprising a second active ingredient optionally in combination with one or more diluents or carriers; (c) optionally one or more further pharmaceutical compositions each comprising a third or further active ingredient optionally in combination with one or more diluents or carriers; and (d) instructions for the administration of the pharmaceutical compositions to a subject in need thereof. The subject in need thereof may suffer from or be susceptible to a mycosis such as Aspergillus fumigatus infection.

The compound of the invention may be administered at a suitable interval, for example once per day, twice per day, three times per day or four times per day.

A suitable dose amount for a human of average weight (50-70 kg) is expected to be around 50 μg to 10 mg/day e.g. 500 μg to 5 mg/day although the precise dose to be administered may be determined by a skilled person.

The compound of the invention is expected to have one or more of the following favourable attributes: potent antifungal activity, particularly activity against Aspergillus spp. such as Aspergillus fumigates, especially following topical administration to the lung or nose;

long duration of action in lungs, preferably consistent with once daily dosing;

low systemic exposure following topical administration to the lung or nose; and

acceptable safety profile, especially following topical administration to the lung or nose.

EXPERIMENTAL SECTION

Abbreviations used herein are defined below (Table 1 ). Any abbreviations not defined are intended to convey their generally accepted meaning.

Table 1 : Abbreviations

ABPA allergic bronchopulmonary aspergillosis

aq aqueous

ATCC American Type Culture Collection

BALF bronchoalveolar lavage fluid

BEAS2B SV40-immortalised human bronchial epithelial cell line

Boc ie f-butyloxycarbonyl

B0C2O di-ie f-butyl dicarbonate

br broad

BSA bovine serum albumin

CC50 50% cell cytotoxicity concentration

COI cut off index

cone concentration

COPD chronic obstructive pulmonary disease

d doublet

DCM dichloromethane

DIPEA Λ/,/V-diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMEM Dulbecco's Modified Eagle Medium

DMF /V,/V-dimethylformamide

DMSO dimethyl sulfoxide

DSS dextran sodium sulphate

ECM extracellular matrix

EDCI 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide

EUCAST European Committee on Antimicrobial Susceptibility Testing

(ES+) electrospray ionization, positive mode

Et ethyl

ΕίβΝ triethylamine

EtOAc ethyl acetate

FBS foetal bovine serum GM galactomannan

HATI 1 1 -[bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3- oxid hexafluorophosphate

hr hour(s)

IA invasive aspergillosis

ip intraperitoneal

i.t. intra-tracheal

LC-MS/MS liquid chromatography-mass spectrometry

Li Hep lithium heparin

□HMDS lithium bis(trimethylsilyl)amide

(M+H)+ protonated molecular ion

MDA malondialdehyde

Me methyl

MeCN acetonitrile

MeOH methanol

MHz megahertz

MICso 50% of minimum inhibitory concentration

MIC75 75% of minimum inhibitory concentration

MIC90 90% of minimum inhibitory concentration

min minute(s)

MMD mass median diameter

MOI multiplicity of infection

MOPS 3-(/V-morpholino)propanesulfonic acid

m/z: mass-to-charge ratio

NCPF National Collection of Pathogenic Fungi

NMR nuclear magnetic resonance (spectroscopy)

NT not tested

OD optical density

PBS phosphate buffered saline

P protective group

q quartet

RT room temperature

RP HPLC reverse phase high performance liquid chromatography

RPMI Roswell Park Memorial Institute medium

RuPhos 2-dicyclohexylphosphino-2', 6'-diisopropoxybiphenyl

6'-diisopropoxybiphenyl)[2-(2'-amino-1 , hanesulfonate

s singlet

sat saturated

sc sub-cutaneous

SDS sodium dodecyl sulphate

t triplet TFA trifluoroacetic acid

THF tetrahydrofuran

General Procedures

All starting materials and solvents were obtained either from commercial sources or prepared according to the literature citation. Unless otherwise stated all reactions were stirred. Organic solutions were routinely dried over anhydrous magnesium sulfate.

Analytical Methods Reverse Phase HPLC: Waters Xselect CSH C18 XP column, 2.5 μηι (4.6 x 30 mm) at 40°C; flow rate 2.5-4.5 mL min"1 eluted with a H20-MeCN gradient containing 0.1 % v/v formic acid over 4 min employing UV detection at 254 nm. Gradient information: 0-3.00 min, ramped from 95% H20-5% MeCN to 5% H20-95% MeCN; 3.00-3.01 min, held at 5% H20-95% MeCN, flow rate increased to 4.5 mL min"1; 3.01 3.50 min, held at 5% H20-95% MeCN; 3.50-3.60 min, returned to 95% H20-5% MeCN, flow rate reduced to 3.50 mL min"1; 3.60-3.90 min, held at 95% H20-5% MeCN; 3.90-4.00 min, held at 95% H20-5% MeCN, flow rate reduced to 2.5 mL min"1.

