WO2010068296A1 - Piperazine carboxamidines as antimicrobial agents - Google Patents

Abstract

The present invention relates to novel piperazine carboxamidine compounds possessing antimicrobial activity and their preparation.

Description

Piperazine Carboxamidines as Antimicrobial Agents

Field of the Present Invention

The present invention relates to novel piperazine 1- carboxamidine compounds possessing antimicrobial activity and their preparation.

Background of the Invention

The most common causes of the fungal infections in humans are due to Candida spp., of which C. albicans accounts for approximately 50%, and filamentous fungi such as Aspergillus spp. [Kremery, V. et al., Hosp. Infect., 2002, 50, 243]. Mortality associated with invasive Candida ranges from around 40% [Edmond, M.B. et al., Clin. Infect. Pis., 1999, 29, 239], while mortality associated with invasive Aspergillus approaches 100% in solid organ transplant recipients [Minari, A, et al., Transpl. Infect. Pis., 2002, 4, 195].

Given the lack of readily available fungal vaccines, the only clinical resource available to combat fungal infections is antifungal therapeutics (antimycotics). The antimycotics currently in clinical use are limited either by their general ineffectiveness and inadequate pharmacological profile, including undesired drug-drug interactions and narrow activity spectrum, or by their high overall cytotoxicity [White, T. C. et al., Clin. Microbiol. Rev. 1998, 11, 382-402]. Accordingly, there is a critical need for new antifungal compounds that could overcome these disadvantages.

It is known that guanidine-containing trityl compounds, such as the compound shown below, exhibit antibacterial activity [Stephenson, K., et al., J.Biol. Chem.. 2000, 275, 38900-38904].

Piaryl piperazine-containing compounds, such as the two compounds shown below, have also been shown to exhibit antifungal activity [Heeres, J., et al. WO 95/19983].

Miconazole has been demonstrated to induce increased endogenous reactive oxygen species (ROS) levels in C. albicans [Borgers, M. et al., 1977, 193-199; De Nollin, et al., Antimicrob. Agents Chemother., 1977, 500-513; and ROS induction capacity has recently been linked to fungicidal activity in antifungal compounds [Francois, I.E. et al., Curr. Med. Chem. Antinfective Agents, 2005, 5, 3-13]. In the present invention, select piperazine carboxamidine derivatives have been identified as promising new chemical entities that possess antimicrobial activity.

Summary of the Invention

One aspect of the present invention is a compound of Formula (I):

wherein

A = -CH₂- or -CH₂CH₂-;

Ri and R₂ are independently selected from -H, -Ci_-6 alkyl, -C_3-6 cycloalkyl, aryl, heterocyclic

Ci -₆ alkyl, heteroaryl, -OCi_-6 alkyl, -C(O)OR₃ or -(CH₂)JNIHR₃ ; D is an optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocyclic Ci-₆ alkyl moiety; m is an integer having a value of 1 , 2, 3 or 4; n is 0 or an integer having a value of 1 , 2, 3 or 4;

R₃ is independently selected from -H, Ci_-6 alkyl, aryl, -C_3-6 cycloalkyl, heteroaryl or heterocyclic Ci-₆ alkyl; or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is a pharmaceutical composition comprising at least one compound of Formula (I), or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable carrier or diluent thereof.

Another aspect of the present invention is a method for the treatment of microbial (i.e., fungal and/or bacterial) disorders to a mammal, suitably a human, in need of such treatment comprising administering the composition to the recipient. In an exemplary embodiment, the microbial disorder is of the skin or mucosae (e.g., oral and/or genital mucosae). In an exemplary embodiment, the route of administration is topically or systemically.

Another aspect of the present invention is a method for killing or inhibiting the growth of Candida spp. comprising administering to a mammal afflicted with a disease associated with Candida spp. (e.g., candidiasis) a composition comprising at least one compound of Formula (I), or a pharmaceutically acceptable salt thereof, in an effective amount, and for an effective duration. In an exemplary embodiment, an effective amount and an effective duration is the amount and duration of administration of at least one compound of Formula (I), or a pharmaceutically acceptable salt thereof, necessary to increase the intracellular reactive oxygen species levels to achieve antifungal activity to the Candida spp. In an exemplary embodiment, the route of administration is topically or systemically.

Brief Description of Drawings

FIG. 1 shows that compound 67 induces apoptosis in S. cerevisiae. S. cerevisiae WT cultures were treated with various concentrations of compound 67 or DMSO in PBS. Percentage survival of compound 67-treated cells was calculated relative to DMSO-treated cells. Data represent mean ± SEM. *^<0.05; **p<0.0\; ***p<0.00\ .

FIG. 2 shows apoptotic features of S. cerevisiae cultures treated with 20 μg/ml compound 67 (grey bars) or DMSO (white bars) were assessed by determining the endogenous ROS levels via DHE staining, DNA fragmentation via TUNEL staining and phosphatidylserine externalization and membrane integrity via annexinV /propidium iodide co-staining. In each experiment, 500 cells were evaluated using fluorescence microscopy (100% represents the number of cells, i.e. 500). Values are the mean of triplicate measurements. Data represent mean ± SEM. ***/Kθ.OOl . FIG. 3 shows compound 67-induced apoptosis in yeast is dependent on Ycal and Fisl . S. cerevisiae WT (black bars), Δycal (white bars) and Δfisl (grey bars) cultures were treated with 20 μg/ml compound 67or DMSO in PBS. (A) Percentage survival of compound 67- treated cells was calculated relative to DMSO-treated cells. Data represent mean ± SEM. **p<0.01.

FIG. 4 shows apoptotic features of the S. cerevisiae cultures treated with 20 μg/ml compound 67 were assessed by determining the endogenous ROS levels via DHE staining and DNA fragmentation via TUNEL staining. In each experiment, 500 cells were evaluated using fluorescence microscopy (100% represents the number of cells, i.e. 500). Values are the mean of triplicate measurements. Data represent mean ± SEM. */Kθ.O5; ***/?<0.001.

FIG. 5A shows compound 67 induces mitochondrial fission in yeast. S. cerevisiae WT cultures were treated with DMSO and mitochondrial morphology was assessed by DΪOC6 mitochondrial membrane straining.

FIG. 5B shows compound 67 induces mitochondrial fission in yeast. S. cerevisiae WT cultures were treated with 20 μg/ml compound 67 in PBS and mitochondrial morphology was assessed by DiOC6 mitochondrial membrane straining.

FIG. 6 shows the functional distribution of the S. cerevisiae compound 67 tolerance genes.

FIG. 7 shows the functional distribution of the S. cerevisiae compound 67 sensitivity genes. Note that the portion of compound 67 tolerance/sensitivity genes implicated in the indicated processes is given as a percentage of the total number of identified compound 67 tolerance genes.

Detailed Description of the Present Invention

Definitions

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present invention belongs.

As defined herein, the phrases an "effective amount" or "an amount effective to" or a "therapeutically effective amount" of an active agent or ingredient, or pharmaceutically active agent or ingredient, which are synonymous herein, refer to an amount of the pharmaceutically active agent sufficient enough to have a net positive effect upon administration. A therapeutically effective amount of the pharmaceutically active agent will cause a substantial relief of symptoms when administered repeatedly over time. Effective amounts of the pharmaceutically active agent will vary with the particular condition or conditions being treated, the severity of the condition, the duration of the treatment, the specific components of the composition being used, and like factors.

As defined herein, the terms "administering", "administration", and like terms refer to any method which, in sound medical or cosmetic practice, delivers the composition to a subject in such a manner as to provide a net positive effect.

As defined herein, "topical administration" is used in the conventional sense to mean delivery of a drug or pharmacologically active agent directly to the skin or mucosa of an individual.

As defined herein, "systemic administration" is used in the conventional sense to mean, e.g., oral, subcutaneous (SC), intraperitoneal (IP), intramuscular (IM) or intravenous (IV) delivery of a drug or pharmacologically active agent to an individual.

As defined herein, an "antimicrobial" is an agent that inhibits the growth of fungal and/or bacterial microorganisms or kills them outright. More specifically, an "antifungal" is an agent that inhibits the growth of fungi {i.e., a fungistat) or kills them outright {i.e., a fungicide) and an "antibacterial" or "antibiotic" is a substance that kills or slows the growth of bacteria.

As used herein, the term "halo" or "halogens" is used herein to mean chloro, fluoro, bromo or iodo.

As used herein, the term "Cj.galkyl" or "alkyl" or "alkylj.g" is used herein to mean both straight and branched hydrocarbon chain containing the specified number of carbon atoms, e.g. Cj.galkyl means a straight or branched alkyl chain of at least 1 , and at most 6, carbon atoms, unless the chain length is otherwise limited. Examples of "alkyl" as used herein include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, isobutyl, isopropyl, sec-butyl, tert-buty\ or t-butyl and hexyl and the like.

As used herein, the term "alkenyl" refers to straight or branched hydrocarbon chains containing the specified number of carbon atoms and containing at least one double bond. For example, C2-6^alkenyl means a straight or branched alkenyl containing at least 2, and at most

6, carbon atoms and containing at least one double bond. Examples of "alkenyl" as used herein include, but are not limited to ethenyl, 2-propenyl, 3-butenyl, 2-butenyl, 2-pentenyl, 3- pentenyl, 3-methyl-2-butenyl, 3-methylbut-2-enyl, 3-hexenyl, l,l-dimethylbut-2-enyl and the like.

As used herein, the term "alkoxy" refers to straight or branched chain alkoxy groups containing the specified number of carbon atoms. For example, Cj.galkoxy means a straight or branched alkoxy containing at least 1 , and at most 6, carbon atoms. Examples of "alkoxy" as used herein include, but are not limited to, methoxy, ethoxy, propoxy, prop-2-oxy, butoxy, but-2-oxy, 2-methylprop-l-oxy, 2-methylprop-2-oxy, pentoxy and hexyloxy.

As used herein, the term "cycloalkyl" refers to non-aromatic hydrocarbon ring containing a specified number of carbon atoms. For example, C3_6cycloalkyl means a non-aromatic ring containing at least three and at most six ring carbon atoms. Representative examples of "cycloalkyl" as used herein include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl and the like.

The term "aryl" is used herein to mean phenyl, naphthyl or indene.

The terms "heteroaryl ring", "heteroaryl moiety", and "heteroaryl" are used herein to mean an aromatic monocyclic five- to seven- membered unsaturated hydrocarbon ring containing at least one heteroatom selected from oxygen, nitrogen and sulfur. Examples of heteroaryl rings include, but are not limited to, furyl (such as 2-furyl, 3-furyl,), pyranyl, thienyl (such as 2-thienyl, 3- thienyl), pyrrolyl (such as 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), oxazolyl (such as 2-oxazolyl, 4- oxazolyl, 5-oxzaolyl) thiazolyl (such as 2-thiazolyl, 4-thiazolyl, 5-thiazolyl), isoxazolyl (such as 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl) isothiazolyl, imidazolyl (such as 2-imidazolyl, 4- imidazolyl), pyrazolyl, oxadiazolyl, oxathiadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridyl (such as 2-pyridyl, 3-pyridyl, 4-pyridyl) pyridazinyl, pyrimidinyl (such as 2-pyrimidyl, 4- pyrimidyl), pyrazinyl, triazinyl, and uracil. The terms "heteroaryl ring", "heteroaryl moiety", and "heteroaryl" shall also used herein to refer to fused aromatic rings comprising at least one heteroatom selected from oxygen, nitrogen and sulfur. Each of the fused rings may contain five or six ring atoms. Examples of fused aromatic rings include, but are not limited to, indolyl, isoindolyl, indazolyl, indolizinyl, azaindolyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl (such as 1-isoquinolyl, 5-isoquinolyl), quinazolinyl, quinoxalinyl (such as 2-quinoxalinyl, 5-quinoxalinyl), naphthyridinyl, cinnolinyl, purinyl, 2,3-dihydrobenzo[l ,4]dioxin-6-yl, benzo[l,3]dioxol-5-yl and phthalazinyl.

The terms "heterocyclic rings", "heterocyclic moieties", "heterocyclo" and "heterocyclyl" are used herein to mean a monocyclic three- to seven-membered saturated or non-aromatic, unsaturated hydrocarbon ring containing at least one heteroatom selected from nitrogen, oxygen, sulphur or oxidized sulphur moieties, such as S(0)m, and m is 0 or an integer having a value of 1 or 2. The terms "heterocyclic rings", "heterocyclic moieties", "heterocyclo" and "heterocyclyl" shall also refer to fused rings, saturated or partially unsaturated, and wherein one of the rings may be aromatic, or heteroaromatic. Each of the fused rings may have from four to seven ring atoms. Examples of heterocyclyl groups include, but are not limited to, the saturated or partially saturated versions of the heteroaryl moieties as defined above, such as tetrahydropyrrole, tetrahydropyran, tetrahydrofuran (such as tetrahydrofuran-2-yl, tetrahydrofuran-3-yl), tetrahydrothiophene (including oxidized versions of the sulfur moiety, and tetrahydrothien-2-yl, tetrahydrothien-3-yl), azepine, diazepine, aziridinyl, pyrrolinyl, pyrrolidinyl, 2-oxo-l-pyrrolidinyl, 3-oxo-l-pyrrolidinyl, l,3-benzdioxol-5-yl, imidazolinyl, imidazolidinyl, indolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl (such as 1-piperidinyl, 2- piperidinyl, 3 -piperidinyl) piperazinyl (such as 1 -piperazinyl, 2-piperazinyl), morpholine (such as 4-morpholinyl, 3-morpholinyl) and thiomorpholino (including oxidized versions of the sulfur moiety).

The term "arylalkyl" or "heteroarylalkyl" or "heterocyclicalkyl" is used herein to mean a Q -6 alkyl (as defined above) attached to an aryl, heteroaryl or heterocyclic moiety (as also defined above) unless otherwise indicated.

