WO1995008341A1

WO1995008341A1 - Antifungal and anti-pneumocystis compounds, compositions containing such compounds, and methods of use

Info

Publication number: WO1995008341A1
Application number: PCT/US1994/010468
Authority: WO
Inventors: James M. Balkovec; Frances A. Bouffard; James F. Dropinski
Original assignee: Merck & Co., Inc.
Priority date: 1993-09-22
Filing date: 1994-09-16
Publication date: 1995-03-30
Also published as: AU7874494A

Abstract

The present invention relates to antifungal and anti-Pneumocystis compounds with an Echinocandin B nucleus and with a polyphenylene side chain attached to the dihydroxyornithine residue at the N2 position and an aminoalkyl ether derivative at the dihydroxyornithine at the C5 position.

Description

TITLE OF THE INVENTION

ANTIFUNGAL AND ANΗ-PNEUMOCYSTIS COMPOUNDS, COMPOSITIONS CONTAINING SUCH COMPOUNDS, AND METHODS OF USE

BACKGROUND OF THE INVENTION

The present invention relates to antifungal and anti- Pneumocystis compounds with a polyphenylene side chain attached to the dihydroxyornithine residue at the N^ position and an aminoalkyl ether derivative at the dihydroxyornithine at the C5 position, subsequently referred to as the "C5 orn" or "hemiaminal" position.

There presently exists a need for new antifungal and anti- Pneumocystis compounds due to an increase in the number of isolates which are resistant to conventional agents. Also, conventional agents show somewhat high levels of toxicity, which limits the usefulness of these agents. Lastly, the incidence of Pneumocystis carinii pneumonia is increasing, particularly in view of the high incidence of immuno- compromised patients, e.g., suffering with AIDS.

SUMMARY OF THE INVENTION

Compounds represented by the Formula I (SEQ ID NOS. 2-11) are disclosed:

as well as pharmaceutically acceptable salts, esters and hydrates thereof.

Rl represents -CH2CH(NH2)CH2R^I, -(CH2)2-4NRHRΠI. -(CH2)1-3CH(NH2)R^IV or -(CH2)2-4NHRV_;

Rl represents -OH, -NH2, -NHC(=NH)NH2 or -NHC(=NH)(CH2)0-3H;

RU and R^ΪD each independently represent H, Cl-4 alkyl or benzyl, or RU and Rill are taken together to represent -(CH2)4-5S

RlV represents Cl-4 alkyl or -C(0)NH2;

RV represents -C(=NH)NH2, -C(=NH)(CH2)0-3H, -(CH2)2-4NH2, -(CH2)2-4θH, -C(0)(CH₂)l-3NH2, -(CH2)2-4NH- (C=NH)NH2,

-(CH2)2-4NH(C=NH)(CH2)0-3H or

R2 represents

p represents 1, 2 or 3; Ra represents -Cl-10 alkyl or -(CH2)qNRt>RC_; q represents 2, 3 or 4; Rb and R^c each independently represent H or -Cl-10 alkyl; or R^D and R^c are taken together with the nitrogen to which they are attached to represent:

wherein Rd represents Cl-16 alkyl, C5-6 cycloalkyl, C5-6 cycloalkyl(Cl-6)alkyl, phenyl or benzyl.

Pharmaceutical compositions are also included, which are comprised of a compound represented by Formula I in combination with a pharmaceutically acceptable carrier.

A method of treating a fungal infection in a mammalian patient in need of such treatment is also included, which is comprised of administering to the patient an effective amount of a compound represented by Formula I to treat the fungal infection.

A method of treating a Pneumocystis infection in a mammalian patient in need of such treatment is also included, which is comprised of administering to the patient an effective amount of a compound represented by Formula I to treat the Pneumocystis infection. A method of preventing a Pneumocystis infection in an immunocompromised mammalian patient in need of such treatment is also included, which is comprised of administering to the patient an effective amount of a compound represented by Formula I to prevent an occurrence of Pneumocystis infection.

DETAILED DESCRIPTION OF THE INVENTION

The invention is described herein in detail using the terms defined below unless otherwise specified.

The term "alkyl" refers to a monovalent alkane

(hydrocarbon) derived radical containing from 1 to 30 carbon atoms unless otherwise defined. It may be straight, branched or cyclic. Preferred alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, cyclopentyl and cyclohexyl. When substituted, alkyl groups may be substituted with up to three substituent groups at any available point of attachment. When the alkyl group is said to be substituted with an alkyl group, this is used interchangeably with "branched alkyl group". When the alkyl group is substituted with a cycloalkyl group, the cycloalkyl group may be at any available point of attachment. The alkyl portion of "alkoxy" is also defined as above.

Cycloalkyl is a specie of alkyl containing from 3 to 15 carbon atoms, without alternating or resonating double bonds between carbon atoms. It may contain from 1 to 4 rings which are fused. The preferred cycloalkyl groups are cyclopentyl and cyclohexyl.

