OA16257A - Novel antifungal 5,6-dihydro-4-[(difluoroethyl)phenyl]-4H-pyrrolo[1,2a][1,4]benzodiazepine and 4-(difluoroethyl)phenyl-6H-pyrrolo[1,2a][1,4]benzodiazepine derivatives. - Google Patents

Abstract

The present invention concerns novel compounds of Formula (I) wherein R1 , R2 , R3 , R4 , R5 and R6 have the meaning defined in the claims. The compounds according to the present invention are active mainly against dermatophytes and systemic fungal infections. The invention further relates to processes for preparing such novel compounds, pharmaceutical compositions comprising said compounds as an active ingredient as well as the use of said compounds as a medicament.

Description

The présent invention is concemed with novel antifungal 5,6-dihydro-4~[(dîfluorocthyl)phcnyI]-4//-pyrrolo[l ,2-û][ l,4]bcnzodiazepinc and 4-(difluorocthyl)phcnyl-6//pyrrolo[l,2'ii][l,4]benzodiazepine dérivatives, active maînly against dermatophytes and systemic fungal infections. The invention further relates to processes for preparing such novel compounds, pharmaceutical compositions comprising said compounds as an active ingrédient as well as the use of said compounds as a médicament.

Background of the invention

Dermatophyte is a common label for a group of 3 types of fungî that commonly causes skin disease in animais and hurnans. These anamorphic (asexual or imperfect fungi) généra are: Microsporum, Epidermophyton and Trichophyton. There are about 40 specîes in these 3 généra,

Dermatophytes cause infections of the skin, haïr and nails due to their ability to obtain nutricnts from kcratinizcd material. The organisms eolonize the keratîn tîssucs and inflammation îs caused by host response to metabolic by-products. They are usually restricted to the comified layer of the epidermis because of their inability to penetrate viable tissue of an immunocompétent host. However, occasionally the organisms do invade subeutaneous tissues, resulting in kerion development. Invasion does elicit a host response ranging from mild to severe. Acid protéinases, elastase, keratinases, and other protéinases reportedly act as virulence factors.

Systemic fungal infections (SFI) are life-threatening conditions that most commonly affect patients with reduced immunity often resulting from therapeutic interventions to treat malignant diseases. The number of SFI’s in modem hospitals keeps increasing, and the number of different fungi that hâve been involved in SFI îs large and still growing. Despite many cases of invasive candidiasis and aspergillosis there has been an increased incidence of infections due to other molds like Scedosporium apiospermum, Fiisariuin spp., and Zygomycetes, Rhizopus and Mucorspp.. Effective therapeutic agents treating ail these infections very well therefore need to hâve very broad spectrum of activity. In the past few décades itraconazole, fluconazole, kétoconazole, and intravenous or liposomal amphotericin B hâve been used in SFI, and ail of these agents hâve their limitations with regard to spectrum, safety or ease of administration. More recently a

-2third génération of azoles hâve been învestigated and introduced to the market, improving the treatment options in intensive care units, Voriconazole (Vfend™) and posaconazole (Noxafîl™) show much improvement of treatment towards life threatening invasive SF1 such as candidiasis, aspergillosis, and infections due to Fusarium species at clinical relevant dosages. Moreover posaconazole shows effîcacy against infections caused by the emerging Zygomycetes spp., Echinocandins, such as anidulafungin, caspofungin, and micafungin, which are non-competîtive inhibitors of l,3-p-glucan synthesis in fungal cell walls, display high effîcacy against Candida spp. and Aspergillus spp., but no activity against Cryptococcus, Fusarium, or Zygotnycetes spp..Of ail antimycotic agents, azoles still rcpresent a unique class of compounds displaying the broadest antifungal spectrum via inhibition of 14-a-demethylase, an enzyme being essential for ergosterol biosynthesis in fungi.

Onychomycosis is the most common disease of the nails and constîtutes about a half of ail nail abnormalities. The prcvalcncc of onychomycosis is about 6-8 % in the adult population. The causative pathogens of onychomycosis include dermatophytes, Candida, and non-dcrmatophytîc moulds. Dermatophytes are the fiingi most commonly rcsponsiblc for onychomycosis in the temperate western countrics; mcanwhile, Candida and non-dermatophytic moulds are more frequently involved in the tropics and subtropics.Trichophyton rubrum is the most common dermathophyte involved in onychomycosis. Other dermatophytes that may be involved arc Trichophyton interdigitale, Epidermophyton floccosum, Trichophyton violaceum, Microsporum gypseum, Trichophyton tonsurans, Trichophyton soudanense and Trichophyton verrucosum. Other causative pathogens include Candida and non-dcrmatophytic moulds, in particular members of the mould génération Scytalidium (also Neoscytalidium), Scopulariopsis, and Aspergillus.

5,6-Dihydro-4/ï-pyrrolo[I,2-a][l,4]benzodiazepines hâve been described in J. Chem. Soc.(C), 2732-2734 (1971); J. Heterocyclic Chem., 13, 711-716 (1976); and J. Heterocyclic Chem., 16, 241-244 (1979), The compounds disclosed in these references ail hâve a different substitution on the phenyl moiety in the 4-position and moreover no biological activitics were reported in any of these rcfcrenccs.

WO02/34752 describes 4-substituted 5,6-dihydro-4W-pyrrolo[l,2-a][l,4]benzodiazepines as a new class of antifungal compounds. However, WO02/34752 does not specifically disclose the présent substitution pattern on the phenyl moiety in the 4position.

Many drug compounds, while possessing desired therapeutic propertîes, are used

-3inefficiently due to their poor water solubility. Thus for example, where such compounds are administered orally, only a small fraction of the drug is taken up into the blood during transit of the gastro-intestinal tract. As a resuit, to achieve adéquate drug uptake it may be necessary to administer high doses of the drug compound, to prolong the period of drug administration or to make frequent administrations of the drug compound. Indeed, the poor solubility and hencc poor bioavailability of a drug may cause an alternative drug, perhaps one with undesired side effects or one which requires invasive administration (e.g. by injection or infusion), to be used in place of the poorly soluble drug.

Thcrcforc it is an objcct of the présent invention to producc more bioavaiiablc compounds with a broad antîfungal spectrum or to provîde useful alternative compounds, maintaining adequately high therapeutic efficacy and adequately low toxicity or other sidc effects.

Unexpectedly, the antîfungal compounds of the présent invention or part of the compounds of the présent invention may hâve improved metabolic stability properties, improved PK (pharmacokinetic) properties, improved solubilities, reduced cytochrome P450 liabilities, or improved bioavailability compared with the compounds disclosed in the prior art.

The compounds of the présent invention are useful as squalene epoxidase inhibitors.

It is accordingly an object of the présent invention to provide novel compounds with antîfungal activity to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Sumrnar,' of the invention

It has been found that the compounds of the présent invention are useful as antîfungal compounds.

The présent invention concerns novel compounds of Formula (I):

(D and stcreoisomeric forms thereof, wherein R¹ is hydrogen, chloro or fluoro;

R² is hydrogen, chloro, fluoro or methyl;

R³ and R⁴ are hydrogen;

or R³ and R⁴ taken together form a bond;

R³ is l,l-difluoroethyl, and R⁶ is hydrogen or fluoro;

or R⁵ is hydrogen or fluoro, and R⁶ is 1,1-difluoroethyl;

and the pharmaceutically acceptable addition salts, and the solvatés thereof.

The présent invention also conccms methods for the préparation of compounds of Formula (I) and pharmaceutical compositions comprising them.

The présent compounds are useful agents for combating fungi in vivo.

The novel compounds described in the présent invention may be useful in the treatment or prévention of infections caused by dermatophytes, systemic fungal infections and onychomycosis.

The novel compounds described in the présent invention may be active against a wide variety of fungi, such as Candida spp., e.g. Candida albicans, Candida glabrata,

Candida kruceï, Candida parapsilosis, Candida kefyr, Candida tropicalis; Aspergillus spp., e.g. Aspergillus fumigatus, Aspergillus niger, Aspergillusflavus; Cryptococcus neoformans; Sporothrix schenckii; Epidermophytonfloccosum; Microsporum spp., e.g. Microsporum canis, Microsporum gypseum; Trichophyton spp., e.g. Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton quinckeanum, Trichophyton tonsurans, Trichophyton verrucosum, Trichophyton violaceum, Trichophyton interdigitale, Trichophyton soudanense', Fusarium spp., e.g. Fusarium solani, Fusarium oxysporum, Fusarium proliferatum, Fusarium verticillioides; Rhizomucor spp., e.g. Rhizomucor miehei, Rhizomucorpusillus·, Mucor circinelloides; Rhizopus spp., e.g. Rhizopus oryzae, Rhizopus microspores; Malassezia furfur; Acremonîum spp.;

Paecilomyces; Scopulariopsis; Arthrographis spp.; Scytalidium; Scedosporium spp., e.g. Scedosporium apiospermum, Scedosporium prolijïcans; Trichoderma spp.; Pénicillium spp.', Pénicillium marneffei; Blastoschizomyces.

In view of the aforementioned pharmacology of the présent compounds, it follows that they are suitable for use as a médicament.

The invention also relates to a compound according to the general Formula (I), the stereoisomeric forms thereof and the pharmaceutically acceptable addition salts and the solvatés thereof, for use in the treatment or prévention of fungal infections.

One advantage of the compounds or a part of the compounds of the présent invention may lie in their enhanced bîoavailability, improved metabolic stability properties,

-5improved PK properties, reduced hERG channel inhibition, or reduced cytochrome P450 Iiabilities compared with the compounds disclosed in the prior art.

The présent invention will now be further described. In the following passages, different aspects of the invention arc defined in more detail. Each aspect so defined may bc combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Detailed description

The chemical names of the compounds of the présent invention werc gcncratcd according to the nomenclature rules agreed upon by the Chemical Abstracts Service, using Advanced Chemical Development, Inc., nomenclature software (ACD/Name product version 10.01; Build 15494, l Dec 2006).

In case of tautomeric forms, it should be clear that the other non-depicted tautomeric form is also încludcd within the scope of the présent invention.

The atoms in the tricyclic system are numbered as shown in the following formula (XX):

It will be appreciated that some of the compounds of Formula (I) and their pharmaceutically acceptable addition salts and solvatés may contain one or more centers of chiralîty and exist as stcrcoisomcric forms.

Hereinbefore and hereinafter, the term “compound of formula (1)” is meant to include the addition salts, the solvatés and the stereoisomers thereof

The ternis “stereoisomers”, “stereoîsomeric forms” or “stereochemically isomeric forms” hereinbefore or hereinafter are used interchangeably.

The invention includes ail stereoisomers of the compound of Formula (I) either as a pure stereoisomer or as a mixture of two or more stereoisomers.

Enantiomers are stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemate or racemic mixture. Dîastereomers (or diastereoisomers) are stereoisomers that are not enantiomers, i.e. they are not related as mirror images. If a compound contains a double bond, the

-6ΙΟ substituents may be in the E or the Z configuration. If a compound contains a disubstituted cycloalkyl group, the substituents may be in the cis or trans configuration. Therefore, the invention includes enantiomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and mixtures thereof, whenever chemically possible. The absolute configuration is specified according to the Cahn-Ingold-Prelog system. The configuration at an asymmetric atom is specified by either R or S. Resolved compounds whose absolute configuration is not known can be designated by (+) or (-) depending on the direction in which they rotate plane polarized light.

When a spécifie stereoisomer is identified, this means that said stereoisomer is substantially free, i.e. associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, in particular less than 2% and most preferably less than l %, of the other isomers. Thus, when a compound of formula (1) is for instance specified as (R), this means that the compound is substantially free of the (S) isomer; when a compound of formula (I) is for instance specified as E, this means that the compound îs substantially free of the Z isomer; when a compound of formula (I) îs for instance specified as cis, this means that the compound is substantially free of the trans isomer.

For therapeutic use, salts of the compounds of Formula (I) are those wherein the counterion is pharmaceutically acceptable. However, salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the préparation or purification of a pharmaceutically acceptable compound. Ail salts, whether pharmaceutically acceptable or not are included within the ambit of the présent invention.

The pharmaceutically acceptable acid and base addition salts as mentioned hereinabove or hereinafter are meant to comprise the therapeutically active non-toxic acid and base addition sait forms which the compounds of Formula (1) are able to form. The pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfurie, nîtric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacettc, lactic, pyruvic, oxalic (i.e. ethanedioic), malonîc, succîntc (i.e. butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonte, cthancsulfonic, bcnzcncsulfonic, p-tolucncsulfonic, cyclamic, salicylîc, p-aminosalicylîc, pamoic and the like acids. Conversely said sait forms can be converted by treatment with an appropriate base into the free base form.

-7The compounds of Formula (I) containing an acidic proton may also be converted into their non-toxic métal or amine addition sait forms by treatment with appropriate organic and înorganic bases. Appropriate base sait forms comprise, for example, the ammonium salts, the alkali and earth alkaline métal salts, e.g. the lithium, sodium, potassium, magnésium, calcium salts and the like, salts with organic bases, e.g. primary, secondary and tertiary aliphatic and aromatic amines such as methylamine, ethylamine, propylamine, isopropylamine, the four butylamine isomers, dimethylamîne, diethylamine, diethanolamine, dipropylamine, diisopropylamîne, di-w-butylamîne, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinoline and isoquinoline; the benzathine, A-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like. Conversely the sait form can be converted by treatment with acid into the frec acid form.

The terrn solvaté comprises the hydrates and solvent addition forms which the 15 compounds of Formula (I) are able to form, as well as the salts thereof. Examples of such forms are e.g. hydrates, alcoholates and the like.

The compounds of Formula (1) as prepared in the processes described below may be synthesized in the form of mixtures of enantiomers, in particular racemic mixtures of enantiomers, that can be separated from one another following art-known resolution 20 procedures, A manner of separating the enantiomeric forms of the compounds of

Formula (1) involves liquid chromatography using a chiral stationary phase. Said pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically, Preferably if a spécifie stéréo isomer is desired, said 25 compound would be synthesized by stereospecifïc methods of préparation. These methods will advantageously employ enantiomerically pure starting materials.

in the Framework of this application, a compound according to the invention is inherently intended to comprise ail isotopic combinations of its chemical éléments. In the framework of this application, a chemical element, in particular when mentioned in 30 relation to a compound according to Formula (I), comprises ail isotopes and isotopic mixtures of this element. For example, when hydrogen is mentioned, it is understood to refer to 'H, ²H, ³H and mixtures thereof.

A compound according to the invention therefore inherently comprises a compound with one or more isotopes of one or more element, and mixtures thereof, including a 35 radioactive compound, also called radîolabelled compound, wherein one or more non16257

radioactive atoms has been replaced by one of its radioactive isotopes. By the terni radio labelled compound is meant any compound according to Formula (I), or a pharmaceutically acceptable sait thereof, which contains at Ieast one radioactive atom. For example, a compound can be labelled with positron or with gamma emitting radioactive isotopes. For radioligand-binding techniques, the ³H-atom or the ¹²ⁱI-atom is the atomof choice to be replaced. For îmaging, the most commonly used positron emitting (PET) radioactive isotopes are ^HC, ^l8F, ^l5O and ^l3N, ail of which are accelerator produced and have half-lîves of 20,100, 2 and 10 minutes respectively. Since the half-lives of these radioactive isotopes are so short, it is only feasible to use them at institutions which have an accelerator on site for their production, thus limîting their use. The most widely used of these are ¹⁸F, ^99mTc, ^2O1T1 and ¹²³I. The handling of these radioactive isotopes, their production, isolation and incorporation in a molécule are known to the skilled person.

In particular, the radioactive atom is selected from the group of hydrogen, carbon, nitrogen, sulfur, oxygen and halogen. In particular, the radioactive isotope is selected from the group of ³H, ^HC, ¹⁸F, ⁱ²²I, ¹²³I, ¹²⁵I, ¹³ⁱI, ⁷⁵Br, ^7SBr, ⁷⁷Br and ⁸²Br.

As used in the spécification and the appendcd claims, the singular forms a, an, and the also include plural referents unless the context clearly dictâtes otherwise. By way of ex ample, a compound means one compound or more than one compound.

The terms described above and others used in the spccification are well understood to those in the art.

Preferred features of the compounds of this invention are now set forth.

The présent invention concems novel compounds of Formula (I):

(D and stereoisomeric forms thereof, wherein

R¹ is hydrogen, chloro or fluoro;

R² is hydrogen, chloro, fluoro or methyl;

R³ and R⁴ are hydrogen;

€

-9or R³ and R⁴ taken together form a bond;

R⁵ is 1,1-difluoroethyl, and R⁶ is hydrogen or fluoro;

or R⁵ is hydrogen or fluoro, and R⁶ is Ι,Ι-difluoroethyl, and the pharmaceutically acceptable addition salts, and the solvatés thereof,

In an embodiment, the invention relates to compounds of Formula (I) and stereoisomeric forms thereof, wherein

R^! is chloro or fluoro;

R² is hydrogen, chloro, fluoro or methyl;

R³ and R⁴ are hydrogen;

or R³ and R⁴ taken together form a bond;

R⁵ is 1,1-difluoroethyl, and R⁶ is hydrogen or fluoro;

or R⁵ is hydrogen or fluoro, and R⁶ is 1,1-difluoroethyl;

and the pharmaceutically acceptable addition salts, and the solvatés thereof.