1H NMR Spectroscopy: 1H NMR spectra were acquired on a Bruker Advance III spectrometer at 400 MHz using residual undeuterated solvent as reference and unless specified otherwise

Preparation of Compound (I) by Route 1 ferf-Butyl 4-(4-hydroxyphenyl)piperazine-1 -carboxylate.

1

To a suspension of 4-(piperazin-1 -yl)phenol (40.0 g, 224 mmol) in a mixture of THF (350 mL) and triethylamine (46.8 mL, 337 mmol) was added dropwise a solution of di-ie f-butyl dicarbonate (52.1 mL, 224 mmol) in THF (50 mL) over 25 min. The reaction mixture, which changed from a pink suspension to a dark orange solution following the addition, was then stirred at RT for 18 hr. Ethyl acetate (400 mL) was added and the organic layer was separated and was washed with sat. aq. NaHCC (200 mL) and with water (200 mL), and then dried. The volatiles were removed in vacuo to give an off-white solid. The crude product was triturated with hexane (1000 mL), collected by filtration, rinsed with hexane (500 mL) and then dried in vacuo to afford the title compound, intermediate 1 as a white solid (58.1 g, 93%); R' 1.39 min; m/z 279 (M+H)+ (ES+); 1H NMR δ: 1 .42 (9H, s), 2.87-2.89 (4H, m), 3.41 -3.44 (4H, m), 6.65 (2H, d), 6.80 (2H, d) and 8.87 (1 H, s). 1 -(4-(((3 ?,5 ?)-5-((1 H-1 ,2,4-triazoM -yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3- yl)methoxy)phenyl)piperazine.

To a solution of intermediate 1 (25.0 g, 89.0 mmol) in DMSO (550 mL) was added aq sodium hydroxide (31.8 mL, 3.5 M, 1 1 1 mmol). The mixture was stirred at RT for 30 min and was then treated portionwise with ((3S,5R)-5-((1 /-/-1 ,2,4-triazol-1 -yl)methyl-5-(2,4-difluorophenyl) tetrahydrofuran-3-yl)methyl4-methylbenzenesulfonate 2 (ex APIChem, Catalogue Number: AC-8330, 40.0 g, 89 mmol). The black reaction mixture was stirred at 40°C for 18 hr, cooled to RT and water (600 mL) and EtOAc (400 mL) were added. After 10 min the phases were separated and the aq layer was extracted with EtOAc (3 x 400 mL). The combined organic extracts were washed with brine (400 mL), dried and the solvent was removed in vacuo to afford a brown oil. Analysis of the crude, Boc-protected product 3 by 1H NMR showed that it contained -10% of the undesired alkene elimination product: (R)-1 -((2-(2,4-difluorophenyl)-4- methylenetetrahydrofuran-2-yl)methyl)-1 H-1 ,2,4-triazole.

The crude urethane 3 was taken up into DCM (350 mL) and treated with TFA (103 mL, 1334 mmol). After 2 hr at RT the reaction mixture was concentrated in vacuo to remove most of the TFA and then diluted with fresh DCM (350 mL). The solution was treated carefully with aq. NaOH (6 M, 100 mL) and the aq phase (pH -12) was separated and extracted with DCM (2 x 400 mL).The combined organic extracts were washed with 2M NaOH solution (400 mL), and with brine (400 mL), and then dried and evaporated in vacuo to afford a light brown solid (38.9 g). The crude product was recrystallised from THF (100 mL), collected by filtration, washed with diethyl ether (2 x 50 mL) and then dried in vacuo to afford the title compound, intermediate 4, as a white solid (21 .6 g, 53%); R' 1 .37 min; m/z 456 (M+H)+ (ES+); 1H NMR δ: 2.13 (2H, dd), 2.36-2.42 (1 H, m), 2.52-2.55 (1 H, m), 2.79-2.81 (4H, m), 2.88-2.90 (4H, m), 3.66 (1 H, dd), 3.71 -3.76 (2H, ddd), 4.02 (1 H, dd), 4.57 (2H, dd), 6.75 (2H, d), 6.83 (2H, d), 6.98 (1 H, td), 7.24-7.32 (2H, m), 7.77 (1 H, s) and 8.34 (1 H, s). The filtrate was concentrated and the residue recrystallised from THF (50 mL) to give a second crop as a white solid (8.85 g, 22%) Methyl 4-(4-(4-(((3/?,5/?)-5-((1 H-1 ,2,4-triazoM -yl)methyl)-5-(2,4-difluorophenyl)tetra hydrofuran-3-yl)methoxyphenyl)piperazin-1 -yl)benzoate.