The term "sulfinyl" is used herein to mean the oxide S(O) of the corresponding sulfide, the term "thio" refers to the sulfide, and the term "sulfonyl" refers to the fully oxidized S(O)2 moiety.

The term "aroyl" is used herein to mean C(O)Ar, wherein Ar is a phenyl, or naphthyl, or an arylalkyl derivative such as defined above, such group including but are not limited to benzyl and phenethyl.

The term "alkanoyl" is used herein to mean C(O)C] -6 alkyl wherein the alkyl is as defined above.

As defined herein, the term "heteroatom" refers to oxygen (O), nitrogen (N), or sulfur (S).

As defined herein, "derivative" or "derivatives" refers to derivative(s) of the active compound(s) which possess the same pharmacological activity as the active compound(s) and which are neither biologically nor otherwise undesirable. Derivatives of the active compounds include, without limitation, polymorphs, solvates, salts, N-oxides, hydrates, dehydrates, crystalline forms, racemates, isomers, enantiomers, prodrugs, metabolites, analogues, esters, anhydrous forms, amorphous forms, and mixtures thereof. The term "derivative" may also mean a modification to the disclosed compounds including, but not limited to, hydrolysis, reduction or oxidation. Hydrolysis, reduction and oxidation reactions are known in the art.

As defined herein, the phrase "pharmaceutically acceptable salts" refers to salts of certain ingredient(s) which possess the same activity as the unmodified compound(s) and which are neither biologically nor otherwise undesirable. A salt can be formed with, for example, organic or inorganic acids. Non-limiting examples of suitable acids include acetic acid, acetylsalicylic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzoic acid, benzenesulfonic acid, bisulfic acid, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, carbonic acid, citric acid, cyclopentanepropionic acid, digluconic acid, dodecylsulfic acid, ethanesulfonic acid, formic acid, fumaric acid, glyceric acid, glycerophosphoric acid, glycine, glucoheptanoic acid, gluconic acid, glutamic acid, glutaric acid, glycolic acid, hemisulfic acid, heptanoic acid, hexanoic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthylanesulfonic acid, naphthylic acid, nicotinic acid, nitrous acid, oxalic acid, pelargonic, phosphoric acid, propionic acid, saccharin, salicylic acid, sorbic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, thioglycolic acid, thiosulfuric acid, tosylic acid, undecylenic acid, and naturally and synthetically derived amino acids.

Non-limiting examples of base salts include ammonium salts; alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; salts with organic bases, such as dicyclohexylamine salts; methyl-D-glucamine; and salts with amino acids, such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides, such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; asthma halides, such as benzyl and phenethyl bromides; and others. Water or oil-soluble or dispersible products are thereby obtained. The salts of the present invention may be acetate, butyrate, hemisuccinate and phosphate salts.

As used herein, the term "optionally" means that the subsequently described event(s) may or may not occur, and includes both event(s) which occur and events that do not occur.

As used herein, the term "substituted" refers to substitution with the named substituent or substituents, multiple degrees of substitution being allowed unless otherwise stated. It is to be understood that the present invention covers all combinations of particular and preferred groups described hereinabove.

With regard to stereoisomers, the compounds of the Formulas herein may have one or more asymmetric carbon atom and may occur as racemates, racemic mixtures and as individual enantiomers or diastereomers. All such isomeric forms are included within the present invention, including mixtures thereof.

Cis (E) and trans (Z) isomerism may also occur. The present invention includes the individual stereoisomers of the compound of the present invention and where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.

Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. A stereoisomeric mixture of the agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.

Exemplified compounds of the compounds of this present invention include the racemates, or optically active forms of the compounds of the working examples herein, and pharmaceutically acceptable salts thereof.

The phrase "pharmaceutically acceptable carrier" as used herein refers to any inactive ingredient present in an amount effective to enhance ease of administration, tolerability, stability, effectiveness, delivery of the dosage form, taste, shape or other characteristic of a pharmaceutical composition. Non-limiting examples of such pharmaceutically acceptable carriers include diluents, excipients, suspending agents, lubricating agents, adjuvants, vehicles, delivery systems, emulsifiers, disintegrants, absorbents, adsorbents, preservatives, surfactants, colorants, flavorants, emollients, buffers, pH modifiers, thickeners, water softening agents, humectants, fragrances, stabilizers, conditioning agents, chelating agents, sweeteners, propellants, anticaking agents, viscosity increasing agents, solubilizers, plasticizers, penetration enhancing agents, glidants, film forming agents, fillers, coating agents, binders, antioxidants, stiffening agents, wetting agents, or any mixture of these components.

As used herein, "subject" or "individual" or "animal" or "patient" or "mammal," refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or therapy is desired, for example, a human.

As used herein, the term "treating" includes any indicia of success in the treatment or amelioration of an injury, pathology, condition, or symptom (e.g., inflammation or redness), including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient, decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom or condition.

Any concentration range, percentage range or ratio range recited herein is to be understood to include concentrations, percentages or ratios of any integer within that range and fractions thereof, such as one tenth and one hundredth of an integer, unless otherwise indicated.

It should be understood that the terms "a" and "an" as used above and elsewhere herein refer to "one or more" of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms "a," "an" and "at least one" are used interchangeably in this application. For example, "a" polymer refers to both one polymer or a mixture comprising two or more polymers.

Throughout the application, descriptions of various embodiments use "comprising" language, however it will be understood by one of skill in the art that in some specific instances an embodiment can alternatively be described using the language "consisting essentially of or "consisting of."

For the purposes of better understanding the present teachings and in no way limiting the scope thereof, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the various methods and materials are described herein. All publications and patent applications cited herein are incorporated herein by reference for the purpose of disclosing and describing specific aspects of the present invention for which the publication is cited.

One aspect of the invention is a compound represented by the formula:

wherein

A = -CH₂- or -CH₂CH₂-;

Ri and R₂ are independently selected from -H, -Ci_-6 alkyl, -C_3-6 cycloalkyl, aryl, heterocyclic

Ci-₆ alkyl, heteroaryl, -OC_1-6 alkyl, -C(O)OR₃ or -(CH₂)_mNHR₃; A is -CH₂-; or -CH₂CH₂-;

D is an optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocyclic Cp₆ alkyl moiety; and wherein the aryl, heteroaryl or heterocyclic Ci-₆ alkyl moieties are optionally substituted 1 or more times, suitably 1 to 5 times by QR₄; and further D may be optionally substituted 1 or more times, suitably 1 to 3 times by a Ci -₆ alkyl, Ci-₆ alkoxy or a halogen; m is an integer having a value of 1, 2, 3 or 4; n is O or an integer having a value of 1 , 2, 3 or 4; R₃ is independently selected from -H, Ci_-6 alkyl, aryl, -C_3-6 cycloalkyl, heteroaryl or heterocyclic Q-₆ alkyl; or Q is selected from S or CH₂;

R₄ is a -(CH₂)_p-(optionally substituted aryl or heteroaryl), -(CH₂)_p-CO-(optionally substituted aryl or heteroaryl); -(optionally substituted aryl or heteroaryl)-(CH₂)_p-(optionally substituted aryl or heteroaryl), -CH(optionally substituted aryl or heteroaryl)₂; and wherein these aryl and heteroaryl rings are each independently optionally substituted 1 or more times, suitably 1 to 5 times by Ci_-6 alkyl, Ci_-6 alkoxy, phenyl, cyano, halogen, NO₂, CF₃, OCF₃, -CH₂SO₂, -S(O)₂CH₃, or (CH₂)_qS0₂aryl; p is O or an integer having a value of 1 , 2, 3, or 4; q is an integer having a value of 1 , 2, or 3; a pharmaceutically acceptable salt thereof.

Suitably, for compounds of Formula (I), R] and R₂ are independently selected from -H, -Ci_-6 alkyl, -C_3-6 cycloalkyl, aryl, heterocyclic Ci-₆ alkyl, heteroaryl, -0C_)-6 alkyl, -C(O)OR₃ or -(CH₂)_mNHR₃. In one embodiment Ri and R₂ are independently -H or cycloalkyl. In another embodiment, Ri and R₂ are suitably selected from hydrogen or -Ci_-6 alkyl. In another embodiment, Ri and R₂ are both hydrogen.

Suitably, for compounds of Formula (I) unless otherwise indicated, n is 0 or an integer having a value of 1, 2, 3 or 4. In one embodiment, n is 1.

Suitably, for compounds of Formula (I) unless otherwise indicated, D is an optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocyclic Ci-₆ alkyl. In one embodiment, D is an optionally substituted phenyl, 1-naphthyl, 2-naphthyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2- oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5- isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2- pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2- benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl, 2,3-dihydrobenzo[l,4]dioxin-6-yl, benzo[l,3]dioxol-5-yl or 6-quinolyl. In another embodiment, D is an optionally substituted phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, quinolyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

In one exemplary embodiment of the present invention, for compounds of Formula I D is

wherein Z is N or CH;

Q is selected from S or CH₂;

R₄ is a -(CH₂)_p-(optionally substituted aryl or heteroaryl), -(CH₂)_p-CO-(optionally substituted aryl or heteroaryl); -(optionally substituted aryl or heteroaryl)-(CH₂)_p-(optionally substituted aryl or heteroaryl), -CH(optionally substituted aryl or heteroaryl)₂; and wherein these aryl and heteroaryl rings are each independently optionally substituted 1 or more times, suitably 1 to 5 times by Ci_-6 alkyl, Ci_-6 alkoxy, phenyl, cyano, halogen, NO₂, CF₃, OCF₃, -CH₂SO₂, -S(O)₂CH₃, or (CH₂)_qS0₂aryl; p is O or an integer having a value of 1 , 2, 3, or 4; q is an integer having a value of 1 , 2, or 3. X is independently selected from d-₆ alkyl, Cp₆ alkoxy or halogen; t is 0 or an integer having a value of 1, 2, or 3.

In another embodiment A is CH₂.

In another embodiment R¹ and R² are independently -H or cycloalkyl.

In another embodiment R¹ and R² are independently -H or cycloalkyl and A is CH₂.

In another embodiment Z is nitrogen.

In another embodiment Z is carbon.

In another embodiment of the present invention, for compounds of Formula I: D is

wherein Z is N or CH;

Q is selected from S or CH₂;

R₄ is a -(CH₂)_p-(optionally substituted aryl or heteroaryl), -(CH₂)_p-CO-(optionally substituted aryl or heteroaryl); -(optionally substituted aryl or heteroaryl)-(CH₂)_p-(optionally substituted aryl or heteroaryl), -CH(optionally substituted aryl or heteroaryl)₂; and wherein these aryl and heteroaryl rings are each independently optionally substituted 1 or more times, suitably 1 to 5 times by Ci_-6 alkyl, Ci_-6 alkoxy, halo- substituted Ci_-6 alkoxy phenyl, cyano, halogen, NO₂, CF₃, OCF₃, -CH₂SO₂, - S(O)₂CH₃, or (CH₂)_qSO₂aryl; p is O or an integer having a value of 1, 2, 3, or 4; q is an integer having a value of 1 , 2, or 3;

X is independently selected from Ci-₆ alkyl, Ci-₆ alkoxy or halogen; and t is O or an integer having a value of 1 , 2, or 3. In another embodiment A is CH₂.

In another embodiment R¹ and R² are independently -H or cycloalkyl. In another embodiment R¹ and R² are independently -H or cycloalkyl and A is CH_2. In another embodiment Z is nitrogen. In another embodiment Z is carbon.

In another exemplary embodiment for compounds of Formula I, Ri and R₂ are independently -H, -Ci_-6 alkyl, -C_3-6 cycloalkyl, aryl, heterocyclic Ci_-6 alkyl, heteroaryl, -OCi_-6 alkyl, -C(O)OR₃ or -(CH₂)_mNHR₃; A is -CH₂-; D is selected from the group consisting of a thiophene and a quinoline; n is 1 ; and R₃ is -H or Ci_-6 alkyl, aryl, C_3-6 cycloalkyl, heteroaryl or heterocyclic Ci_-6 alkyl. as thiophene and/or quinoline are:

, respectively.

In one embodiment, D is a thiophene, or quinoline substituted by methoxy, Q is CH₂, and p is 0.

In one embodiment, Q is selected from S or -CH₂-. In one embodiment when Q is S, p is 1. In another embodiment, when Q is -C H₂, p is 0.

In one embodiment the R₄ aryl rings are unsubstituted, or are independently substituted 1 or more times, suitably 1 to 3 times by halogen, cyano, Ci_-6 alkoxy, halo-substituted Ci_-6 alkoxy, CF₃, nitro, phenyl, or Ci_-6 alkyl.

In another embodiment the R₄ aryl rings are unsubstituted, or are independently substituted 1 or more times, suitably 1 to 3 times by chlorine, bromine or fluorine, cyano, methoxy, O-CF₃,cyano, nitro, CF₃, nitro, phenyl, mesylate, tosylate, methyl or ethyl.

In another embodiment the R₄ aryl rings are unsubstituted or are independently substituted 1 or more times, suitably 1 to 3 times by halogen, cyano, or Ci_-6 alkyl. In another embodiment, the R4 aryl ring is unsubstituted or substituted one or more times independently by halogen.

In one embodiment of the present invention R₄ is an -(CH₂)_P - (optionally substituted phenyl).

Exemplary possibilities within the scope of this invention, include but are not limited to R⁴ moieties selected from the group consisting of

In one embodiment of the present invention, for the specific R4 moieties noted above, Q is S. In another embodiment Q is -CH₂- and p is O.

Specific compounds of Formula I include, but are not limited to:

or a pharmaceutically acceptable salt thereof.

Suitable pharmaceutical acceptable salts are those of organic or inorganic acids, including, but not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, trifluoroacetic acid, phosphoric acid, acetic acid, succinic acid, oxalic acid, malic acid and the like.