The term "C5-6 cycloalkyl(Ci-6)alkyl" refers to an alkyl group substituted with a cycloalkyl group. The preferred substituent is the following:

Aryl refers to aromatic rings e.g., phenyl, substituted phenyl and the like, as well as ring systems which are fused, e.g., naphthyl, phenanthrenyl and the like. Aryl thus contain at least one ring having at least 6 atoms, with up to five such rings being present, containing up to 22 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms. The preferred aryl groups are phenyl and naphthyl. Aryl groups may likewise be substituted. Preferred substituted aryls include phenyl and naphthyl substituted with one or two such groups.

The term "heteroaryl" refers to a monocyclic aromatic hydrocarbon group having 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing at least one heteroatom, O, S or N, in which a carbon or nitrogen atom is the point of attachment, and in which one additional carbon atom is optionally replaced by a heteroatom selected from O or S, and in which from 1 to 3 additional carbon atoms are optionally replaced by nitrogen heteroatoms, said heteroaryl group being optionally substituted with up to four groups. Examples of this type are pyrrole, pyridine, piperidine, piperazine, oxazole, thiazole and oxazine.

The term "heterocycloalkyl" refers to a cycloalkyl group (non-aromatic) in which one of the carbon atoms in the ring is replaced by a heteroatom selected from O, S, NH, or N(Cl-C4 alkyl), and in which up to three additional carbon atoms may be replaced by hetero groups.

The term "heteroatom" means O, S or N, selected on an independent basis.

Alkylene (alkylidene or alkanediyl) and arylene refer to the groups noted above with divalent points of attachment. For example, phenylene is an arylene group, attached at any of the 1 , 2- 1, 3- or 1, 4- positions. Examples of alkylene include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CHCH3 and -CHCH2CH3.

I I

Aralkyl is a specie of substituted alkyl, containing up to three aryl groups substituted on a straight, branched or cycloalkyl group. The most preferred aralkyl group is benzyl (-CH2C6H5).

Halogen, or "halo" refers to bromine, chlorine, fluorine and iodine. Alkoxy refers to C1-C4 alkyl-O-, with the alkyl group optionally substituted.

The term "alkoxyaryl" thus refers to a Cl-4 alkyl-O- group, except where it is specified that the alkyl portion thereof is a different number of carbon atoms, e.g., C9-20 alkyl. The alkoxy portion thereof is substituted on an aryl group, e.g., a phenyl ring, at any available point of attachment.

The term "echinocandin B nucleus" (SEQ ID NO. 1) refers to the structure:

where X refers to a side chain and a point of attachment for a lipophilic side chain. In echinocandin B, X represents -NHC(0)-(CH2)7-CH=CH- CH2-CH=CH-(CH2)4CH3.

This lipophilic side chain can be removed enzymatically or through fermentative means. The nucleus typically is missing the side chain referred to above, and this is replaced with the aryl side chain in accordance with the chemical synthesis described herein.

Where a particular substituent group is shown with a bond attached, e.g., when R¹ is equal to -CH2CH(NH2)CH2R^I the bond is shown drawn to the substituent group for purposes of identifying the point of attachment, and is not to be taken as an indication with the generic structure that a double bond is intended. Double bonds are plainly shown, such as in the imino group (=NH) attached to a carbon atom via a double bond when Rl represents -NHC(=NH)(CH2)0-3H.

A preferred Compound I-A (SEQ ID NO. 2) has the following formula:

The compounds of the present invention are useful per se and in their pharmaceutically acceptable salt and hydrate forms in the treatment of fungal and Pneumocystis infections in animal and human subjects. The term "pharmaceutically acceptable salt and hydrate," refers to those salts and hydrated forms of the compounds of the present invention which would be apparent to the pharmaceutical chemist i.e., those which are substantially non-toxic and which may favorably effect the pharmacokinetic properties of said compounds, their palatability, absorption, distribution, metabolism and excretion. Other factors, more practical in nature, which are also important in the selection, are cost of the raw materials, ease of crystallization, yield, stability, hygroscopicity, and flowability of the resulting bulk drug. Conveniently, pharmaceutical compositions may be prepared from the active ingredients in combination with pharmaceutically acceptable carriers. Thus, the present invention is concerned with pharmaceutical compositions and methods of treating infections utilizing as an active ingredient the novel cyclic peptide compounds. The pharmaceutically acceptable salts referred to above also includes substantially non-toxic acid addition salts. The Formula I compounds can be used in the form of salts derived from inorganic or organic acids. Included among such salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.

The compounds of the present invention are valuable antifungal agents active against various fungal organisms, and accordingly will likely find utility in human and veterinary medicine. The compounds of the invention are not limited to utility as medicaments; they may be used in all manner of industry, for example: additives to animal feed, preservation of food, disinfectants, and in other industrial systems where control of fungal growth is desired. For example, they may be employed in compositions in concentrations ranging from about 0.01 to about 100 parts of antibiotic per million parts of solution in order to destroy or inhibit the growth of harmful fungi on medical and dental equipment and as fungicides in industrial applications, for example in water based paints and in the white water of paper mills to inhibit the growth of harmful bacteria.

In vitro antifungal activity determined in accordance with the protocol set forth below is predictive of in vivo activity, when the compounds are administered to a mammal infected with a susceptible fungal organism.