A flask charged with intermediate 4 (21.6 g, 47.4 mmol), methyl-4-bromobenzoate (12.2 g, 56.9 mmol), RuPhos (0.44 g, 0.95 mmol, 2 mol%), RuPhosG3 (0.79 g, 0.95 mmol, 2 mol%) and cesium carbonate (24.7 g, 76.0 mmol) was evacuated and refilled with nitrogen three times before DMF (150 mL) was added. The mixture was heated at 80°C for 18 hr and then, whilst still hot, was poured into water (600 mL). The mixture was stirred for 20 min to give an orange suspension. The solid was collected by filtration, washed with water (100 mL) and with diethyl ether (3 x 100 mL) and then re-suspended in THF (250 mL). The mixture was heated at reflux for 15 min to give a white suspension which was allowed to cool to RT and treated with diethyl ether (300 mL). The resulting precipitate was collected by filtration, and the cake washed with diethyl ether (3 x 50 mL) and then dried in vacuo to afford the title compound intermediate 5 as an off-white solid (25.6 g, 91 %); R' 2.52 min; m/z 590 (M+H)+ (ES+); 1H NMR δ: 2.13 (1 H, dd), 2.36-2.42 (1 H, m), 2.51 -2.56 (1 H, m), 3.12-3.15 (4H, m), 3.44-3.47 (4H, m), 3.66 (1 H, dd), 3.72-3.75 (2H, m), 3.78 (3H, s), 4.02 (1 H, dd), 4.58 (2H, dd), 6.80 (2H, d), 6.93 (2H, d), 6.99 (1 H, td), 7.04 (2H, d), 7.26-7.32 (2H, m), 7.78 (1 H, s), 7.81 (2H, d) and 8.35 (1 H, s).

4-(4-(4-(((3/?,5/?)-5-((1 H-1 ,2,4-triazoM -yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran- 3-yl)methoxyphenyl)piperazin-1 -yl)benzoic acid.

To a solution of lithium hydroxide (4.72 g, 197 mmol) in water (100 mL) was added a suspension of intermediate 5 (24.0 g, 39.4 mmol) in DMSO (1000 mL). The mixture was heated at 80°C for 22 hr and was then cooled to RT, diluted with water (800 mL) and acidified (to ~ pH 3) by the addtion of 1 M aq HCI (250 mL). The mixture was stirred for 30 min and the resulting precipitate was collected by filtration. The filter cake was washed with water (3 x 150 mL) and with diethyl ether (3 x 50 mL). The solid was triturated with THF (300 mL) and was collected by filtration, washed with diethyl ether (3 x 50 mL) and dried in vacuo at 50°C to give the title compound, intermediate 6 as a white solid (20.7 g, 90%); R' 2.18 min; m/z 576 (M+H)+ (ES+); 1H NMR δ: 2.13 (1 H, dd), 2.37-2.42 (1 H, m), 2.53-2.54 (1 H, m), 3.12-3.15 (4H, m), 3.42- 3.45 (4H, m), 3.66 (1 H, dd), 3.72-3.77 (2H, m), 4.02 (1 H, dd), 4.58 (2H, dd), 6.80 (2H, d), 6.93 (2H, d), 6.95-7.04 (3H, m), 7.26-7.32 (2H, m), 7.78 (1 H, s), 7.79 (2H, d), 8.35 (1 H, s) and 12.33 (1 H, s). 4-(4-(4-(((3 ?,5 ?)-5-((1 H-1 ,2,4-triazoM -yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran- 3-yl)methoxy)phenyl)piperazin-1 -yl)-yV-(4-fluorophenyl)benzamide.

Compound (I) To a suspension of intermediate 6 (40.0 mg, 0.07 mmol), HATU (39.6 mg, 0.10 mmol) and DIPEA (49.0 μΙ_, 0.28 mmol) in DMF (1.2 mL) was added 4-fluoroaniline (10.0 μΙ_, 0.10 mmol). The mixture was heated at 80°C for 2 hr and was then cooled to RT and diluted with water (4.0 mL). The resulting precipitate was collected by filtration and the crude product so obtained was purified by flash column chromatography (S1O2, 4 g, 0-2% MeOH in DCM, gradient elution) to afford the title compound, Compound (I) as a white solid (26.0 mg, 54%); R' 2.51 min; m/z 669 (M+H)+ (ES+); 667 (M-H)" (ES"); 1H NMR δ: 2.14 (1 H, dd), 2.37-2.43 (1 H, m), 2.51 -2.57 (1 H, m), 3.15-3.17 (4H, m), 3.43-3.45 (4H, m), 3.67 (1 H, dd), 3.72-3.78 (2H, m), 4.02 (1 H, dd), 4.58 (2H, dd), 6.81 (2H, d), 6.95 (2H, d), 6.99 (1 H, td), 7.08 (2H, d), 7.16 (2H, t), 7.25-7.33 (2H, m), 7.76-7.80 (3H, m), 7.89 (2H, d), 8.34 (1 H, s) and 10.00 (1 H, s).

Preparation of Compound (I) by Route 2 4-Bromo-/V-(4-fluorophenyl)benzamide.