In an embodiment, the invention relates to compounds of Formula (I) and stereoisomeric 15 forms thereof, wherein

R¹ is chloro or fluoro;

R² is chloro, fluoro or methyl;

R³ and R⁴ are hydrogen;

or R³ and R⁴ taken together form a bond;

R^s is 1,1-difluoroethyl, and R⁶ is hydrogen or fluoro;

or R⁵ is hydrogen or fluoro, and R⁶ is 1,1-difluoroethyl;

and the pharmaceutically acceptable addition salts, and the solvatés thereof.

In an embodiment, the invention relates to compounds of Formula (Γ) and stereoisomeric forms thereof, wherein

R¹ is chloro or fluoro;

R² is hydrogen, chloro, fluoro or methyl;

R³ and R⁴ arc taken together to form a bond;

R⁵ is 1,1-difluoroethyl, and R⁶ is hydrogen or fluoro;

or R⁵ is hydrogen or fluoro, and R⁶ is 1,1-difluorocthyl;

and the pharmaceutically acceptable addition salts, and the solvatés thereof.

In another embodiment, the invention relates to compounds of Formula (I) and stereoisomeric forms thereof, wherein

R¹ is chloro or fluoro;

R² is hydrogen;

R³ and R⁴ taken together form a bond;

-ΙΟR⁵ îs 1,1-difluoroethyl;

R⁶ is hydrogen;

and the pharmaceutically acceptable addition salts, and the solvatés thereof.

In a ncxt embodiment, the invention relates to any of the other embodiments or any combination of the other embodiments, wherein R⁵ îs 1,1-difluoroethyl and R⁶ is hydrogen or fluoro.

In a further embodiment, the invention relates to any of the other embodiments or any combination of the other embodiments, wherein R⁵ îs hydrogen or fluoro and R⁶ is 1,1difluoroethyl.

In a next embodiment, the invention relates to any of the other embodiments or any combination of the other embodiments, wherein at least one of R¹ and R² is other than hydrogen.

In a particular embodiment, the invention relates to any of the other embodiments or any combination of the other embodiments, wherein R² is hydrogen, chloro or fluoro.

In another embodiment, the invention relates to any of the other embodiments or any combination of the other embodiments, wherein R³ and R⁴ are hydrogen.

In another embodiment, the invention relates to any of the other embodiments or any combination of the other embodiments, wherein R¹ or R² îs in the 7-position and is other than hydrogen.

In another embodiment, the invention relates to any of the other embodiments or any combination of the other embodiments, wherein R¹ is in the 7-position and is chloro or fluoro; in particular R¹ is in the 7-position and is chloro.

In another embodiment, the invention relates to any of the other embodiments or any combination of the other embodiments, wherein

R¹ is in the 7-position and is chloro or fluoro; in particular R¹ is in the 7-position and is chloro; and

R² is in any of the other positions and is hydrogen, chloro, fluoro or methyl; în particular chloro, fluoro or methyl; more in particular chloro or fluoro; even more in particular chloro.

In another embodiment, the invention relates to any of the other embodiments or any combination of the other embodiments, wherein R² is in the 7-position and is chloro, fluoro or methyl.

-llΙΟ

In another embodiment, the invention relates to any of the other embodiments or any combination of the other embodiments, wherein

R² is in the 7-position and is chloro, fluoro or methyl; and

R^l is in any of the other positions and is hydrogen, chloro or fluoro; in particular chloro or fluoro; more in particular chloro.

In a further embodiment, the invention relates to any of the other embodiments or any combination of the other embodiments, wherein R³ and R⁴ arc taken together to form a bond.

In a next embodiment the cornpound of Formula (I) is selected from the group comprising:

7-chloro-4-[4-(l,l-difluoroethyr)phenyi]-5,6-dihydro-4H-pyrrolo[I,2-a][l,4]benzodiazepine,

7-chloro-4-[4-( 1,1 -difluoroethyl)phenyl]-5,6-dihydro-4H-pyrrolo[ 1,2-a][ 1,4]benzodîazepine .HCl, (4S)-7-chloro-4-[4-( 1, l-difluoroethyl)phenyl]-5,6-dihydro-4H-pyrrolo[l ,2-a][l,4]benzodiazepîne, (4S)-7-cliloro-4-[4-(l,l-difluoroethy[)phenyl]-5,6-dihydiO-4H-pyrro[o[l,2-a][l,4]' benzodiazépine .HCl, (4R)-7-chloro-4-[4-(l,l-difluoroethyl)phenyl]-5,6-dihydro-4H-pyrrolo[ 1,2-a][l ,4]benzodiazépine, (4R)-7-chloro-4-[4-(l, l-difluoroethyl)phenyl]-5,6-dihydro-4H-pyrrolo[l ,2-a][ 1,4]benzodiazepine .HCl,

8.10- dichloro-4-[4-( 1,1 -difluoroethyl)phenyl]-5,6-dihydro-4H-pyrrolo[ 1,2-a] [ 1,4]benzodiazepine,

8.10- dichloro-4-[4-(l, l-difluoroctliyl)phenyl]-5,6“dihydro-4H-pyrrolo[ 1,2-a][l ,4]benzodiazepine .HCl,

8.10- dichloro-4-[4-( 1, l-difluorocthyl)phcnyl]-6H-pyrrolo[ 1,2-a][l ,4]benzodiazepine,

7.8- dichloro-4-[4-(l₎l-difluoroethyl)phenyl]-5,6-dihydro-4H-pyrrolo[l,2-a][ 1,4]benzodiazepine,

7.8- dichloro-4-[4-(l,l-difluoroethyl)phenyl]-5,6-dihydro-4H-pyrrolo[l,2-a][I₎4Jbenzodiazepine .HCl,

7.10- dîchloro-4-[4-(l ,1 -difluoroethyl)phenyl]-6H-pyrrolo[ 1,2-a][l ,4]benzodiazepine,

7.9- dichloro-4-[4-(l,l-difluorocthyl)phcnyl]-6H-pyrrolo[l,2-a][l,4]bcnzodiazcpînc, 7,8-dichloro-4-[4-(l,l-difluoroethyl)phenyl]-6H-pyrrolo[l,2-a][l,4]benzodiazepine,

7.10- dichloro-4-[4-( 1,1 -difluoroethyl)phenyl]-5,6-dihydro-4H-pyrrolo[l ,2-a][ 1,4] benzodiazépine,

7,l0-dichloro-4-f4-(l,I-difluoroethyl)phenyl]-5,6-dihydro-4H-pyrrolo[l,2-a]fl,4]benzodiazepine .HCl,

7.9- dichloro-4-[4-(l,l-difiuoroethyl)phenyl]-5,6-dihydro-4H-pyrrolo[l,2-a][l,4]benzodiazepine,

7.9- dichloro-4-[4-( l, l-difluoroethyl)phenyl]-5,6-dihydro-4H-pyrrolo[l,2-a][l,4]benzodiazepîne .HCl,

7-chloro-4-[4-( 1,1 -difiuoroethyl)phenyl]-6H-pyrrolo[ 1,2-a] [ 1 ,4]benzodiazepine, 7-chloro-4-[3-(l,l-difluoroethyl)plicnyl]-6H-pyrrolo[l,2-a][l,4]benzodiazepine, 4-(4-(1,1-difluo roethyl)phenyl]-7-fluoro-6H-pynOlo[l,2-a][l,4]benzodiazepine, 9-chloro-4'[4-( 1,1 -difiuoroethyl)phenyl]-7-methyl-6H-pyrrolo[l ,2-a][ 1,4]benzodiazepine,

9- chloro-4-[4-(l ,1 -difluo roethyl)phenyl]-6H-pyrrolo[ 1,2-a][ 1,4]benzodiazepine,

4-[4-( 1,1 -difluo roethyl)phenyl]-7,9-difluoro-6H-pyrrolo[ 1,2-a][ 1,4]benzodiazepîne,

10- chloro-4-[4-( 1, 1-difluo roethyl)phenyl]-6H-pyrrolo[ l,2-a][l,4]benzodiazepine, 4-(3-(1,1 -difluo roethyl)-4-fl uorophenyl]-7-fluoro-6H-pyrrolo[ 1,2-a][l ,4]benzodiazepine, 7-chloro-4-f3-( 1,1 -difluo rocthyl)-4-fluorophcnyl]-6H-pyrro lof 1,2-a][ 1,4]bcnzodiazepinc,

9.10- dichloro-4-[4-( 1, l-difluoroethyl)phenyl]-6H-pyrrolo[ 1,2-a][l,4]beuzodiazepine, 4-(3-(1,1 -difluoroethyÎ)-4-fluorophenyl]-7,9-difluoro-6H-pyrrolof 1,2-a][ 1,4]bcnzodiazcpinc,

7-chloro-4-[4-( 1,1 -difluoroethyl)phenyl]-9-fluoro-5,6-dihydro-4H-pyrrolo( 1,2-a] [1,4]benzodiazepine,

7-chloro-4-[4-(l ,1 -difluorocthyl)phcnyl]-9-fluoro-5,6-dihydro-4H-pyrrolo(l,2-a][l ,4]benzodiazepine .HCl,

7,9-dichloro-4-[3-(l ,1 -difluo rocthyl)-4-fl uoropheuyl]-6H-pyrrolo[1,2-a][ 1,4]bcnzodiazcpinc,

7-chloro-4-[3-(l ,1 -difluoroethyl)-4-fluorophenyl]-9-fluoro-6H-pyrrolo[ l,2-a][ 1,4]benzodiazepine,

7-chloro-4-[4-( 1,1 -difluo rocthyl)phcnyl]-9-fluoro-6H-pyrro lof 1,2-a] [ 1,4]bcnzodiazcpinc, 10-chloro-4-[4-( 1, l-difluoroethyl)phenyl]-7-methyl-6H-pyrrolo[l ,2-a][l,4]benzodiazepine,

7-chloro-4-[3-(l, l-difluorocthyl)phcnyl]-5,6-dihydro-4/7-pyirolo[l ,2-a][l ,4]benzodiazepine,

7-chloro-4-[3-( 1,1 -difluoroethyl)phenyl]-5,6-dihydro-4?/-pyrrolo[ 1,2-a] [ 1,4]benzodiazepine .HCl,

7-chloro-4-[4-( 1,1 -difluorocthyl)-3-fluorophenyl]-6H-pyrrolo[ 1,2-a][ 1,4]benzodiazepine, 4-(4-(1, l-difluoroethyl)-3-fluorophenyl]-7-fluoro-6H-pyrrolo[l,2-a][l,4]benzodiazepine, 7-chloro-4-[4-( 1,1 -difluoroethyl)-3-fluoropheny l]-9-fluoro-6H-pyrrolo[ 1,2-a] [1,4]16257

-13benzodîazepine,

9-chloro-4-[4-( 1,1-difluoroethyl)phenyl]-7-fluoro-6H'pyrrolo[ 1,2-a][ 1,4]benzodiazepine, 4-[4-(1,1 -difluoroethyl)-3-fluorophenyl]-7,9-difluoro-6H-pyrrolo[ 1,2-a][l ,4]benzodiazepine,

9- chloro-4-[3-(l,l-difIuoroethyl)-4-fluorophenyl]-6H-pyrrolo[l_!2-a][l,4]benzodiazepine,

10- chloro-4-[3-(l, I-difluoroethyl)-4-fluorophenyl]-6H-pyrrolo[ 1,2-a][l ,4]benzodiazepine, including any stereochemically isomcric form thereof, and the pharmaceutically acceptable addition salts and the solvatés thereof.

In an cvcn other embodiment the compound of Formula (I) is 7-chloro-4-[4-(l,ldifhioroethyl)pheny!]-6H-pyrrolo[l,2-a][l,4]benzodiazepine.

Ail possible combinations of the abovc-indîcated înteresting embodiments are considered to be embraced within the scope of this invention.

The présent invention also encompasses processes for the préparation of compounds of Formula (1) and subgroups thereof

The compounds of Formula (1) and the subgroups thereof can be prepared by a succession of steps as described hereunder. They are generally prepared from starting materials which are either commercially available or prepared by standard means obvious to those skilled in the art. The compounds of the présent invention can be also prepared using standard synthetic processes commonly used by those skilled in the art of organic chemistry.

The compounds of the présent invention can be prepared according to Scheme 1 :

-14Scheme 1

1,4-dioxane R¹ R²

-15The compounds of Formula (I) wherein R’ and R⁴ together form an extra bond, said compounds being represented by formula (I-b), can be prepared froin the compounds represented by the formula (I-a), following art-known amine to imine oxidation reactions. These oxidation reactions may be conducted by reacting a compound of formula (I-a) with an oxidant such as, for example, lead tetra-acetate or manganèse dioxide, in a reaction inert solvent such as a halogenated hydrocarbon e.g. dichloromethane (DCM) or trîchloromethane. The reaction rate can be enhanced by stirring and optionally heating the reaction mixture.

Altematively, a compound of formula (I-b) can be prepared by an intramolecular cyclization of an intermediate of formula (II). In the présence of an acid such as, for example, POClj, the amide in the intermediate of formula (II) can fonction as a Celectrophîle, resulting in a ring closure. The reaction may be performed in a suitable solvents such as, for cxamplc, DCM. Stirring and heating may cnhancc the rate of the reaction.

A compound of formula (I-a) can be prepared from an intermediate of formula (IV) by converting it in a sait (III) by reaction with an acid H⁺X of formula (XI), and reacting said sait of formula (III) with an aldéhyde of formula (XII) in an appropriaie solvent such as an alcohol, e.g. methanol (MeOH), éthanol (EtOH), isopropanol, at an elevated température, preferably at reflux température.

Altematively, the intermediate of formula (IV) may be reacted first with the aldéhyde of formula (XII) and the thus formed imine may be cyclizcd in the présence of an acid H⁺X‘ of formula (XI) to a compound of formula (I-a).

Altematively, a compound of formula (I-a) may be obtained by the réduction of a compound of formula (I-b) by using methods well-known to those skîlled in the art. An intermediate of formula (II) may be prepared by a coupling reaction between an intermediate of formula (111) and (XIII). Said reaction may be performed in the presence of coupling agents such as typically l-hydroxy-177-benzotriazole (HOBt) and M-(ethylcarbonimidoyl)-V,N-dimethyl-l ,3-propanediamine monohydrochloride (EDCI). The reaction may be performed in the presence of a base such as trîetylamine (EtjN) and a suitable solvent such as, for example, DCM. Altematively, an acid chloride dérivative of (XIII) or a reactive ester dérivative of (ΧΠΤ) can also be used in this type of reaction to prépare an intermediate of formula (11).

An intermediate of formula (XIII) or its acid chloride or ester dérivative, can be easily prepared by those skîlicd in the art.

-16Intermediates of formula (III) and (IV) are prepared by reducing a I-(2-cyanophenyl)pyrrole dérivative of formula (V), Several procedures well-known to those skilled în the art may be used to reduce the nitrile function such as, for example:

1. L1AIH4/THF [S. Raines, S.Y. Chai and F.P. Palopoli; J, Heterocyclic Chem., 13, 711-

716(1976)]

2. i. sodium bis(2-methoxyethoxy)aluminate (Red-Al⁵) 70% w/w Toluene, RT :

ii. NaOH 10%, RT [G.W.H. Cbeeseman and S.G. Greenberg; J. Heterocyclic Chem., 16, 241-244(1979)]

3a. i. KBH₄/CF₃COOH, THF; ii. H₂O; iii. HCl [P. Trinka, P. Slégel and J. Reiter;

J. Prakt. Chem., 338, 675-678(1996)]

3b. Borane-dimethyl sulfide (1:1), THF

4a, RaNi (Raney Nickel) / H₂

4b. RaNi / thiophene solution / (MeOH/NHj)

Even other well-known methods for reducing the nitrile function may also be used.

An intermediate of formula (V) in turn is commercially available or alternatively can be easily prepared by, for example, treating a 2-aminobenzonitrile dérivative of formula (VI) with tetrahydro-2,5-dimethoxyftiran în an inert solvent such as dioxane or tetrahydrofuran (THF) in the présence of an acid such as 4-chloropyridine hydrochloride, or in an acidic solvent such as glacial acetic acid, at an elevated température, preferably at reflux température. Alternatively, an intermediate of formula (V) can also be prepared from an intermediate of formula (X). Typically, an intermediate of formula (X) wherein Halo is defined as Br, I, Cl or F, is reacted with pyrrole in the presence of a base such as, for example, Cs₂COj or NaH, in a suitable solvent such as typically DMF.

Alternatively, an intermediate of formula (IV) may be prepared by treating an intermediate of formula (VIï) with borane-dimethyl sulfide (1:1) in a suitable solvent such as, for example, THF. The reaction typically can be performed in the presence of an acid such as HCl. After the reaction has proceeded, the reaction mixture can be basified with a suitable base such asNaOH. The réaction can be performed at an elevated température, preferably at reflux température.