It is intended that the present invention include within its scope the stereochemically pure isomeric forms of the piperazine carboxamidine compounds as well as their racemates. Stereochemically pure isomeric forms may be obtained by the application of known principles. For example, diastereoisomers may be separated by physical separation methods such as fractional crystallization and chromatographic techniques, and enantiomers may be separated from each other by the selective crystallization of the diastereomeric salts with optically active acids or by chiral chromatography. Pure isomers may also be prepared synthetically from appropriate stereochemically pure starting materials, or by using stereospecific reactions.

Formulations for the compounds of the present invention include, for example, ointments, salves, tablets, creams, gels, solutions, lotions, foams, dressings, shampoos, tinctures, pastes, powders and the like. Other suitable embodiments may be anhydrous formulations for some applications. Application of the compositions of the present invention may also be by aerosol, e.g., with a propellant such as nitrogen, carbon dioxide, a freon, or without a propellant such as a pump spray, drops, lotions, or a semisolid such as a thickened composition which can be applied by a swab. In addition, the compounds of the present invention may be applied as a transdermal patch. Further suitable routes of administration include intravenous and oral delivery.

Other suitable formulations include toilet waters, packs, skin milks or milky lotions. Such formulations often include therapeutically inactive components such as, for example, oils, fats, waxes, surfactants, humectants, thickening agents, antioxidants, viscosity stabilizers, chelating agents, buffers, preservatives, perfumes, dyestuffs and the like. If desired, additional ingredients may be incorporated in the compositions of the present invention such as anti-infiamatory agents, antibacterials (antibiotics), antifungals, disinfectants, vitamins, sunscreens or anti-acne agents.

Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, kaolin. Liquid carriers include sterile water, propylene glycol, glycerin, ethylene glycol, polyethylene glycol, lower alcohols (e.g., ethanol, propanol, isopropanol and butanol) and edible oils such as corn, peanut and sesame oils. The carriers may be present in an amount of from about 5 to about 80 weight percent, about 10 to about 70 weight percent, about 15 to about 60 weight percent, about 15 to about 50 weight percent, about 15 to about 35 weight percent, about 15 to about 20 percent, about 20 to about 40 weight percent, about 25 to about 40 weight percent, about 30 to about 40 weight percent.

The pharmaceutical compositions of the present invention may also optionally include other carriers, diluents, stabilizers, preservatives or adjuvants. For typical examples of these classes of compounds, see Remington: The Science and Practice of Pharmacy, Lippincott, Williams & Wilkins (2005), which is incorporated by reference in its entirety.

A therapeutically effective amount of the piperazine carboxamidine compounds of the present invention may vary depending on the particular compound used; the mode of administration; the identity and severity of the disease state; the age, sex, weight and general physical condition of the particular recipient; and other medications that the recipient may be concurrently taking. Furthermore, it is evident that the effective frequency of administration of the combination therapy may be lowered or increased depending on the response of the treated patient and/or depending on the evaluation of the physician prescribing the therapeutic agent being used. In general, satisfactory results are obtained when the amount of the active compound used ranges from about 0.01 weight percent to about 10 weight percent, about 0.01 to about 5 weight percent, about 0.05 to about 3 weight percent or about 0.1 to about 3 weight percent.

Dosage levels on the order of about 0.001 mg to about 5,000 mg per kilogram body weight of the active ingredient components are known to be useful in the treatment of the diseases, disorders, and conditions contemplated herein. Typically, this effective amount of the active agent will generally comprise from about 0.001 mg to about 100 mg per kilogram of patient body weight per day. Moreover, it will be understood that this dosage of ingredients can be administered in a single or multiple dosage units to provide the desired therapeutic effect.

It may be appropriate to administer the compounds of the invention either alone or in a combination therapy, once daily or as two, three, four or more sub-doses at appropriate intervals throughout the day. Sub-doses may be formulated as unit dosage forms, for example, containing 0.001 mg to 500 mg of active ingredient per unit dosage form.

Additional therapeutic agents that may be used in combination with the compounds of the invention include, but are not limited to, antimicrobial agents (e.g., amphotericin B, clotrimazole, econazole nitrate, fluconazole, flucytosine, haloprogin, itraconazole, ketoconazole, miconazole and nystatin), anti-allergic agents (e.g., astemizole, betamethasone, carbinoxamine maleate, chlorpheniramine maleate, clemastine fumarate, dexbrompheniramine maleate, dexchlorpheniramine maleate, diphenhydramine hydrochloride, diphenylpyraline hydrochloride and trimeprazine tartrate), anti-inflammatory agents (e.g., ibuprofen, fenoprofen, ketoprofen, naproxen, diclofenac, etodolac, meclofenamate sodium phenylbutazone, indomethacin, piroxicam, sulindac and tolmetin), anti -proliferating agents (e.g., mycophenolate mofetil and evodiamine), anti-acne agents (e.g., tretinoin, isotretinoin, salicylic acid, benzoyl peroxide and azelaic acid), anti-pruritic agents (e.g., azelastine, cetirizine permethrin and lindane), anti-aging agents and combinations thereof. Method of Manufacture

The compounds of this present invention may be made by a variety of methods, including standard chemistry. Any previously defined variable will continue to have the previously defined meaning unless otherwise indicated. Illustrative general synthetic methods are set out below and then specific compounds of the present invention are prepared in the working

Examples.

The compounds of Formula (I) may be obtained by applying the synthetic procedures described herein. The synthesis provided for is applicable to producing compounds of the Formulas herein having a variety of different Ri, R₂, R₃- D, Q, etc. groups which are reacted, employing optional substituents which are suitably protected, to achieve compatibility with the reactions outlined herein. Subsequent deprotection, in those cases, then affords compounds of the nature generally disclosed. While a particular formula with particular substituent groups is shown herein, the synthesis is applicable to all formulas and all substituent groups herein.

Scheme 1: General parallel synthesis procedure for compounds such as those illustrated

below and labeled as 1-38, (wherein the D ring is represented by

and X is carbon or nitrogen) and Y is N; the phenyl ring substituted by R₂-R₆ is representative of an optionally substituted R₄ moiety as defined herein, and Q is S. In the first step the representative tertiary amine disisopropyl amine may be replaced with any other suitable tertiary amine or solvent.

7, 26-38)

(Y"hal<ςcn)

X = N (23-25)

(Y = haloeBi)

Scheme 2: Synthesis procedure for compounds such as those illustrated below (Compounds

39-44 and 14 (X is nitrogen in the D ring represented by

; Y is N; the R₄ is an optionally substituted -(CH)(aryl)₂ ring, etc. The same procedure may be employed for compounds where X=CH, and Y is C.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples describe embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Experimental

The present invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention. All temperatures are given in degrees centigrade.

Analytic Method A:

A HPLC gradient was supplied by an Alliance HT 2790 (Waters) system comprising a quaternary pump with degasser, an autosampler, a column oven (set at 40 ⁰C), a diode-array detector (DAD) and a column as specified in the respective methods below. Flow from the column was split to a MS detector. The MS detector was configured with an electrospray ionization source. Mass spectra were acquired by scanning from 100 to 1000 in 1 second using a dwell time of 0.1 second. The capillary needle voltage was 3 kV and the source temperature was maintained at 140 °C. Nitrogen was used as the nebulizer gas. Data acquisition was performed with a Waters-Micromass MassLynx-Openlynx data system.

Reversed phase HPLC was carried out on an Xterra MS C18 column (3.5 μm, 4.6 x 100 mm) with a flow rate of 1.6 mL/min. Three mobile phases (mobile phase A: 95% 25 mM ammoniumacetate + 5 % acetonitrile; mobile phase B: acetonitrile; mobile phase C: methanol) were employed to run a gradient condition from 100 % A to 1 % A, 49 % B and 50 % C in 6.5 minutes, to 1 % A and 99 % B in 1 minute and hold these conditions for 1 minute and reequilibrate with 100 % A for 1.5 minutes. An injection volume of 10 μl was used. Cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode.

Analytic Method B:

The LC gradient was supplied by an Acquity UPLC (Waters) system comprising a binary pump, a sample organizer, a column heater (set at 55 °C), a diode-array detector (DAD) and a column as specified in the respective methods below. Flow from the column was split to a MS detector. The MS detector was configured with an electrospray ionization source. Mass spectra were acquired by scanning from 100 to 1000 in 0.18 seconds using a dwell time of 0.02 seconds. The capillary needle voltage was 3.5 kV and the source temperature was maintained at 140 °C. Nitrogen was used as the nebulizer gas. Data acquisition was performed with a Waters-Micromass MassLynx-Openlynx data system.

Reversed phase UPLC was carried out on a bridged ethylsiloxane/silica (BEH) Cl 8 column (1.7 μm, 2.1 x 50 mm) with a flow rate of 0.8 mL/min. Two mobile phases (mobile phase A: 0.1 % formic acid in H₂0/methanol 95/5; mobile phase B: methanol) were used to run a gradient condition from 95 % A to 5 % A, 95 % B in 1.3 minutes and hold for 0.2 minutes. An injection volume of 0.5 μl was used. Cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode.

Melting points were generally obtained with a Kofler hot bench, consisting of a heated plate with linear temperature gradient, a sliding pointer and a temperature scale in degrees Celsius.

General parallel synthesis procedure for compounds of Scheme 1 :

All reactions were conducted in Argonaut QUEST 210™ under a nitrogen atmosphere. The reaction tubes were loaded with a suspension of Hunigs base polystyrene resin (type, loading JOAQUIN 0.6 mmol) in anhydrous DMF (quantity JOAQUIN). Upon agitation compound A (1 18.7 mg, 0.2 mmol) and substituted benzylbromides (0.22 mmol) were added (Scheme 1). The reaction mixture was heated at 65 °C for 24 hours. The reaction mixture was filtered and the resin was washed with DMF. The combined filtrates were evaporated using a Genevac HT-4 yielding the target compounds 1 - 38 as trifluoroacetic acid salts analysed by LCMS.

Synthesis procedure for compounds 39-44 and 14 (X=N), illustrated in Scheme 2: Compound 39: (2-r[(4-bromophenyl^')phenylmethyl]thio]-3-Dyridinecarboxylic acid) A mixture of 2-mercapto-nicotinic acid (17.8g, 0.115 mol) and 4-bromo-benzhydrol (0.1 15 mol) in sodium bisulfite (15OmL) was stirred at room temperature for 18 hours and then poured out into water (80OmL). The mixture was stirred for 30 min. The resulting precipitate was filtered off, washed with water and hexane, and then dried in vacuo. Yield: 42.6g; TSMGJ)163_024_l ; (93%). ¹HNMR 6.47 (s, 1 H); 7.06 (dd, J=I.8, 4.7 Hz, 1 H); 7.22 (t, J=I.3 Hz, 1 H); 7.30 (t, J=7.4 Hz, 2 H); 7.37 (d, J=8.8 Hz, 2 H); 7.38 - 7.44 (m, 4 H); 8.28 (dd, J=7.8, 1.9 Hz, 1 H); 8.48 (dd, J=4.7, 1.9 Hz, 1 H). Anal. Calcd for C₉H₁₄BrNO₂S: C, 57.01 ; H, 3.53; N, 3.50. Found: C, 55.71; H, 3.22; N, 3.87.

Compound 40: (2-[[(4-bromophenyl)phenylmethyl1thio1-3-pyridinemethanol) Approximately 1OM borane dimethylsulfide complex in THF (12.7mL, 0.127 mol) was added carefully to a solution of compound 39 (42.6g, 0.106 mol) in dry THF (10OmL). The mixture was stirred at 50°C for 2.5 hours and then brought to room temperature. A saturated NaHCO₃ solution was added carefully. The mixture was treated with CH₂Cl₂/ sat.NaHCO₃. The organic layer was dried (Na₂SO₄), filtered and the solvent was evaporated. Yield: 37g; TSMGj) 163_029_l ; (90%). IH NMR (300MHz, DMSO-^₆) δ ppm 4.45 (d, J=5.4 Hz, 2H), 5.50 (t, J=5.3 Hz, IH), 6.42 (s, IH), 7.12 (dd, J=7.6, 4.7 Hz, IH), 7.22 (t, J=7.2 Hz, 1 IH), 7.30 (t, J=7.2 Hz, 2H), 7.37 - 7.51 (m, 6H), 7.68 (d, J=7.5 Hz, IH), 8.25 (d, J=4.9 Hz, IH); Anal. Calcd. (C₁₉H ₁₆BrNOS.2H₂O) C 54.03; H 4.77; N 3.32; Exp. C 59.31; H 4.72; N 3.17.

Compound 41 : (2-[f(4-bromophenyl)phenylmethyllthio1-3-pyridinemethylchloride) A solution of compound 40 (37g, 0.096 mol) in CH₂Cl₂ (40OmL) was cooled to 0⁰C. MeSO₂Cl (HmL, 0.144 mol) and Et₃N (15.8mL) were added. The mixture was stirred at room temperature for 2 hours. A saturated NH₄Cl was added. The mixture was treated with CH₂Cl₂/sat. NH₄Cl solution. The organic layer was dried (Na₂SO₄), filtered, and the solvent was evaporated. The resulting residue was used without further purification. IH NMR (CDCl₃) δ ppm 4.60 (s, 2H), 6.51 (s, IH), 7.00 (dd, J=7.3, 4.8 Hz, IH), 7.22 (t, J=7.3 Hz, I H), 7.29 (t, J=7.5 Hz, 2H), 7.33 - 7.43 (m, 6H), 7.58 (dd, J=7.5, 1.6 Hz, IH), 8.32 (dd, J=4.9, 1.6 Hz, IH)

Compound 42: (4-([2-([[(4-bromophenyl)phenyl)methyllthiol-3-pyridinvnmethyl)-l-tert- boc-piperazine) A mixture of compound 41 (0.096 mol), N-tert-boc piperazine (17.9g, 0.096 mol), and K₂CO₃ (13.2g, 0.096 mol) in acetonitrile (40OmL) was stirred at 80°C overnight. The solvent was evaporated in vacuo. The resulting residue was dissolved in CH₂Cl₂ and then washed with water. The organic layer was dried (Na₂SO₄), filtered and the solvent was evaporated under reduced pressure to give 53.5g of residue 1. A portion (27g) of residue 1 was purified by column chromatography over silica gel (eluent: CH₂Cl₂:hexane (1 :3)). The pure fractions were collected and the solvent was evaporated. Yield: 19g; TSMG_0163_034_l.