The compounds of the present invention are active against many fungi and particularly against Candida, Aspergillus and Cryptococcus species. The antifungal properties may be illustrated with the minimum fungicidal concentration (MFC) determination against certain Candida and Cryptococcus organisms in a microbroth dilution assay carried out in a Yeast Nitrogen Base (Difco) medium with 1 percent dextrose (YNBD).

In a representative assay, Compound I-A is solubilized in 100 percent dimethyl sulfoxide (DMSO) at an initial concentration of 5 mg/ml. Once dissolved, the drug stock is brought to a concentration of 512 mcg/ml by dilution in water such that the final DMSO concentration is about 10 percent. The solution is dispensed via a multichannel pipette into the first column of a 96-well plate (each well containing 0.075 ml of YNBD), resulting in a drug concentration of 256 mcg/ml. Compounds in the first column are diluted 2-fold across the rows yielding final drug concentrations ranging from 256 mcg/ml to 0.12 mcg/ml.

Four hour broth cultures of organisms to be tested are adjusted using a spectrophotometer at 600 nm to equal a 0.5 McFarland Standard. This suspension is diluted 1:100 in YNBD to yield a cell concentration of 1-5 x 10^ colony forming units (CFU) per ml.

Aliquots of the suspension (0.075 ml) are inoculated into each well of the microtiter plate resulting in a final cell inoculum of 5- 25 x lθ3 CFU/ml and final drug concentrations ranging from 128 mcg/ml to 0.06 mcg/ml. Each assay includes one row for drug-free control wells and one row for cell-free control wells.

After 24 hours of incubation, the microtiter plates are gently shaken on a shaker to resuspend the cells. The MIC-2000 inoculator is used to transfer a 1.5 microliter sample from each well of the 96-well microtiter plate to a single reservoir inoculum plate containing Sabouraud dextrose agar (SDA). The inoculated SDA plates are incubated for 24 hours at 35 °C. However, for Cryptococcus neoformans strains, SDA plates are inoculated at 48 hours after being spotted on SDA before making minimum fungicidal concentration (MFC) readings.

The compounds also show in vivo effectiveness against fungi which can be demonstrated as follows: Growth from an overnight SDA culture of Candida albicans MY 1055 is suspended in sterile saline and the cell concentration determined by hemocytometer count and the cell suspension adjusted to 3.75 x 10^ cells/ml. 0.2 ml of this suspension is administered LV. in the tail vein of mice so that the final inoculum is 7.5 x lθ4 cells/mouse. The assay is then carried out by administering aqueous solutions of the compounds at various concentrations intraperitoneally (LP.) twice daily (b.i.d.) for four consecutive days to 18-20 gram female DBA/2 mice, which previously had been infected with Candida albicans in the manner described above. Distilled water is administered LP. to C. albicans challenged mice as controls. After seven days, the mice are sacrificed by carbon dioxide gas, paired kidneys are removed aseptically and placed in sterile polyethylene bags containing 5 mis of sterile saline. The kidneys are homogenized in the bags, serially diluted in sterile saline and aliquots spread on the surface of SDA plates. The plates are incubated at 35°C for 48 hours and yeast colonies are enumerated for determination of colony forming units per gram of kidney tissue.

The following in vitro antifungal activity against Candida for Compound I-A is representative of the utility and potency of these compounds.

Organism MFC (mc^/ D

C. albicans (MY 1055) 0.125

C. tropicalis (MY1012) 0.5

C. pseudotropicalis (MY2099) 1

The in vivo anticandida activity of Compound I-A was determined in the manner described above and showed >99 reduction in CFUs when dosed IP BID at 0.375 mg/kg.

The compounds of the present invention may also be used to inhibit or alleviate Pneumocystis carinii infections in immunocompromised patients. Pneumocystis carinii may become opportunistic in mammals which are immunocompromised, such as in AIDS patients. The efficacy of the compounds for therapeutic or anti- infective purposes may be demonstrated in studies on immuno¬ suppressed rats.

In a representative study, Sprague-Dawley rats (weighing approximately 250 grams) are immunosuppressed with dexamethasone in the drinking water (2.0 mg/L) and maintained on a low protein diet for seven weeks to induce the development of Pneumocystis pneumonia from a latent infection. Before drug treatment, two rats can be sacrificed to confirm the presence of Pneumocystis carinii pneumonia (PCP).

Five rats (weighing approximately 150 grams) are injected twice daily for four days subcutaneously (sc) with a compound of the invention in 0.25 ml of vehicle (water). A vehicle control is also carried out. All animals are continued on dexamethasone in the drinking water and a low protein diet during the treatment period. At the completion of the treatment, all animals are sacrificed, the lungs are removed and processed, and the extent of disease determined by microscopic analysis of stained slides.

With respect to Pneumocystis, the compounds of the invention can thus be used to treat an infection which has been diagnosed or the compound can be used in those mammalian patients who are immunocompromised and predisposed to developing a Pneumocystis infection, to prevent the organism from becoming pathogenic and causing an infection. As used herein, both treatment modalities are included in the invention.

The compounds of this invention may be used in a variety of pharmaceutical preparations. The pharmaceutical composition is comprised of a compound of Formula I in combination with a pharmaceutically acceptable carrier.