An intermediate of formula (VII) can be prepared from an intermediate of formula (VIII). An intermediate of formula (VIII) can be reacted with a nitrogen source such as, NHj.HjO in the presence of HOBt and EDCI. This type of reaction typically can be performed in a suitable solvent like DMF. Stirring of the reaction mixture may enhance 35 the rate of reaction.

Ο

-17Αη intermediate of formula (VIII) can be easily prepared by treating an intermedîate of formula (IX) with tetrahydro-2,5-dimethoxyfuran in an inert solvent such as dioxane in the presence of an acid such as 4-chloropyridine hydrochloride at an elevated température, preferably at reflux température. Altematively, a reactive ester derivatîve 5 of (IX) can also be used in this type of reaction to préparé an intermediate of formula (VIII).

Ail starting materials arc commcrcially availablc or can be easily prepared by those skîlled în the art.

In ail these préparations, the reaction products may be isolated from the reaction medium and, if necessary, further purified according to méthodologies generally known in the art such as, for example, extraction, crystallization, trituration and chromatography. In particular, stereoisomers can be isolated chromatographically using a chiral stationary phase such as, for example, Chiralpak® AD (amyiose 3,5 dimethylphenyl carbamate) or Chiralpak® AS, both purchased from Daicel Chemical

Industries, Ltd, in Japan.

Pure stéréoisomeric forms of the compounds and the intermediates of this invention may be obtained by the application of art-known procedures. Enantiomers may be separated from each other by the sélective crystallization of their dîastcrcomcric salts with optically active acids. Altematively, enantiomers may be separated by chromatographie 20 techniques using chiral stationary phases. Said pure stereoisomeric forms may also be derived from the corresponding pure stereoisomeric forms of the appropriatc starting materials, provided that the reaction occurs stereoselectively or stereospecifically. Preferably if a spécifie stereoisomer is desired, said compound will be synthesized by stcrcosclcctivc or stcrcospccific methods of préparation. These methods will advantageously employ chirally pure starting materials. Stereoisomeric forms of the compounds of Formula (I) are obviously intended to be included within the scope ofthe invention.

The chirally pure forms of the compounds of Formula (1) form a preferred group of compounds. It is therefore that the chirally pure forms of the intermediates and their sait 30 forms are partie ularl y useful in the préparation of chirally pure compounds of Formula (I). Also enantiomeric mixtures of the intermediates are useful in the préparation of compounds of Formula (I) with the corresponding configuration.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may bc active

-18against dimorphic pathogens, dermatophytes, zygomycetes, hyaline hyphomycetes, dematiaceous hyphomycetes, yeasts and yeastlike organisms.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may bc active against dimorphic pathogens, yeasts and yeastlike organisms.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against moulds.

The compounds of Formula (!) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against a wîde variety of fungi, such as Candida spp., e.g. Candida albicans, Candida glabrata, Candida kruceï, Candida parapsilosis, Candida kefyr, Candida tropicalis; Aspergillus spp., e.g. Aspergillus fumigatus, Aspergillus niger, AspergillusJlavus; Cryptococcus neoformans; Sporothrix schenckii; Epidermophyton floccosum; Microsporum spp., e.g. Microsporum canis, Microsporum gypseum; Trichophyton spp., e.g. Trichophyton mentagrophytes, Trichophyton rubruni, Trichophyton quinckeanum, Trichophyton tonsurans, Trichophyton verrucosum, Trichophyton violaceum, Trichophyton interdigitale, Trichophyton soudanense; Fusarium spp., e.g. Fusarium solani, Fusarium oxysporum, Fusarium proliferatum, Fusarium verticillioides; Rhizomucor spp., e.g. Rhizomucor miehei, Rhizomucorpusillus; Mucor circinelloides; Rhizopus spp., e.g. Rhizopus oryzae, Rhizopus microspores; Malassezia furfur; Acremonium spp.; Paecîlomyces; Scopulariopsis; Arthrographis spp.; Scytalidium; Scedosporium spp., e.g. Scedosporium apiospermum, Scedosporium prolificans; Trichoderma spp.; Pénicillium spp.; Pénicillium marneffei;

Blastoschizomyces.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against a wide variety of fungi, such as Candida parapsilosis; Aspergillus spp., e.g. Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus; Cryptococcus neoformans; Sporothrix schenckii; Epiderniophytonfloccosum; Microsporum spp., e.g. Microsporum canis, Microsporum gypseum; Trichophyton spp., e.g. Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton quinckeanum, Trichophyton tonsurans, Trichophyton verrucosum, Trichophyton violaceum, Trichophyton interdigitale, Trichophyton soudanense; Fusarium spp., e.g. Fusarium solani, Fusarium oxysporum, Fusarium proliferatum, Fusarium verticillioides; Rhizomucor spp., e.g.

©

-19Rhizomucor miehei, Rhizomucor pusillus; Mucor circinelloides; Rhizopus spp., e.g. Rhizopus oryzae, Rhizopus microspores; Acremonium spp.; Paecilomyces; Scopulariopsis; Arthrographis spp.; Scytalidium; Scedosporium spp., e.g.

Scedosporium apiospermum, Scedosporium prolificans; Trichoderma spp.; Pénicillium 5 spp. ; Pénicillium marneffei; Blastoschizomyces.

The compounds of Formula (I) and stereoisomerîc forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may bc active against a wide variety of fungi, such as Candidaparapsîlosis; Aspergillus spp., e.g. Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus; Cryptococcus neoformans; Epidermophyton floccosum; Microsporum spp., e.g. Microsporum canis,

Microsporum gypseum; Trichophyton spp., e.g. Trichophyton mentagrophytes, Trichophyton iitbrum, Trichophyton quinckeanum, Trichophyton tonsurans, Trichophyton verrucosum, Trichophyton violaceum, Trichophyton interdigitale, Trichophyton soudanense; Fusarium spp., e.g. Fusarium solani, Fusarium oxysporum,

Fusarium proliferatum, Fusarium verticillioides; Rhizomucor spp., e.g. Rhizomucor miehei, Rhizomucorpusillus-, Mucor circinelloides; Rhizopus spp., e.g. Rhizopus oryzae, Rhizopus microspores; Acremonium spp.; Paecîlomyces; Scopulariopsis;

Arthrographis spp.; Scytalidium; Scedosporium spp., e.g. Scedosporium apiospermum, Scedosporiumprolificans-, Trichoderma spp.; Pénicillium spp.; Pénicillium marneffei;

Blastoschizomyces; in particular Aspergillus spp., e.g. Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus; Cryptococcus neoformans; Epidermophyton floccosum; Microsporum spp., e.g. Microsporum canis, Microsporum gypseum; Trichophyton spp., e.g. Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton quinckeanum, Trichophyton tonsurans, Trichophyton verrucosum,

Trichophyton violaceum, Trichophyton interdigitale, Trichophyton soudanense', Fusarium spp., e.g. Fusarium solani, Fusarium oxysporum, Fusarium prolifération, Fusarium verticillioides; Rhizomucor spp., e.g. Rhizomucor miehei, Rhizomucor pusillus; Mucor circinelloides; Rhizopus spp., e.g. Rhizopus oryzae, Rhizopus microspores; Acremonium spp.; Paecilomyces; Scopulariopsis; Arthrographis spp.;

Scytalidium; Scedosporium spp., e.g. Scedosporium apiospermum, Scedosporium prolificans; Trichoderma spp.; Pénicillium spp.; Pénicillium marneffei; Blastoschizomyces.

The compounds of Formula (1) and stcrcoisomcric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against Candida parapsilosis B66I26, Aspergillus fumigatus B42928, Cryptococcus neoformans B66663, Sporoihrix schenckii B62482, Microsporum canis B68I28,

-20Trichophyton mentagrophvtes B70554, Trichophyton rubrum B68183, Scedosporium apiospermum IHEM3817 and Scedosporium prolificans IHEM21157.

The compounds of Formula (T) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatcs thereof, may bc active against Candidaparapsilosis B66126, Aspergillus fumigatus B42928, Cryptococcus neoformans B66663, Sporothrix schenckii B62482, Microsporum canis B68128, Trichophyton nientagrophytes B70554, Trichophyton rubrum B68183, Scedosporium apiospermum IHEM3817, Scedosporiumprolificans IHEM21157, Rhizopus oryzae IHEM5223, Rhizomucor miehei IHEM13391 and Mucor circinelloides IHEM21105.

The compounds of Formula (1) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against Candida parapsilosis, Aspergillus fumigatus, Cryptococcus neoformans, Sporothrix schenckii, Microsporum canis, Trichophyton nientagrophytes, Trichophyton rubrum, Scedosporium apiospermum, Scedosporium prolificans, Rhizopus oryzae,

Rhizomucor miehei and Mucor circinelloides.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may bc active against Candida parapsilosis, Aspergillus fumigatus, Cryptococcus neoformans, Sporothrix schenckii, Microsporum canis, Trichophyton nientagrophytes, Trichophyton rubrum, Scedosporium apiospermum and Scedosporium prolificans', in parti cul ar Aspergillus fumigatus, Microsporum canis, Trichophyton nientagrophytes, Trichophyton rubrum, Scedosporium apiospermum and Scedosporium prolificans.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against a wide variety of fungi, such as Candida parapsilosis; Aspergillus spp.; Cryptococcus neoformans; Sporothrix schenckii; Microsporum spp.; Fusarium spp.; Scedosporium spp.;

in particular Candida parapsilosis; Aspergillus spp.; Cryptococcus neoformans; Microsporum spp.; Fusarium spp.; Scedosporium spp.;

more in particular Aspergillus spp.; Cryptococcus neoformans; Microsporum spp.; Fusarium spp.; Scedosporium spp.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against a wide variety of fungi, such as Candidaparapsilosis; Aspergillus spp.;

Cryptococcus neoformans; Trichophyton spp.; Sporothrix schenckii; Microsporum

-21spp.; Fusarium spp.; Scedosporium spp.;

in particular Aspergillus spp.; Microsporum spp.; Trichophyton spp.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatcs thereof, may bc active against fungi such as Candîda parapsilosis; Aspergillus spp., e.g. Aspergillus fimiigatus, Aspergillus niger, Aspergillus Jlavus; Cryptococcus neoj'onnans; Sporothrix schenckii; Epidermophyton floccosum; Microsporum canis; Trichophyton spp., e.g. Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton quinckeanum;

in particular Candîdaparapsilosis; Aspergillus spp., e.g. Aspergillus Jumigatus, Aspergillus niger, Aspergillus Jlavus; Cryptococcus neoformans; Epidermophyton floccosum; Microsporum canis; Trichophyton spp., e.g. Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton quinckeanum;

more in particular Aspergillus spp., e.g. Aspergillus fumigatus, Aspergillus niger, Aspergillus Jlavus; Cryptococcus neoformans; Epidermophyton floccosum; Microsporum canis; Trichophyton spp., e.g. Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton quinckeanum.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against fungi such as Candîdaparapsilosis', Aspergillus spp.; Cryptococcus neoformans; Microsporum spp.; Trichophyton spp.; Scedosporium spp..

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against Candîda parapsilosis, Aspergillus fumigatus, Cryptococcus neojbrmans, Sporothrix schenckii, Microsporum canis, Trichophyton mentagrophytes, Trichophyton rubrum, Scedosporium apiospermum and Scedosporium proliflcans;

in particular Candîda parapsilosis, Aspergillus fumigatus, Cryptococcus neoformans, Microsporum canis, Trichophyton mentagrophytes, Trichophyton rubrum, Scedosporium apiospermum and Scedosporium proliflcans;

more in particular Aspergillus fumigatus, Cryptococcus neoformans, Microsporum canis, Trichophyton mentagrophytes, Trichophyton rubrum, Scedosporium apiospermum and Scedosporium proliflcans.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against a variety of fungi that infect the skin, haïr and nails, as well as subeutaneous and systemic fungal pathogens.

-22The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against the 3 dermatophyte généra: Trichophyton, Microsporum and Epidermophytow, in particular against Trichophyton and Microsporum.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against dcrmatophytcs and Aspergillus spp.; in particular Microsporum canis, Trichophyton mentagrophytes, Trichophyton rubrum and Aspergillus fumigatus; more in particular Microsporum canis, Trichophyton mentagrophytes and Trichophyton rubrum.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against Trichophyton mentagrophytes, Trichophyton rubrum and Aspergillus spp.; in particular Trichophyton mentagrophytes, Trichophyton rubrum and Aspergillus fumigatus.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against Trichophyton mentagrophytes; Trichophyton rubrum', Aspergillus spp., e.g. Aspergillus fumigatus', Fusarîum spp.; Mucor Spp.; Zygomycetes spp.; Scedosporium spp.; Microsporum canis; Sporothrix schenckii; Cryptococcus neoformans and Candida parapsilosis.

The compounds of Formula (1) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against dermatophytes.

The compounds of Formula (1) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, may be active against Aspergillus fumigatus.

The compounds of Formula (I) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatcs thereof, may bc active against a wide variety of fungi, such as one or more ofthe fungi mentioned hereinbefore.

The compounds of Formula (1) and stereoisomeric forms thereof, and the pharmaceutically acceptable addition salts, and the solvatés thereof, are potent antifungals when administered orally or topically.

The compounds of the présent invention may bc useful as crgostcrol synthesis inhibitors.

-23In view of the utility of the compound of Formula (I), there is provided a method of treating warm-blooded animais, including humans, suffering from, or a method of preventing warm-blooded animais, including humans, to suffer from any one of the diseases mentioned hereinbefore. Hence, compounds of Formula (l) are provided for use as a medicîne. Also the use of a compound of Formula (I) in the manufacture of a médicament useful in treating fungal infections is provided. Further compounds of Formula (!) are provided for use in the treatment of fungal infections.

As used herein, the term treatment is intended to refer to ail processes, wherein there may be a slowing, interrupting, arresting, or stopping of the progression of an infection, but does not ncccssarily indîcatc a total élimination of ail symptoms.

The invention relates to a compound according to the general Formula (I), the stereoîsomeric forms thereof and the pharmaceutically acceptable acid or base addition salts and the solvatés thereof, for use as a médicament.

The invention also relates to a compound according to the general Formula (I), the stereoîsomeric forms thereof and the pharmaceutically acceptable acid or base addition salts and the solvatés thereof, for the treatment or prévention of fungal infections; în particular fungal infections caused by one or more of the fungi mentioned hereinbefore, The invention also relates to a compound according to the general Formula (I), the stereoîsomeric forms thereof and the pharmaceutically acceptable acid or base addition salts and the solvatés thereof, for the treatment of fungal infections; in particular fungal infections caused by one or more of the fungi mentioned hereinbefore,

The invention also relates to a compound according to the general Formula (1), the stereoîsomeric forms thereof and the pharmaceutically acceptable acid or base addition salts and the solvatés thereof, for use in the treatment or prévention of fungal infections; in particular fungal infections caused by one or more of the fungi mentioned hereinbefore.

The invention also relates to a compound according to the general Formula (I), the stereoîsomeric forms thereof and the pharmaceutically acceptable acid or base addition salts and the solvatés thereof, for use în the treatment or prévention, in particular treatment, of fungal infections; in particular fungal infections caused by one or more of the fungi selected from a group consisting of fungi mentioned hereinbefore.

The invention also relates to a compound according to the general Formula (I), the stereoîsomeric forms thereof and the pharmaceutically acceptable acid or base addition

-24salts and the solvatés thereof, for use in the treatment of fiingal infections; in particular fungal infections caused by one or more of the fungi mentioned hereinbefore.

The invention also relates to a compound according to the general Formula (I), the stcrcoisomcric forms thereof and the pharmaceutically acceptable acid or base addition salts and the solvatés thereof, for use în the treatment or prévention of a fungal infection, in particular a fungal infection caused by one or more ofthe fungi mentioned hereinbefore.

The invention also relates to a compound according to the general Formula (I), the stereoisomeric forms thereof and the pharmaceutically acceptable acid or base addition 10 salts and the solvatés thereof, for use in the treatment or prévention of a fungal infection, wherein the fungal infection is caused by one or more of the fungi selected from the group consisting of Candida spp.; Aspergillus spp.; Cryptococcus neofortnans;

Sporothrix schenckii; Epidermophyton floccosum; Microsporum spp.; Trichophyton spp; Fusarium spp.; Rhizomucor spp.; Mucor circinelloides; Rhizopus spp.; Malassezia furfur; Acremonium spp.; Paecilomyces; Scopulariopsis; Arthrographis spp.;

Scvtalidium; Scedosporium spp.; Trichoderma spp.; Pénicillium spp.; Pénicillium marneffei; and Blastoschizomyces;

in particular wherein the fungal infection is caused by one or more of the fungi selected from the group consisting of Candidaparapsilosis; Aspergillus spp.; Cryptococcus neoformans; Sporothrix schenckii; Epidermophyton floccosum; Microsporum spp.;

Trichophyton spp.; Fusarium spp.; Rhizomucor spp.; Mucor circinelloides; Rhizopus spp.; Acremonium spp.; Paecilomyces; Scopulariopsis; Arthrographis spp.;

Scytalidinm; Scedosporium spp.; Trichodenna spp.; Pénicillium spp.; Pénicillium marneffei; and Blastoschizomyces;

even more in particular wherein the fungal infection is caused by one or more of the fungi selected from the group consisting of Microsporum cards; Trichophyton mentagrophytes, Trichophyton rubrum and Aspergillus fumigatus.

