WO1999066878A2

WO1999066878A2 - Use of bis(diazo) compound as antifungal agents

Info

Publication number: WO1999066878A2
Application number: PCT/US1999/013779
Authority: WO
Inventors: Gary R. Ostroff
Original assignee: Alpha-Beta Technology, Inc.
Priority date: 1998-06-25
Filing date: 1999-06-17
Publication date: 1999-12-29
Also published as: AU4577599A

Abstract

The present invention relates to a method for treating a fungal infection in a patient. The method comprises the step of administering to the patient an effective amount of a compound comprising a bis(diazo)biphenyl moiety. The compound to be administered can, for example, comprise a 4,4'-bis(diazo)biphenyl moiety wherein the diazo groups are additionally bonded to a substituted or unsubstituted aryl group.

Description

USE OF BIS(DIAZO) COMPOUNDS AS ANITFUNGAL AGENTS

RELATED APPLICATIONS

The application claims priority to U.S. Application No. 60/090,662, filed June 25, 1998, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The incidence of fungal infection continues to increase in a variety of patient populations, particularly among those patients with compromised immune systems.

These include patients infected with human immunodeficiency virus and patients undergoing treatment with cytotoxic chemotherapeutic agents. Although the demand for effective antifungal agents continues to increase, few effective agents are available in the clinic. The most commonly prescribed drugs for the treatment of mycotic infections are the azoles and the polyenes, both of which inhibit sterol biosynthesis. These drugs, however, have several drawbacks. For example, the development of resistance to the azoles has been observed in Candida albicans.

Further, the polyene amphotericin B, the most commonly prescribed antifungal drug, causes toxic side effects.

The development of new drugs depends upon the discovery of new therapeutic targets and new assays for assessing the desired biological activity.

SUMMARY OF THE INVENTION

The present invention relates to a method for treating a fungal infection in a patient. The method comprises the step of administering to the patient an effective amount of a compound comprising a bis(diazo)biphenyl moiety. The compound to be administered can, for example, comprise a 4,4'-bis(diazo)biphenyl moiety wherein the diazo groups are additionally bonded to a substituted or unsubstituted aryl group. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that compounds comprising a bis(diazo)biphenyl moiety inhibit one or more activities which are necessary for fungal cell wall formation. The cell wall of fungi has a complex composition and structure. In general human pathogenic fungi contain β-l,3-glucans, mannoproteins, peptides and lipids.

Cell wall components are structural, providing mechanical strength to the wall, or cementitious, keeping structural components glued together. Structural materials are fibrous and include chitin and β-l,3-linked glucans. Cementitious materials are amorphous and include β-l,6-glucans and glycoproteins. The requirement of normal cell wall assembly for growth and viability of fungi has been demonstrated.

One potential target of antifungal agents is fungal cell wall biosynthesis. The assembly of the fungal cell wall is a complex, incompletely understood process.

Interference with cell-wall assembly with micro fibril inhibitors, such as Congo Red and calcofluor, interferes with fungal cell growth. In addition, the inhibition of the chitin and β-l,3-glucan assembly processes by Congo Red and calcofluor results in aberrant morphology and reduced growth.

Cell wall assembly is believed to proceed via the extracellular formation of chitin and β-glucan fibrils, a process which is best understood for β-glucan. Formation of β-glucan fibrils proceeds via the interaction of three β-glucan chains to form a triple helical structure. As the growing β-glucan chains assemble into this triple helical structure they are cross-linked by the enzyme-catalyzed formation of β-

1,6 branches. The branched triple helical β-glucan chains are incorporated into the cell wall as parallel triple helix aggregates, thereby forming the basic β-glucan fibril structure. These β-glucan fibrils are then interwoven with chitin microfibrils to form the fungal cell wall inner layer.

Targeting enzymatic processes for cell wall biosynthesis has proven to be difficult. From genome sequencing of Saccharomyces cerevisiae, it is known that the fungal genome includes redundant genes for many critical enzymatic activities. The resulting isozymes enable the fungal cell to synthesize a cell wall even if a particular enzyme is inhibited or a particular gene is knocked out. This redundancy complicates the drug discovery process and results in significant difficulties in the development of new antifungal drugs.

