AU751213B2 - Substituted aurone derivatives - Google Patents

Description

WO 99/04789 PCT/US98/15388 SUBSTITUTED AURONE DERIVATIVES Background of the Invention The invention relates to methods of inhibiting microbial infections with substituted aurone derivatives.

Microbial infections, such as fungal infections and bacterial infections, can contribute to and complicate many diseases, including meningitis, pulmonary diseases, and respiratory tract diseases. Opportunistic infections have proliferated, particularly in immunocompromised patients, such as those with AIDS, those undergoing chemotherapy for cancer, and those undergoing therapy to prevent graft rejection following organ transplant surgery.

Fungal infections (mycoses) may be cutaneous, subcutaneous, or systemic.

Superficial mycoses include tinea capitis, tinea corporis, tinea pedis, onychomycosis, perionychomycosis, pityriasis versicolor, oral thrush, and other candidoses such as vaginal, respiratory tract, biliary, eosophageal, and urinary tract candidoses. Systemic mycoses include systemic and mucocutaneous candidosis, cryptococcosis, aspergillosis, mucormycosis, paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, and sporotrichosis.

Pathogenic organisms include dermatophytes Microsporum canis and other M. spp.; and Trichophyton spp. such as T. rubrum, and T. mentagrophytes), yeasts Candida albicans or C. tropicalis), Torulopsis glabrata, Epidermophyton floccosum, Malasseziafurfur (Pityropsporon orbiculare, or P. ovale), Clyptococcus neoformans, Aspergillusfumigatus and other Aspergillus spp., Zygomycetes Rhizopus, Mucor), Paracoccidioides brasiliensis, Blastomyces dermatitidis, Histoplasma capuslatum, Coccidioides immitis, and Sporothrix schenckii.

Summary of the Invention In one aspect, the invention features a method for treating a microbial WO 99/04789 PCT/US98/15388 -2infection. The method includes administering to a patient a pharmaceutical composition containing a compound selected from formula (IA):

Y

R

R

R z

(IA)

wherein each R is independently H, OH, Br, Cl, I, amino, thiol, nitro, C,.

4 alkoxy,

C,.

4 alkenyloxy, C 2 6 alkoxyalkyleneoxy, C,,4 alkylthio, 8 alkyl, or C 3 1 8 alkenyl; or two adjacent Rs, taken together, are a C 2 8 bivalent moiety containing at least one oxgen atom, substituted or disubstituted with A or B, or both, A being H, OH, Br, Cl, I, amino, or thiol, and B being H, CI-0o alkyl, C 2 .18 alkenyl, or C 61 g aryl; provided that at least two Rs are not H; further provided that when each of two Rs is one of OH,

C,.

4 alkoxy, C 14 alkenyloxy. or C 2 alkoxyalkyleneoxy, and X is phenyl substituted with two substituents independently selected from OH, alkoxy, and alkenyloxy, the remaining R cannot be prenyl; further provided that when each of two Rs is one of OH, C- 4 alkoxy, C, 4 alkenyloxy, or C2- 6 alkoxyalkyleneoxy, and X is phenyl substituted with three substituents independently selected from OH, alkoxy, and alkenyloxy, the remaining R cannot be prenyl; further provided that when each of two Rs is one of OH, C 14 alkoxy, C 14 alkenyloxy, or C 2 6 alkoxyalkyleneoxy, and X is phenyl substituted with a prenyl substituent and with two additional substituents independently selected from OH, alkoxy, and alkenyloxy, the remaining R cannot be H or OH; further provided that when each of two Rs is one of OH, C,.

4 alkoxy, C,.

4 alkenyloxy, or alkoxyalkyleneoxy, and X is phenyl substituted with a substituent containing three rings and with two additional substituents independently selected from OH, alkoxy, and alkenyloxy, the remaining R cannot be prenyl; X is C 4 1 0 alkyl, C 4 20 alkenyl, or a C 4 20 single, C6 20 bridged, or C 6 2 0 fused 04/06 2002 10:14 FAX 61 3 92438333 GRIFFITH HACK 0006 3 ring moiety containing cycloalkyl, cycloalkenyl, aryl, heterocycle, or heteroaryl, wherein X is substituted with H, OH, Cl, Br, I, amino, cyano, nitro, alkyl, alkoxy, alkenyl, or alkenyloxy; provided that if X is a heteroaryl S or heterocyclic moiety where two of R and each OH and meta to each other, the remaining R is H are ortho to each of the two hydroxyls, Y and Z are each 0, and a ring atom of X is linked directly to the sp 2 carbon atom adjacent to X, then substituted with H, OH, Cl, Br, I, amino, cyano, alkyl, alkoxy, alkenyl, or alkenyloxy; and each of Y and Z is independently selected from 0, S, and NH; or a pharmaceutically acceptable salt or ester thereof. The infection can be, for example, a fungal infection or a bacterial infection.

oo 15 The invention also provides use of a compound of the formula (IA) as defined above for the manufacture of a medicament for treating a microbial infection.