The compound may be employed in solid, powder or crystalline form, in liquid solution, or in suspension. They may be administered by a variety of means; those of principal interest include: topically, orally, parenterally by injection (intravenously or intramuscularly) and for purposes of treating Pneumocystis pneumonia, via inhalation as a powder or liquid.

Compositions for injection, one preferred route of delivery, may be prepared in unit dosage form in ampoules, or in multidose containers. The injectable compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain various formulating agents. Alternatively, the active ingredient may be in powder (lyophilized or non- lyophilized) form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water.

Topical applications are also preferred for the treatment of candidiasis, and may be formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints, or powders.

Compositions administered orally may take such forms as tablets, capsules, oral suspensions and oral solutions. The oral compositions may utilize conventional formulating agents, and may include sustained release properties as well as rapid delivery forms.

The dosage to be administered depends to a large extent upon the condition and size of the subject being treated, the route and frequency of administration, the sensitivity of the pathogen to the particular compound selected, the virulence of the infection and other factors. Such matters, however, are left to the routine discretion of the physician according to principles of treatment well known in the medical arts. Another factor influencing the precise dosage regimen, apart from the nature of the infection and peculiar identity of the individual being treated, is the molecular weight of the compound.

The compositions for human use per unit dosage, whether liquid or solid, may contain from about 0.01% to as high as about 99% of active material, the preferred range being from about 10-60%. The composition will generally contain from about 1.5 mg to about 2.0 g of the active ingredient; however, in general, it is preferable to employ a dosage amount in the range of from about 1 mg to 500 mg. In parenteral administration, the unit dosage will typically include the pure compound in sterile water solution or in the form of a soluble powder intended for solution, which can be adjusted to neutral pH and isotonic.

The preferred methods of administration of the Formula I compounds include oral and parenteral, e.g., i.v. infusion, i.v. bolus and i.m. injection.

For adults, about 0.5-50 mg of the compound per kg of body weight given one to four times daily is preferred. The preferred dosage is 2.5 mg to 1000 mg of the compound given one to four times per day. More specifically, for mild infections a dose of about 2.5 to 100 mg administered two or three times daily is recommended. For moderate infections against highly susceptible organisms a dose of about 500 mg three or four times daily is recommended. For severe, life-threatening infections against organisms at the upper limits of sensitivity to the compound, a dose of about 1000-2000 mg three to four times daily may be recommended.

For the treatment of vaginal candidiasis, the compound may be administered in a predetermined amount which is not necessarily adjusted for body weight, e.g., 50 mg given once daily for seven to ten days. Typically compounds used for the treatment of vaginal candidiasis will be formulated in a cream or suppository form which is administered intravaginally.

For the treatment of Pneumocystis carinii pneumonia, the compound may preferably be administered via the pulmonary route, such as through the use of an intermittent positive pressure breathing (IPPB) apparatus, or in the form of an aerosol or unit dose spray powder. Typically the compound is administered three or four times daily, in an amount sufficient to treat the infection which has developed, or to prevent the development of infection.

As used herein, prevention of a Pneumocystis infection in an immunocompromised mammalian patient involves administering a compound in accordance with Formula I to the patient prior to the development of symptoms, based upon an expectation that the immunocompromised patient is more likely to develop a Pneumocystis infection than an immune competent patient. Immunocompromised patients are readily recognized by the skilled artisan by reviewing the overall condition of the patient, taking into account blood chemistry and cellular component values, e.g., the CD4 lymphocyte count.

For children, a dose of about 0.5-25 mg/kg of body weight given 2, 3, or 4 times per day is preferred; a dose of about 10 mg/kg is typically recommended.

The invention is further illustrated in connection with the following non-limiting examples.

EXAMPLE I

A. Preparation of 4-(n-Pentyloxyphenyl)-4'-carboxylbiphenyl pentafluorophenyl ester

4-(4-n-Pentoxyphenyl bromobenzene

To a stirred solution of 4-(4-bromophenyl)phenol (25.5 g, 0.102 mol) in 400 mL of dimethylsulf oxide was added 2.5N NaOH (40.9 mL, 0.102 mol) followed by n-pentyl bromide (12.7 mL, 0.102 mol). The resulting mixture was heated at 70°C for a period of 18h. After cooling, the yellow solution was partitioned between ethyl acetate (1000 mL) and water (500 mL). The organic phase was washed with water (3X) and brine and dried with magnesium sulfate. The solvent was removed in vacuo to give 4-(4-/2-pentoxyphenyl)bromobenzene (30.9 g, 95% yield). lH NMR (400MHz, DMSO-d6) δ 0.93 (t, J=7.2 Hz, 3H), 1.41 (m, 4H), 1.79 (m, 2H), 3.97 (t, J=6.6 Hz, 2H), 6.94 (d, J=8.8 Hz, 2H), 7.39 (d, J=8.6 Hz, 2H), 7.45 (d, J=8.8 Hz, 2H), 7.51 (d, J=8.6 Hz, 2H).