One cell wall synthesis target that avoids the difficulties presented by enzyme redundancy is the non-enymatic formation of β-glucan microfibrils, which, as discussed above, is a necessary step in fungal cell wall synthesis. Thus, compounds which inhibit β-glucan microfibril formation are potential antifungal agents. An in vitro method for assessing the ability of a compound to inhibit β- glucan microfibril formation is described in Example 1. See also U.S. Patent Application Serial No. 09/104,914, filed June 25, 1998, entitled "β-Glucan Microfibril Assembly Assay", the entire teachings of which are incorporated herein by reference.

Methods for preparing soluble β-glucans are disclosed in U.S. Serial Nos. 08/400,488, 08/432,303, 08/373,251 and 08/469,233 and U.S. Patent Nos. 5,322,841, 5,488,040, 5,532,223, 5,622,939 and 5,633,369, each of which is incorporated by reference herein in its entirety. In general, soluble β-glucans can be prepared by hydro lyzing β-l,3-glucan microfibrils derived from yeast cell walls. These soluble β-glucans mimic the conformational properties of fungal cell wall β- glucan. The soluble glucan exists in a single chain form, or conformation, under denaturing conditions, such as high pH, for example in 0.1 M NaOH, or high temperature, for example, about 85 °C, at neutral pH. Under native and renatured conditions, the soluble glucan forms triple helix aggregates comprising 6 to 9 chains each. Soluble β-glucan conformations are described, for example, in U.S. Serial No. 08/902,586, incorporated herein by reference in its entirety.

PGG-glucan is a polysaccharide composed of glucopyranose units linked in chains via β-1 ,3-glycosidic bonds, with branches intermittently linked to the main chain via β-l,6-glycosidic bonds. Single chains can be isolated, i.e., not substantially interacting with another chain. Three single helix chains can also combine to form a triple helix structure which is held together by interchain hydrogen bonding. Two or more β-glucan triple helices can join together to form a triple helix aggregate. A β-glucan polysaccharide can exist in at least four distinct conformations: single disordered chains, single helix, single triple helix and triple helix aggregates. Preparations of the β -glucan can comprise one or more of these forms, depending upon such conditions as pH and temperature. Methods for preparing a PGG-glucan composition enriched in the triple helix and higher-order conformations are described in U.S. Serial No. 08/902,586, incorporated herein by reference.

The present invention provides a method of treating a fungal infection in a patient. The method comprises the step of administering to the patient a therapeutically effective amount of a compound comprising a bis(diazo)biphenyl moiety. For example, the compound to be administered can comprise a 4,4"- bis(diazo)biphenyl moiety. The diazo groups can also be bonded to a substituted or unsubstituted aryl group.

As used herein, a "therapeutically effective amount" is an amount sufficient to inhibit or prevent, partially or totally, a fungal infection or to reverse the development of a fungal infection or prevent or reduce its further progression. The patient can be any animal which suffers from or is potentially subject to a fungal infection, for example, a human or an animal. In one embodiment, the patient is a mammal. Animals which can be treated using the method of the invention include domestic animals, such as dogs, cats, cows, horses, pigs, and avian animals (birds), for example, poultry, such as chickens, turkeys, geese and ducks. The patient can be an immunocompromised individual, which is generally defined as a person who exhibits an attenuated or reduced ability to mount a normal cellular or humoral defense to challenge by infectious agents, e.g., viruses, bacteria, fungi and protozoa. The method of the invention can be used, for example, to prevent or treat fungal infections in malnourished patients, patients undergoing surgery and bone marrow transplants, patients undergoing chemotherapy or radiotherapy, neutropenic patients, HIV-infected patients, trauma patients, burn patients, patients with chronic or resistant infections such as those resulting from myelodysplastic syndrome, and the elderly, all of who may have weakened immune systems. A protein malnourished individual is generally defined as a person who has a serum albumin level of less than about 3.2 grams per deciliter (g/dL) and/or unintentional weight loss of greater than 10% of usual body weight. More particularly, in one embodiment, the method of the invention can be used to therapeutically or prophylactically treat animals or humans who are at a heightened risk of fungal infection due to imminent surgery, injury, illness, radiation or chemotherapy, or other condition which deleteriously affects the immune system. The method is useful for treating patients who have a disease or disorder which causes the normal metabolic immune response to be reduced or depressed, such as HIV infection (AIDS).