The novel compounds described in the présent invention may be useful in the treatment or prévention of diseases or conditions selected from the group consisting of infections 30 caused by dermatophytes, systemic fungal infections and onychomycosis.

The novel compounds described in the présent invention may be useful in the treatment or prévention of diseases or conditions such as for example infections caused by dermatophytes, systemic fungal infections or onychomycosis.

-25The invention also relates to the use of a compound according to the general Formula (I), the stereoisomeric forms thereof and the pharmaceutically acceptable acid or base addition salts and the solvatés thereof, for the manufacture of a médicament.

The invention also relates to the use of a compound according to the general Formula (I), the stereoisomeric forms thereof and the pharmaceutically acceptable acid or base addition salts and the solvatés thereof, for the manufacture of a médicament for the treatment or prévention, in particular treatment, of fungal infections, in particular fungal infections caused by one or more of the fungi mentioned hereinbefore.

The compounds of the présent invention can be administered to mammals, preferably humans, for the treatment or prévention, in particular treatment, of fungal infections, in particular fungal infections caused by one or more of the fungi mentioned hereinbefore. In view of the utility of the compound of Formula (I), there is provided a method of treating warm-blooded animais, including humans, suffering from or a method of preventing warm-blooded animais, including humans, to suffer from fungal infections, in particular fungal infections caused by one or more of the fungi mentioned hereinbefore.

Said methods comprise the administration, i.e. the systemîc or topical administration, preferably oral administration, of an effective amount of a compound of Formula (I), a stereoisomeric form thereof and a pharmaceutically acceptable addition sait or solvatc thereof, to warm-blooded animais, including humans.

Those of skill in the treatment of such diseases could détermine the effective therapeutic daily amount from the test results presented hereinafter. An effective therapeutic daily amount would be from about 0.005 mg/kg to 50 mg/kg, in particular 0.01 mg/kg to 50 mg/kg body weight, more în particular from 0.01 mg/kg to 25 mg/kg body weight, preferably from about 0.01 mg/kg to about 15 mg/kg, more preferably from about 0.01 mg/kg to about 10 mg/kg, even more preferably from about 0.01 mg/kg to about 1 mg/kg, most preferably from about 0.05 mg/kg to about 1 mg/kg body weight. The amount of a compound according to the présent invention, also referred to here as the active ingrédient, which is rcquired to achieve a therapeutically effect will of course, vaty on case-by-case basis, for example with the particular compound, the route of administration, the âge and condition of the récipient, and the particular disorder or disease being treated.

A method of treatment may also include administering the active ingrédient on a regimen ofbetween one and four intakes per day. In these methods of treatment the compounds according to the invention are preferably formulated prior to administration. As

-26ÎO described herein below, suitable pharmaceutical formulations are prepared by known procedures using well known and readily available ingrédients.

While it is possible for the active ingrédient to be administered alone, it is préférable to présent it as a pharmaceutical composition.

The présent invention also provides compositions for treating or preventing fungal infections comprising a therapeutically effective amount of a compound of Formula (I) and a pharmaceutically acceptable carrier or diluent.

The carrier or diluent must be “acceptable” in the sense of being compatible with the other ingrédients of the composition and not deteterious to the récipients thereof.

The compounds of the présent invention,that are suitable to treat or prevent fungal infections, may be administered alone or in combination with one or more additional therapeutic agents. Combination therapy includes administration of a single pharmaceutical dosage formulation which contains a compound of Formula (I) and one or more additional therapeutic agents, as well as administration of the compound of Formula (I) and each additional therapeutic agents in its own separate pharmaceutical dosage formulation. For example, a compound of Formula (I) and a therapeutic agent may be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent may be administered in separate oral dosage formulations.

In view of their useful pharmacological properties, the subject compounds may be formulated into various pharmaceutical forms for administration purposes. The compounds according to the invention, in particular the compounds according to Formula (1), a pharmaceutically acceptable acid or base addition sait thereof, a stereochemically isomeric form thereof, or any subgroup or combination thereof may be formulated into various pharmaceutical forms for administration purposes. As appropriate compositions there may be cited all compositions usually employed for systemically administering drugs.

To préparé the pharmaceutical compositions of this invention, an effective amount of the particular compound, optionally in addition sait form, as the active ingrédient is combined in intimate admîxture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of préparation desired for administration. These pharmaceutical compositions are désirable in unitary dosage form suitable, in particular, for administration orally, rcctally, percutaneously, by parentéral injection or by inhalation. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed such as, for example,

C

-27water, glycols, oils, alcohols and the like in the case of oral liquid préparations such as suspensions, syrups, élixirs, émulsions and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their easc in administration, tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed. For parentéral compositions, the carrier will usually comprise stérile water, at least in large part, though other ingrédients, for example, to aid solubility, may be includcd. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable solutions containing compounds of Formula (I) may be formulated in an oil for prolonged action. Appropriate oils for this purpose are, for example, peanut oil, sesame oil, cottonseed oil, corn oil, soybean oil, synthetic glycerol esters of long chain fatty acids and mixtures of these and other oils. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspendîng agents and the like may be employed. Also included are solid form préparations that are intended to be converted, shortly before use, to liquid form préparations. In the compositions suitable for percutancous administration, the carrier optionally comprises a pénétration cnhancîng agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minorproportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitatc the administration to the skîn and/or may bc helpful for preparing the desired compositions. These compositions may be administered in various ways, e.g., as a transdcrmal patch, as a spot-on, as an ointment.

Acid or base addition salts of compounds of Formula (I) duc to their incrcascd water solubility over the corresponding base or acid form, are more suitable in the préparation of aqueous compositions.

Transungual compositions arc in the form of a solution and the carrier optionally comprises a pénétration enhancing agent which favours the pénétration of the antifungal into and through the keratinized ungual layer of the nail. The solvent medium comprises water mixed with a co-solvent such as an alcohol having from 2 to 6 carbon atoms, e.g. éthanol.

In order to cnhancc the solubility and/or the stability of the compounds of Formula (I) in pharmaceutical compositions, it can be advantageous to employ α-, β- or γ-cyclodextrms or their dérivatives, in particular hydroxyalkyl substituted cyclodextrins, e.g. 2-hydroxypropyl-P-cyclodcxtrin or sulfobutyl-p-cyclodcxtrin. Also co-solvents such as

-28alcohols may improve the solubility and/or the stability of the compounds according to the invention in pharmaceutical compositions.

The ratio of active ingrédient over cyclodextrin may vary widely. For example ratios of l/l 00 to 100/1 may bc applied. Intcrcsting ratios of active ingrédient over cyclodextrin 5 range from about 1/10 to 10/1. More interesting ratios of active ingrédient over cyclodextrin range from about 1/5 to 5/1.

Dépend ing on the mode of administration, the pharmaceutical composition will preferably comprise from 0.05 to 99 % by weight, more preferably from 0.1 to 70 % by weight, even more preferably from 0.1 to 50 % by weight of the compound of Formula 10 (I), and, from 1 to 99.95 % by weight, more preferably from 30 to 99.9 % by weight, even more preferably from 50 to 99.9 % by weight of a pharmaceutically acceptable carrier, ail percentages being based on the total weight of the composition.

For parentcral compositions, also other ingrédients, to aid solubility for example, e.g. cyciodcxtrins, may bc includcd. Appropriate cyclodcxtrins arc a-, β-, γ-cyclodcxtrins or 15 ethers and mixed ethers thereof wherein one or more of the hydroxy groups of the anhydroglucose unîts of the cyclodextrin are substituted with Cj.galkyl, particularly methyl, ethyl or isopropyl, e.g. randomly methylated β-CD; hydroxyCj.galkyl, particularly hydroxyethyl, hydroxy-propyl or hydroxybutyl; carboxyC ] ^alkyl, particularly carboxymethyl or carboxy-ethyl; Ci-^alkylcarbonyl, particularly acetyl.

Espccially noteworthy as complexants and/or solubilizers arc β-CD, randomly methylated β-CD, 2,6-dîmethyl-h-C'D, 2-hydroxyethyl-J5-CD, 2-hydroxycthyl-y-CD, 2-hydroxypropyl-y-CD and (2-carboxymethoxy)propylHl-CD, and in particular 2-hydroxypropyl-|l-CD (2-HP^-CD).

The terrn mixed ether dénotés cyclodextrin dérivatives wherein at least two cyclodextrin 25 hydroxy groups are etherified with different groups such as, for example, hydroxypropyl and hydroxyethyl.

The average molar substitution (M.S.) is used as a measure of the average number of moles of alkoxy units per mole of anhydroglucose. The average substitution degree (D.S.) refers to the average number of substituted hydroxyls per anhydroglucose unit.

The M.S. and D.S. value can be determined by various analytical techniques such as nuclear magnetic résonance (NMR), mass spectrometry (MS) and infrared spectroscopy (IR). Dcpcnding on the technique used, slightly different values may bc obtained for onc gîven cyclodextrin dérivative. Preferably, as measured by mass spectrometry, the M.S. ranges from 0.125 to 10 and the D.S. ranges from 0.125 to 3.

Ο

-29Other suitable compositions for oral or rectal administration comprise particles consisting of a solid dispersion comprising a compound of Formula (I) and one or more approprîate pharmaceutically acceptable water-soluble polymers.

The term ”a solid dispersion” used hcrcinaftcr dcfincs a System in a solid statc (as opposed to a liquid or gaseous state) comprising at least two components, in casu the compound of Formula (I) and the water-soluble polymer, wherein one component is dispersed more or less cvcnly throughout the other component or components ( in case additional pharmaceutically acceptable formulatîng agents, generally known în the art, are included, such as plasticizers, preservatives and the like). When said dispersion of the components is such that the System is chcmically and physicaliy uniform or homogenous throughout or consists of one phase as defined in thermo-dynamics, such a solid dispersion will be called “a solid solution”. Solid solutions are preferred physical Systems because the components thcrcin arc usually rcadily bioavailablc to the organisms to which they are administered. This advantage can probably be explained by the ease with which said solid solutions can form liquid solutions when contacted with a liquid medium such as the gastro-intestinal juices. The ease of dissolution may be attributed at least in part to the fact that the energy required for dissolution of the components from a solid solution is less than that required for the dissolution of components from a crystalline or microcrystalline solid phase.

The term “a solid dispersion” also comprises dispersions which are less homogenous throughout than solid solutions. Such dispersions are not chemically and physicaliy uniform throughout or comprise more than one phase. For example, the term “a solid dispersion” also relates to a System having domains or small régions wherein amorphous, microcrystalline or crystalline compound of Formula (I), or amorphous, microcrystalline or crystalline water-soluble polymer, or both, are dispersed more or less evcnly in another phase comprising w^ater-soluble polymer, or compound of Formula (I), or a solid solution comprising compound of Formula (I) and water-soluble polymer. Said domains are régions within the solid dispersion distinctively marked by some physical feature, small in size, and evcnly and randomly distributed throughout the solid dispersion.

It may further be convenient to formulate the présent antifungal compounds în the form of nanoparticles which hâve a surface modifier ad,sorbed on the surface thereof in an amount sufïicient to maintain an effective average particle size of less than 1000 nm. Useful surface modifiers arc believed to include those which physicaliy adhéré to the surface of the antifungal agent but do not chemically bond to the antifungal agent.

-30Suitable surface modifîers can preferably be selected from known organic and inorganîc pharmaceutical excipients. Such excipients include various polymers, low molecular weight oligomers, natural products and surfactants. Preferred surface modifîers include nonionic and anionic surfactants,

Yet another interesting way of formulating the présent compounds involves a pharmaceutical composition whereby the présent antifùngals are incorporated in hydrophilic polymers and applying this mixture as a coat film over many small bcads, thus yielding a composition which can conveniently be manufactured and which is suitable for preparing pharmaceutical dosage forms for oral administration.

Said beads comprise a central, roundcd or spherical core, a coating film of a hydrophilic polymer and an antifungal agent and a seal-coating layer.

Materials suitable for use as cores in the beads are manifold, provided that said materials are pharmaceutically acceptable and hâve appropriate dimensions and fïrmness. Examples of such materials are polymers, înorganic substances, organic substances, and saccharidcs and dérivatives thereof.

It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for case of administration and uniformity of dosage. Unît dosage form as used in the spécification and claims herein refers to physically discrète units suitable as unitary dosages, each unit containing a predetermined quantity of active ingrédient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, suppositories, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.

Since the compounds according to the invention are potent orally administrable compounds, pharmaceutical compositions comprising said compounds for administration orally are especially advantageous.

The following examples illustrate the présent invention.

Experimental part

Hereinafter, the term “DCM” means dichloromethane; “MeOH” means methanol; “LCMS” means Liquid Chromatography/Mass spcctromctry; “HPLC” means highperformance liquid chromatography; “r.t.” means room température; “m.p.” means melting point; “min” means mînute(s); “h” means hour(s); “LD.” means internai diameter; “EtOAc” means ethyl acetate; “Et_?N” means tricthylaminc; “EtOH” means

-31éthanol; “eq.” means équivalent; “r.m.” means réaction mixture(s); “q.s.” quantum sufficit; “SFC” means supercritîcal fluid chromatography; “THF” means tetrahydrofuran; “HOAc” means acetic acid; “DEA” means diethylamine, “HOBt” means 1-hydroxy-1Hbcnzotriazole; “Me₂S” means dimethyl sulfide; “Pd(PPhi)₂Cl₂” means dichlorobis(triphenylphosphine)palladium; and “EDCI” means jV-(etliylcarbonimîdoyl)-7V,7V-dimetliy 1-1,3-propanedîamine monohydrochloride.

A. Préparation of the intermediates

Example Al a-1) Préparation of intermediate 1 NH

Cl

N .HCl

2-Chloro-6-(lZ7-pyrrol-l-yl)-bcnzonitrilc (50 g, 0.2467 mol) was dissolvcd in THF (500 mi). A 10.0 M solution of BH.i in MeîS (27.1 ml, 0.2714 mol) was added dropwisc to this solution at r.t. The r.m. was then stirred and refluxed for 12 h. Subsequently, the mixture was coolcd to r.t. and a 6 N HCl solution (47 ml) was added dropwisc. The r.m. was heated under reflux température for 30 min. The clear solution is cooled to 0 °C and NaOH (55.5 g) was added. The mixture was extracted with EtOAc and saturated brine. The organic layer was dried (MgSCh), filtered and the solvent was evaporated, yiclding 46.5 g of the product as a free base. Subsequently, the product was acidified by HCL/dioxane (q.s.) to obtain 55 g of intermediate 1 (97 % yield; .HCl).

a-2) Alternative procedure for the préparation of intermediate 1

A mixture of 2-Chloro-6-(lJY-pyrtol-l-yl)-benzonitrile (0.08 mol) in MeOH/NHj (250 ml) was hydrogenated at 14 °C with Raney Nickel (2 g) as a catalyst, in the presence of a 4 % thîophenc solution in MeOH (2 ml). After uptake of H₂ (2 eq.), the catalyst was filtered off and the fîltrate was evaporated. Subsequently, a 6 N 2propanol/HCl solution (14 ml) was added. Then, the solvent was evaporated, yielding a mixture of2-(l//-pyrrol-l-yO-benzenemethanamine and intermediate 1 as a HCI-salt form (.HCl). This mixture was used as such without further purification in the next reaction step.

-32Example A2

Préparation of intermediate 2

4-(l,l-difluoroethyl)-benzoic acid (5.05 g, 27.1 mmol) was dissolved in DCM (100 ml). Et₃N (10 ml, 60.9 mmol), HOBt (3.6 g, 27.1 mmol), EDCI (5.18 g, 27.1 mmol) and intermediate I (6.0 g, 24.7 mmol) were added to this solution and the r.m. was stirred ovemight. Subsequently, water (q.s.) was added and the mixture was extracted with DCM. The separated organic layer was dried (NajSOô, filtered, and the solvent was evaporated. The residue was purified by column chromatography (petrol etherZEtOAc gradient elution from 15/1 to 10/1). The product fractions were collected and the solvent was evaporated in vacuo to yield 8.2 g of intermediate 2 (88.6 % yield).