Compound 43 : (4-([^"2-([[(4-bromophenyl)phenyl)methyllthiol-3-pyridinyllmethyl)-l - piperazine)

A mixture of compound 42 (17.2g, 0.031 mol) in 20% TFA/CH₂C1₂ (15OmL) was stirred at room temperature for 5 hours. A 2N NaOH solution was added until the mixture was alkaline. The mixture was treated with CH₂C1₂/2N NaOH. The organic layer was separated, dried (Na₂SO₄), filtered and the solvent was evaporated in vacuo to give 12.6g of residue 1. This residue was purified by column chromatography over silica gel (eluent: CH₂C1₂:(CH₃OH:NH₃) (7:1)). Two fractions were collected and their solvents were evaporated. Yield: 7.5g of Fl (pure) and 4.4g of F2 (impure). F2 was purified again by HPLC. The pure fractions were collected and the solvent was evaporated. Yield: 1.7g of F2. Fl and re-purified F2 were combined. Yield: 9.2g; TSMGJ)169_023_l.

Compound 44: (4-(r2-(rr(4-bromophenyl)phenyl)methyl]thio-3-pyridinyllmethyl)-l- bis-tert- boc piperazine carboxamidine)

A mixture of compound 43 (7.5g, 0.0165 mol), N,N'-bis-tert-butoxycarbonylthiourea (4.5g, 0.0165 mol), EDCI (3.8g, 0.0198 mol) and Hunig's base (3.5mL, 0.0198) in ether (q.s.) was stirred for 16 hours at room temperature. The mixture was partitioned between IN NH₄Cl solution and Et₂O. The organic layer was separated, dried (Na₂SO₄), filtered and the solvent was evaporated in vacuo. The resulting residue was purified by flash column chromatography over silica gel (eluent: CH₂Cl₂:hexane (1:3 to 2:3)). The product fractions were collected and the solvent was evaporated. Yield: 3g of (I) (26%), 6.5g of (II) (56%) and 0.40Og of (III) (yellow foam; impure). IH NMR (DMSO-J₆) δ ppm 1.35 (s, 9H), 1.40 (s, 9H), 2.33 - 2.43 (m, 4H), 3.33 - 3.41 (m, 4H), 3.49 (s, 2H), 6.41 (s, IH), 7.10 (dd, J=7.5, 4.9 Hz, IH), 7.23 (t, IH), 7.31 (t, J=7.5 Hz, 2H), 7.40 - 7.49 (m, 6H), 7.64 (dd, J=7.5, 1.6 Hz, IH), 8.29 (dd, J=4.8, 1.8 Hz, IH), 9.54 (s, IH); Anal. Calcd. (C₃₄H₄₂BrN₅O₄S-H₂O) C 57.14; H 6.21 ; N 9.80; Exp. C 57.83; H 6.22; N 9.49.

Compound 14: (4-r(2-mercapto-3-pyridinyl)methyll-l -piperazine carboxamidine^")

A mixture of compound 44 (0.0136 mol) and triethylsilane (1OmL) in 50% TFA/CH₂C1₂ (100 mL) was stirred for 3 hours at room temperature. Hexane and Et₂O were added to start precipitation. The solvent was then decanted. More Et₂O was added and the mixture was stirred overnight. The resulting yellow solid was filtered off, washed with Et₂O and dried. Yield: 6.3g; TSMG_0169_026_l (used in next reaction step without further purification). TSMGJ)180_024_l. IH NMR (DMSO-J₆, 125°C) δ ppm 2.82 (t, J=5.14 Hz, 4 H) 3.57 (t, J=5.17 Hz, 4 H) 3.92 (s, 2 H) 6.78 (t, J=6.69 Hz, 1 H) 7.36 (br. s., 4 H) 7.63 (d, J=6.23 Hz, 1 H) 7.66 (d, J=7.18 Hz, 1 H); Anal. Calcd. (CnH,₇N₅S.2C₂HF₃O₂) C 37.58; H 3.99; N 14.61 ; Exp. C 37.46; H 3.61 ; N 14.80.

BIOLOGICAL EXAMPLES: Mode of Action

Mitochondria represent an important source of ROS in microorganisms. It is known that cellular stresses such as irradiation and cytotoxic drugs result in cell growth inhibition and death via endogenous (i.e., intracellular) ROS production. The discovery of the presence of a ROS scavenger in fungi such as Candida albicans may indicate that fungi need protection against endogenous ROS. The present invention demonstrates that the piperazine carboxamidine compounds of the invention can induce endogenous ROS in Candida spp., and further, that the compounds with high ROS induction properties can be fungicidal, while most of those compounds with lower ROS induction properties are fungistatic.

In an exemplary embodiment, the compounds of the present invention exhibit antifungal properties against, for example, Candida spp. For the most part, Candida spp. are ubiquitous fungi found throughout the world as normal body flora. Candidiasis is a common mycotic infection, especially in immunocompromised hosts, that contributes to a variety of diseases, such as, but not limited to, vaginitis, vulvovaginitis, vulvar rash, oral thrush, conjunctivitis, oropharyngeal candidiasis, endophthalmitis, diaper rash, nail infections, infections of skin folds, systemic candidiasis, oral candidiasis, gastrointestinal candidiasis and red macerated intertriginous areas. Exemplary species of Candida include, but are not limited to, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsiliosis, Candida guilliermondi, Candida lusitaniae and Candida krusei.

Additional diseases caused by fungi other than Candida that may be treated by the compounds of the invention include, for example, aspergillosis, blastomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis, sporotrichosis and zygomycosis.

Other fungi that are succeptible to treatment with the presently described compounds include, for example, associated with Pityrosporum spp., Malassezia spp. and Trichophyton spp. Exemplary embodiments of Trichophyton include, but are not limited to, Trichophyton mentagrophytes and Trichophyton rubrum. Exemplary embodiments of Pityrosporum include, but are not limited to, Pityrosporum orbiculare, Pityrosporum ovale, Pityrosporum canis and Pityrosporum pachydermatis. Exemplary embodiments of Malassezia include, but are not limited to, Malassezia sympodialis, Malassezia globosa, Malassezia restricta, Malassezia slooffiae, Malassezia furfur, Malassezia obtusa and Malassezia pachydermatis.

In an aspect of the invention, the compounds of the present invention exhibit antibacterial properties against Gram-positive bacteria such as e.g., Staphylococcus spp. Staphylococcus aureus and Staphylococcus epidermidis and are particularly significant in their interactions with humans. Staphylococcus aureus typically causes a variety of suppurative (pus-forming) infections and toxinoses in humans, including superficial skin lesions such as boils, styes and furuncles. More serious infections such as pneumonia, mastitis, phlebitis, meningitis, and urinary tract infections as well as deep-seated infections, such as osteomyelitis and endocarditis may also result. S. aureus is a major cause of hospital-acquired (nosocomial) infection of surgical wounds and infections associated with indwelling medical devices. S. aureus also causes food poisoning by releasing enterotoxins into food, and toxic shock syndrome by release of superantigens into the blood stream.

In another embodiment of the present invention, novel fungicidal compounds with potent anti-biofilm activity were identified. Previously, a target-based screening of a compound library was conducted in which compounds with antifungal activity and ROS accumulation capacity in C. albicans were identified. This screening resulted in the identification of a class of fungicidal piperazine-1-carboxamidine derivatives. Based on chemical similarity between these piperazine-1-carboxamidines and the antifungal arylguanidine abafungin, a series of related benzylsulfanyl-phenylamines were synthesized, this to identify more potent fungicidal compounds against the human fungal pathogens C. albicans and C. glabrata. Four compounds with MFC < lOμM for both pathogens were selected for subsequent biological evaluation. Their bactericidal activity against Staphylococcus epidermidis was assessed, as well as their potential to eradicate single (C albicans) and mixed (C albicans + S. epidermidis) species biofilms. Furthermore, to gain preliminary insight in their in vivo efficacy performance, these molecules were tested in an in vivo Caenorhabditis elegans model system for C. albicans infection.

Antifungal activity of the compound library was tested at a single concentration (100 μg/mL). Compounds with antifungal activity at higher minimal concentrations were not considered sufficiently potent or specific for potential use as an antimycotic. Compounds were dispensed in microtiter plates at a final concentration of 500 μg/mL in 10% DMSO. Approximately 80 μL of a Candida albicans cell culture grown overnight in YPD (diluted in fresh YPD to approximately 10⁶ cells/mL) was added to 20 μL of the compounds in order to assess their antifungal activity at 100 μg/mL in 2% DMSO. After 24 h of incubation at 37°C, the antifungal activity of the compounds on Candida albicans was assessed by microspectrophotometry of liquid cultures grown in microtiter plates by known procedures [Thevissen, K.; Terras, F.R.G.; Broekaert, W., "Permeabilization of Fungal Membranes by Plant Defensins Inhibits Fungal Growth," Appl. Envir. Microbiol.. 1999, 65, 5451-5458; Thevissen, K.; Cammue, B.P.A.; Lemaire, K.; Winderickx, J.; Dickson, R.C.; Lester, R.L.; Ferkert, K.K.; Van Even, F.; Parret, A.H.; Broekaert, W.F., "A Gene Encoding a Sphingolipid Biosynthesis Enzyme Determines the Sensitivity of Saccharomyces cerevisiae to an Antifungal Plant Defensin from Dahlia {Dahlia merckii) " Proc. Natl. Acad. Sci. USA, 2000, 97, 9531-9536]. Compounds displaying antifungal activity were further analyzed to determine their MIC₅₀ (i.e., the concentration of the antifungal compound that is required to inhibit 50% of the yeast growth). The MIC₅₀ concentration was calculated from dose- response curves with two-fold dilution steps. To this end, 20 μL of two-fold dilution series of the compounds were prepared in DMSO, after which 180 μL of MiIIiQ was added, leading to a dilution series of the compounds in 10% DMSO. Approximately 80 μL of a Candida albicans cell culture grown overnight in YPD (diluted in fresh YPD to approximately 10⁶ cells/mL) was added to 20 μL of these dilution series (leading to a dilution series of compounds in 2% DMSO) and incubated for 24 h at 37°C. Subsequently, antifungal activity of the compounds on Candida albicans was assessed by microspectrophotometry of liquid cultures grown in microtiter plates as before.

Strains and media. The yeast strains used in this study were Candida albicans SC5314 (Fonzi, W. A., and M. Y. Irwin. 1993. Isogenic strain construction and gene mapping in Candida albicans. Genetics. 134:717-728) and C glabrata BG2 (Kaur, R., B. Ma, and B. P. Cormack. 2007. A family of glycosylphosphatidylinositollinked aspartyl proteases is required for virulence of Candida glabrata. Proc Natl Acad Sci U S A. 2007 104:7628-7633); the bacterial strain was Staphylococcus epidermidis LMGl 0474. The media used were YPD (1% yeast extract, 2% peptone, 2% glucose) and 1/20 TSB (5% Tryptic Soy Broth; BD Diagnostics, MD, USA).

Fungicidal and bactericidal activity assay. The fungicidal activity of the compounds against Candida sp. was determined in PBS (5*105 CFU/mL) and the MFC for each compound was calculated as the minimal concentration resulting in less than 0.1 % survival of the yeast culture relative to DMSO control (Graybill, J. R., D. S. Burgess, and T. C. Hardin. 1997. Key issues concerning fungistatic versus fungicidal drugs. Eur J Clin Microbiol Infect Dis. 16:42-50; Thevissen, K., U. Hillaert, E. M. Meert, K. K. Chow, B. P. Cammue, S. Van Calenbergh, and I. E. Francois. 2008. Fungicidal activity of truncated analogues of dihydrosphingosine. Bioorg Med Chem Lett. 18:3728-3730). To assess the bactericidal activity of the compounds against S. epidermidis in a similar setup as for MFC determination, an overnight culture in TSB was 1/10,000 diluted in PBS (2*105 CFU/mL) and the MBC (minimal bactericidal concentration) for each compound was determined as the minimal concentration resulting in less than 0,1 % survival of the bacterial culture as compared to DMSO control treatment.

Measurement of ROS production

Endogenous ROS levels were measured by a fluorometric assay with 2',7'-dichlorofluorescin diacetate (DCFHDA; Molecular Probes, Inc., Eugene, OR) as described [Francois et al., 2005]. Briefly, 5 mL of an early log phase yeast culture in YPD (grown at 37°C) was centrifuged. The cell pellet was washed with PBS and resuspended in 5 mL PBS. Approximately 40 mL aliquots of the yeast cell suspension were mixed with 20 μL of a compound at a final concentration of 100 μg/mL in 2% DMSO (initial screening to identify compounds that induce ROS at 100 μg/mL) or with 20 μL of a two-fold dilution series of compounds (prepared as described above) or DMSO and incubated in white 96-well microtiter plates (PE white, Perkin-Elmer, Norwalk, CT). After 1 h of incubation at 37°C, 40 μL aliquots of DCFHDA stock solution (25 μM in PBS) were added to the cell suspensions. Fluorescence emitted by the cells in the microtiter plates was measured with a Perkin-Elmer LS 50 B fluorescence spectrometer at an excitation wavelength of 485 nm (2.5 nM slit) and an emission wavelength of 540 nm (2.5 nm slit). Fluorescence was measured after 1, 3 and 5 hrs of incubation at 37⁰C in the dark. Fluorescence values of the samples were corrected by subtracting the fluorescence values of the antifungal compound in the corresponding concentration without cells but with DCFHDA. The corrected fluorescence values (CFVs) can hence be considered as a measure to determine the extent of increased endogenous ROS levels.