The fungal infection can be an infection by any fungus which is pathogenic in the patient. Fungi which can be pathogenic under certain circumstances include Microsporum species, such as M. canis, M. audouini, and M gypseum; Trichophyton species, such as T. tonsurans, T. rubrum, T. mentagrophytes and T. violaceum; Candida species, such as C. albicans, C. krusei, C. parapsilosis, C. tropicalis, C. glabrata and C. guilliermondii; Aspergillus species, such as A.fumigatus, A. niger and A. flavus; Epidermophyton floccosum, Pityrosporum orbiculare, Cladosporium werneckii, Trichosporon cutaneum, Piedraia hortae, Torulopsis glabrata,

Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, Sporothrix schenckii, Petriellidium boydii, Pneumocystis carinii and species of Absidia, Mucor, Phialophora and Rhizopus. In one embodiment, the compound to be administered is a bis(diazo)biphenyl compound of Formula I,

and pharmaceutically acceptable salts thereof. R₃-R₁₀ are each, independently, a hydrogen atom, a - -alkyl group, a C,-C₆-alkoxy group, a sulfonate group, or an HO(0)CCH₂0- group. and R₂ are each, independently, an aryl or substituted aryl group. For example, R, and R₂ can each be a substituted or unsubstituted phenyl, naphthyl or heteroaryl group. Suitable aryl group substituents include one or more halogen atoms, such as fluorine, chlorine, bromine or iodine atoms; alkyl groups and acyl groups. Preferred aryl substituents include one or more hydroxy, amino, sulfonate, carboxylate, trimethylsilyl or C_j-Cg-alkoxy groups. In one embodiment, R_j and R₂ are each a substituted naphthyl group comprising at least one sulfonate substituent. R, and R₂ can further comprise, independently, one or more additional substituents selected from among hydroxy, amino or carboxylate groups. For example, in one embodiment the bis(diazo)biphenyl compound to be administered is Direct Red, which has the formula below, or pharmaceutically acceptable salts thereof.

In another embodiment, at least one of R_} and R₂ is a aryl group which is substituted by a substituted or unsubstituted aryldiazo group. Suitable aryldiazo group substituents include sulfonate groups, carboxylate groups, hydroxy groups, amino groups and alkoxy groups. For example, the bis(diazo)biphenyl compound to be administered can be of Formula JJ or pharmaceutically acceptable salts thereof.

In Formula II, ^R_JQ are as defined above for Formula I, Ar is a substituted or unsubstituted aryl group, such as a substituted or unsubstituted phenyl group or naphthyl group. R_u-R₁₅ are each, independently, a halogen atom, an alkyl group or, preferably, a hydrogen atom or a hydroxy, amino, sulfonate, carboxylate or C,-C₆- alkoxy group. Suitable aryl substituents include one or more halogen atoms, such as fluorine, chlorine, bromine or iodine atoms; alkyl groups and acyl groups. Preferred aryl substituents include one or more hydroxy, amino, sulfonate, carboxylate, trimethylsilyl or C_rC₆-alkoxy groups.

Examples of preferred bis(diazo)compounds are shown below. In Tables 1- 6, each of these compounds is referred to by the reference number indicated.

Acids which can be used to form pharmaceutically acceptable acid addition salts of compounds of Formulas I and II include hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid, mucic acid, benzoic acid, glucuronic acid, oxalic acid, ascorbic acid, N-acetylglycine and others known in the art.

Compounds of Formulas I and II having one or more acid functional groups, such as carboxylate groups or sulfonate groups, can also be used as salts with a pharmaceutically acceptable cation. Such cations include sodium, potassium, ammonium, calcium, ferric ion and those derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The quantity of a given bis(diazo)biphenyl compound to be administered will be determined on an individual basis and will be determined, at least in part, by consideration of the individual's size, the severity of symptoms to be treated and the result sought. The bis(diazo)biphenyl compound can be administered alone or in a pharmaceutical composition comprising the bis(diazo)biphenyl compound, an acceptable carrier or diluent and, optionally, one or more additional drugs.