Example A3

a) Préparation of intermediate 3

A mixture of 2-amino-4,6-difluorobenzoic acid (60 g, 346 mmol), tetrahydro-2,5dimethoxyfuran (45.7 g, 346 mmol) and pyridine hydrochloride (1:1) (40 g, 346 mmol) în dîoxanc (500 ml) was heated to reflux ovemight. Subsequently, the solvent was evaporated and the residue was dissolved in EtOAc (100 ml). This solution was washed with brine and water. The organic layer was separated, dried (MgSOi), filtered and the solvent was evaporated. Yield: 70 g of crude intermediate 3, which was used as such in the next reaction step.

b) Préparation of intermediate 4

O

-33NHî.HîO (l 00 ml) was added to a solution of intermediate 3, HOBt (47 g, 346 mmol) and EDC1 (70 g, 346 mmol) in DMF (300 ml). The r.m. was stirred ovemight. Subsequently the solvent was evaporated, and the residue was dissolved in EtOAc. This organic solution was first washed with brine and water, then dried (MgSO₄) and filtered, and finally the solvent was evaporated. Yield: 55 g of crude intermediate 4, which was used as such in the next réaction step.

c) Préparation of intermediate 5

.HCl

BHj.MezS (10 M solution; 40.5 ml, 405 mmol) was added to a mixture of intermediate 4 (45 g, 202.5 mmol) in THF (500 ml). The r.m. was refluxed ovemight under Ni atmosphère. Subsequently the mixture was cooled on an ice-water bath and a 6 N HCl solution (10 ml) was added. The mixture was refluxed again for 30 minutes. The mixture was cooled on an ice-water bath and solid NaOH was added until pH > 9. The mixture was extracted with DCM (2 times 300 ml). The organic layers were separated, combined, dried (MgSOf), filtered and the solvent was evaporated. The brown residue was converted to its HCl sait form (1:1 ) with HCl/2-propanol, yielding 35 g of intermediate 5 (71 % yield).

d) Préparation of intermediate 6

4-(l,l-difluorocthyl)benzoic acid (1.17 g, 6.13 mmol) was dissolved in DCM (50 ml). Et₃N (7.3 ml), HOBt (0.828 g, 6.13 mmol), EDCI (1.17 g, 6.13 mmol) and intermediate 5 (1.5 g, 6.13 mmol) were added to the solution, and the r.m. was stirred ovemight at r.t. Subsequently, the solvent was evaporated and water was added to the residue. The aqueous mixture was extracted with DCM. The organic layer was separated, dried (Na₂SO₄), filtered and the solvent was evaporated. Yield: 1.4 g of intermediate 6 (60 %

-34Example A4

a) Préparation of intermediate 7

CN

3-acetyl-4-fluorobenzonitrile (11.0 g, 67.4 mmol) was dissolved in (diethylamino)sulfur trifluoride (25 ml) under Nï flow. The solution was heated to 50 °C for 16 h. and was then poured into a NaHCOi solution (q.s.). This mixture was extracted with EtOAc. The organic layer was separated, dried (NaîSOO, filtered and the solvent was evaporated under reduced pressure. The residue was purifîed by column chromatography over silica gel (eluent: petroleum ether/EtOAc from 30/1 to 20/1). The desired fractions were collected and the solvent was evaporated in vacuo. Yield: 10.5 g of intermediate 7 (84 % yield).

A mixture of intermediate 7 (8.2 g, 5.7 mmol) in a 20 % NaOH solution (50.0 ml) and EtOH (20.0 ml) was refluxed and stirred overnight. Subsequently, the mixture was concentrated in vacuo. HCl was added till pH 2 and a precipitate was formed, The prccipitatc was filtered off and dried in vacuo. The solid was used as such in the next reaction step. Yield: 9.2 g of intermediate 8.

A mixture of intermediate 1 (1.0 g, 4.2 mmol), intermediate 8 (1.0 g, 5.0 mmol), EtjN (5.0 ml, 36 mmol), HOBt (0.62 g) and EDCI (0.96 g) in DCM (40 ml) was stirred overnight at r.t. The resulting mixture was washed with water (3 times 100 ml), dried (MgSO₄), filtered and the solvent was evaporated under reduced pressure. The residue

«

-35was purified by column chromatography over silica gel (eluent: petroleum ether/EtOAc from 20/l toi0/1). The desired fractions were collected and the solvent was evaporated in vacuo. Yîeld: 0.9 g of intermediate 9 (59.6 % yield).

Example A5

a) Préparation of intermediate 10

Tert-butyl nitrite (25 g, 242 mmol) was added to a suspension of CuCN (21.5 g,

242 mmol) in CHjCN (500 ml). The mixture was hcatcd to 70 °C and was stirred for 15 min. Subsequently, a mixture of 2-chloro-4-fluoro-6-nitro-benzenamine (23 g,

121 mmol) and CHjCN (q.s.) was added, and the resulting brown solution was heated 10 ovemight at 70 °C. The solvent was removed in vacuo. Water (q.s.) was added to the residue and the aqueous mixture was extracted with EtOAc. The separated organic layer was dried (MgSO₄), filtered, and the solvent was evaporated. The residue was purified by column chromatography over silica gel (clucnt: EtOAc/pctrolcum ether 1/4). The desired fractions were collected and the solvent was evaporated. Yield: 10 g of intermediate 10 (41 % yield; yellow solid).

b) Préparation of intermediate 11

Fe (11.6 g, 200 mmol) was added portionwise to a solution of intermediate 10 (10 g, 50 mmol) in HOAc (250 ml). The r.m. was heated to 70 °C for 1 h, and was then cooled to r.t. The solid was filtered off and the solvent was evaporated. The residue was dissolved 20 in EtOAc, washed with aqueous NaHCO₃ and water. The organic layer was dried (MgSO₄), filtered and the solvent was evaporated. Yield: 6.2 g of intermediate 11 which was used as such in the next reaction step.

c) Préparation of intermediate 12

A solution of înteimediate 11 (6 g, 35 mmol) and tetrahydro-2,5-dimethoxy-furan (4.66 g, 35 mmol) in HOAc (50 ml) was hcated at reflux température for 6 h. Then, the solvent was evaporated and the residue was purified by column chromatography over silica gel (eluent: DCM). The desired fractions were collected and the solvent was evaporated. Yield: 4.6 g of intermediate 12 (90 % yield).

d) Préparation of intermediate 13

A 10 M solution of BHj.Me₂S (2 ml; 20 mmol) was added to a mixture of intermediate 12 (4.4 g, 20 mmol) in THF (50 ml). The r.m. was refluxed overnight under N₂ atmosphère. Subsequently, the mixture was cooled on an ice-water bath and a 6 N HCl solution (10 m!) was carefully added to the mixture. The solution was then refluxed again for 30 min, and was then cooled again on an ice-water bath. Solid NaOH was added till pH > 9. The mixture was extracted with DCM (2 times 300 ml). The separated organic layer was dried (MgSO₄), filtered and the solvent was evaporated, The brown residue was convcrtcd to the HCl sait (1:1) with HCl/2-propanol. Yield: 5 g of intermediate 13 (96 % yield).

Examplc A6

a) Préparation of intermediate 14

Reaction under N₂ atmosphère: A mixture of 4-bromo-3-fluorobenzonitrile (6,0 g, 30.00 mmol) and (l-ethoxyethenyl)tributyltin (J 1.2 ml, 33.00 mmol) in toluene (100 ml) was stirred at r.t. Pd(PPh₂)₂Cl₂ (0.72 g, 1.03 mmol) was added to the mixture, and the r.m. was heated at refluxed température and stirred overnight. The mixture was cooled and a

-372 N KF solution (50 ml) was added to the mixture while stirring. After 30 min, a 6 N HCl solution was added (100 ml) to the mixture, and the mixture was heated to 70 °C and stirred for 2 h. The insoluble substance was filtered off, and the fïltrate was extracted with EtOAc (3 times 80 ml). The combined organic layers were washed with brine, dried (MgSO₄), filtered and the solvent was evaporated in vactio. Yield: 4,3 g of intermediate 14 (88 % yield).

Intcrmcdiatc 14 can bc rcactcd further analogous to the réaction protocol described in Example A4, to obtain compound 29.

Example A7

a) Préparation of intermediate 15

A mixture of 4-chloro-2,6-difluorobcnzonitrilc (1.66 g, 9.56 mmol), pyrrolc (0.67 g, mmol) and CszCCE (3.7 g, 12 mmol) in DMF (20 ml) was stirred overnight. Then, the solid was filtered off and the solvent was removed by reduced pressure. The residue was purifîcd by column chromatography over siiica gel (clucnt: EtOAc/pctrolcum ether 10/1). The desired fractions were collected and the solvent was evaporated. Yield: i .1 g of intermediate 15 (15 % yield; whîte solid).

Intermediate 15 can be reacted further analogous to the reaction protocol described in Example Al, to obtain compound 32.

B. Préparation of the compounds

Example B1

a) Préparation of compound 1

.HCl

A mixture of intermediate 1 (9.5 g, 39.2 mmol) and 4-(l,l-difluoroethyl)-benzaldehyde (8.0 g, 47.0 mmol) in EtOH (15 ml) was stirred and refluxed for 4 h. Subsequently, the mixture was cooled and crystallized overnight. The precipitate was filtered off, washed with isopropylether, and dried in vacuo. Yield: 8.0 g of compound 1 (52.2 % yield).

-38b) Préparation of compound 2 and compound 3

.HCl

Compound 2: S enantiomer; Compound 3: R enantiorner

Compound l was separated into its enantiomers by chiral SFC (Column AD 300 mm*50 mm, 20 μΜ; Mobile phase: CO₂/MeOH(0.2 % DEA) 60/40; Flow: 200 ml/min). Two different fractions were collected, and both fractions were converted to the HCl sait form with HCl/EtOH l/l.

Yield: 3.2 g of compound 2 (20.8 % yield; S configuration).

Yield: 3.0 g of compound 3 (19.6 % yield; R configuration).

Example B2

a) Préparation of compound 4

A mixture of compound l (0.334 g, 0.0009 mol) and MnCh (2.6 g, 0.0299 mol) in DCM (15 ml) was stirred for 96 h at r.t. Subsequently, the precipitate was filtered off over diatomaceous earth and the filtrate was evaporated. The residue was purified by préparative HPLC, yiclding 0.19 g of compound 4 (59 %).

b) Alternative préparation of compound 4

Intcrmcdiate 2 (4.0 g, 10.67 mmol) was dissolvcd in POCl₃ (12 ml). The mixture was stirred and refluxed overnight. Subsequently, the mixture was cooled and poured into water. The aqueous mixture was neutralized with NaOH to pH 7, and was then extracted with DCM. The separated organic layer was dried (Na^SCL), filtered and the solvent was evaporated. The residue was crystallized from EtOH, yielding compound 4 (56.3 % yield).

-39Example_B3 aj Préparation of compound 18

F intcrmcdiatc 6 (1,4 g, 3.71 mmol) was dissolved in POCL (10 ml). The mixture was stirred and refluxed overnight. Subsequently, the mixture was cooled and poured into water. The aqueous mixture was neutralized with NaOH till pH 7, and was then cxtractcd with DCM. The organic layer was separated, dried (MgSOj, filtered and concentrated. The residue was purified by flash column chromatography over silica gel (eluent: petroieum ether/EtOAc 4/1). The product fractions were collected and the solvent was evaporated to yicld 150 mg of compound 118 (21.6 % yîcld).

Example B4

a) Préparation of compound 21

Intermediate 9 (0.80 g, 2.0 mmol) was dissolved in POCij (3 ml). The mixture was stirred overnight at 100 °C. Subsequently, the r.m. was poured out into ice water. NaOH was added to pH 8-9. The mixture was then extracted with DCM. The organic layers were separated, dried (Na₂SO₄), filtered and the solvent was evaporated in vacuo. The residue was purified by column chromatography over silica gel (eluent: petroieum ether/EtOAc from 10/1 to 5/1 ). The desired fractions were collectcd and the solvent was evaporated in vacuo. Yield: 0.510 g of compound 21 (yield 68 %).

Example B5

a) Préparation of compound 24

-40A solution of intermediate 13 (0.52 g, 2 mmol) and 4-(l,l-difluoroethyI)benzaldehyde (0.374 g, 2 mmol) in EtOH (5 ml) was heated to reflux for 4 h. Subsequently, the mixture was cooled to room température and the solid was collected and dried. Yield: 0.6 g of compound 24 (80 % yield; .HCl).

By using analogous reaction protocole as described in the foregoing examples, the following compounds hâve been prepared. ‘Co. No? means compound number. ‘Pr? refers to the Example number according to which protocol the compound was synthesized. In case no sait form is indicated, the compound was obtained as a free base. Compounds wherein R³ and R⁴ are hydrogen, and for which no spécifie stereochemistry is indicated in Table I, were obtained as mixtures of enantiomers.

In case a final compound obtained by using an analogous protocol as described in B1 or B5, was not fully converted to the HCl sait form, the compound was converted to the HCi sait form by using procedures known to those skilled în the art. In a typical procedure, the compound was dissolved in a solvent such as, for cxamplc, 2-propanol, and subsequently a HCl solution în a solvent such as, for example, 2-propanol was added drop wise. Stirring for a certain period of time, typically about 10 minutes, could enhancc the rate of the reactions.

Table 1:

(D

Co. No.	Pr.	R1	R2	R3	R4	R5	R6	Sait Form / Optical rotation (OR)/ Stereochemistry
1	Bl.a	7-C1	H	H	H	cf2ch3	H	.HCl
2	Bl.b	7-C1	H	H	H	cf2ch3	H	.HCl OR:-172.64° (589 nm; 20 °C; 0.3684 w/v %; MeOH) S-confïguration
3	Bl.b	7-C1	H	H	H	cf2ch3	H	.HCl

Co. No.	Pr.	R1	R2	R3	RJ	Rs	R6	Sait Form / Optical rotation (OR)/ Stereochemistry
								OR:+168.90° (589 mn; 20 °C; 0.3434 w/v %; MeOH) R-configuration
4	B2.a/ B2.b	7-CI	H	bond	CF2CH3	H
5	Bl.a	8-C1	10-C1	H	H	CFjCHj	H	.HCl
6	B2.a	8-C1	10-C1	bond	CF2CH3	H
7	Bl.a	7-CI	8-C1	H	H	cf2ch3	H	.HCl
8	B2,a	7-CI	J0-C1	bond	CF2CHi	H
9	B2.a	7-C1	9-Ci	bond	cf2ch3	H
10	B2.a	7-CI	8-C1	bond	cf2ch3	H
11	B5	7-CJ	10-CI	H	H	cf2ch3	H	.HCl
12	Bl.a	7-CI	H	H	H	H	cf2ch3	.HCl
13	B5	7-CJ	9-C1	H	H	cf2ch3	H	.HCl
14	B2,a	7-CI	H	bond	H	cf2ch3
15	B2.a	7-F	H	bond	cf2ch3	H
16	B3	9-C!	7-CH3	bond	cf2ch3	H
17	B3	9-C1	H	bond	cf2ch3	H
18	B3	7-F	9-F	bond	cf2ch3	H
19	B3	10-C1	H	bond	cf2ch3	H
20	B4	7-F	H	bond	F	CF2CH3
21	B4	7-CI	H	bond	F	cf2ch3
22	B4	9-C1	10-C1	bond	cf2ch3	H
23	B4	7-F	9-F	bond	F	CF2CH3
24	B5	7-CI	9-F	H	H	cf2ch3	H	.HCl

ï

Co. No.	Pi.	R1	R2	r'	R4	Rs	R6	Sait Form / Optical rotation (OR)/ Stereochemîstry
25	B4	7-C1	9-C1	bond	F	cf2ch3
26	B4	7-C1	9-F	bond	F	cf2ch3
27	B2.a	7-C1	9-F	bond	CF2CH3	H
28	B4	10-Cl	7-CH;	bond	cf2ch3	H
29	B4	7-C1	H	bond	cf2ch3	F
30	B4	7-F	H	bond	cf2ch3	F
31	B4	7-C1	9-F	bond	cf2ch3	F
32	B4	7-F	9-C1	bond	cf2ch3	H
33	B4	7-F	9-F	bond	cf2ch3	F
34	B4	9-Ci	H	bond	F	cf2ch3
35	B4	H	10-CI	bond	F	cf2ch3

C. Analytical results

LCMS

The HPLC measurement was performed using an Agilent 1100 module comprising a pump, a diodc-array dctector (DAD) (wavclcngth used 220 nm), a column heater and a column as specified in the respective methods below. Flow from the column was splît to a Agilent MSD Sériés G1946C and G1956A. MS detector was configured with API-ES (atmospheric pressure clcctrospray ionization). Mass spcctra were acquired by scannîng from 100 to 1000. The capillary needle voltage was 2500 V for positive and 3000 V for négative ionization mode. Fragmentation voltage was 50 V. Drying gas température was maintaincd at 350 °C at a flow of 10 1/mm. Rcvcrscd phase HPLC was carried out on a YMC-Pack ODS-AQ, 50x2.0 mm 5 pm column with a flow rate of 0.8 ml/mîn. 2 mobile phases (mobile phase A: water with 0.1 % TFA; mobile phase B: CHjCN with 0.05 % TFA) were used. First, 90 % A and 10 % B was hold for 0.8 min. Then a gradient was applied to 20 % A and 80 % B in 3.7 min and hold for 3 min. Typical injection volumes of 2 μΐ were used. Oven température was 50 °C. (MS polarity: positive)

Melting Points

For a number of compounds, melting points (m,p.) were determmed with a WRS-2A

I

melting point apparatus purchased from Shanghai Précision and Scientific Instrument Co. Ltd. Melting points were measured with a linear heating up rate of 0.2-5.0 °C/min The reported values are melt ranges. The maximum température was 300 °C.