4-(4- 2-Pentoxyphenyl phenylboronic acid

To a stirred suspension of 4-(4-n-pentoxyphenyl)bromo- benzene (1.0 g, 3.13 mmol) in anhydrous tetrahydrofuran (20 mL) at -78°C under a nitrogen atmosphere was added «-butyllithium in hexanes (2.5M, 1.32 mL, 3.30 mmol). After a period of 15 min, triisopropyl- borate (760 mL, 3.30 mmol) was added. Stirring at -78°C was continued for 15 min and then at 25°C for 40 min. The mixture was acidified with 0.5N HCl (20 mL) and then partitioned between ether (50 mL) and water (40 mL). The organic phase was washed with water (3X) and brine and dried with magnesium sulfate. The solvent was removed in vacuo to give 4-(4-«-pentoxyphenyl)phenylboronic acid (750 mg, 84% yield) as a white solid. lH NMR (400MHz, DMSO-d6) δ 0.89 (t, J=7.2 Hz, 3H), 1.38 (m, 4H), 1.72 (m, 2H), 3.99 (t, J=6.5 Hz, 2H), 6.99 (d, J=8.8 Hz, 2H), 7.57 (d, J=8.2 Hz, 2H), 7.60 (d, J=8.8 Hz, 2H), 7.83 (d, J=8.2 Hz, 2H). 4-(n-PentyloxyphenylV4'-methoxycarbonylbiphenyl

To a stirred solution of 4-(4-n-pentyloxyphenyl)phenyl- boronic acid (1.45 g, 5.11 mmol) and methyl 4-bromobenzoate (1.21 g, 5.62 mmol) in dimethylformamide (12 mL) was added triethylamine (1.43 mL, 10.21 mmol) followed by triphenylphosphine (536 mg, 2.04 mmol) and palladium(II) acetate (230 mg, 1.02 mmol). The reaction was heated at 80°C for a period of 18h. The cooled mixture was partitioned between methylene chloride and water. The organic phase was washed with 1M sodium bicarbonate, water (2X) and brine, dried 0 over magnesium sulfate and filtered through a bed of celite. The solvent was removed in vacuo to give crude product which was purified by pre-adsorption flash silica gel chromatography to provide 4-(n- pentyloxyphenyl)-4'-methoxycarbonylbiphenyl (220 mg). MS (El) = 374 5 lH NMR (400MHz; CDCI3) δ 0.93 (t, J=7.1 Hz, 3H), 1.41 (m, 4H), 1.83 (m, 2H), 3.93 (s, 3H), 3.99 (t, J=6.6 Hz, 2H), 6.97 (d, J=8.8 Hz, 2H), 8.10 (d, J=8.5 Hz, 2H).

4-(n-Pentyloxyphenyl -4'-carboxybiphenyl ⁰ A mixture of 4-(n-pentyloxyphenyl)-4'-methoxycarbonyl- biphenyl (220 mg, 0.58 mmol) and 5N sodium hydroxide (1.17 mL, 5.88 mmol) in tetrahydrofuran (20 mL) was heated at 80°C under reflux for a peridd of 18h. The cooled mixture was acidified with IN hydrochloric acid (35 mL) and partitioned between ethyl acetate and ⁵ water. The organic suspension was washed with water (3X) and brine. The organic layer was then filtered to give 4-(n-pentyloxyphenyl)-4'- carboxybiphenyl (210 mg). MS (El) = 360.2 lH NJVIR (400MHz; DMSO-d6) δ 0.90 (t, J=7.1 Hz, 3H), ° 1.37 (m, 4H), 1.73 (m, 2H), 4.0 (t, J=6.6 Hz, 2H), 7.02 (d, J=8.9 Hz, 2H), 8.02 (d, J=8.5 Hz, 2H). 4-(/2-Pentyloxyphenyl -4'-Carboxylbiphenyl Pentafluorophenyl Ester

To a mixture of 4-(n-pentyloxyphenyl)-4'-carboxybiphenyl (380 mg, 1.05 mmol) and dicyclohexylcarbodiimide (327 mg, 1.58 mmol) in dimethylformamide at 0°C was added pentaflourophenol (388 mg, 2.10 mmol). The mixture was stirred at 25 °C for a period of 18h. The mixture was filtered prior to partitioning between ethyl acetate and water. The organic phase was washed with water (2X) and brine, dried over magnesium sulfate. The solvent was removed in vacuo to give crude product (750 mg), contaminated with traces of pentaflourophenol and dicyclohexylurea, which was used directly in the acylation.

B. Reacylation of Echinocandin B Nucleus

The preparation of the echinocandin B nucleus, where the linoleic acid sidechain has been removed, can be prepared according to Abbott, et al. U. S. Pat. No. 4,287,120, issued on September 1, 1981, and hereby incorporated by reference.

The nucleus (880 mg, 43% pure) and diisopropylethyl amine (0.090 mL, 1.1 eq) was mixed with the pentafluorophenyl ester (385 mg, 1.5 eq) prepared above in 10 mL of dry dimethyl¬ formamide. The mixture was stirred overnight by which time HPLC analysis (Zorbax RX-C18, 45% CH3CN/55% H2θ/0.1% trifluoro- acetic acid; 1.5 mL/min) showed that the reaction had proceeded substantially. The reaction mixture was diluted with an equal volume of water and pre-purified by chromatography on 40 g of Lichroprep Flash C18 reverse phase silica gel (40% CH3CN/60% H2θ -> 60% CH3CN/40% H2θ). The appropriate fractions as determined by HPLC analysis were frozen and lyophilized to give 500 mg of semi-pure product. The material was further purified by preparative HPLC (C18 Delta-Pak, 30X300 mm; 50% CH3CN/50% H2O/0.1 % TFA; 20 mL/min; 1=220 nm) to give 120 mg of the desired product after lyophilization that was >93% pure by analytical HPLC.