To a solution of 4-fluoroaniline (0.85 mL, 9.00 mmol), triethylamine (1 .88 mL, 13.5 mmol) and DMAP (0.1 1 g, 0.90 mmol) in THF (15 mL) was added 4-bromobenzoyl chloride (2.37 g, 10.8 mmol). The reaction mixture was maintained at RT for 1 hr and was then partitioned between EtOAc (100 mL) and 1 M aq HCI (100 mL). The phases were separated and the organic phase was washed sequentially with 1 M aq HCI (100 mL), sat. aq. NaHCC (100 mL) and brine (100 mL) and then dried and evaporated in vacuo. The crude residue was triturated from warm DCM (100 mL) and the mixture was heated at reflux to give a white suspension which was allowed to cool to RT. The resulting precipitate was collected by filtration to afford the title compound, intermediate 7 as white solid (1 .81 g, 65%); R' 2.23 min; m/z 294/296 (M+H)+ (ES+); 1H NMR δ: 7.20 (2H, t), 7.74-7.79 (4H, m), 7.90 (2H, d) and 10.36 (1 H, s). yV-(4-fluorophenyl)-4-(4-(4-hydroxyphenyl)piperazin-1 -yl)benzamide.

A flask charged with 4-(piperazin-1 -yl)phenol (0.51 g, 2.86 mmol), intermediate 7 (1.01 g, 3.43 mmol), RuPhos (10 mg, 0.03 mmol) and RuPhosG3 (20 mg, 0.03 mmol) was evacuated and backfilled with nitrogen three times. A solution of LiHMDS (1 M in THF, 10.0 mL, 10.0 mmol) was added and the reaction mixture was heated at 70°C for 3 hr. After cooling to RT the mixture was diluted with EtOAc (20 mL) and 1 M aq. HCI (20 mL). The residue was triturated in the mixture and the resulting solid was collected by filtration, washed with EtOAc (10 mL) and dried in vacuo to give the title compound, intermediate 8, as a tan solid: (1 .02 g, 87%); R' 1 .63 min; m/z 392 (M+H)+ (ES+); 390 (M-H)" (ES"); 1H NMR δ: 3.03-3.05 (4H, m), 3.39-3.42 (4H, m), 6.54 (2H, d), 6.75 (2H, d), 7.06 (2H, d), 7.16 (2H, t), 7.78 (2H, dd) and 7.89 (2H, d).

4-(4-(4-(((3K, 5 ?)-5-((1 H-1 ,2,4-triazoM -yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran -3-yl)methoxy)phenyl)piperazin-1 -yl)-yV-(4-fluorophenyl)benzamide.

Compound (I)

To a solution of intermediate 8 (1 .09 g, 2.79 mmol) in DMSO (10.0 mL) at 30°C was added aq. NaOH (0.34 mL, 12.5 M, 4.19 mmol). After 40 min a solution of intermediate 2 (1.38 g, 3.07 mmol) in DMSO (5.0 mL) was added and the reaction mixture was stirred at 30°C for 18 hr, then cooled to RT and poured into water (30 mL). The resulting precipitate was collected by filtration, and was washed sequentially with water (20 mL), aq. 2M NaOH (20 mL), water (10 mL) and /-propanol (20 mL). The crude solid was triturated with MeCN (10 mL), collected by filtration and dried in vacuo to give the title compound, Compound (I) as an off-white solid (1 .05 g, 56%); R' 2.50 min; m/z 669 (M+H)+ (ES+); 667 (M-H)" (ES"); 1H NMR δ: 2.14 (1 H, dd), 2.37-2.43 (1 H, m), 2.51 -2.57 (1 H, m), 3.15-3.17 (4H, m), 3.42-3.45 (4H, m), 3.67 (1 H, dd), 3.72-3.78 (2H, m), 4.02 (1 H, dd), 4.58 (2H, dd), 6.81 (2H, d), 6.94 (2H, d), 6.99 (1 H, td), 7.08 (2H, d), 7.16 (2H, t), 7.25-7.33 (2H, m), 7.76-7.80 (3H, m), 7.89 (2H, d), 8.34 (1 H, s) and 10.00 (1 H, s). Biological Testing: Experimental Methods