The results of the analytical measurements are shown in table 2.

Table 2: Rétention time (R_t) in min., [M+H]~ peak (protonated molécule), and m.p. (melting point in °C). (“n.d.” means not determined; “dcc” means decomposed).

Co. No.	R(	[M+H]+	m.p. (°C)
1	3.57	359	n.d.
2	3.38	359	206.7-207.6
3	3.33	359	206.3-206.8
4	3.59	357	215.2-217.6
5	4.00	393	n.d.
6	4.03	391	132.1-134.2
7	3.95	393	253.5-254.2
8	3.99	391	162.3-163.5
9	4.03	391	n.d.
10	4.03	393	n.d.
11	4.04	393	188.5-191.1
12	n.d.	n.d.	n.d.
13	4.11	393	233.9-235.0
14	3.51	357	106.9-109.7
15	3.27	341	n.d.
16	3.97	371	89.6-91.2
17	3.53	357	115.3 dcc
18	3.31	359	n.d.

Co. No.	K	[M+Hf	m.p. (°C)
19	3.52	357	247.5 dec
20	3.46	359	n.d.
21	3.56	375	n.d.
22	3.84	391	140.9-142.1
23	3.48	377	143.2-144.3
24	3.53	377	240.0-240.7
25	3.77	409	124.2-125.4
26	3.53	393	84.9-85.9
27	3.84	375	n.d.
28	3.71	371	139.3-141.2
29	3.44	375	186.3-186.6
30	3.25	359	170.8-171.5
31	3.44	393	162.5-164.8
32	3.31	375	145.3-J47.4
33	3.23	377	158.2-161.3
34	3.95	375	n.d.
35	3.97	375	n.d.

’HNMR

For a number of compounds, *H NMR spectra were recorded on a Bruker DPX-300, or 10 on a Bruker DPX-400 spectrometer with standard puise sequences, operating at 300

MHz and 400 MHz respective ly, using CHLOROFORM-tf (deuterated chloroform, CDCl_?) or DMSO-dû (deuterated DMSO, dimethyl-d6 sulfoxide) as solvents. Chemical shifts (Ô) are reported in parts per million (ppm) relative to tetramethylsilane (TMS), which was used as internai standard.

Co. No. I: (300 MHz, DMSO-c/fi) δ ppm 2.01 (t, J=\8.9 Hz, 3 H) 3.87 (d, J=14.3 Hz, 1 H) 4.57 (d, .7=13.9 Hz, 1 H) 5.35 (br. s„ 1 H) 5.84 (br. s., 1 H) 6.30 (t, .7=3.2 Hz, 1 H) 7.43 (br. s., 1 H) 7.57-7.77 (m, 5 H) 7.90 (d, .7=8.1 Hz, 2 H) 10.45 (br. s., 2 H).

Co. No. 2; (300 MHz, DMSO-</_e) δ ppm 2.01 (t, 7=18.93 Hz, 3 H) 3.88 (d, 7=14.32 Hz, 1 H) 4.58 (d, 7=13.94 Hz, 1 H) 5.35 (br. s., 1 H) 5.84 (br. s., 1 H) 6.31 (t, 7=3.20 Hz, 1 H) 7.44 (br. s., 1 H) 7.57-7.78 (m, 5 H) 7.90 (d, 7=8.10 Hz, 2 H) 10.45 (br. s„ 2

-44H).

Co. No. 3: (300 MHz, DMSO-î/₆) δ ppm 2.01 (t, 7=18.93 Hz, 3 H) 3.88 (d, 7=14.13 Hz, 1 H) 4.58 (d,>13.94 Hz, 1 H) 5.35 (br. s., 1 H) 5.84 (d,>2.64 Hz, 1 H) 6.31 (t, >3.20 Hz, 1 H) 7.43 (dd, 7=2.64, 1.51 Hz, 1 H) 7.56-7.79 (m, 5 H) 7.90 (d, >7.91 Hz, 2 H) 10.40 (br. s., 2 H).

Co. No. 4: (300 MHz, CDC10 δ ppm 1.84 (t, >18.08 Hz, 3 H) 4.13 (br. s., 1 H) 5.44 (br. s., 1 H) 6.25-6.54 (m, 2 H) 7.19-7.24 (m, 2 H) 7.25-7.35 (m, 2 H) 7.42 (m, >8.29 Hz, 2 H) 7.69 (m,7=8.10 Hz, 2 H).

Co. No. 5: (400 MHz, DMSO-J₆) δ ppm 2.01 (t, >18.97 Hz, 3 H) 3.62 (d, >13.32 Hz, 1 H) 4.39 (d, >13.32 Hz, 1 H) 5.41 (br. s., 1 H) 5.88 (d, >2.83 Hz, 1 H) 6.30 (t, >3.23 Hz, 1 H) 7.40 (dd, 7=3.03, 1.41 Hz, 1 H) 7.70 (m, 7=8.07 Hz, 2 H) 7.80 (d, >2.42 Hz, 1 H) 7.91 (m, 7=8.07 Hz, 2 H) 8.07 (d, >2.42 Hz, 1 H) 10.26 (br. s., 1 H) 10.66 (br. s., 1 H).

Co. No. 6: (400 MHz, CDCR) δ ppm 1.93 (t, >18.1 Hz, 3 H) 4.28 (d, >11.0 Hz, 1 H) 4.87 (d, 7=11.0 Hz, 1 H) 6.47 (br. s., 1 H) 6.57 (br. s., 1 H) 7.41-7.66 (m, 5 H) 7.88 (d, >7.8 Hz, 2 H).

Co. No. 8: (400 MHz, CDCh) δ ppm 1.92 (t, >18.2 Hz, 3 H) 4.08 (d, >11.3 Hz, 1 H) 5.46 (d, 7=11.3 Hz, 1 H) 6.38-6.46 (ni, 1 H) 6.51 (d, >2.5 Hz, 1 H) 7.34 (d, 7=8.5 Hz, 1 H) 7.39 (d, 7=8.5 Hz, 1 H) 7.46-7.58 (m, 3 H) 7.87 (d, 7=8.3 Hz, 2 H).

Co.No. 9: (400 MHz, CDClj) δ ppm 1.92 (t, 7=18.2 Hz, 3 H) 4,16 (br. s., 1 H) 5.49 (br. s., 1 H) 6.38-6.57 (m, 2 H) 7.30 (d, 7=2.0 Hz, 1 H) 7.34 (br. s., 1 H) 7.41 (d, 7=1.8 Hz, 1 H) 7.51 (m, 7=8.3 Hz, 2 H) 7.76 (m, 7=8.3 Hz, 2 H).

Co.No. 10: (400 MHz, CDC1₃) δ ppm 1.95 (t, 7=18.3 Hz, 3 H) 4.46 (br. s., 1 H) 5.82 (br.s., 1 H) 6.80 (dd, 7=4.3, 2.8 Hz, 1 H) 7.04 (dd, 7=4.3, 1.5 Hz, 1 H) 7.29 (d, 7=8.8 Hz, 1 H) 7.66 (d, 7=8.5 Hz, 1 H) 7.69 (d, 7=8.3 Hz, 2 H) 7.79-7.85 (m, 1 H) 7.90 (d, 7=8.3 Hz, 2 H).

Co. No. 11 : (300 MHz, DMSO-7₆) δ ppm 2.01 (t, 7=18.8 Hz, 3 H) 3.59 (d, 7=14.3 Hz, 1 H) 4.57 (d, 7=14.3 Hz, 1 H) 5.44 (br. s., 1 H) 5.91 (br.s., 1 H) 6.32 (t, 7=3.2 Hz, 1 H) 7.42 (br. s., 1 H) 7.65-7.79 (m, 3 H) 7.83 (d, 7=7.9 Hz, 2 H) 7.89 (d, 7=8.7 Hz, 1 H).

Co. No. 13: (300 MHz, DMSO-7₆) δ ppm 2.01 (t, 7=19.0 Hz, 3 H) 3.83 (d, 7=14.3 Hz, 1 H) 4.53 (d, 7=13.9 Hz, 1 H) 5.42 (br. s., I H) 5.83 (br. s., 1 H) 6.30 (t, 7=3.0 Hz, 1 H) 7.49 (br. s., 1 H) 7.70 (d, 7=7.9 Hz, 2 H) 7.77-7.94 (m, 4 H) 10.42 (br. s., 2 H).

Co. No. 14: (300 MHz, CDCh) Ô ppm 1.90 (t, 7=18.1 Hz, 3 H) 4.17 (br. s., 1 H) 5.50 (br. s., 1 H) 6.43-6.55 (m, 2 H) 7.22-7.30 (m, 2 H) 7.30-7.45 (m, 3 H) 7.56 (d, 7=7.5 Hz, 1 H) 7.76 (d, 7=7.9 Hz, 1 H) 7.84 (s, 1 H).

-45Co. No. ï5: (400 MHz, CDClj) δ ppm l .92 (t, >18.2 Hz, 3 H) 4.74 (s, 2 H) 6.43-6.51 (m, 1 H) 6.53 (dd, >3.8, 1.5 Hz, 1 H) 7.09 (t, >8.3 Hz, 1 H) 7.18 (d, >8.3 Hz, 1 H) 7.35 (td, >8.1, 5.9 Hz, 1 H) 7.39-7.43 (m, 1 H) 7.52 (m,>8.3 Hz, 2 H) 7.78 (m, >8.3 Hz, 2 H).

Co. No. 16: (300 MHz, CDCh) Ô ppm 1.93 (t, >18.18 Hz, 3 H) 2.60 (s, 3 H) 4.13 (br. s., 1 H) 5.17 (br. s., 1 H) 6.42-6.53 (m, 2 H) 7.20 (d, >1.88 Hz, 1 H) 7.24 (d, >1.88 Hz, 1 H) 7.33-7.40 (m, 1 H) 7.52 (m, >8.29 Hz, 2 H) 7.75 (m, >8.29 Hz, 2 H).

Co. No. 18: (400 MHz, CDCb) 5 ppm 1.92 (t, >18.1 Hz, 3 H) 4.69 (br. s., 2 H) 6.456.49 (m, I H) 6.49-6.53 (m, 1 H) 6.84 (td, >9.0, 2.4 Hz, 1 H) 6.95 (dt, >9.0, 1.9 Hz, 1 H) 7.33 (dd, >2.8, 1.5 Hz, 1 H) 7.51 (m,>8.3 Hz, 2 H) 7.75 (m, >8.3 Hz, 2 H). Co. No. 20; (400 MHz, CDCl₃) δ ppm 2.00 (t, >18.6 Hz, 3 H) 4.77 (br. s., 2 H) 6.53 (br. s., 1 H) 6.59 (br. s., 1 H) 7.06-7.26 (m, 3 H) 7.33-7.44 (m, 1 H) 7.47 (br. s., 1 H) 7.90 (br. s., 1 H) 7.95 (d, 7=6.5 Hz, 1 H).

Co. No. 21: (300 MHz, CDCb) δ ppm 2.01 (t, >18.6 Hz, 3 H) 4.24 (br. s„ 1 H) 5.62 (br. s., 1 H) 6.50-6.79 (m, 2 H) 7.16-7.33 (m, 2 H) 7.38 (t, >7.9 Hz, 1 H) 7.43-7.62 (m, 2 H) 7.85-8.15 (m, 2 H).

Co. No. 22: (400 MHz, CDC 1₃) δ ppm 1.92(1, >18.1 Hz, 3 H) 4,24 (d, >11.3 Hz, 1 H) 4.82 (d, >11.3 Hz, 1 H) 6.39-6.46 (m, 1 H) 6.49 (dd, >3.9, 1.4 Hz, 1 H) 7.37 (d, >8.0 Hz, 1 H) 7.44 (d,>8.3 Hz, 1 H) 7.51 (m,>8.3 Hz, 2 H) 7.55 (dd,>2.8, 1.5 Hz, I H) 7.83 (m,>8.5 Hz, 2 H).

Co. No. 23: (400 MHz, CDCk) δ ppm 2.00 (t, >18.6 Hz, 3 H) 5.03 (br. s., 2 H) 6.456.52 (m, 2 H) 6.85 (td, >9.0, 2.4 Hz, 1 H) 6.95 (d, >9.3 Hz, 1 H) 7.07-7.18 (m, 1 H) 7.33 (t, >2.1 Hz, 1 H) 7.69-7.81 (m, 1 H) 7.92 (dd, >7.3, 2.0 Hz, 1 H).

Co. No. 24: (400 MHz, DMSO-t/₆) δ ppm 2.01 (t, >18.97 Hz, 3 H) 3.83 (d, >14.13 Hz, 1 H) 4.54 (d, >14.13 Hz, 1 H) 5.41 (br. s., 1 H) 5.85 (br. s., 1 H) 6.32 (t, >3.43 Hz, 1 H) 7.46 (br. s., 1 H) 7.65 (dd, >9.28, 2.42 Hz, 1 H) 7.67-7.74 (m, 3 H) 7.85 (d, >807.00 Hz, 2 H) 10.15 (br. s„ 1 H) 10.44 (br. s., 1 H).

Co. No. 25: (400 MHz, CDCb) δ ppm 1.99 (t, >18.4 Hz, 3 H) 4.14 (br. s., 1 H) 5.45 (br.s., 1 H) 6.44-6.55 (m, 2 H) 7.05-7.17 (m, 1 H) 7.30 (d, >2.0 Hz, 1 H) 7.34 (t, ./=2.3 Hz, 1 H) 7.41 (d, >2.0 Hz, 1 H) 7.71-7.83 (m, 1 H) 7.91 (dd,>7.3, 2.3 Hz, 1 H).

Co. No. 26: (400 MHz, CDClj) δ ppm 1.99 (t,>18.3 Hz, 3 H) 4.12 (br. s., 1 H) 5.44 (br. s., 1 H) 6.42-6.55 (m, 2 H) 7.05 (dd,>8.9, 2.4 Hz, 1 H) 7.09-7.20 (m, 2 H) 7.33 (t, .7=2.1 Hz, 1 H) 7.72-7.83 (m, 1 H) 7.92 (dd, ./=7.3, 2.3 Hz, 1 H).

Co. No. 27; (400 MHz, CDCh) δ ppm 1.92 (t, >18.2 Hz, 3 H) 4.16 (br. s., 1 H) 5.47 (br.s., 1 H) 6.42-6.55 (m, 2 H) 7.04 (dd, >8.9, 2.4 Hz, 1 H) 7.16 (dd, >8.3, 2.5 Hz, 1 H) 7.29-7.36 (m, 1 H) 7.51 (m, >8.3 Hz, 2 H) 7.76 (m, >8.0 Hz, 2 H).

-46Co. No. 28: (300 MHz, CDCI₃) δ ppm l.9l (t, 7=18.2 Hz, 3 H) 2.59 (s, 3 H) 4.04 (d, J=ll.l Hz, 1 H) 5.11 (d, J=\ 1.1 Hz, 1 H) 6.33-6.44 (m, 1 H) 6.44-6.53 (m, l H) 7.12 (d, 7=8.3 Hz, 1 H) 7.33 (d, 7=8.1 Hz, 1 H) 7.45-7.58 (m, 3 H) 7.84 (d, 7=8.1 Hz, 2 H). Co. No. 29: (300 MHz, CDClj) δ ppm 2.00 (t, 7=18.9 Hz, 3 H) 4.20 (br. s., 1 H) 5.53 (br. s., 1 H) 6.42-6.50 (m, 1 H) 6.50-6.56 (m, 1 H) 7.28-7.35 (m, 2 H) 7.37 (dd, ,7=2.8, 1.7 Hz, 1 H) 7.38-7.43 (m, 1 H) 7.46-7.63 (m, 3 H).

Co. No. 30: (400 MHz, CDClj) δ ppm 2.00 (t, 7=18.6 Hz, 3 H) 4.73 (br. s., 2 H) 6.426.49 (m, 1 H) 6.50-6.57 (m, 1 H) 7.09 (t, >8.5 Hz, 1 H) 7.18 (d, >8.0 Hz, 1 H) 7.31-

7.41 (m, 2 H) 7.47-7.61 (m, 3 H).

Co. No. 31: (400 MHz, CDClj) Ôppm 2.00 (t, >18.4 Hz, 3 H) 4.14 (br. s„ 1 H) 5.48 (br. s., 1 H) 6.44-6.51 (m, 1 H) 6.51-6.59 (m, 1 H) 7.04 (dd, >8.9,2.4 Hz, 1 H) 7.17 (dd, >8.3, 2.3 Hz, I H) 7.34 (dd, >2.8, 1.5 Hz, 1 H) 7.54 (d, >3.3 Hz, 3 H).

Co. No. 32: (300 MHz, CDClj) δ ppm 1.92 (t, >18.1 Hz, 3 H) 4.70 (br. s., 2 H) 6.436.55 (m, 2 H) 7.11 (dd, >8.6, 1.8 Hz, 1 H) 7.18-7.23 (m, 1 H) 7.31-7.39 (m, 1 H) 7.51 (m, >8.3 Hz, 2 H) 7.74 (m, >8.1 Hz, 2 H).