Partial 1H-NMR (CD3OD, 500 MHz): δ 7.93 (d), 7.73

(m), 7.68 (m), 7.59 (m), 7.15 (d), 6.99 (d), 6.75 (d), 5.35 (dd), 5.01 (dd), 4.65 (m), 3.40 (m), 2.53 (m), 2.43 (m), 2.23 (m), 1.8 (m), 1.28 (d), 1.25 (d), 1.05 (d), 0.96 (t).

C. Preparation of .Aminoethyl Ether

The material from Step B above (70 mg, 0.061 mmol) was dissolved in 1.5 mL of dimethylformamide and 239 mg (40 eq) of ethanolamine hydrochloride was added followed by 14.3 mg (1.0 eq) of (- -)-camphorsulfonic acid. After 24 hours of stirring under N2, the cloudy reaction mixture was made clear by adding 0.5 mL of dimethylsulfoxide. Analytical HPLC (RX-C18 Zorbax, 50/50 CH3CN/H2O 0.1% TFA, 1.5 mL/min) indicated >80% conversion to a new product. The reaction was diluted with water to about 10 mL and filtered. The filtrate was injected onto a preparative HPLC column (C18 DELTAPAK, 42/58 CH3CN/H2O 0.1 % TFA, 12 mL/min) and the appropriate fractions as determined by UV absorption at 210/277nm were combined, frozen and lyophilized to obtain 27 mg (37%) of a white floculent solid that was >98% pure by HPLC.

Partial iH NMR (400 MHz, CD3OD): δ 7.96 (dd), 7.82- 7.67 (m), 7.65-7.55 (m), 7.14 (d), 6.99 (d), 6.77 (m), 5.36 (d), 3.12 (m), 1.08 (d), 0.97 (t).

FAB MS (M+Li): 1189.2.

EXAMPLE II

A. Preparation of Pentafluorophenyl 4-(4-«-Octyloxyphenyl)- benzoate

4-(4-/x-Octyloxyphenyl benzoic Acid

To a solution of 4-(4-hydroxyphenyl)benzoic acid (0.102 mol) in dimethylsulf oxide and 2.5N NaOH (0.102 mol) is added n-octyl bromide (0.102 mol). The mixture is stirred at 70°C for a period of 18h. After cooling, the reaction mixture is acidified to pH3 by the addition of 2N HCl and partitioned between ethyl acetate and water. The organic phase is washed with water and brine and dried with magnesium sulfate. The solvent is removed in vacuo to give 4-(4-n- octyloxyphenyl)benzoic acid: C21H26O3, mol. wt. = 326.4.

Pentafluorophenyl 4-(4-/2-Octyloxyphenyl benzoate

To a mixture of 4-(4-«-octyloxyphenyl)benzoic acid (10.5 mmol) and dicyclohexylcarbodiimide (10.5 mmol) in ethyl acetate at 0°C is added pentafluorophenol (11.5 mmol). The mixture is stirred at 25°C for a period of 18 h, producing a precipitate. The mixture is filtered. The filtrate is washed with water and brine and dried with magnesium sulfate. The solvent is removed in vacuo to give pentafluoφhenyl 4-(4-n-octyloxyphenyl)benzoate: C27H25F5O3, mol. wt. = 492.5.

B. Reacylation of Echinocandin B Nucleus

In a manner similar to Part B of Example I but substituting pentafluorophenyl 4-(4-«-Octyloxyphenyl)benzoate as the acylating agent, the echinocandin B nucleus may be reacylated to give the octyloxybiphenyl derivative.

C. Preparation of Aminoethyl Ether

In a manner analogous to Part C of Example I, the aminoethyl ether of the reacylated echinocandin B nucleus from Part B above may be obtained.

EXAMPLE m

A. Preparation of 4'-(l-oxy-2-(l-N-(4-methylcyclohexyl)- piperidinyl)ethyl-p-biphenyl 4-carboxylic acid pentafluoro- phenyl ester

Preparation of 4-Methylcvclohexylpiperidine.

4-Benzylpiperidine is dissolved in glacial acetic acid containing Ptθ2 (approximately 50 wt percent). A Paar hydrogenator is used and the reaction vessel is flushed with H2 and pressurized to 3 atm. The mixture is shaken for sufficient time to give reduction of the aromatic ring to the fully saturated product which is determined by the uptake of 3 molar equivalents of H2. The black solid is filtered and the acetic acid removed by evaporation under reduced pressure to obtain the product as an acetate salt.

Preparation of 1 -N-(2-hvdroxyethylV4-methylcvclohexyl-piperidine

The product from above (1.0 eq) is dissolved in dichloromethane containing an equimolar amount of diisopropylethyl amine. Ethylene oxide (10 eq) is added and the mixture is stirred until starting material is consumed. The desired product is obtained by removal of the solvent in vacuo followed by purification by column chromatography.