An antifungal agent of this invention or two or more such antifungal agents can be used for the manufacture of a medicament for simultaneous, separate or sequential use in managing fungal infection or prophylaxis thereof. The agents can be administered subcutaneously, intravenously, parenterally, intraperitoneally, intradermally, intramuscularly, topically, enteral (e.g., orally), rectally, nasally, buccally, vaginally, by inhalation spray, by drug pump or via an implanted reservoir in dosage formulations containing conventional non-toxic, physiologically acceptable carriers or vehicles. The preferred method of administration is by oral delivery. The form in which it is administered (e.g., syrup, elixir, capsule, tablet, solution, foams, emulsion, gel, sol) will depend in part on the route by which it is administered. For example, for mucosal (e.g., oral mucosa, rectal, intestinal mucosa, bronchial mucosa) administration, nose drops, aerosols, inhalants, nebulizers, eye drops or suppositories can be used. The compounds and agents of this invention can be administered together with other biologically active agents.

In a specific embodiment, it may be desirable to administer the agents of the invention locally to a localized area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application (e.g., for skin infections), transdermal patches, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes or fibers. For example, the agent can be injected into the joints. The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically (or prophylactically) effective amount of the agent, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile. The formulation should suit the mode of administration.

Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc. The pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as tri glycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrolhdone, sodium saccharine, cellulose, magnesium carbonate, etc.

The composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. For topical application, there are employed as nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. The drug may be

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Na+ incorporated into a cosmetic formulation. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.

The amount of agents which will be effective in the treatment of a particular fungal infection will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. As described in the examples below, a number of compounds comprising a bis(diazo)biphenyl moiety have been assessed in assays measuring the ability to inhibit processes which are critical to fungal cell wall synthesis. As discussed below, these compounds exhibited surprisingly high activity against a number of fungal activities. For example, compounds were assessed for the ability to inhibit the fungal enzymes chitin synthase, glucan synthase and glucanase. Chitin synthase and glucan synthase catalyze the synthesis of chitin and β-glucan, respectively. As discussed above, both chitin and β-glucan are essential fungal cell wall structural elements, and inhibition of chitin and β-glucan synthesis, thus, impairs fungal cell wall construction. Glucanase catalyzes the breakdown of β-glucan, a key process in the fungal cell wall remodeling required for fungal cell growth and division. Compounds were also assessed for the ability to inhibit β-glucan microfibril formation, a non-enzymatic process which, as discussed above, is also necessary for fungal cell wall formation. Compounds were also assessed for fungicidal and fimgistatic activity in vitro and for the ability to inhibit cell wall synthesis by C. albicans protoplasts. The results presented in Tables 1-7 indicate that many of the bis(diazo)biphenyl compounds tested were active against multiple cell wall targets, indicating that these compounds target more than one fungal cell wall activity and are, thus, highly effective antifungal agents. The invention is further illustrated by the following Examples.

EXAMPLES

Example 1 Glucan microfibril formation assay

To a sterile eppendorf tube was added 100 μL of a 1 mg/mL aqueous solution of a triple helix PGG-glucan composition described in Serial No. 08/902,586. The tube was heated in a water bath to 90-100°C for 30 minutes. A solution (1 μL of a lOmg/ml solution of the test compound in water or dimethylsulfoxide was immediately added to the tube. After the tube had cooled to room temperature over about 1 hour, the solution was diluted with sterile water to a final volume of 500 μL. The solution was then analyzed by gel permeation chromatography to determine the amounts of single chain PPG-glucan and triple helix PGG-glucan. The ratio of triple helix PGG-glucan to single chain PGG-glucan was compared to a control solution to which the test compound was not added. The %inhibition of glucan microfibril formation was calculated using the relationship

%inhibition = [l-(TH/SC)s/(TH/SC)c] x 100

where (TH/SC)s is the triple helix/single chain ratio in the test sample and (TH/SC)c is the triple helix/single chain ratio in the control. The results of this assay for a variety of bis(diazo)biphenyl compounds are presented in Table 1. Example 2 Glucan synthase inhibition assay

The ability of a test compound to inhibit fungal glucan synthase was assessed in an in vitro assay performed on a 96 well microtiter plate. To each well was added 3 μL of a 16.7 mg/mL solution of -amylase, 2 μL of an 8 mg/mL solution of uridine diphosphate glucose (UDP-glucose) and

1 μL of an 8 mg/mL solution of ¹ C-UDP-glucose. To each well was also added either 1 μL of a test compound solution (1 mg/mL), 1 μL water or DMSO (solvent control) or 1 μL of cilofungin solution (3.75, 15 and 60 μM, positive control).