Co. No. 33: (400 MHz, DMSO-7₆) δ ppm 2.02 (t, 7=] 8.97 Hz, 3 H) 4.57 (br. s., 2 H) 6.55 (t, 7=3.23 Hz, 1 H) 6.59 (dd, 7=3.84, 1.41 Hz, 1 H) 7.36 (td, 7=9.49, 2.42 Hz, 1 H) 7.40-7.48 (m, 1 H) 7.51-7.58 (m, 2 H) 7.62 (t, 7=7.87 Hz, 1 H) 7.81 (dd, 7=2.62,

1.41 Hz, 1 H).

Co. No. 34: (400 MHz, CDCh) δ ppm 1.88-2.10 (m, 3 H) 4.42 (br. s., 1 H) 4.78 (br. s., 1 H) 6.45-6.50 (m, 2 H) 7.12 (dd, 7=10.0, 8.8 Hz, 1 H) 7.29 (dd, 7=8.0, 2.0 Hz, 1 H)

7.36 (t, 7=2.3 Hz, 1 H) 7.40 (d, 7=2.0 Hz, 1 H) 7.42 (d, 7=8.0 Hz, 1 H) 7.69-7.79 (m, 1 H) 7.93 (dd, 7=7.4, 2.1 Hz, 1 H).

Co. No. 35: (400 MHz, CDCh) δ ppm 1.93-2.06 (m, 3 H) 4.27 (d, 7=11.3 Hz, 1 H) 4.86 (d,7=11.0 Hz, I H) 6.39-6.45 (m, 1 H) 6.49 (dd,7=3.6, 1.4 Hz, 1 H) 7.13(dd, 7=10.2, 8.9 Hz, 1 H) 7.24 (t, 7=8.0 Hz, 1 H) 7.41-7.46 (m, 1 H) 7.48 (dd, 7=8.2, 1.4 Hz, 1 H) 7.59 (dd, 7=2.6,1.4 Hz, 1 H) 7.79-7.89 (m, 1 H) 8.03 (dd, 7=7.3, 2.3 Hz, 1 H).

D, Pharmacological examples

Examplc D, 1 : Measurement of antlfungal activity in vitro

The standard susceptîbîlity screen was performed in 96-well plates (U-bottom, Greiner Bio-One). Serial dilutions (2-fold or 4-fold) of 20 mM compound stock solutions were 5 made în 100 % DMSO, followcd by an intermediate dilution step in water. These serial dilutions (10 μΐ) were then spotted onto test-plates that could be stored in the dark at 4°C for a maximum period of 2 weeks. An adéquate broad dose-range was included with 64 μΜ as the highest in-test concentration. The culture medium RPMI-1640 was

-47supplemented with L-glutamine, 2% glucose and buffered with 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.0 ±0.1.

The different fungal species/isolates (Table 3a) were cryopreserved and 1/1000 diluted in medium just prior to use. A standard inoculum of 200 μΐ containing 10³ colony5 formîng unit (cfu) was then added to each well. A positive control (100 % growth = fungal culture without antifungal) and a négative control (0 % growth = RPM1-MOPS medium) wcrc includcd on each plate. Optimal incubation time and température wcrc dépendent on the fungal species and vary from 24 h for yeasts (37 °C) to one week or more for dermatophytes (27 °C). Inhibition of fungal growth was measured after adding 10 10 μΐ of 0.005% (w/v) resazurin (Sigma Aldrich) to cach well, based on the principlc that livîng cells convert the non-fluorescent blue resazurin into the pink and fluorescent resorufin, allowing fluorimetric reading (λ^_χ 550 nm and λ™ 590 nm) after an additional incubation period (‘rcsa’ time mentioned in Table 3a). Results arc shown in Table 3b as pIC_;o values.

Table 3a: Incubation conditions for the different fungal species. ‘Resa time’ represents the additional incubation time after the addition of resazurin to the test system.

Species	Température (°C)	Time	Resa time
Microsporum canis	27	9 days	24 hours
Trichophyton mentagrophytes	27	7 days	24 hours
Trichophyton rubrum	27	7 days	24 hours
Scedosporium apiospermum	37	48 hours	17 hours
Scedosporium prolificans	37	48 hours	17 hours
Sporothrix schenkii	27	4 days	24 hours
Aspergillus fumigatus	27	48 hours	17 hours
Candida parapsilosis	37	24 hours	4 hours
Cryptococcus neoformans	37	24 hours	4 hours
Rhizopus oryzae	37	24 hours	6 hours
Rhizomucor miehei	37	48 hours	17 hours
Mucor circinelloides	27	48 hours	17 hours

Table 3b: Activities of the test compounds in vitro (‘n.d.’ means not determined; ‘Inf.’ means infection; values are pICso values)

Inf. ‘A’: Sporothrix schenkii B62482

Inf. ‘B’: Microsporum canis B68128

Inf. ‘C’: Trichophyton rubrum B68783

Inf. ‘D’: Candida parapsilosis B66126

Inf. Έ’: Aspergillus fumigatus B42928

Inf. ‘F’: Cryptococcus neoformans

Inf. ‘G’: Trichophyton mentagrophytes B70554

Inf. ΤΓ: Scedosporium apiospermum IHEM3817

Inf. T’: Scedosporium protificans IHEM21157

Inf. ‘J’ : Rhizopus oryzae IHEM5223

Inf. ‘K’: Rhizomucor miehei IHEM13391

Inf. ‘L’: Mucor circinelloides 1HEM21105

Co. No.	Inf. A	Inf. B	Inf. C	Inf. D	Inf. E	Inf. F	Inf. G	Inf. H	Inf. I	Inf. J	Inf. K
1	4.68	6.45	6.91	5.70	6.72	5.30	6.89	<4.19	n.d.	<4.19	<4.19
5	<4.19	<4.19	4.22	<4.19	<4.19	4.46	<4.19	n.d.	n.d.	n.d.	n.d.
6	5.58	4.76	5.23	<4.19	6.03	5.85	5.61	<4.19	n.d.	<4.19	<4.19
7	<4.19	5.40	5.40	<4.19	<4.19	<4.19	<4.19	n.d.	n.d.	n.d.	n.d.
8	<4.19	5.40	5.52	<4.19	<4.19	<4.19	4.22	n.d.	n.d.	n.d.	n.d.
9	4.96	5.40	6.00	<4.19	5.10	6.06	4.89	5.02	5.22	<4.19	<4.19
10	<4.19	<4.19	<4.19	<4.19	<4.19	<4.19	<4.19	n.d.	n.d.	n.d.	n.d.
11	<4.19	<4.19	4.86	<4.19	<4.19	<4.19	<4.19	n.d.	n.d.	n.d.	n.d.
13	<4.19	4.23	5.70	<4.19	4.81	<4.19	5.14	n.d.	n.d.	n.d.	n.d.
4	4.92	6.32	6.63	5.51	5.65	4.67	6.05	<4.19	6.83	<4.19	<4.19
14	4.36	6.28	6.43	5.52	5.70	4.43	6.22	5.76	5.88	<4.19	4.34
15	<4.19	6.31	7.37	6.20	6.65	<4.19	6.60	<4.19	5.60	4.47	<4.19
2	<4.19	5.09	5.82	<4.19	<4.19	<4.19	5.43	n.d.	n.d.	n.d.	n.d.
3	<4.19	6.30	6.96	5.58	5.70	5.26	6.36	5.13	6.15	<4.19	4.59
16	5.42	5.70	6.21	4.97	5.77	6.14	5.78	4.99	5.92	<4.19	<4.19
17	5.25	5.58	6.34	5.61	6.26	6.49	5.79	<4.19	6.12	5.08	<4.19
18	5.09	5.67	6.83	5.63	6.30	5.79	5.96	<4.19	6.10	<4.19	4.30
19	5.55	5.69	6.67	4.88	5.69	5.79	6.19	5.63	6.29	5.01	4.84
20	<4.19	6.29	6.84	4.89	6.95	<4.19	6.10	6.24	6.80	<4.19	4.57
21	5.43	6.32	6.71	5.61	6.90	5.80	6.16	7.05	7.30	<4.19	5.18
22	<4.19	5.11	5.11	<4.19	<4.19	<4.19	<4.19	n.d.	n.d.	n.d.	n.d.
23	5.02	6.38	6.89	5.44	6.27	<4.19	6.10	6.74	7.00	<4.19	<4.19
24	<4.19	5.11	6.21	5.58	4.94	<4.19	5.77	n.d.	n.d.	n.d.	n.d.
25	4.85	5.80	5.90	<4.19	5.99	<4.19	5.66	6.39	6.30	<4.19	<4.19
26	4.99	6.33	6.33	5.42	6.23	<4.19	5.92	6.80	6.72	<4.19	4.87
27	5.61	6.29	6.54	5.74	6.30	5.99	5.82	6.36	7.00	<4.19	5.08
28	4.55	4.85	5.13	4.34	4.19	4.78	4.57	6.15	5.64	<4.19	4.38
29	<4.19	6.96	7.07	<4.19	6.30	<4.19	6.72	<4.19	<4.19	<4.19	<4.19
30	<4.19	6.33	6.79	5.11	6.30	5.85	6.44	5.05	5.34	<4.19	4.91
31	5.63	6.36	6.33	5.46	6.30	6.53	5.90	4.98	5.06	<4.19	<4.19
32	5.21	5.78	5.78	5.56	5.70	5.89	5.78	<4.19	<4.19	<4.19	<4.19
33	5.03	5.71	6.14	4.87	5.72	5.58	5.67	6.19	4.98	<4.19	<4.19
34	5.10	5.09	5.10	5.53	5.10	5.24	5.09	n.d.	n.d.	n.d.	n.d.
35	5.37	6.28	6.52	4.83	5.84	6.28	5.80	n.d.	n.d.	n.d.	n.d.
12	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.

The pIC₅o va ues for Inf. ‘L’ were determined for compounds 1,3,4, 6, 9, 14-21, 23, 25-33, and were <4,19.

Example D.2 : Solubility ^CUPLC’ means_Ultra Performance Liquid Chromatography; ‘HPBCD’ means 2hydroxypropyl-p-cyclodcxtrin.

-49An amount of the compound to be tested, typically between 2 mg and 4 mg, was accurately weighed using a microbalance and added to 0.5 ml of one of the buffer Systems below in a 20 ml clear glass vial:

A) 0.01 N HCl

B) buffer pH 4: phosphate (0.2 M) - citrate mixture (0.1 N) in accordance to Mc Ilvaine’s buffer solutions)

C) buffer pH 4, containing 10 % (w/v) of HPBCD

D) buffer pH 4, containing 20 % (w/v) of HPBCD

E) buffer pH 7.4: phosphate (0.2 M) - citrate mixture (0.1 N) in accordance to Mc Ilvaine’s buffer solutions)

F) buffer pH 7.4, containing 10 % (w/v) of HPBCD

G) buffer pH 7.4, containing 20 % (w/v) of HPBCD

A magnetic stirring bar was added to the mixtures. The mixtures were stirred at ambient température for at least 2 hours and then visually inspected.

If the compound was dissolved, the resuit was reported (twice the weighed amount of compound/ml).

If the compound was not dissolved, 0.5 ml of extra solvent was added to the suspension. The mixtures were stirred at ambient température for at least another 2 hours, followed by inspection.

If the compound was dissolved, the result was reported (weighed amount of compound/ml).

If the compound was not dissolved, 1 ml of extra solvent is pipetted into the suspension. The mixtures were shaken (Edmund Buhler SM25 175 SPM) at ambient température ovemight and inspected afterwards.

If the compound was dissolved, the resuit was reported (half the weighed amount of compound/ml).

If the compound was not dissolved, the suspension was filtered over a filter dise. An aliquot of the filtrate was diluted with an appropriate solvent (0.1 N HCl/acetonitrile 1/1) and submîttcd for concentration mcasurcmcnt (mg/ml) using a gcncric UPLC method.

-50Table4:

	p-o Cl Compound 23 of WO02/34752	\/F fp Cl Compound 17 of the présent invention
Buffer System	UPLC (mg/ml)	UPLC (mg/ml)
A	1.878	7.10>X>3.55
B	<0.001	0.087
C	0.048	1.229
D	1.49	n.d.
E	<0.001	<0.001
F	0.035	0.191
G	0.11	0.603

Example D.3 : In vivo Pharmacokinetic déterminations (bioavailability)

Three animais (mean weight 20 ± 7 g for mice and 275 ± 20 for Guinea pigs) were used 5 per dose route, time point and formulation.

For the oral (PO) solution formulation, compound was dissolved in a 20 % hydroxypropyl-P-cyclodextrîn (ΗΡ-β-CD) solution at a final concentration of l mg/ml. HCl was added to facilitate dissolution. After dissolution, the pH was brought up to 3.7 with NaOH. Maunitol was added to makc the solution isotonie. For the PO suspension 10 formulation, compound was suspended in 0.5 % methocel at a final concentration of I mg/ml. The formulations were stored at room température, protected from lîght and analysed quantîtatively with LC-MS/MS on the day of préparation. Stability of the formulations was checked on the day of dosing.

The animais were dosed orally by gastric intubation at 10 ml/kg to obtain a final dose 10 15 mg/kg. Three dosed animais were sacrificed for blood sampling at 15 and 30 min, 1, 2,

3, 4, 6, 8 and 24 h after PO dose administration as a solution and at 30 min, 1, 2, 4, 6, 8 and 24 h after PO dose administration as a suspension.

Blood was collected by multiple sampling from a tail vein into Multivette® 600 K3E tubes (Sarstcdt). Samplcs were placcd immcdiatcly at approximately 4 °C and plasma 20 was obtained following centrifugation at 4 °C for 10 minutes at approximately 1900 x g.

Ail samples were shielded from daylight and stored at < -18 °C prior to analysis.

-51Plasma samples were analysed for dosed compound using a qualifîed research LCMS/MS method. The key anaiytical performance (linearity, upper and lower lirnit of quantification, accuracy and précision) of the method was reported together with the plasma concentrations.

A limited pharmacokinetic analysis was pcrformed using WÎnNonlin™ Professional (Version 5.2.l). A non-compartmental analysis using the lin/log trapézoïdal rule with lin/log interpolation was used for all data.

Table 5:

Cl J	Cl I
	f=\ F
Γ Ά υΤχρ		— F
	o
Compound 22 of WO02/34752	Compound 4 of the présent application
Bioavailability in mice: 11 % (PO 10 mg/kg;	Bioavailability in mice: 20 % (PO 10 mg/kg;
IV 2.5 mg/kg)	IV 2.5 mg/kg)
Bioavailability in guinea pigs: 2.7 %(PO 40	Bioavailability in guinea pigs: 35 % (PO 10
mg/kg; IV 2.5 mg/kg)	mg/kg; IV l .25 mg/kg)

H3C 3 \ ^ch3 r 11 CH3 -A ch3 Terbinafine	Cl
JXJCH Compound 9 of the présent applica	—F dion
Bioavailability in mice: 19 % (PO 10 mg/kg;	Bioavailability in mice: 100 % (PO 20
IV 2.5 mg/kg)	mg/kg; IV 2.5 mg/kg)
Bioavailability in guinea pigs: 14 % (PO 10	Bioavailability in guinea pigs: not soluble
mg/kg; IV 2.5 mg/kg)	enough

		—F
Compound 23 of WO02/34752	Compound 17 of the présent application
Bioavailability in guinea pigs: 7 % (PO 40	Bioavailability in guinea pigs: 70 % (PO 10
mg/kg; IV 2.5 mg/kg)	mg/kg; IV 2.5 mg/kg)

-52Example D.4: Measurement of antifungal activity in vivo

General

The animais used in the tests described beiow were female Guînea pigs (DuncanHartlcy, Charles River, 200 g). Thèse animais were kept in groups of 2 or 3 în cages of 5 56x33x20 cm³. Food pellets (Carfil Quality) and water were available ad libitum în addition to a small daily amount of hay. Standard husbandry conditions were adopted: room température: 22 °C, humidity level: 60 % and a day-night cycle of 12 h.

For the préparation of inoculums, Microsporum canis (strain B68128) was grown on Sabouraud Dextrose Agar (SDA) plates for at least one week (7-10 days) before 10 infection (27 °C). On the day of infection, spores were harvested by adding 4-5 ml stérile water on the plate and suspending the înfectious material with a bend Pasteur pipette. From the recoliected fluid, 10 μΐ was added to a KOVA counting chamber for détermination of the number of spores in 1 square (average of several squares). If needed, additional dilutions (10 x or 100 x) were prepared. The average number of 15 spores in one square x dilution x 90.000 = cfu/ml. An inoculum of 1 x 10⁷ cfu/ml was prepared in a honey-water mixture (50 %-50 %).

In the tests described below, semi-quantitative lésion scores were given based on different clinical parameters. Scores between 0 and 3.5 were assigned based on the severity and size of the lésion. The crîteria are lîsted in detail in Table 6.