Assessment of planktonic fungus growth

a. Resazurin-microtitre assay

This assay was conducted using a modified, published method (Monteiro, etjal., 2012). Spores of Aspergillus fumigatus (NCPF2010, Public Health England, Wiltshire) were cultured in Sabouraud dextrose agar for 3 days. A stock spore suspension was prepared from a Sabouraud dextrose agar culture by washing with PBS-tween (10 ml_; phosphate buffered saline containing 0.05% Tween-20, 100 U/mL Penicillin and 100 U/mL Streptomycin). The spore count was assessed using a Neubauer haemocytometer and adjusted to 106 spores/mL with PBS. A working suspension of spores (104 spores/mL) was prepared in filter sterilised MOPS RPMI (50 ml_; RPMI-1640 containing 2 mM L-glutamine, 2% glucose and 0.165 M MOPS, buffered to pH 7 with NaOH). Resazurin sodium salt (100 μΙ_ of 1 % solution; Sigma- Aldrich, Dorset, UK) was added to the spore suspension and mixed well. The spore suspension-resazurin mixture (100 L/well) was added to 384-well plates (Catalogue number 353962, BD Falcon, Oxford, UK). Simultaneously, test compounds (0.5 μΙ_ DMSO solution) were added to 100 μΙ_ of the spore-resazurin mixture in quadruplicate to provide a final DMSO solution of 0.5% using an Integra VIAFLO 96 (Intergra, Zizers, Switzerland). For non-spore control wells, MOPS-RPMI-resazurin solution (100 μΙ_) was added instead of the spore- resazurin mixture. The plate was covered with a Breathe Easier membrane (Catalogue No Z763624, Sigma-Aldrich, Dorset, UK), and incubated (35°C, 5% C02) until fluorescence in the inoculated wells was double that of the control wells (around 24 hr). The fluorescence of each well (545 nm (excitation) / 590 nm (emission), gain 800, focal height 5.5mm) was determined using a multi-scanner (Clariostar: BMG, Buckinghamshire, UK). The percentage inhibition for each well was calculated and the MIC50, MIC75 and MIC90 values were calculated from the concentration-response curve generated for each test compound.

b. Broth microdilution assay

This assay was conducted using a modified method published by EUCAST (Rodriguez- Tudela, et ai, 2008). Spores of Aspergillus fumigatus (NCPF2010, Public Health England, Wiltshire) were cultured in Sabouraud dextrose agar for 3 days. A stock spore suspension was prepared from a Sabouraud dextrose agar culture by washing with PBS-tween (10 ml_; phosphate buffered saline containing 0.05% Tween-20, 100 U/mL Penicillin and 100 U/mL Streptomycin). The spore count was assessed using a Neubauer haemocytometer and then adjusted to 106 spores/mL with PBS. A working suspension of spores (2 x 105 spores/mL) was prepared in filter sterilised, BSA MOPS RPMI (50 mL; RPMI-1640 containing 2 mM L- glutamine, 0.5% BSA, 2% glucose, 0.165 M MOPS, buffered to pH 7 with NaOH). For the assay, BSA MOPS RPMI (50 μίΛ/νβΙΙ) was added throughout the 384-well plate (Catalogue number 353962, BD Falcon, Oxford, UK) first. Test compounds (0.5 μί DMSO solution) were then added in quadruplicate using an Integra VIAFLO 96 (Integra, Zizers, Switzerland), and mixed well using a plate mixer. Subsequently 50 μί of the working spore suspension prepared above was added to all wells except non-spore control wells. For non-spore control wells, BSA MOPS-RPMI solution (50 μί/ννβΙΙ) was added instead. The plate was covered with a plastic lid, and incubated (35°C with ambient air) for 48 hr. The OD of each well at 530 nm was determined using a multi-scanner (Clariostar: BMG, Buckinghamshire, UK). The percentage inhibition for each well was calculated and the MIC50, MlCzs and MIC90 values were calculated from the concentration-response curve generated for each test compound.

Assessment of Biofilm formation

This assay was conducted using a modified, published method (Pierce et al., 2008). Spores of Aspergillus fumigatus (NCPF2010, Public Health England, Wiltshire) were cultured in Sabouraud dextrose agar for 3 days. A spore suspension was prepared from a Sabouraud dextrose agar culture by washing with PBS-tween (10 ml_; phosphate buffered saline containing 0.05% Tween-20, 100 U/mL Penicillin and 100 U/mL Streptomycin). The spore count was assessed using a Neubauer haemocytometer and then adjusted to 106 spores/mL with PBS. A working suspension of spores at 105 spores/mL was prepared in filter sterilised MOPS RPMI (50 ml_; RPMI-1640 containing 2 mM L-glutamine, 2% glucose, 0.165 M MOPS, buffered to pH 7 with NaOH,). The spore suspension (100 [\Uwe\\) was added to the 384-well plates (Catalogue number 353962, BD Falcon, Oxford, UK), covered with a Breathe Easier membrane (Catalogue No Z763624, Sigma-Aldrich, Dorset, UK) and incubated (35°C and 5% CO2). For non-spore control wells, MOPS-RPMI solution (100 μΙ_) was added instead of spore suspension. After 24 hr the cover membrane was removed and test compounds (0.5 μΙ_ DMSO solution) were added to the wells in quadruplicate to provide a final DMSO solution of 0.5% using an Integra VIAFLO 96 (Integra, Zizers, Switzerland). The plate was covered with a Breathe Easier membrane, and incubated (35°C, 5% CO2) for 20hr or 44 hr. When the incubation was complete, the cover membrane was removed and resazurin sodium salt solution (5 μΙ_ of 0.08% solution; Sigma-Aldrich, Dorset, UK) was added to each well. The plate was incubated (35°C, 5% CO2) until fluorescence in the inoculated wells was double that of the control wells (around 4 hr). The fluorescence of each well (545 nm (excitation) / 590 nm (emission), gain 800, focal height 5.5mm) was determined using a multi-scanner (Clariostar: BMG, Buckinghamshire, UK). The percentage inhibition for each well was calculated and the MIC50, MIC75 and MIC90 values were calculated from the concentration-response curve generated for each test compound.