Table 1 : Results of Glucan Microfibril formation assay I = inhibition; N = no effect

Finally, 19 μL of a 2 mg/mL C. albicans lysate was added to each well. To a time 0 well was then immediately added 50 μL of 10% trichloroacetic acid. The plate was then incubated for 30 minutes at room temperature and then filtered on a Millipore filter vacuum apparatus. Each well was then washed twice with 200 μL sterile water and filtered again. The plate was then read on a Top Count Scintillation counter. The net counts per minute (CPM) of each well is defined as

measured cpm - time 0 cpm.

The %inhibition was calculated using the relationship

%inhibition = 100 - ((net cpm sample/net cpm DMSO) x 100).

The results of this assay for a variety of bis(diazo)biphenyl compounds are presented in Table 2.

Example 3 Chitin synthase inhibition assay

The ability of a test compound to inhibit fungal chitin synthase was assessed in an in vitro assay performed on a 96 well microtiter plate. A solution containing 1.9 mg/mL uridine-5-diphospho-N-acetylglucosamine (UDP-NAG), 19 mg/mL N- acetylglucosamine (NAG) and ^I C-UDP-NAG was prepared. To each well was added 1 μL of either a solvent control (water or 50%> DMSO), test sample (1 mg/mL) or nikkomycin solution (400 μM). 3.5 μL of the UDP-NAG/NAG/¹⁴C-UDP-NAG solution was then added to each well, followed by 15.5 μL of a C. albicans cell lysate. To a time 0 well was then immediately added 50 μL of 10% Table 2: Results of Glucan Synthase inhibition assay

trichloroacetic acid. The plate was then incubated for 3 hours at room temperature and then to each well was added 50 μL of 10% trichloroacetic acid followed by 200 μL water. The plate was then filtered on a Millipore filter vacuum apparatus. Each well was then washed twice with 200 μL sterile water and filtered again. Microscint 20 (50 μL) was then added to each well and the plate was covered with a plate sealer. The plate was then read on a Top Count Scintillation counter.

The net counts per minute (CPM) of each well is defined as

measured cpm - time 0 cpm.

The %inhibition is calculated using the relationship

%inhibition = 100 - ((net cpm sample/net cpm DMSO) x 100).

The results of this assay for a variety of bis(diazo)biphenyl compounds are presented in Table 3.

Example 4 Trichoderma Glucanase assay

Assessment of in vitro inhibition of Trichoderma glucanase was performed using a 96-well microtiter plate β-l,3-glucanase assay using a crude Trichoderma extract as the enzyme and a large soluble β-l,3-glucan derived from S. cerevisiae as the substrate. The test compound was mixed with the substrate, then the enzyme was added. The resulting mixture was maintained at 37° C for 30 minutes. The enzymatic reaction was then stopped by the addition to the mixture of an alkaline Cu(II) solution for spectrophotometric reducing sugar analysis (Breuil et al. Enzyme Microb. Technol. 7 : 327-332 (1985)). After development of the reducing sugar color reaction, the absorbance of the solution at 520 nm was measured. The amount of reducing sugar released by the enzymatic reaction was then determined by comparing the measured absorbance with a standard curve. This result was compared to a control reaction which is performed in the absence of test compound and reported as percent inhibition relative to the control. The results of this assay for two bis(diazo)biphenyl compounds are given in Table 4. Example 5 Candida albicans growth inhibition assay

Assessment of C. albicans growth inhibition was performed using a 96 well microtiter plate agar diffusion assay. A 2.5 μl volume of solvent controls (water or dimethylsulfoxide), positive controls (cilofungin, nikkomycin Z, amphotericin B, fluconazole; each at 10 mg/mL), and test compounds (10 mg/mL) were added to the individual wells of a 96 well microtiter plate. 75 μL of molten yeast extract-peptone- dextrose agar (YPDA, 1.5% agar) medium cooled to 50°C was then added to each well and allowed to solidify at room temperature. C. albicans (ATCC 20402) grown to mid-log phase of growth in YPD liquid medium at 30 °C was washed twice with sterile water by centrifugation at 3,000 x g for 10 minutes, adjusted to 5 x 10⁶ cells/mL, then mixed with YPD soft agar (YPDS A, 0.4%