Antibacterial activity of the compounds was tested with the following dose range in μM: 64 - 16 - 4 - 1 - 0.25 - 0.0625 - 0.015625 - 0.00390625. Compounds with antibacterial activity at higher minimal concentrations were not considered sufficiently potent or specific for potential use as an antibacterial (antibiotic). Assays were performed by adding 190 μL of the bacteria suspension (5x10⁵ CFU/mL). After 17 hours at 37°C, bacteria growth was assessed fluorimetrically after addition of resazurin. Fluorescence was measured after 20 minutes. The results were expressed as % reduction in bacteria growth/viability compared to control wells.

Biofilm activity assay. The activity of the compounds against 24h-old C. albicans SC5314 biofilms was assessed using the crystal violet quantification method as shown herein. The biofilmeradicating capacity of a compound was determined as the concentration resulting in 50% killing of the Candida biofilm (BEC50). To assess the activity of the compounds against mixed biofilms composed of C. albicans SC5314 and S. epidermidis in a fifty/fifty ratio, overnight cultures of both organisms were suspended in 1/20 TSB at

0.5 and 50μL of both cultures were mixed in the wells of a 96-well plate. After an adhesion phase of 24 hours, planktonic cells were 6 removed and fresh 1/20 TSB-medium was added for the 48- hour growth phase. Mature biofilms were incubated for 24 hours with the fungicidal compounds in PBS and biofilm mass was quantified using the crystal violet staining. To assess the number of bacterial and fungal cells in these biofilms, biofilm cells were resuspended and plated on media promoting growth of both C. albicans and S. epidermidis (YPD) or of C. albicans alone (YPD+lOOμg/mL ampicillin) after which the colony forming units (CFUs) were determined.

C. elegans model for C. albicans infection. The in vivo efficacy of the compounds was assessed in a C. elegans model for Candida infection as described by Breger et al., 2007 (Breger, J., et al., 2007. PIoS Pathog. 3:el8). To this end, L4 larvae of a double mutant (glp- 4Δsek-lΔ) of C. elegans were used and fed for 4 h on C. albicans SC5314 agar plates (YPD agar plates on the surface inoculated with lOOμl of an overnight culture in YPD and incubated for 16 h at 37°C). Worms were collected and washed with M9 buffer containing 3g/L KH2PO4, 6g/L Na2HPO4, 5g/L NaCl, ImM MgSO4, lOμg/mL cholesterol and lOOμg/mL Kanamycin. Fourty-to-fifty worms were suspended in ImL in each well of 24-well microtiterplates, in the presence or absence (DMSO control) of the fungicidal compounds. Survival of the worms was monitored daily. The percentage survival of the worms in the presence or absence of antifungal compounds was calculated each day relative to the survival at day 0. Miconazole (60μM) was used as a positive control. Data are means of duplicate measurements and experiments were performed at least twice.

Table 1 exhibits the observed antifungal activities of specific embodiments of the present invention. The antifungal activity demonstrated by the listed MIC values shows the fungistatic activity of the compounds. Several compounds also demonstrated fungicidal activity, even without combined ROS activity. Compounds with a combined ROS/MIC activity (ROS >300) were observed to have fungicidal activity. The contribution and safety of ROS together with antifungal activity leads to compounds with superior therapeutic properties.

Table 1: Antifungal activity of specific compounds of the present invention.

Table 2 exhibits the observed antibacterial activities of specific embodiments of the present invention. This table demonstrates the chemical diversity of the compounds of the present invention along with their IC_5O values. The antibacterial activity demonstrated by the IC₅₀ values shows the bacteriostatic activity of the compounds.

Table 2: Antibacterial activity of specific embodiments of the claimed compounds.

Table 3 exhibits the observed antifungal activities of specific embodiments of the present invention. The antifungal activity demonstrated by the listed MIC₅₀ values (in μg/mL), MFC values (in μg/mL), and Corrected Fluorescence Value (CFV) at 100 μg/mL shows the fungistatic activity of the compounds.

Table 3. Antifungal activity of the piperazine-1-carboxamidine derivatives on C. albicans

Comp R₂ R₃ R₄ Rs R₆ MIC₅₀ MFC CFV

26 CN H H H H >100 >100 0

27 CH₃ H H H H >100 >100 47

11 CH₂SO₂Ph H H H H 12.5 >100 155

8 H Ph H H H 20 50 761

7 H H Br H H 75 100 467

5 H OCF₃ H H H 80 100 338

28 H CN H H H >100 >100 0

2 H Cl H H H 75 >100 31 1

29 H NO₂ H H H >100 >100 0 6 H H Cl H H 75 100 780

13 H H CF₃ H H 75 83 552

30 H H CN H H >100 >100 0

12 H H Et H H 37.5 60 645

4 H H Ph H H 20 50 780

31 H H MeSO₂ H H >100 >100 0

32 F F H H H >100 >100 62

33 F H F H H >100 >100 114

34 F H H F H >100 >100 87

35 F H H H F >100 >100 1

36 F H H H Cl >100 >100 0

37 F H F H F >100 >100 0

38 OCH₃ H H NO₂ H >100 >100 59

1 H Br H Br H 9.4 50 780

3 H F H F H 50 >100 171

Additional specific compounds of Formula I include, but are not limited to:

54;

or pharmaceutically acceptable salts thereof.

A systematic structure activity relationship (SAR) analysis of a family of piperazinecarboxamidines was carried out. Designated compounds were evaluated for fungicidal properties and their ability to induce reactive oxygen species (ROS). These fungicidal compounds with ROS inducing capacity (FROS compounds) were tested for their toxicity and spectrum and selectivity of their antifungal activity.

The investigation aimed for compounds that were not toxic for human cells, active against C. albicans, C. glabrata, azole-resistant clinical Candida isolates, and/or Candida biofilms. In addition, compounds that are active against pathogenic bacteria such as Staphylococcus spp. but not active against beneficial organisms such as Lactobacillus spp. may be used in the present invention.

Selected compounds in vitro were then tested in vivo in a rat model for vulvovaginal candidosis. The following findings relate to the potential of the certain compounds to induce apoptosis and the subsequent evaluation of the apoptotic signaling pathways involved are described. The nature of the piperazinecarboxamidine backbone is important for its fungicidal activity and/or ROS inducing capacity. From the compounds synthesized, various compounds with a good selective fungicidal in vitro activity were selected. All synthesized compounds were analyzed for (i) fungicidal activity, (ii) ROS inducing activity, (iii) in vitro toxicity, and (iv) antifungal activity spectrum.

In order to identify FROS compounds among the synthesized piperazinecarboxamidine derivatives/hybrids, the ROS inducing capacity and fungicidal character of the compounds on Candida albicans were analyzed and the results shown in Table 3.

ROS inducing capacity in C. albicans

The synthesized derivatives were analyzed for their ROS inducing capacity by determining the endogenous ROS levels induced at various compound concentrations in two-fold dilution series in a 2% DMSO solution. Compounds with a high corrected fluorescence value (CFV>350), medium (50<CFV<350) and low (CFV<50) ROS inducing capacity are indicated by High (H), Medium(M), and Low (L), respectively.

Analysis of the fungicidal character of the synthesized compounds on C. albicans and C glabrata

In order to determine the fungicidal activity of the compounds, an overnight C. albicans or C. glabrata culture was incubated in the presence of the respective compounds (in a range of 1 - 200 μM) or DMSO. Percentage survival is calculated as the ratio of the number of colony forming units (CFUs) after treatment with the compound as compared to the number of CFUs of the cells treated with DMSO. The minimal fungicidal concentration (MFC) of a compound is defined as the minimal compound concentration leading to a reduction of 99.9% of the viability of the inoculum (Graybill, J. R. et al., Eur. J. Clin. Microbiol. Infect. Pis., 1997, 16, 42). The results of these analyses, in addition to the above mentioned ROS values, are shown in the central and last columns of Table 4.

Table 4

Analysis of toxicity of the selected FROS compounds

HaCaT (a human keratinocyte cell line), NHEK (normal human epidermal keratinocytes) and MRC-5 (secondary human lung fibroblasts) cells may be used to assess the in vitro toxicity of a selection of FROS compounds. Since the outcome of the toxicity assays using the different cell lines is similar, only one cell line, namely MRC-5 cells, can be used for in vitro toxicity/selectivity tests. By testing the synthesized compounds using the MTTlike resazurin method for potential toxicity on MRC-5 cells, a significant subset of compounds possessed in vitro toxicity.

Activity against mixed species biofilms of C. albicans and Staphylococcus epidermidis Biofilm formation of C. albicans, whether or not mixed with S. epidermidis, was achieved in 96-well polystyrene microtiter plates. A protocol to obtain these mixed fungal-bacterial biofilms was adapted from Adam, B. et al., (2002) J Med Microbiol 51(4): 344-349. Based on their preliminary experiments, Tryptic Soy Broth (TSB) was selected as the best liquid medium to support the growth requirements of both fungi and bacteria. However, a number of research groups appears to assume an equal distribution of bacterial and fungal cells in the biofilms, referring to the similar growth rates of the organisms in the selected medium and the mixing of equal volumes of a standardized suspension of each organism (Adam, B. et al., (2002). J Med Microbiol 51(4): 344-349; Al-Fattani, M. A. et al., (2006) J Med Microbiol 55(Pt 8): 999-1008; Thein, Z. M. et al., (2006) Arch Oral Biol 51(8):672-680; Kawarai, T. et al., (2007) Appl Environ Microbiol 73(14): 4673-4676). However, no quantitative data are available yet regarding the ratio bacteria/fungus in mixed species biofilms. As such, we fine- tuned the protocol to obtain a fifty-fifty ratio.

Therefore, biofilm cells were resuspended and plated on media promoting growth of both C. albicans and S. epidermidis (YPD and LB-medium) or of C. albicans alone (LB-medium + 100 μg/ml ampicillin) after which the colony forming units (CFUs) could be determined.

Briefly, the formation of the mixed species biofilms occurred by mixing diluted overnight cultures of both organisms (OD590nm 0,5; diluted in 20 times diluted TSB (1/20 TSB)) and subsequent addition of the suspension to the wells of a 96-well plate. After an adhesion phase of 24 hours, planktonic cells were removed and fresh 1/20 TSB-medium was added for the 48-hour growth phase. Mature biofilms were incubated for 24 hours with the fungicidal compounds after which biofilm mass was quantified using crystal violet staining.

Elucidation of the mode of antifungal action of FROS compounds with selective activity

Compound 67, a piperazine carboxamidine derivative, was investigated for its activity, a Saccharomyces cerevisiae deletion mutant library (Invitrogen) was screened for enhanced resistance and hypersensitivity towards this compound. As such, genes involved in sensitivity or tolerance mechanisms, respectively, could be detected and the mode of action delineated.

Compound 67

Screening of the S. cerevisiae deletion mutant library yielded 86 hypersensitive and 51 compound 67-resistant mutants. Most of the affected genes were involved in either (i) gene expression, (ii) mitochondrial functionality, (iii) vacuolar functionality, (iv) vesicular transport and (v) (sphingo)lipid metabolism (FIGS. 6 and 7). Based on the facts that (i) compound 67 induces endogenous ROS in yeast, (ii) ROS species are generated in mitochondria and (iii) ROS production can be involved in apoptosis, whether compound 67 is able to induce apoptosis in S. cerevisiae was analyzed. To address this issue, the presence of molecular markers for apoptosis in compound 67-treated S. cerevisiae wild type (WT) cells was investigated. As such, compound 67 was able to induce apoptosis, in the aforementioned cells, as shown by the presence of chromatin condensation/fragmentation and phosphatidylserine externalization.

Furthermore, the use of yeast deletion mutants, affected in different apoptotic pathways, enabled the analysis of the compound 67-induced apoptotic process. The deletion of a pro- or an anti-apoptotic gene, involved in compound 67-induced apoptotic signalling, rendered the mutants less or more susceptible, respectively, to compound 67 in comparison with WT cells. Additionally, the deletion of genes involved in mitochondrial fusion and fission (DNMl, MDVl, FISl) exhibited an altered compound 67 sensitivity phenotype. Using fluorescence microscopy, an altered mitochondrial morphology of WT yeast cells could be visualized upon compound 67 treatment.

Moreover, the interference of caspases (Ycal) and the apoptosis-inducing factor Aiflp in compound 67 antifungal activity was demonstrated. Another subset of deletion mutants, displaying an altered sensitivity phenotype, was affected in sphingolipid metabolism. Based on the results obtained, compound 67-induced apoptosis is dependent on the presence of sphingolipids. Conclusively, compound 67 induces apoptosis in the model yeast S. cerevisiae. Moreover, mitochondrial morphology is affected by compound 67 treatment and different apoptotic pathways play a role in its antifungal activity and the compound 67- induced apoptosis.

Furthermore, the induction of apoptosis by compound 67 is dependent on the presence of sphingolipids. The mode of action of another piperazine carboxamidine derivative, compound 1, was investigated in parallel. However, since compound 67 (MFC = 8 μM) proved to be more potent than compound 1 (MFC = 50 μM), the unravelling of the mode of action of compound 67 was the focus (see supra).

The research did demonstrate that the initial mechanistic approach on induction of intracellular reactive oxygen in fungi notably Candida species several new in vitro models have been established and some existing in vivo models were improved.