Preparation of the carboxylic acid

4-Carbomethoxy-4'-hydroxybiphenyl (1.0 eq) is dissolved in dichloromethane and triphenylphosphine (1.3 eq) and the hydroxy- ethyl compound (1.0 eq) from above is added. Next, diethylazodi- carboxylate (1.3 eq) is added and the mixture is stirred until starting material is consumed. The mixture is diluted with dichloromethane and washed with water. The organic layer is dried with MgS04 and filtered. The solvent is removed in vacuo and the residue is dissolved in ethanol. An excess of 3N sodium hydroxide is added and the mixture stirred for several hours. The reaction is neutralized with 2N HCl and is extracted with ethyl acetate. The ethyl acetate layer is dried with MgS04, filtered and rotovaped. The desired product is obtained pure by column chromatography.

Preparation of the pentafluorophenyl ester

The carboxylic acid (1.0 eq) and dicyclohexylcarbodiimide (1.0 eq) are dissolved in ethyl acetate and the solution is cooled to 0°C. Pentafluorophenol (1.05 eq) is added, the ice bath is removed and the reaction stirred at ambient temperature for 18-24 h. An equal volume of ether is added, the mixture is filtered and the solvent removed in vacuo. The product (MW = 587.64) is sufficiently pure to be utilized "as is" for nucleus acylation.

B. Reacylation of echinocandin B nucleus

In a manner similar to Part B of Example I but substituting 4'-(l-oxy-2-(l-N-(4-methylcyclohexyl)-piperidinyl)- ethyl-p-biphenyl 4-carboxylic acid pentafluorophenyl ester as the acylating agent, the echinocandin B nucleus may be reacylated to give the aminoalkoxybiphenyl derivative.

C. Preparation of aminoethyl ether

EXAMPLE IV

A. Preparation of 4χi-oxy-2-N-(4-N-undecylpiperazinyl))ethyl-p- biphenyl-4-carboxylic acid pentafluorophenyl ester

Preparation of 4-N-undecylpiperazine

Excess piperazine (5 eq) and 1-bromoundecane (1.0 eq) are dissolved in dichloromethane and allowed to react overnight. The mixture is washed with aqueous sodium bicarbonate and the organic layer dried with sodium sulfate. The mixture is filtered, the solvent removed from the filtrate in vacuo and the residue purified by column chromatography .

Preparation of l-N-hvdroxyethyl-4-N-undecylpiperazine

The substituted piperazine from above (1.0 eq) is dissolved in n-propanol and bromoethanol (1.0 eq) is added along with diisopropylethyl amine (1.1 eq). After several hours, the solvent is removed in vacuo and the residue dissolved in dichloromethane. The organic layer is washed with water and then aqueous sodium bicarbonate. The organic layer is dried with MgS04 and filtered. Removal of the solvent in vacuo is followed by purification by column chromatography.

Preparation of the carboxylic acid

4-carbomethoxy-4'-hydroxybiphenyl (1.0 eq) is dissolved in dichloromethane and triphenylphosphine (1.3 eq) and the hydroxy- ethyl compound (1.0 eq) from above is added. Next, diethylazodi- carboxylate (1.3 eq) is added and the mixture is stirred until starting material is consumed. The mixture is diluted with dichloromethane and washed with water. The organic layer is dried with MgS04 and filtered. The solvent is removed in vacuo and the residue is dissolved in ethanol. An excess of 3N sodium hydroxide is added and the mixture stirred for several hours. The reaction is neutralized with 2N HCl and is extracted with ethyl acetate. The ethyl acetate layer is dried with MgSθ4, filtered and the solvent is removed by rotary evaporation. The desired product is obtained pure by column chromatography.

Preparation of the pentafluorophenyl ester

The carboxylic acid from above (1.0 eq) and dicyclohexyl- carbodiimide (1.0 eq) are dissolved in ethyl acetate and the solution is cooled to 0°C. Pentafluorophenol (1.05 eq) is added, the ice bath is removed and the reaction stirred at ambient temperature for 18-24 h. .An equal volume of ether is added, the mixture is filtered and the solvent removed in vacuo. The product (MW = 646.75) is sufficiently pure to be utilized "as is" for nucleus acylation.

B. Reacylation of echinocandin B nucleus

In a manner similar to Part B of Example I but substituting 4'-(l -oxy-2-N-(4-N-undecylpiperazinyl))ethyl-p- biphenyl-4-carboxylic acid pentafluorophenyl ester as the acylating agent, the echinocandin B nucleus may be reacylated to give the aminoalkoxybiphenyl derivative. C. Preparation of aminoethyl ether

EXAMPLES V-X

In a manner analogous to the above examples, the following materials may also be prepared:

Example Rl SEQ ID NO.

V -(CH2)3NH2 6

VI -CH2CH(NH2)CH2NH2 7

VII -CH2CH(NH2)CONH2 8

VIII -(CH2)2NHC(=NH)NH2 9

IX -(CH2)2NH(CH2)2NH2 10

X -(CH2)2NHCO(CH2)2NH2 11 While certain preferred embodiments of the invention have been described herein in detail, numerous alternative embodiments are contemplated as falling within the scope of the invention. Consequently, the claims are not to be limited to the specific teachings contained herein.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: BALKOVEC, JAMES M.