Table 3: Results of Chitin Synthase inhibition assay

Table 4: Results of Trichoderma Glucanase inhibition assay

agar) medium and 30 μL of YPDSA containing approximately 1500 cells is added to each well. Plates were then incubated at 30°C and scored for growth by visual and microscopic video imaging at 20 and 48 hours. Data were recorded as "no effect", fimgicidal or fungistatic. Fungicidal and fungistatic compounds were then analyzed at 10-fold and at 2-fold dilutions to determine a minimum inhibitory concentration (MIC). Results of this assay for a series of bis(diazo)biphenyl compounds are presented in Table 5.

Example 6 C. albicans protoplast regeneration assay

Assessment of C. albicans protoplast regeneration inhibition was performed using a 96 well microtiter plate agar diffusion assay. A 2.5 μL volume of solvent controls (water or dimethylsulfoxide), positive controls (cilofungin, nikkomycin Z, amphotericin B, fluconazole; each at 10 mg/mL), and test compounds (10 mg/mL) were added to the individual wells of a 96 well microtiter plate. 75 μL of molten yeast extract-peptone-dextrose agar (YPD A, 1.5% agar) medium cooled to 50°C was then added to each well and allowed to solidify at room temperature. A preparation of C. albicans (ATCC 20402) protoplasts was adjusted to 5 x 10⁶ cells/mL, then mixed with YPD soft agar (YPDSA, 0.4% agar) medium and 30 μL of YPDSA containing approximately 1500 cells is added to each well. Plates were then incubated at 30 °C and scored for growth by visual and microscopic video imaging at 20 and 48 hours. Data were recorded as "no effect", fungicidal, fungistatic or cell wall active. Fungicidal, fungistatic and cell wall active compounds are then analyzed at 10-fold and at 2-fold dilutions to determine a minimum inhibitory concentration (MIC). Results of this assay for a series of bis(diazo)biphenyl compounds are presented in Table 6. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Table 5: Results of C. albicans growth in inhibition assay

K = kills; N = no effect; RCS = reduced colony si2;e

Table 6: Results of C. albicans protoplast regeneration assay

Wall = inhibits wall regeneration; RCS = reduced colony size; N = no effect; K ^: kills

Claims

CLAIMSWe claim:

1. A method for treating a fungal infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound comprising a 4,4'-bis(diazo)biphenyl moiety.

2. The method of Claim 1 wherein the compound comprising a 4,4'- bis(diazo)biphenyl moiety is of Formula I,

or a pharmaceutically acceptable salt thereof, wherein R_j and R₂ are each, independently, an aryl or substituted aryl group; and R₃-R_I0 are each, independently, a hydrogen atom, a C_r

C₆-alkyl group, a C_rC₆-alkoxy group, a sulfonate group or an

HO(O)CCH₂0- group.

3. The method of Claim 2 wherein R_j and R₂ are each, independently, a substituted or unsubstituted phenyl, naphthyl or heteroaryl group.

4. The method of Claim 3 wherein at least one of R_j and R₂ is a phenyl, naphthyl or heteroaryl group substituted by one or more substituents selected from the group consisting of hydroxy, amino, sulfonate, carboxylate, trimethylsilyl and C,-C₆-alkoxy.

5. The method of Claim 4 wherein R_j and R₂ are each, independently, a substituted naphthyl group.

6. The method of Claim 5 wherein the compound comprising a 4,4'- bis(diazo)biphenyl moiety is of the formula

or a pharmaceutically acceptable salt thereof.

7. The method of Claim 1 wherein the compound comprising a 4,4'- bis(diazo)biphenyl moiety is of Formula II,

or a pharmaceutically acceptable salt thereof, wherein R_j is a substituted or unsubstituted aryl group; R₃-R₁₀ are each, independently, a hydrogen atom, a C,-

C₆-alkyl group, a C_rC₆-alkoxy group, a sulfonate group or an HO(O)CCH₂O- group; Ar is a substituted or unsubstituted aryl group; and R_╧Ç-R₁₅ are each, independently, a hydrogen atom or a hydroxy, amino, sulfonate, carboxylate or - -alkoxy group.

8. The method of Claim 7 wherein Ar is a substituted or unsubstituted phenyl or naphthyl group.