Experimental Studies of Compound 67 In the current study, the fungicidal piperazine-1-carboxamidine 4-{[3-(4-chlorobenzyl)-2- methoxyquinolin-6-yl]methyl}piperazine-l-carboximidamide (compound 67) induces caspase-dependent apoptosis and mitochondrial fragmentation in Saccharomyces cerevisiae. Furthermore, the mitochondrial fission protein, Fisl, antagonizes the compound 67-induced apoptosis, as the mutant ΔfisJ is hypersensitive to compound 67 and characterized by increased apoptosis induced by compound 67. These data point to the induction of yeast apoptosis by compound 67 which is dependent on mitochondrial fission protein Fisl and caspase Ycal .

To treat fungal infections, fungicidal compounds are favored over fungistatic compounds since the latter probably contribute to the development of resistance [Onyewu, C. et. al., (2003) Antimicrob. Agents Chemother. 47, 956-964]. As it has been demonstrated that accumulation of endogenous ROS and fungicidal activity in C. albicans are linked [Francois, I.E. et al., (2006) Anti- Infect. Agents Med. Chem. 5, 3-13], a compound library for compounds inducing ROS in C. albicans has been screened herein in an attempt to identify new fungicidal compounds. The various piperazine-1-carboxamidine derivatives have such ROS accumulation capacity that these compounds are fungicidal, i.e. their minimal fungicidal concentration, MFC, range between 50 and 100 μg/ml. ROS involvement in fungicidal activity of these compounds is further demonstrated by the abolishment of the fungicidal activity of these compounds in combination with an antioxidant.

An excess of endogenous ROS levels is a phenotypical marker of yeast cells undergoing apoptosis [Madeo, F. et al., (1997) J Cell Biol. 139, 729-734; Madeo, F. et al., (1999) J. Cell Biol. 145, 757-767]. Whether fungicidal piperazine-1-carboxamidine derivatives induce apoptosis in yeast was investigated in this study using the model yeast S. cerevisiae and the fungicidal piperazine-1-carboxamidine quinoline 4-{[3-(4-chlorobenzyl)-2-methoxyquinolin- 6-yl]methyl}piperazine-l-carboximidamide (compound 67). Moreover, a putative involvement of caspases and the mitochondrial fission pathway in fungal cell death induced by compound 67 was investigated.

Materials and Microorganisms

4-{[3-(4-chlorobenzyl)-2-methoxyquinolin-6-yl]methyl}piperazine-l-carboximidamide (compound 67) was purchased from Johnson & Johnson Pharmaceutical Research & Development (VaI de Reuil, France). Yeast strains used in this study are Saccharomyces cerevisiae BY4741 and the corresponding deletion mutants Aycal and Afisl (EUROSCARF). Medium used was YPD (1% yeast extract, 2% peptone, 2% glucose).

Statistical analysis Statistical analysis was performed using paired T-test.

Antifungal activity assay

S. cerevisiae cultures, grown overnight in YPD, were diluted in PBS (2*107 cells/ml) and incubated with various concentrations of compound 67 or DMSO in the presence or absence of 80 μM z-VAD-FMK caspase inhibitor (Promega, Madison, WI, USA). After 0 h and 2.5 h of incubation at 30 °C, 100 μL aliquots were plated on YPD plates and colony forming units (CFUs) were counted after 2 days of incubation at 30 °C. Percentage survival was calculated based on the ratio of the number of CFUs after treatment with compound 67 as compared to DMSO-treated controls. Data are the mean of three independent experiments, each consisting of duplicate measurements.

Yeast apoptosis and caspase assays

S. cerevisiae cultures, grown overnight in YPD, were diluted in PBS (2*107 cells/ml) and incubated with 20 μg/ml compound 67 or DMSO 2.5 h at 30 °C. Apoptotic markers, including ROS levels, phosphatidylserine externalization and DNA fragmentation of yeast cultures (n=l 00-500 cells per measurement; values presented in the text are mean of triplicate measurements), were visualized via staining with 2',7'-dichlorofluorescin diacetate (DCFHDA), FITC-labelled annexin V in combination with propidium iodide and TUNEL, respectively, as described previously [Madeo, F. et al., (1997) J Cell Biol. 139, 729-734; Madeo, F. et al., (1999) J. Cell Biol. 145, 757-767 '. Caspase activity was measured using the CaspACE FITC-VAD-FMK In Situ Marker (Promega). For image acquisition, a fluorescence microscope (model Axio Imager Zl), a digital camera [Madeo, F. et al., (2002) MoI. Cell 9, 911-917] (model Axio Cam Mrm), and Axio Vision ReI. 4.6 software were used. The percentage positive cells for the different treatments was calculated relative to the total number of cells (i.e. 100-500).

Visualization of mitochondrial morphology

S. cerevisiae cultures, grown overnight in YPD, were diluted in PBS (2*107 cells/ml) and incubated with 20 μg/ml compound 67 or DMSO 2.5 h at 30 °C. Mitochondria were visualized using 100 ng/ml 3,3'-dihexyloxacarbocyanine iodide (DiOC6) (Acros Organics, Geel, Belgium) in PBS as described previously [Aerts, A.M. et al., (2008) Cell MoI Life Sci. 65, 1933-1942].

Various fungicidal piperazine-1-carboxamidine derivatives are shown herein to induce ROS accumulation in yeast species. Since ROS accumulation is an early marker of induction of apoptosis in yeast, the fungicidal piperazine-1 -carboxamidine 4-{[3-(4-chlorobenzyl)-2- methoxyquinolin-6-yl]methyl}piperazine-l-carboximidamide (compound 67) was demonstrated to induce apoptosis in yeast. In general, apoptosis in yeast is best studied at rather high survival rates, since severe killing results in a high necrotic yeast population [Phillips, A.J., et al., (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 726-731 ; Kitagaki, H. et al., (2007) FEBS Lett. 581, 2935-2942]. The percentage survival of S. cerevisiae cultures after treatment were first demonstrated with different concentrations of compound 67 (ranging from 15 μg/ml to 30 μg/ml) in PBS (FIG. 1). Treatment of S. cerevisiae cultures with 20 μg/ml BAR0329 in PBS for 2.5 h resulted in 40.7 ± 3.0% survival as compared to DMSO treatment. In order to clarify if the compound 67-induced cell death is of apoptotic nature, the same cultures were assessed for other apoptotic markers, including DNA fragmentation (visualized via the deoxynucleotidyltransferase-mediated dUTP nick end laeling (TUNEL) assay) and phosphatidylserine (PS) translocation from the inner leaflet to the extracellular side of the plasma membrane. PS translocation and loss of membrane integrity were simultaneously analyzed in order to discriminate between apoptotic and necrotic death (visualized via costaining with FITC-labeled annexin V and propidium iodide (PI)) [Madeo, F. et. al., (1999) J. Cell Biol. 145, 757-767; Madeo, F. et al., (2002) MoI. Cell 9, 91 1 -917]. Compound 67-treated cultures were characterized by increased ROS and DNA fragmentation levels, and by an excess of annexin V positive/PI negative cells as compared to water-treated cultures (FIG. 2). Apoptosis in yeast automatically leads to secondary necrosis after time [Phillips, AJ. et al., (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 726-731 ; Phillips, A.J. et al., (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 14327-14332]. The amount of annexin V negative/PI positive ('PI only') cells that represent necrotic cells (i.e. 1.0 ± 0.2%) was approximately 35-fold lower than the amount of annexin V positive/PI negative cells after compound 67 treatment (i.e. 35.0 ± 3.0%), indicating a predominant apoptotic cell death of the S. cerevisiae culture induced by compound 67.

In S. cerevisiae, yeast caspase Ycalp is a key player of apoptosis. To examine the involvement of active caspases in compound 67 antifungal activity, survival of AycaJ and wild type (WT) S. cerevisiae cultures treated with compound 67 was assessed. AycaJ was found to be resistant to compound 67: treatment of Aycal with 20 μg/ml compound 67 in PBS for 2.5 h resulted in 100% survival as compared to DMSO treatment. Moreover, Aycal was characterized by decreased accumulation of endogenous ROS and chromosomal fragmentation upon compound 67 incubation as compared to WT (FIGS. 3 AND 4), pointing to increased resistance of Aycal to compound 67-induced apoptosis as compared to WT.

In order to clarify a possible caspase activation in S. cerevisiae upon compound 67 treatment, S. cerevisiae cultures were treated with 20 μg/ml compound 67 in PBS for 2.5 h and afterwards incubated with 10 μM of the FITC-labeled analog of the pancaspase inhibitor z- VADFMK, which binds to the active site of caspases in yeast [Madeo, F., et al., (2002) MoI. Cell 9, 911-917]. Compound 67 treatment led to an increase in the number of fluorescent cells (37.0 ± 3.0%) as compared to DMSO-treated control cultures (2.0 ± 0.1%), indicating that compound 67 induced caspase activation. All these data point to a compound 67 mode of antifungal action that is dependent on caspase activity.

To get more insight in the molecular mechanism underlying compound 67-induced caspasedependent apoptosis in yeast, a putative involvement of FISl in this process was assessed. FISl encodes a mitochondrial fission protein and has been implicated in caspasedependent apoptosis in yeast [Fannjiang, Y. et al., (2004) Genes Dev. 18, 2785-2797]. To this end, survival of Afisl and WT S. cerevisiae cultures treated with compound 67 was assessed. Δfisl was found to be hypersensitive to compound 67: treatment of Δfisl with 20 μg/ml compound 67 in PBS for 2.5 h resulted in 3.5 ± 1.1% survival as compared to DMSO treatment. Moreover, Δfisl was characterized by increased induction of endogenous ROS and chromosomal fragmentation upon compound 67 incubation as compared to WT (FIGS. 3 AND 4), pointing to increased apoptosis of Δfisl culture induced by compound 67 as compared to WT.

As Fisl has been shown to be involved in mitochondrial fragmentation and apoptosis in yeast [Aerts, A.M. et al. (2008) Cell MoI Life Sci. 65, 1933-1942; Kitagaki, H. et al., (2007) FEBS Lett. 581, 2935-2942; Fannjiang, Y. et al., (2004) Genes Dev. 18, 2785-2797], mitochondrial morphology in WT yeast upon incubation with 20 μg/ml compound 67 for 1 h in PBS was next assessed. To this end, mitochondria were visualized using the potential-dependent fluorescent dye DiOC6 [Aerts, A.M. et al., (2008) Cell MoI Life Sci. 65, 1933-1942]. Balanced fusion and fission of mitochondria results in tubular mitochondrial morphology, as is the case for untreated cultures (Fig. 5A). In compound 67-treated yeast cultures however, excessive fission of tubular mitochondria into short punctuate structures, also referred to as mitochondrial fragmentation [Scott, S.V. et al., (2003) Curr. Opin. Cell. Biol. 15, 482-488], was observed (Fig. 5B). These data indicate that compound 67 induces mitochondrial fragmentation in yeast.

The fungicidal activity of a series of piperazine-1-carboxamidines involving the induction of ROS generation in yeast is demonstrated herein, which is one of the phenotypical markers of apoptosis in yeast [Madeo, F. et al., (1997) J Cell Biol. 139, 729-734; Madeo, F. et al., (1999) J. Cell Biol. 145, 757-767]. In this study, the piperazine-1-carboxamidine 4-{[3-(4- chlorobenzyl)-2-methoxyquinolin-6-yl]methyl}piperazine-l-carboximidamide (compound 67) indeed induces apoptotic cell death in S. cerevisiae, and concomitantly triggers caspase activation. Moreover, the compound 67-induced apoptotic death mediated by the mitochondrial fission pathway and is accompanied by increased mitochondrial fission. A broad range of stress conditions and drugs induce caspase-dependent apoptosis of S. cerevisiae [reviewed in: Mazzoni, C. et al., (2008) Biochim Biophys Acta. 1783, 1320-1327], including low doses of H2O2, acetic acid, hyperosmotic stress induced by glucose or NaCl, the short-chained fatty acid valproic acid, arsenic, caffeine and metal ions and ethanol. The involvement of the mitochondrial fission machinery, and more specifically Fislp, in apoptosis-induction by these stimuli has only been described for ethanol [Kitagaki, H. et al.,

(2007) FEBS Lett. 581, 2935-2942] and acetic acid [Fannjiang, Y. et al., (2004) Genes Dev. 18, 2785-2797; Cheng, W.C. et al., (2008) Cell Death Differ. 15, 1838-1846]. Note that there exist several reports on stimuli that cause mitochondrial fragmentation and apoptosis in yeast, independent of a functional yeast caspase, like pheromone treatment [Zhang, N.N. (2006) MoI Biol Cell. 17, 3409-3422] and overexpression of alkaline ceramidase [Aerts, A.M.

(2008) Cell MoI Life Sci. 65, 1933-1942].

The mechanism by which mitochondria fragment in response to apoptotic stimuli is not fully understood. Kitagaki et al. propose that Fisl is required for mitochondrial fragmentation which occurs within short time span (30 min) after treatment with the apoptotic stimulus ethanol [Kitagaki, H. (2007) FEBS Lett. 581, 2935-2942]. However, Fannjiang et al. found that Fisl is not required for mitochondrial fission during cell death. To the contrary, Fisl limits mitochondrial fission and death [Fannjiang, Y. (2004) Genes Dev. 18, 2785-2797]. Thus, in the absence of Fisl, mitochondria fail to regain their ability to fuse following a death stimulus, subsequently leading to loss of mitochondria that is concomitant with cell death.

In conclusion, this study presents evidence for a fungicidal piperazine-1 -carboxamidine quinoline to induce caspase-dependent apoptosis in yeast, involving mitochondrial fission pathway. Interestingly, cytotoxicity of these piperazine-1-carboxamidine compounds, tested in vitro on HELAM cells, was low: their pIC50 (i. e. -log of the half maximal (50%) inhibitory concentration) was typically lower than 4, pointing to selective fungicidal activity as described herein. Thus, all these data shed light on possible medical applications involving piperazine-1-carboxamidines in general and compound 67 in particular for the treatment of fungal infections.