BOUFFARD, FRANCES, A. DROPINSKI, JAMES F.

(ii) TITLE OF INVENTION: ANTIFUNGAL AND ANTIPNEUMOCYSTIS

COMPOUNDS, COMPOSITIONS CONTAINING SUCH COMPOUNDS, AND METHODS OF USE

(iii) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Richard C. Billups

(B) STREET: P.O. BOX 200, EAST LINCOLN AVE.

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(v) COMPUTER READABLE FORM:

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(D) SOFTWARE: Microsoft word 5.0a

(vi) CURRENT APPLICATION DATE:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: NONE

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: RICHARD C. BILLUPS (B) REGISTRATION NUMBER: 31,916

(C) REFERENCE/DOCKET NUMBER: 1909IP

(ix) TELECOMMUNICATION INFORMATION

(A) TELEPHONE: 908-594-4683

(B) TELEFAX: 908-594-4720

(C) TELEX: (2) INFORMATION FOR SEQ ID NO: 1

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: NA

(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:

(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 Xaa Thr Xaa Xaa Thr Xaa

1 5

(2) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: NA

(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:

(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Xaa Thr Xaa Xaa Thr Xaa 1 5

(2) INFORMATION FOR SEQ ID NO: 3

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: NA

(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:

(2) INFORMATION FOR SEQ ID NO: 4

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: NA

(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:

(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 Xaa Thr Xaa Xaa Thr Xaa 1 5

(2) INFORMATION FOR SEQ ID NO: 5

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: NA

(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:

(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 Xaa Thr Xaa Xaa Thr Xaa 1 5

(2) INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: NA (D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:

(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 Xaa Thr Xaa Xaa Thr Xaa 1 5

(2) INFORMATION FOR SEQ ID NO: 7

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: NA

(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:

(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 Xaa Thr Xaa Xaa Thr Xaa 1 5

(2) INFORMATION FOR SEQ ID NO: 8

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: NA

(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:

(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 Xaa Thr Xaa Xaa Thr Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 9

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: NA

(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:

(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 Xaa Thr Xaa Xaa Thr Xaa 1 5

(2) INFORMATION FOR SEQ ID NO: 10 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: AMINO ACID (C) STRANDEDNESS: NA

(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:

(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10

Xaa Thr Xaa Xaa Thr Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 11 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6

(B) TYPE: AMINO ACID

(C) STRANDEDNESS: NA

(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:

(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 Xaa Thr Xaa Xaa Thr Xaa 1 5

Claims

WHAT IS CLAIMED IS:

1. A compound represented by the Formula I:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

Rl represents -CH2CH(NH2)CH2R^!, -(CH2)2-4N HRΠI.

-(CH2)1-3CH(NH2)R^IV or -(CH2)2-4NHRV;

Rl represents -OH, -NH2, -NHC(=NH)NH2 or -NHC(=NH)(CH2)0-3H;

RU and RID each independently represent H, Cl-4 alkyl or benzyl, or R^ and RΪH are taken together to represent -(CH2)4-5-;

R^IV represents Cl-4 alkyl or -C(0)NH2;

R represents -C(=NH)NH2, -C(=NH)(CH2)0-3H, -(CH2)2-4NH2, -(CH2)2-4θH, -C(0)(CH2)l-3NH2, -(CH2)2- 4NH(C=NH)NH2, -(CH2)2-4NH(C=NH)(CH2)0-3H

R2 represents

p represents 1, 2 or 3; Ra represents -Cl-10 alkyl or -(CH2)qNRbRC_; q represents 2, 3 or 4; Rb and R^c each independently represent H or -Ci_io alkyl; or R^D and R^c are taken together with the nitrogen atom to which they are to represent:

wherein Rd represents Ci-16 alkyl, C5-6 cycloalkyl, C5-6 cycloalkyl(Ci-6)alkyl, phenyl or benzyl.

2. A compound in accordance with Claim 1 represented by the structural formula:

3. A compound represented by the structural formula:

or a pharmaceutically acceptable salt thereof, wherein Rl is selected from the group consisting of:

-(CH2)3NH2, -CH2CH(NH2)CH2NH2, -CH2CH(NH2)CONH2, -(CH2)2NHC(=NH)NH2, -(CH2)2NH(CH2)2NH2 and -(CH2)2NHCO(CH2)2NH2.

4. A pharmaceutical composition comprised of a compound in accordance with Claim 1 in combination with a pharmaceutically acceptable carrier.

5. A method of treating a fungal infection in a mammalian patient in need of such treatment comprising administering a compound in accordance with Claim 1 to said patient in an amount effective to treat said fungal infection.

6. A method of treating a Pneumocystis infection in a mammalian patient in need of such treatment comprising administering a compound in accordance with Claim 1 to said patient in an amount effective to treat said Pneumocystis infection.

7. A method of preventing a Pneumocystis infection in an immunocompromised mammalian patient in need of such treatment comprising administering a compound in accordance with Claim 1 to said patient in an amount effective to prevent said Pneumocystis infection.