Cell Viability: Resazurin Assay

BEAS2B cells were seeded in 384-well plates (100 μΙ_; 3000 / well /; BD Falcon, Catalogue No 353962) in RPMI-LHC8 (RPMI-1640 and LHC8 media combined in equal proportions) one day before experimentation. For cell-free control wells, RPMI-LHC8 (100 μΙ_) was added. Test compounds (0.5 μΙ_ DMSO solution) were added to give a final DMSO concentration of 0.5% using an Integra VIAFLO 96 (Integra, Zizers, Switzerland). BEAS2B cells were incubated with each test compound for 1 day (37°C / 5% C02 in RPMI-LHC8). After addition of resazurin stock solution (5 μΙ_, 0.04%) the plates were incubated for a further 4 hr (37°C / 5% C02). The fluorescence of each well at 545 nm (excitation) and 590 nm (emission) was determined using a multi-scanner (Clariostar: BMG Labtech). The percentage loss of cell viability was calculated for each well relative to vehicle (0.5% DMSO) treatment. Where appropriate, a CC50 value was calculated from the concentration-response curve generated from the concentration-response curve for each test compound.

In Vivo Anti-fungal Activity

Aspergillus fumigatus (ATCC 13073 [strain: NIH 5233], American Type Culture Collection, Manassas, VA, USA) was grown on Malt agar (Nissui Pharmaceutical, Tokyo, Japan) plates for 6-7 days at RT (24 ± 1 °C). Spores were aseptically dislodged from the agar plates and suspended in sterile distilled water with 0.05% Tween 80 and 0.1 % agar. On the day of infection, spore counts were assessed by haemocytometer and the inoculum was adjusted to obtain a concentration of 1 .67 108 spores mL"1 of physiological saline.

To induce immunosuppression, A J mice (males, 5 weeks old) were dosed with hydrocortisone (Sigma H4881 ; 125 mg/kg, sc,) on days 3, 2 and 1 before infection, and with cyclophosphamide (Sigma C0768; 250 mg/kg, ip) 2 days before infection. On day 0, animals were infected with the spore suspension (30 uL intra-nasally).

Test substances were administered intra-nasally (35 μΙ_; a suspension, 0.08-2 mg/mL, prepared using physiological saline) once daily, 30 min before infection on day 0, and then on days 1 , 2 and 3. Animal body weight was monitored daily and if it was reduced by 20% compared with the body weight on day 0, the animal was culled. Six hr after the last dose, animals were anesthetized, the trachea was cannulated and BALF was collected. The total number of alveolar cells was determined using a haemocytometer, and the numbers of alveolar macrophages and neutrophils were determined by FACS analysis (EPICS® ALTRA II, Beckman Coulter, Inc., Fullerton, CA, USA) using anti-mouse MOMA2-FITC (macrophage) or anti-mouse 7/4 (neutrophil), respectively, as previously reported (Kimura et al., 2013). The levels of IFN-γ and IL-17 in BALF were determined using Quantikine® mouse IFN-γ or IL-17 ELISA kit (R&D systems, Inc., Minneapolis, MN, USA) respectively. MDA, an oxidative stress marker, was assayed using OxiSelect® TBARS Assay Kits (MDA Quantitation; Cell Biolabs Inc, San Diego, CA, USA). Aspergillus GM in serum was determination using Platelia GM-EIA kits (Bio-Rad Laboratories, Redmond, WA, USA). Cut-off index was calculated by the formula: Cut-off index = OD in sample / OD in cut-off control provided in kit. For tissue fungal load assays, 100 mg of lung tissue was removed aseptically and homogenized in 0.2 mL of 0.1 % agar in sterile distilled water. Serially diluted lung homogenates were plated on Malt agar plates (50 Up\ate), and incubated at 24±1 °C for 72 to 96 hr. The colonies of A. fumigatus on each plate was counted and the fungal titre presented as CFU per gram of lung tissue.

In vivo Pharmacokinetic studies

Mice were briefly sedated (20 μί of ketamine (0.06 mg/mL) / xylazine (3 mg/mL)) before i.t. dosing of either the compound of formula (I) or posaconazole (20 μί; 2 mg/mL suspension in physiological saline) using a PennCentury device (FMJ-250). Blood samples were collected (cardiac puncture under gaseous anesthesia with isoflurane) into pre-chilled Li Hep tubes 24 hr after dosing. Each sample was mixed gently and kept on wet ice, for a maximum of 15 min, before centrifugation (1500 g, 10 min at 4°C) to prepare plasma. Samples were then aliquoted and stored at -80°C prior to analysis.