Additional Experimental Section and Synthetic Schemes

Additional synthetic schemes useful for making the herein described compounds are as described below. All compound numbers referred to in this specific "Additional Experimental Section and Synthetic Schemes" section refer only to the compounds described in this section and nowhere else in the present application. Scheme 3:

Scheme 4. General parallel synthesis procedure for compounds 1-24

Scheme 5. Synthesis procedure for compounds 30-36

d)

37 36 35 34

Chemistry

General methods. Melting points were measured in open capillaries on a Buchi B545 instrument and are uncorrected. IH NMR spectra were recorded with Bruker Avance DPX 400, 360 and 300 spectrometers and chemical shifts (d) are expressed in parts per million (ppm) with TMS as internal standard Elemental analyses were performed with a Carlo-Erba EAl I lO analyzer Silica gel thin-layer chromatography was performed on precoated plates Kieselgel 6OF 254 (Merck, Germany). Silica gel column chromatography was performed with Kiesel gel 60 (0.063 to 0 200 mm, Merck, Germany) Mass spectra were obtained on a Waters-Micromass ZQ mass spectrometer with an electrospray ionization (ESI) source operated in positive and negative ionization modes. Mass spectra were acquired by scanning from 100 to 1000 mass units in 1 s using a dwell time of 0.1 s The capillary needle voltage was 3 kV and the source temperature was maintained at 1408C. Nitrogen was used as the nebulizer gas Cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode HPLC was performed on a Waters Alliance HT 2790 system with a column heater set at 408C. Flow from the column was split to a Waters 996 photodiode array (PDA) detector and a Waters Micromass ZQ mass spectrometer with an ESI source. Reversed phase HPLC was carried out on a Xterra MS Cl 8 column (3.5 mm, 4.6x100 mm) with a flow rate of 1.6 mLmin^"1. Three mobile phases (A: ammonium acetate (25 mm)/CH3CN, 95:5; B: CH3CN; C: CH3OH) were employed to run a gradient condition from A (100%) -> B/C (1:1) over 6.5 min, -> B (100%) over 1 min, -> C (100%) for 1 min and re-equilibrate with A (100%) for 1.5 min. An injection volume of 10 mL was used. MsCl (methanesulfonyl chloride), EDC (l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), DIPEA (N,N-Diisopropylethylamine), TFA (trifluoroacetic acid), and DIPE (diisopropyl ether).

General parallel synthesis procedure for compounds 1-24: All reactions were conducted using an Argonaut QUEST 210 under a N2 atmosphere. The reaction tubes were loaded with a suspension of polystyrene-bound DIPEA (1.3 mmolg^"1, 0.6 mmol) in anhydDMF (8 mL). Upon agitation, 37 (1 18.7 mg, 0.2 mmol) and the appropriate substituted benzylbromide, or benzylchloride in the case of 9, 13, 14, and 20, (0.22 mmol) were added. The reaction mixture was heated at 65C for 24 h. After cooling, the mixture was filtered and the resin was washed with DMF. The combined filtrates were concentrated to give the target compounds 1-24 as TFA salts. LCMS analysis confirmed high levels of purity (81-100%; see Supporting Information).

4-[[2-[[(4-Chlorophenyl)methyl]thio]-3-pyridinyl]methyl]-l-piperazinecarboximidamide trifluoroacetate (1 :2) (10): A solution of TFA in CH2C12 (10 mL, 50%) was added to 30 (111 mg, 0.143 mmol). The mixture was stirred at RT for 2 h. The solvent was removed and the residue was stirred in 2-propanol/DIPE (1 :3). The precipitate was filtered off and recrystallized (CH3OH/Et2O; 1 :10) yielding compound 10 (0.042 g, 60%) as a solid; mp: 181.6-183.8C; ¹H NMR (400 MHz, [Do]DMSO): 6=2.40-2.47 (m, 4H), 3.36-3.43 (m, 4 H), 3.46 (s, 2 H), 4.43 (s, 2 H), 7.17 (dd, J=7.5, 4.8 Hz, 1 H), 7.35 (d, J=8.6 Hz, 2 H), 7.37 (br s, 3H), 7.42 (d, J=8.6 Hz, 2 H), 7.66 (dd, J=7.5, 1.8 Hz, IH), 8.42 ppm (dd, J=4.8, 1.7 Hz, IH); Anal, calcd for C18H22C1N5S-2C2HF3O2 : C 43.75, H 4.01, N 14.61, found: C 37.68, H 3.61, N 14.80.

2-[[(4-Chlorophenyl)methyl]thio]-3-pyridinecarboxylic acid (25): A mixture of 2-mercapto nicotinic acid (5 g, 0.032 mol) and Na2CO3 (3.8 g, 0.035 mol) in acetone (50 mL) was stirred at RT. 4-Chlorobenzyl chloride (5.1 g, 0.032 mol) was added dropwise and the reaction mixture was stirred at RT for 30 min. The mixture was poured into water (250 mL) and the solution was acidified to pH 6 with aq HCl (10 %), and extracted with CH2C12 (x2). The combined organic layer were washed with H2O, the combined organic layers were dried (MgSO4), filtered and concentrated. The residue was crystallized from 2-propanol to give 25 (3.46 g, 38.9%); ¹H NMR (300 MHz, [DO]DMSO): δ=4.36 (s, 2H), 7.26 (dd, J=7.8, 4.8 Hz, IH), 7.35 (d, J=8.8 Hz, 2 H), 7.44 (d, J=8.8 Hz, 2 H), 8.22 (dd, J=7.8, 1.8 Hz, IH), 8.65 (dd, J=4.8, 1.9 Hz, IH), 13.48 ppm (br s, IH).

Methyl 2-[[(4-chlorophenyl)methyl]thio]-3-pyridinecarboxylate (27): A mixture of 25 (3.36 g, 0.012 mol) in thionyl chloride (40 mL) was refluxed while stirring for 2 h. The reaction mixture was concentrated in vacuo. Dry toluene (50 mL) was added then removed in vacuo to give 26 (3.55 g, 99%), which was used in the next step without purification. A mixture of 26 (3.1 g, 10 mmol) in dry CH3OH (150 mL) was stirred at RT for 16 h. The solvent was evaporated and the residue was purified by column chromatography (CH2C12/hexane; 50:50). The resultant product was recrystallized from DIPE/hexane yielding 27 (2.7 g, 92%); mp: 778C; ¹H NMR (360 MHz, CDC13): δ=3.91 (s, 3H), 4.40 (s, 2 H), 7.07 (dd, J=7.78, 4.74 Hz, 1 H), 7.24 (d, J=8.41 Hz, 2H), 7.37 (d, J=8.40 Hz, 2H), 8.21 (dd, J=7.82, 1.87 Hz, 1 H), 8.57 ppm (dd, J=4.74, 1.88 Hz, IH); Anal, calcd for C14H12C1NO2S: C 57.24, H 4.12, N 4.77, found: C 57.21, H 4.18, N 4.67.

2-[[(4-Chlorophenyl)methyl]thio]-3-pyridine methanol (28): A solution of 27 (3.4 g, 0.012 mol) in THF (200 mL) was treated with LiBH4 (29 mL, 0.058 mol, 2m in THF) and the solution was refluxed for 16 h. The reaction was poured into water (300 mL), acidified with aq HCl (In) and extracted with CH2C12. The organic layer was separated, dried (MgSO4), filtered and concentrated. The residue was recrystallized from petroleum ether (PE) and CH2C12 (1 : 1) yielding 28 (1.68 g, 52%); mp: 89.68C; ¹H NMR (400 MHz, [Do]DMSO): δ=2.06 (s, IH), 4.46 (s, 2H), 4.64 (s, 2H), 7.06 (dd, J=7.5, 4.9 Hz, IH), 7.24 (d, J=8.4 Hz, 2H), 7.33 (d, J=8.4 Hz, 2H), 7.63 (dd, J=7.5, 1.8 Hz, IH), 8.39 ppm (dd, J=4.9, 1.8 Hz, IH). Anal, calcd for C13H12C1NOS: C 58.75, H 4.55, N 5.27, found: C 58.74, H 4.54, N 5.15.

2-[[(4-Chlorophenyl)methyl]thio]-3-pyridine methylchloride (29): Et3N (1 mL) was added at 08C under a flow of N2 to a solution of 28 (1.6 g, 0.012 mol) in dry CH2C12 (30 mL). After stirring for 15 min, MsCl (0.93 mL, 0.012 mol) was added dropwise. The mixture was warmed to RT and stirred for an additional 16 h. The reaction mixture was poured into water (100 mL) and extracted with CH2C12 (x2). The combined organic layer was dried (MgSO4), filtered, and concentrated. The residue was recrystallized from PE and CH2C12 (15:1) yielding 29 (1.7 g, 99%); mp: 678C; ¹H NMR (360 MHz, CDC13): δ=4.48 (s, 2 H), 4.58 (s, 2 H), 7.06 (dd, J=7.6, 4.8 Hz, IH), 7.25 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.4 Hz, 2 H), 7.61 (1 H, dd, J=7.6, 1.7 Hz, 1 H), 8.42 ppm (dd, J=4.8, 1.7 Hz, IH); Anal, calcd for C13H11C12NS: C 54.94, H 3.90, N 4.93, found: C 55.10, H 3.99, N 4.97. bis(l,l-Dimethylethyl)[(Z)-[4-[[2-[[(4-chlorophenyl)methyl]thio]-3-pyridinyl]methyl]-l- piperazinyl]methylidyne]biscarbamate (30): A solution of 29 (0.43 g, 0.00152 mol) in CH3CN (25 mL) was added dropwise to a solution of l-[N,N'-bis(tert- butoxycarbonyl)amidino]-piperazine (0.5 g, 0.00152 mol) in CH3CN (25 mL) and Et3N (0.25 mL). KI (25 mg) was added and the mixture was stirred at 6OC for 16 h. The reaction mixture was poured into water (200 mL) and extracted with CH2C12. The organic layer was separated, dried (MgSO4), filtered and concentrated. The residue (0.75 g) was purified by HPLC using a gradient elution (0.5% NH4OAc in H2O/CH3CN (9:1)/CH3OH/CH3CN; 23 :42:35->0:30:70->0: 100:0) to give 30 (0.1 11 g, 13%); mp: 70.4-73.8 8C; ¹H NMR (400 MHz, CDC13): δ=1.49 (br s., 18H), 2.51 (t, J=4.89 Hz, 4H), 3.46 (s, 2H), 3.59 (br s., 4 H), 4.42 (s, 2 H), 7.01 (dd, J=7.40, 4.89 Hz, 1 H), 7.25 (d, J=8.3O Hz, 2H), 7.34 (d, J=8.28 Hz, 2 H), 7.54 (dd, J=7.40, 1.38 Hz, 1 H), 8.38 (dd, J=4.77, 1.51 Hz, 1 H), 10.17 ppm (br s., IH); Anal, calcd for C28H38C1N5O4S: C 58.37, H 6.65, N 12.16, found: C 58.84, H 6.64, N 12.24.

2-[[(4-bromophenyl)phenylmethyl]thio]-3-pyridinecarboxylic acid (31): A mixture of 2- mercapto nicotinic acid (17.8 g, 0.115 mol) and 4-bromo benzhydrol (0.115 mol) in MsOH (150 mL) was stirred at RT for 18 h and then poured into water (800 mL). The mixture was stirred for 30 min. The precipitate was filtered off, washed with H2O and hexane, and recrystallized from EtOAc yielding compound 31 as a white solid (42.6 g, 93%); mp: 208.1— 208.6 8C; ¹H NMR (360 MHz, CDC13): δ=6.47 (s, 1 H), 7.06 (dd, J=7.8, 4.7 Hz, 1 H), 7.22 (t, J=7.3 Hz, 1 H), 7.30 (t, J=7.4 Hz, 2H), 7.37 (d, J=8.8 Hz, 2 H), 7.38-7.44 (m, 4 H), 8.28 (dd, J=7.8, 1.9 Hz, 1 H), 8.48 ppm (dd, J-4.7, 1.9 Hz, IH); Anal, calcd for C19H14BrNO2S: C 57.01, H 3.53, N 3.50, found: C 55.71, H 3.22, N 3.87.

2-[[(4-bromophenyl)phenylmethyl]thio]-3-pyridine methanol (32): BH3 SMe2 (12.7 mL, 0.127 mol, 10m in THF) was added carefully to a solution of 31 (42.6 g, 0.106 mol) in dry THF (100 mL). The mixture was stirred at 50C for 2.5 h and then brought to RT. Saturated aq NaHCO3 (100 mL) was added carefully and the mixture was diluted with CH2C12 (200 mL). The organic layer was separated,dried (Na2SO4), filtered and concentrated to yield compound 32 as a brown foam (37 g, 90%); ¹H NMR (300 MHz, [DO]DMSO): δ=4.45 (d, J=5.4 Hz, 2H), 5.50 (t, J=5.3 Hz, 1 H), 6.42 (s, IH), 7.12 (dd, J=7.6, 4.7 Hz, IH), 7.22 (t, J=7.2 Hz, 1 H), 7.30 (t, J=7.2 Hz, 2 H), 7.37-7.51 (m, 6 H), 7.68 (d, J=7.5 Hz, 1 H), 8.25 ppm (d, J=4.9 Hz, IH); Anal, calcd for C19H16BrNOS.2H2O: C 54.03, H 4.77, N 3.32, found: C 59.31, H 4.72, N 3.17.