Table 6: Crîteria for the semi-quantitative dermal lésion scores __________________

Type of lésion	<0.5 cm	0.5-1.5 cm	1.5-3 cm	>3 cm
only scarification lésion or no lésion	0	0	0	0
erythema	0	0.5	1	1.5
pilo-erection	0	0.5	I	1.5
thin white scales (separate)	0.5	1.5	2	2.5
thick white crusts (1 larger crust)	I	2	2.5	3
erosion/red crusts	1.5	2.5	3	3.5
no more crusts, alopecia, irregular skin	0.5	0.5	1	1.5
irregular skin, without alopecia	0.5	0.5	0.5	1
smooth skin (alopecia or not)	0	0	0	0

Test D.4.1 : Activity of compound 4 and compound 14 against M. canis in guinea-pigs after oral treatment at different dosages for 7 consecutive days.

Artificial infection procedure

The dorsum of the guinea pigs was shaved and further depiiated for 3 minutes with

Vcct*’ crcmc before scarification with a steel brush. An inoculum of 10⁶ cfu în 150 μΐ (75 μΐ mQ + 75 μΐ honey) was applied to the wound using a micropipette with

-53disposable tips. The animal was kept immobilîzed until the inoculum had completely dried.

Formulations vchicle: methocel 0.5 %, Twecn 80® (polysorbate 80), demineralised water reference compound: itraconazole at 6.25 mg/ml test compounds: prepared at 25 mg/ml, subsequently further diluted

Dosing and Experimental groups

Oral treatment (gavage 0.16 ml/100 g once daily) was started about 2 hours before infection and continued once daily for 7 consecutive days. The animais were weighed after 3 days of treatment to adjust for the dose.

G1 : Vehîcle-treated infected control (VIC) (2 animais)

G2: Terbinafine (10 mg/kg)

G3: Itraconazole (10 mg/kg) G4: Compound 4 (40 mg/kg)

G5: Compound 4 (10 mg/kg) G6: Compound 4 (5 mg/kg)

G7: Compound 14 (40 mg/kg) G8: Compound 14 (20 mg/kg) G9: Compound 14(10 mg/kg) (mean value of large number of experiments) (2 animais) (2 animais) (3 animais) (3 animais) (3 animais) (3 animais) (3 animais)

Monitoring of infection

Developments of lésions were evaluated following the semi-quantitative lésion scoring System (see Table 6) on days 3, 5, 7, 10, 12, 14, 17 and 20. The results are show in Table 7 (“DPI means days post infection). Reported values in Table 7 are mean values.

Table 7

DPI	G1	G2	G3	G4	G5	G6	G7	GS	G9
3	0	0	0	0	0	0	0	0	0.33
5	1.00	0	0	0.17	0	0.67	0.17	0.33	1.17
7	2.00	0.08	0	0.17	0.33	1.50	0.67	0.83	2.00
10	2.25	0.33	0	0.17	1.00	2.00	1.17	1.67	2.17
12	2.75	0.80	0	0.17	1.50	2.17	0.83	2.00	2.33
14	1.75	0.75	0	0	1.50	1.83	0.50	1.67	2.17
17	1.00	0.50	0	0.17	0.50	1.33	0.17	1.50	1.67
20	0.75	0.42	0	0.17	0.33	1.17	0.17	0.67	1.17

-54From Table 7, can be concluded that the vehicle-treated infected controls (Gl) developed a normal course of infection, i.e._ first lésions after about 5 days with a maximal severity around day 12. Both guinea pigs still showed some white scales at the end of the experiment (day 20).

The animais treated with itraconazole (I0 mg/kg) (G3) never developed any lésions. As for the test compounds, a clear dose-response was seen. Compound 4 is, at 40 mg/kg) (G4), remarkably more active than the terbinafine group (G2). The activity of groups G5 and G7 are comparable to G2.

For the sake of comparison, 2 compounds not having the l, l -difluoroethyl moiety in the 10 R^s or R⁶ position, were tested in an analogous protocol:______________________________

Q5T dérivative of compound 14	àg-o-/ Compound 22 of WO02/34752 dérivative of compound 4
G10; 50 mg/kg	Gll: 50 mg/kg

Artificial infection procedure

The dorsum of the guinea pigs was shaved and further depilated for 7 minutes with Veef⁵' creme before scarification with a steel brush. An înoculum of 10⁶ cfu in 200 μΐ ( 100 μΐ mQ + 100 μΐ honey) was applied to the wound using a micropipette with disposable tips. The animal was kept immobiltzed until the inoculum had completely dried.

Formulations

Vehicle: methocel F4M Premium EP, Tween 80® (polysorbate 80), demineralised water.

Test compounds: prepared at 30 mg/ml, subsequently further diluted until 6 mg/ml.

Dosing and Experimentalgroups

Oral treatment (gavage 0.40 ml/guinea pig once daily) was started about 2 hours before infection and continued once daily for 7 consecutive days.

-55Monitoring of infection

Developments of lésions were evaluated following the semi-quantitative lésion scoring System (see Table 6) on days 3, 5, 7, 10, 12, 14, 18 and 21. The results are show in Table 8. Reported values in Table 8 are mean values.

Table 8

DPI	G10	Gll
3	0	0
5	0.5	0
7	1	0.5

DPI	G10	Gll
10	1	0.5
12	2	0.5
14	1	1

DPI	G10	Gll
18	1	0.5
21	0.5	0.5

The compounds used in G10 and G11 showed partial activity. Howcver even at a higher dose (50 mg/kg), the compounds used in G10 and G1I turned out to be less active than présent compounds 14 and 4 used in G7 and G4 (both at 40 mg/kg).

Example D.5: Squalene-epoxidase inhibition

Using subcellular fractions of Candida albicans, a method was developed enabling to quantify the effects of chemical compounds on different steps in the ergosterol biosynthesis.

C. albicans, strain B2630, was grown for 24 hours (h) in a 500 ml Erlenmeyer flask containing 100 ml CYG medium (0.5 % casein hydrolysate, 0.5 % yeast extract and 0.5 15 % glucose) at 37 °C aerobically in a rotating shaker. After this incubation period, 1 ml aliquots were used to inoculate another 100 ml CYG medium. Cells were grown as above for 8 h. 5 ml ofthis 8 h-culture was used to inoculate 200 ml PYG medium (1 % polypeptone, 1 % yeast extract and 4 % glucose) in a 500 ml Erlenmeyer flask. Cells were grown at 30 °C for 8 h as a standing culture and for another 8 h in a rotating 20 shaker at 100 rpm.

After incubation, the yeast cells were collected by centrifugation (5 min at 1500g) and washed twice with ice cold physiological saline. The cells were resuspended in 15 ml homogenîzation buffer (30 mM nicotinamide, 5 mM MgCl₂ and 5 mM reduced glutathione in 100 mM potassium phosphate buffer pH 7.4) and brought into an ice cold 25 80 ml Bead-Beater récipient containing 40 ml glass beads. The outer jacket of the BeadBeater was filled with îce-cold water. Cells were homogenized for 3 times 1 min with întermitting cooling. The homogenate was centrifuged at 4 °C for 20 min at 8000g. Protein concentration of the 8000g supematant was measured by the Bio-Rad method. The Bio-rad method is a rapid protein measurement. It is a dye-binding assay based on 30 the differential color change of a dye in response to various concentrations of protein based on the method ofBradford. In a typical assay 0.1 ml properly diluted sample was mixed with 5.0.ml dye reagent. A blue-green color resulted which was read by a

-56spectrophotometer at 595 nm. A standard curve was prepared using 10-150 micrograms gamma globuiin.

¹⁴C mevalonate incorporation was measured în a reaction mixture containing in a final volume of 1 ml: 900 μΐ S 8000 fraction (protein 4 mg/ml), 3 mM MgCl2, 2 mM MnCI2, 5.4 mM ATP, 1.38 mM NADH, 1.51 mM NADPH, 0.3 gCi ^I4C-mevalonate and 10 μΐ drug and/or solvent. After an incubation period of 2 h at 30 °C in a reciprocating shaker at 120 spm (scintillations per minute), the reaction was stopped by the addition of 1 ml 15 % KOH in 90 % éthanol. After saponification for 1 h at 80°C and cooling, nonsaponifiable lipids were extracted with 3 ml n-heptane and the extracts dried under a stream of nitrogen. Lipids were separated by TLC (Silicagel 60F25_4î Merck) using a solvent System consisting of 75 volumes HIA (n-heptaan/di-iospropylether/acetic acid, 60/40/4, v/v/v) and 25 volumes ethyl acetate. Lipid fraction were visualized by phosphor-imaging, scanned with a Typhoon 9200 Variabel Mode Imager and quantified using the ImageQuant 5.0 software. Using the above described method, 14ademethylase as well as squalene-epoxidase as Δ14 reductase- and the Δ7-Δ8 isomerase inhibitors could be detectcd.

By following the protocol described hereabove, it was determined that the compounds according to Formula(T) are squalene epoxidase inhibitors.

E. Composition example “Active ingrédient” as used throughout these examples, relates to a compound of Formula (I), including any stereochemically isomeric form thereof, a pharmaceutically acceptable sait thereof or a solvaté thereof; in particular to any one of the exemplified compounds.

Example El : Injectable solution,

1.8 Grams methyl 4-hydroxybenzoate and 0.2 grams sodium hydroxide were dissolved in about 0,5 1 of boiling water for injection. After cooling to about 50°C there were added while stîrring 0.05 grams propylene glycol and 4 grams of the active ingrédient. The solution was cooled to room température and supplemented with water for injection q.s. ad 1 1, giving a solution comprising 4 mg/ml of active ingrédient. The solution was sterilized by filtration and filled in stérile containers.

Example E2 : Transungual composition,

0.144 g KH₂PO₄, 9 g NaCl, 0,528 g Na₂HPO₄.2H₂O was added to 800 ml H₂0 and the mixture was stirred. The pH was adjusted to 7.4 with NaOH and 500 mg NaNj was added. Ethanol (42 v/v %) was added and the pH was adjusted to 2.3 with HCl.

mg active ingrédient was added to 2.25 ml PBS (Phosphate Buffer Saline)Æthanol (42 %; pH 2.3) and the mixture was stirred and treated with ultrasound. 0.25 ml

-57PBS/Ethanol (42 %; pH 2.3) was added and the mixture was further stirred and treated with ultrasound until ail active ingrédient was dissolved, yielding the desired transungual composition.

Examplc E3 : Oral drops

500 Grams of the A.I. was dissolved in 0.5 l of a sodium hydroxide solution and 1.5 I of the polyethylene glycol at 60-80 °C. After cooling to 30-40°C there were added 35 1 of polyethylene glycol and the mixture was stirred well. Then there was added a solution of 1750 grams of sodium saccharin in 2,5 1 of purified water and while stirring there were added 2.5 1 of cocoa flavor and polyethylene glycol q.s. to a volume of 501, providing an oral drop solution comprising 10 mg/ml of AJ.. The rcsulting solution was filled into suitable containers.

Example E4 : Capsules

Grams of the A.I., 6 grains sodium lauryl sulfate, 56 grams starch, 56 grams lactose, 0.8 grams colloïdal sîlicon dioxide, and 1.2 grams magnésium stéarate were vigorously stirred together. The resulting mixture was subsequently filled into 1000 suitable hardened gelatin capsules, comprising each 20 mg of the active ingrédient.

Example E5 : Film-coated tablets

Préparation of tablet core

A mixture of 100 grams of the AJ., 570 grams lactose and 200 grams starch was mixed well and thereafter humidified with a solution of 5 grams sodium dodecyl sulfate and 10 grams polyvinylpyrrolidone in about 200 ml of water. The wet powder mixture was sieved, dried and sieved again. Then there was added 100 grams microcrystalline cellulose and 15 grams hydrogenated vegetable oil. The whole was mixed well and compressed into tablets, giving 10.000 tablets, each containing 10 mg of the active ingrédient.

Coating

To a solution of 10 grams methyl cellulose in 75 ml of denaturated éthanol there was added a solution of 5 grams of ethyl cellulose in 150 ml of dichloromethane. Then there were added 75 ml of dichloromethane and 2.5 ml 1,2,3-propanetrîol. 10 Grams of polyethylene glycol was molten and dissolved in 75 ml of dichloromethane. The lattcr solution was added to the former and then there were added 2.5 grams of magnésium octadecanoate, 5 grams of polyvinylpyrrolidone and 30 ml of concentrated colour suspension and the whole was homogenated. The tablet cores were coated with the thus obtained mixture in a coating apparatus.

-58Examnle E6 : 2 % Cream

Stearyl alcohol (75 mg), cetyl alcohol (20 mg), sorbitan monostearate (20 mg) and isopropyl myrîstate (10 mg) are introdnced in a doublewall jacketed vessel and heated until the mixture has completely molten. This mixture is added to a seperately prepared mixture of purified water, propylene glycol (200 mg) and polysorbate 60 (15 mg) having a température of 70 to 75 °C while using a homogenizer for liquids. The resulting mixture is allowed to cool to below 25 °C while continuously mixing. A solution of A. 1.(20 mg), polysorbate 80 (1 mg) and purified water q.s. ad lg and a solution of sodium sulfite anhydrous (2 mg) in purified water are next added to the émulsion while continuously mixing. The cream is homogenized and ïïlled into suitable tubes.

Example E7 : 2 % Cream

A mixture of A.I. (2 g), phosphatidyl choline (20 g), cholestérol (5 g) and ethyl alcohol (10 g) is stirred and heated at 55-60 °C until complété solution and is added to a solution of methyl paraben(0.2 g), propyl paraben (0.02 g), disodium edetate (0.15 g) and sodium chloride (0.3 g) in purified water (ad 100 g) while homogenizing.

Hydroxypropylmethylceliulose (1.5 g) in purified water îs added and the mixing is continued until swelling is complété.

Claims (13)

1. A compound of Formula (I) or a stereoisomeric form thereof, wherein

R¹ is hydrogen, chloro or fluoro;

R² is hydrogen, chloro, fluoro or methyl;

R³ and R⁴ are hydrogen;

or R³ and R⁴ taken together form a bond;

R⁵ is 1,1-difluoroethyl, and R⁶ is hydrogen or fluoro;

or R⁵ is hydrogen or fluoro, and R⁶ is 1,1-difluoroethyl;

or a pharmaceutically acceptable addition sait or a solvaté thereof.

2. The compound according to daim 1, wherein R¹ is chloro or fluoro.

3. The compound according to claim 1, wherein R¹ is chloro or fluoro; and wherein R² is chloro, fluoro or methyl.

4. The compound according to claim 1, wherein

R¹ is chloro or fluoro;

R² is hydrogen, chloro, fluoro or methyl;

R³ and R⁴ are taken together to form a bond;

R⁵ is 1,1-difluoroethyl, and R⁵ is hydrogen or fluoro;

or R⁵ is hydrogen or fluoro, and R^fi is 1,1-difluorocthyl.

5. The compound according to claim 1, wherein

R¹ is chloro or fluoro;

R² is hydrogen;

R³ and R⁴ taken together form a bond;

R⁵ is 1,1-difluorocthyl;

R⁶ is hydrogen.

6. The compound according to claim 1, wherein

R¹ is in the 7-position and is chloro or fiuoro; and

R² is in any of the other positions and is hydrogen, chloro, fiuoro or methyl.

7. The compound according to daim l, wherein the compound is 7-chloro-4-[4-(l,ldifluoroethyl)phenyI]-6H-pyrrolo[ l ,2-a] [ 1,4]benzodiazepine.

8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as active ingrédient, a therapeutically effective amount of a compound as defined in any one of claims 1 to 7.

9. A compound as defined in any one of claims I to 7 for use as a médicament,

10. A compound as defined in any one of claims 1 to 7 for use in the treatment or prévention of a fungal infection.

11. The compound for use according to claim 10 wherein the fungal infection is caused by one or more of the fungi selected from the group consisting of Candida spp.; Aspergillus spp.; Cryptococcus neoformans; Sporothrix schenckii; Epidermophyton floccosum; Microsporum spp.; Trichophyton spp; Fusarium spp.; Rhizomucor spp.; Mucor circinelloides; Rhizopus spp.; Malasseziafurfur; Acremonium spp.; Paecilomyces; Scopulariopsis; Arthrographis spp.; Scytalidium; Scedosporium spp.; Trichoderma spp.; Pénicillium spp.·;, Pénicillium mameffei; and Blastoschizomyces.

12. The compound for use according to claim 10 wherein the fungal infection is caused by one or more of the fungi selected from the group consisting of Candidaparapsilosis; Aspergillus spp.; Cryptococcus neoformans; Sporothrix schenckii; Epidermophyton floccosum; Microsporum spp.; Trichophyton spp.; Fusarium spp.; Rhizomucor spp.; Mucor circinelloides; Rhizopus spp.; Acremonium spp.; Paecilomyces; Scopulariopsis; Arthrographis spp.; Scytalidium; Scedosporium spp.; Trichoderma spp.; Pénicillium spp. ; Pénicillium marneffei; and Blastoschizomyces.

13. The compound for use according to claim 10 wherein the fungal infection is caused by one or more of the fungi selected from the group consisting tâ Microsporum canis, Trichophyton mentagrophytes, Trichophyton rubrum and Aspergillus fumigatus.