In preparation for analysis, a calibration line was prepared in control mouse plasma over the range of 0.5-10000 ng/mL. QC samples were prepared at three concentrations: 10, 100 and 1000 ng/mL. Control blanks, zero sample, standards, QCs and unknown samples (50 μΙ_) were aliquoted into a 96 well plate. Acetonitrile (150 μΙ_) was added to control blanks and internal standard working solution (150 μΙ_; Chrysin, 1000 ng/mL) was added to all other wells. The plate was vortex mixed and centrifuged and the supernatant transferred to a fresh 96 well plate for analysis by LC-MS/MS.

Extracts were analysed using a Thermo Vantage LC-MS/MS and Waters Acquity UPLC. Mobile phases used were 0.1 % formic acid (aq) and 0.1 % formic acid (ACN). The column used was an Acquity HSS T3 50x2.1 mm Ι .δμηη.

The lower limit of quantification for the assay for compound of formula (I) was 0.5 ng/mL. Summary of Screening Results Compound (I), as disclosed herein, demonstrates potent inhibitory activity against planktonic fungal growth evaluated by both the resazurin assay and the broth microdilution assay (Table A). In these assay systems Compound (I) showed significantly greater potency than voriconazole and Amphotericin B, and somewhat greater potency than posaconazole. Incubation with Compound (I) had no detectable effects on the viability of BEAS2B bronchial epithelial cells.

Table A The effects of treatment with Voriconazole, Posaconazole, Amphotericin B and Compound (I) on Aspergillus fumigatus planktonic fungal growth and on BEAS2B bronchial epithelial cell viability.

MIC5o / MIC75 / CC50 Values in assay system indicated (nM)

Test Material Resazurin Broth Microdilution Cell Viability

MICso MIC75 MICso MIC75 CC50

Voriconazole 80.8 169 512 839 > 28,600

Posaconazole 3.96 21 .8 10.8 12.1 > 14,300

Amphotericin B 58.1 > 108 200 317 559

Compound (I) 0.76 3.97 2.59 3.94 > 15,000

Furthermore, Compound (I), as disclosed herein, exhibited potent inhibition of biofilm formation by Aspergillus fumigatus after 48 hr (Table B). The fact that Compound (I) showed much greater potency than voriconazole and greater potency than posaconazole in this in vitro biofilm assay is notable. Unlike Compound (I), voriconazole and posaconazole, the activity of Amphotericin B showed a marked drop-off in potency as the incubation time was extended from 24 hr to 48 hr.

Table B The effects of treatment with Compound (I) on Aspergillus fumigatus biofilm formation at 24 hr and 48 hr incubation time points.

MIC5o / MIC75 Values at incubation time indicated (nM)

Test Materials 24hr incubation 48hr incubation

MICso MIC75 MIC 50 MIC75

Voriconazole > 28,600 > 28,600 > 286 > 286

Posaconazole >14,300 >14,300 2.02 > 143

Amphotericin B 43.2 103 > 108 > 108

Compound (1) > 15,000 > 15,000 1 .18 > 150

In vivo, Compound (I), as disclosed herein, showed superior effects to posaconazole given intranasally on galactomannan concentrations in serum and on fungal load in lung (Table C, and Figures 1A and 1 B respectively); on inflammatory cells (Table D, Figure 2A) and on cytokine concentrations in BALF (Table E, Figures 2B and 2C) in Aspergillus fumigatus infected, immunocompromised, neutropenic mice.

Table C: The Effects of Treatment with Posaconazole, and Compound (I) on CFU in lung and serum galactomannan concentrations in Aspergillus fumigatus infected, immunocompromised, neutropenic mice.

Drug CFU / mg of lung Galactomannan in

Treatment Suspension serum (COI)

Cone (mg/mL) (% inhibition) (% inhibition)

Vehicle + Spores None 21.0 ± 7.3 3.7 ± 0.39

0.08 13.8 ± 9.7 (34) 3.0 ± 0.61 (19)

Posaconazole 0.4 9.5 ± 6.3 (55) 2.0 ± 1.3 (46)

2 1 .6 ± 0.9 (92) 0.30 ± 0.24 (92)

0.08 14.6 ± 8.6 (30) 3.2 ± 1.1 (14)

Compound (I) 0.4 5.6 ± 2.7 (73) 0.34 ± 0.23 (91 )

2 1 .6 ± 1.0 (92) 0.26 ± 0.1 1 (93)

The data for fungal load are shown as the mean ± standard error of the mean (SEM; n = 6-9) Table D: The Effects of Treatment with Compound (I) on inflammatory cell accumulation into BALF of Aspergillus fumigatus infected, immunocompromised, neutropenic mice.