2-[[(4-Bromophenyl)phenylmethyl]thio]-3-chloromethyl-pyridine (33): A solution of 32 (37 g, 0.096 mol) in CH2C12 (400 mL) was cooled to 0 8C. MsCl (1 1 mL, 0.144 mol) and Et3N (15.8 mL) were added and the mixture was stirred at RT for 2 h. The reaction was quenched with saturated aq NH4C1 (50 mL). The organic layer was separated, dried (Na2SO4), filtered and concentrated. The crude residue was used without further purification in the next reaction; ¹H NMR (400 MHz, CDC13): 5=4.60 (s, 2H), 6.51 (s, IH), 7.00 (dd, J=7.3, 4.8 Hz, IH), 7.22 (t, J=7.3 Hz, 1 H), 7.29 (t, J=7.5 Hz, 2 H), 7.33-7.43 (m, 6 H), 7.58 (dd, J=7.5, 1.6 Hz, IH), 8.32 ppm (dd, J=4.9, 1.6 Hz, IH).

4-{2-[(4-Bromo-phenyl)-phenyl-methyl]thio]-pyridin-3-ylmethyl}-piperazine-l-carboxylic acid tert-butyl ester (34): A mixture of 33 (-0.096 mol), tert-butyl 1 -piperazinecarboxylate (17.9 g, 0.096 mol) and K2CO3 (13.2 g, 0.096 mol) in CH3CN (400 mL) was stirred at 80C overnight. The solvent was removed in vacuo. The residue was dissolved in CH2C12 (200 mL) and then washed with H2O (2x100 mL). The organic layer was dried (Na2SO4), filtered and concentrated in vacuo. The residue was purified by column chromatography (CH2C12/hexane; 1 :3) to yield 34 as a light yellow foam (28 g, 52%).

1 - {2-[(4-Bromo-phenyl)-phenyl-methyl]thio]-pyridin-3-ylmethyl}-piperazine (35): A solution of 34 (17.2 g, 0.031 mol) in TFA (25% in CH2C12 v/v; 150 mL) was stirred at RT for 5 h. The mixture was basified to pH 10-12 with aq NaOH (2n), then CH2C12 (200 mL) was added. The organic layer was separated, dried (Na2SO4), filtered and concentrated. The residue was purified by column chromatography (CH2C12/(CH3OH/NH3); 7:1) and then by preparative HPLC to yield 35 (9.2 g, 65%).

bis(l,l-Dimethylethyl)[(Z)-[4-[[2-[[(4-bromophenyl)phenylmethyl]thio]-3- pyridinyl]methyl]-l-piperazinyl]methylidyne]biscarbamate (36): A mixture of 35 (7.5 g, 0.0165 mol), N,N'-bis-tert-butoxycarbonylthiourea (4.5 g, 0.0165 mol), EDC (3.8 g, 0.0198 mol) and DIPEA (3.5 mL, 0.0198) in DMF (60 mL) was stirred for 16 h at RT. The mixture was partitioned between aq NH4C1 (50 mL, In) and Et2O (200 mL). The organic layer was separated, dried (Na2SO4), filtered and concentrated in vacuo. The residue was purified by column chromatography (CH2C12/hexane; l :3->2:3) to yield 36 as a white foam (9.5 g, 82%); ¹H NMR (400 MHz, [Do]DMSO): δ=1.35 (s, 9H), 1.40 (s, 9 H), 2.33-2.43 (m, 4 H), 3.33-3.41 (m, 4H), 3.49 (s, 2H), 6.41 (s, IH), 7.10 (dd, J=7.5, 4.9 Hz, 1 H), 7.23 (t, IH), 7.31 (t, J=7.5 Hz, 2H), 7.40-7.49 (m, 6 H), 7.64 (dd, J=7.5, 1.6 Hz, 1 H), 8.29 (dd, J=4.8, 1.8 Hz, 1 H), 9.54 ppm (s, IH); Anal, calcd for C34H42BrN5O4S.H2O: C 57.14, H 6.21, N 9.80, found: C 57.83, H 6.22, N 9.49.

4-[(2-mercapto-3-pyridinyl)methyl]-l -piperazinecarboximidamidetrifluoroacetate (1 :2) 37: A solution of 36 (0.0136 mol), triethylsilane (10 mL) in TFA (50% in CH2C12 v/v; 100 mL) was stirred for 3 h at RT. Hexane and Et20 were added and the deprotected product was precipitated as a yellow solid, which was filtered off, washed with Et2O and dried to yield compound 37 (6.3 g, 65%); ¹H NMR (400 MHz, [D6]DMSO, 1258C): 5=2.82 (t, J=5.14 Hz, 4 H), 3.57 (t, J=5.17 Hz, 4H), 3.92 (s, 2H), 6.78 (t, J=6.69 Hz, IH), 7.36 (br s., 4 H), 7.63 (d, J=6.23 Hz, 1 H), 7.66 ppm (d, J=7.18 Hz, IH); Anal, calcd for C11H17N5S.2C2HF3O2 : C 37.58, H 3.99, N 14.61, found: C 37.46, H 3.61, N 14.80.

Biological evaluation

Strains and media: The yeast strain used in this study was C. albicans strain SC5314 CAI.

The medium used was YPD (1 % yeast extract, 2% peptone, 2% glucose) unless stated otherwise.

Antifungal activity: The antifungal activity of the compounds was determined at 100 μg mL^" '. Compounds were dispensed in microtiterplates at a final concentration of 500 μg mL^" in 10% DMSO. C. albicans cell culture grown overnight in YPD (diluted in fresh YPD to -106 cells mL^'1) was added to 20 mL of the compound solution in 2% DMSO. After 24 h incubation at 37C, antifungal activity of the compounds on C. albicans was assessed by microspectrophotometry of liquid cultures grown in microtiterplates as described previously. Compounds displaying antifungal activity were analyzed further to determine their MIC50 (concentration of compound required to inhibit 50% yeast growth). A twofold dilution series (20 mL) of the test compounds were prepared in DMSO. Subsequently 180 mL of MiIIiQ was added giving a dilution series of compound in 10% DMSO. C. albicans cell culture (80 mL) grown overnight in YPD (diluted in fresh YPD to -106 cells mL^"1) was added to 20 mL of these dilution series (leading to a dilution series of compound in 2% DMSO) and incubated for 24 h at 37C. Subsequently, antifungal activity of the compounds against C. albicans was assessed by microspectrophotometry of liquid cultures grown in microtiterplates as described previously.

Fungicidal action of antifungal compounds: An overnight C. albicans culture in YPD was diluted in 200 mL PBS to a cell density of -10⁶ cells mL^"1 and incubated in the presence of a compound or DMSO. To examine whether ROS is involved in the fungicidal process (at the MFC value of the compounds), incubations were conducted in parallel in the absence and presence of 8 mm ascorbic acid (AA). Administration of AA resulted in a PBS pH decrease from 7.2 to 6.0. After 0 h and 5 h of incubation at 37C, 100 mL aliquots were plated on YPD plates and colony forming units (CFUs) were counted after 2 d of incubation at 378C. Percentage survival was calculated as the ratio of the number of CFUs after treatment with the compound as compared to the number of CFUs of the initial inoculum. The minimal fungicidal concentration (MFC) of a compound is defined as the concentration leading to reduction of 99.9% of the viability of the initial inoculum. Cytotoxic activity in vitro: A volume of 180 mL of Eagle's minimum essential medium (supplemented with 5% fetal calf serum and 20 mm Hepes buffer) was dispensed in flat- bottomed 96-well plates. Test compound (45 mL) was added and serial fivefold compound dilutions were made. Additionally, 50 mL of medium and 50 mL of HeLaM cell suspension (2x10⁵ cells mL^"1) were added. The microtiterplates were incubated at 37C over 7 d in a 5% CO2 atmosphere. The viability of the cells was quantified spectrophotometrically by a tetrazolium colorimetric method (MTT assay). Briefly, to each well of the microtiterplate, 25 mL of a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added, followed by incubation for 2 h at 37 8C and removal of the medium. The formazan crystals were solubilized by adding 200 mL 2-propanol and shaking. Finally, the absorbance was measured at 690 nm and 540 nm. To eliminate the effects of nonspecific absorption, the absorbance at 690 nm was subtracted from the values at 540 nm. The pIC50 was defined as -log of the half maximal (50%) inhibitory concentration for cytotoxicity of a compound.

Measurement of ROS production: Endogenous ROS levels were measured by a fluorometric assay with 2',7'-dichlorofluorescin diacetate (DCFHDA; Molecular Probes Inc. USA) as described previously. Briefly, 5 mL of an early log-phase yeast culture in YPD (grown at 37C) was centrifuged. The cell pellet was washed with PBS and resuspended in 5 mL PBS. Aliquots of the yeast cell suspension (40 mL) were mixed with 20 mL of compound at a final concentration of 100 μg mL^"1 in 2% DMSO (initial screening to identify compounds that induce ROS at 100 mgmL^"1) or with 20 mL of a twofold dilution series of compounds (prepared as described above) or DMSO and incubated in white 96-well microtiterplates (PE white;

Perkin-Elmer, USA). After incubation for 1 h at 378C, 40 mL aliquots of DCFHDA stock solution (25 mm in PBS) were added to the cell suspensions. Fluorescence emitted by the cells in the microtiterplates was measured with a Perkin-Elmer LS 50 B fluorescence spectrometer at an excitation wavelength of 485 nm (2.5 nm slit) and an emission wavelength of 540 nm (2.5 nm slit). Fluorescence was measured after incubation for 1 , 3 and 5 h at 37C in the dark. Fluorescence values of the samples were corrected by subtracting the fluorescence value of the antifungal compound in the corresponding concentration without cells but with DCFHDA. These corrected fluorescence values (CFVs) can hence be considered as a measure to determine the extent of increased endogenous ROS levels.

It is appreciated that certain features of the presently described invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the presently described invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

All publications cited in the specification are indicative of the level of skill of those skilled in the art to which the presently described invention pertains. All of these publications are hereby incorporated by reference herein to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

The present invention being thus described, it will be apparent that the same may be modified or varied in many ways. Such modifications and variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications and variations are intended to be included within the scope of the following claims.

Claims

What is claimed is:

1. A compound of Formula I:

wherein

A = -CH₂- or -CH₂CH₂-;

Ri and R₂ are independently selected from -H, -Cj_-6 alkyl, -C_3-6 cycloalkyl, aryl, heterocyclic

Ci-₆ alkyl, heteroaryl, -OC_1-6 alkyl, -C(O)OR₃ or -(CH₂)_mNHR₃ D is an optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocyclic Cp₆ alkyl moiety; m is an integer having a value of 1 , 2, 3 or 4; n is O or an integer having a value of 1 , 2, 3 or 4; R₃ is independently selected from -H, Ci_-6 alkyl, aryl, -C_3-6 cycloalkyl, heteroaryl or heterocyclic Ci-₆ alkyl; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein D is optionally substituted 1 or more times, by QR₄, and wherein Q is selected from S or CH₂;

R₄ is a -(CH₂)_p-(optionally substituted aryl or heteroaryl), -(CH₂)_p-CO-(optionally substituted aryl or heteroaryl); -(optionally substituted aryl or heteroaryl)-(CH₂)_p- (optionally substituted aryl or heteroaryl), -CH(optionally substituted aryl or heteroaryl)₂; and wherein these aryl and heteroaryl rings are each independently optionally substituted 1 or more times, suitably 1 to 5 times by Ci_-6 alkyl, Ci_-6 alkoxy, phenyl, cyano, halogen, NO₂, CF₃, OCF₃, -CH₂SO₂, -S(O)₂CH₃, or (CH₂)_qSO₂aryl; p is O or an integer having a value of 1 , 2, 3, or 4; q is an integer having a value of 1 , 2, or 3.

3. The compound according to claim 1 or 2 wherein Q is sulfur.

4. The compound according to any one of claims 1 to 3 wherein A is CH₂.

5. The compound according to any one of claims 1 to 4 wherein R¹ and R² are independently -H or cycloalkyl.

6. The compound according to any one of claims 1 to 4 wherein R¹ and R² are both hydrogen.

7. The compound according to any one of claims 1 to 6, wherein D is

Z is N or CH;

X is independently selected from Cp₆ alkyl, Ci-₆ alkoxy or halogen; t is 0 or an integer having a value of 1 , 2, or 3.

8. The compound according to claim 7 wherein R¹ and R² are independently -H or cycloalkyl and A is CH_2.

9. The compound according to claim 7 or 8 wherein Z is nitrogen.

10. The compound according to claim 7 or 8 wherein Z is carbon.

1 1. The compound according to any one of claims 1 to 6 wherein D is

wherein

Z is N or CH;

X is independently selected from Cp₆ alkyl, Ci-₆ alkoxy or halogen; t is 0 or an integer having a value of 1 , 2, or 3.

12. The compound according to claim 11 wherein R¹ and R² are independently -H or cycloalkyl and A is CH₂.

13. The compound according to claim 1 1 or 12 wherein Z is nitrogen.

14. The compound according to claim 11 or 12 wherein Z is carbon.

15. The compound according to claim 1 wherein D is selected from the group consisting of an optionally substituted thiophene or optionally substituted quinoline, and n is 1

16. The compound n the D-QR4 moiety is

17. The compound ac the D-QR4 moiety is

18. The compound according to claim 1 which is:

or a pharmaceutically acceptable salt thereof.

19. A pharmaceutical composition comprising a compound according to any one of claim 1 to 18, and a pharmaceutically acceptable carrier or diluent.

20. A method for killing Candida spp. or inhibiting the growth of Candida spp., comprising: administering to a mammal afflicted with a disease associated with Candida spp. a compound according to any one of claims 1 to 18 in an amount and for a duration effective to increase the intracellular reactive oxygen species levels in the Candida spp.

21. The method according to claim 20, wherein the Candida spp. is selected from the group consisting of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsiliosis, Candida guilliermondi, Candida lusitaniae and Candida krusei.

22. The method according to claim 21, wherein the Candida spp. is Candida albicans.