Cell numbers in BAL x105/ml_

Drug

(% inhibition)

Treatment Concentration

(mg/mL) Macrophage Neutrophil

Vehicle + Spore 0.62 ± 0.19 0.42 ± 0.21

0.08 0.46 ± 0.14 (26) 0.37 ± 0.09 (12)

Posaconazole 0.4 0.35 ± 0.07 (44) 0.24 ± 0.05 (43)

2 0.27 ± 0.07 (56) 0.18 ± 0.04 (57)

0.08 0.37 ± 0.1 1 (40) 0.29 ± 0.08 (31 )

Compound (1) 0.4 0.29 ± 0.10 (53) 0.24 ± 0.10 (43)

2 0.16 ± 0.05 (74) 0.14 ± 0.05 (67) The data for cell number are shown as the mean ± standard error of the mean (SEM), N = 6-9

Table E: The Effects of Treatment with Compound (I) on biomarkers in the BALF of Aspergillus fumigatus infected, immunocompromised, neutropenic mice

Concentrations of Biomarkers (%inhibition)

Drug

Treatment Concentration IFNY IL-17 MDA

(mg/mL) (pg/mL) (pg/mL) (Mg/mL)

Vehicle + Spores None 1 1 ± 3.4 24.7 ± 13.3 1 .3 ± 0.4

0.08 8.4 ± 3.5 (24) 22.5 ± 8.8 (9) 1 .4 ± 0.9 (-8)

Posaconazole 0.4 4.1 ± 1.3 (63) 15.2 ± 5.9 (38) 0.7 ± 0.3 (46)

2 2.3 ± 0.50 (79) 4.5 ± 1.6 (82) 0.5 ± 0.2 (62)

0.08 5.9 ± 2.6 (46) 10.5 ± 3.0 (57) 1 .4 ± 0.4 (-8)

Compound (I) 0.4 3.2 ± 1.2 (71 ) 8.4 ± 3.1 (66) 1 .0 ± 0.4 (23)

2 2.0 ± 0.60 (82) 5.8 ± 2.4 (77) 0.4 ± 0.2 (69)

The data for biomarker concentrations are shown as the mean ± standard error of the mean (SEM), N = 6-9

Finally the plasma concentration of the compound of formula (I) was found to be <0.5 ng/i 24 hr after i.t. dosing (n = 6). In summary, the compound of formula (I) has been found to be a potent inhibitor of Aspergillus fumigatus planktonic growth and biofilm formation in in vitro assays. In vivo, in Aspergillus fumigatus infected, immunocompromised, neutropenic mice, Compound (I), as disclosed herein, demonstrated potent inhibition of Aspergillus fumigatus infection accompanied by an inhibition of the lung immune response. This anti-aspergillus effect apparently occurred in the absence of any significant systemic exposure, as judged by the absence of detectable compound in the plasma 24 hr after topical dosing. Given the significant burden resulting from the systemic effects of present azole therapy, this represents a very significant advance over currently available treatments.

References

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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.

Claims

1 . A compound, namely Compound (I),

Compound (I) which is:

4-(4-(4-(((3R,5R)-5-((1 H-1 ,2,4-triazol-1 -yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran- 3-yl)methoxy)phenyl)piperazin-1 -yl)-/V-(4-fluorophenyl)benzamide;

or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 for use as a pharmaceutical.

3. A compound according to claim 1 for use in the treatment of mycoses or for use in the prevention or treatment of disease associated with mycoses.

4. Use of a compound according to claim 1 in the manufacture of a medicament for the treatment of mycoses or for the prevention or treatment of disease associated with mycoses.

5. A method of treatment of a subject with a mycosis which comprises administering to said subject an effective amount of a compound according to claim 1.

6. A method of prevention or treatment of disease associated with a mycosis in a subject which comprises administering to said subject an effective amount of a compound according to claim 1.

7. A compound for use, use or method according to any one of claims 3 to 6 wherein the mycosis is caused by Aspergillus spp.

8. A compound for use, use or method according to claim 7 wherein the Aspergillus sp. is Aspergillus fumigatus.

9. A compound according to claim 1 for use as a pharmaceutical in combination with a second or further active ingredient.

10. A pharmaceutical composition comprising a compound according to claim 1 optionally in combination with one or more pharmaceutically acceptable diluents or carriers.

1 1 . A pharmaceutical composition according to claim 10 which comprises a second or further active ingredient.

12. A compound for use according to claim 9 or a pharmaceutical composition according to claim 1 1 wherein the second or further active ingredient is selected from other anti-fungal agents (such as voriconazole or posaconazole), amphotericin B, an echnocandin (such as caspofungin) and an inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (such as lovastatin, pravastatin or fluvastatin).

A compound of formula (5')

wherein R1 represents d-salkyl;

or a salt thereof.

A compound of formula (6)

or a salt thereof. 15. A compound of formula (8)


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