In another aspect, the invention features a compound selected from formulae (IV) below:

Y

VX

N.X

I* V 25 I

II

W Y Rb Y :"Ra R C -Re v z w z III TV III

IV

where V is a bivalent C2-18 moiety containing at least one oxygen atom and substituted with A, B, or both; each of W and W' is independently selected from the values for A, cyano, nitro, C1- 4 alkoxy, C 1 -4 H;\u,in.a.t \K1ee\Sieci\85R7E-'g.1 SPECI.doc 3/06/02 04/06 2002 10:14 FAX 61 3 92438333 GRIFFITH HACK oo007 4 alkenyloxy, C2-6 alkyloxyalkyleneoxy, C2-7 carboxyalkyloxy, C7-15 arylalkoxy, and C..4 alkylthio; Ra is H, C3-18 alkyl, C3-i1 alkenyl, C5-e1 cyclohexenyl, or C6-1s aryl; each of Rb and Re is independently selected from H and Ci-4 alkyl; X is substituted or unsubstituted C3-15 alkyl, C3-18 alkenyl, C3-15 cycloalkyl, C4-1s cycloalkenyl, C4-20 bicyclo[a.b.c]alkyl, Cs- 20 bicyclola.b.c]alkenyl, tricyclo[a-b.c.d]alkyl, Co-20 tricycloalkenyl, or C2-20 heterobicyclo[a.b.c]alkyl, or a combination thereof, where each of a, b, c, and d is independently 0 to 10 0 to 4, 0 to 6, or 1 to and each of Y and Z is independently selected frpm 0 15 and S.

The invention further provides a pharmaceutical composition comprising an effective amount of a compound of formulae (IV) as defined above together with a pharmaceutically acceptable carrier.

The invention also features synthetic methods suitable for combinatorial synthetic strategies for the production of diverse libraries of structurally related compounds. Other features and advantages of the invention will be apparent from the following detailed description, M 25 and from the claims.

Detailed Description In one aspect, the invention features a method of inhibiting a microbial infection, wherein the compound of formula (IA) is selected from formulae (IV): Y W1 X

X

H:\ummamnet.\Ku..\Sp.ci\B5876-98 .gpzet.l.le 3/06/02 04/06 2002 10:15 FAX 61 3 92438333 GRIFFITH HACK [4008 4a

III

In one aspect, V is a bivalent C 2 -is moiety containing at least one oxygen atom and substituted with A, B, or both. v can contain between 1 and 3 rings, e.g., 1 ring, 2 rings, or three rings. For example, V can be selected from the following five formulae; 0 0000 0*00 0* 0 S 00 S *00 S 00 0 0 05 0* S 0*

OS

.00 0 0 5000 00S0 0005 0 5000 H~nuann~%K~p~p~c\8S7G-8.1SpWf.Oad. 3/06/0:2 WO 99/04789 PCT/US98/15388 B A A 0 Each of W and W' is independently selected from the values for A, cyano, nitro, C 14 alkoxy, C 14 alkenyloxy, C 2 6 alkyloxyalkyleneoxy, C 27 carboxyalkyloxy, C7., arylalkoxy, and CI4 alkylthio.

R, is H, C 3 alkyl, C 3 8 alkenyl, C 5 1 8 cyclohexenyl, or C 6 aryl. For example, R, is H, prop-2-enyl, cinnamyl, 2-methylprop-2-enyl, but-2-enyl, 3-methylbut-2-enyl, 3,7-dimethylocta-2,6-dienyl, (cyclohexenyl)methyl, 3,7,1 1-trimethyldodeca-2,6.10-trienyl, or benzyl. In some cases, R, is not prenyl or isoprenyl.

Each of Rb and R, is independently selected from H and C,.

4 alkyl. In one method, the compound can be of the formula Q=(CHX) where Q is derived from the benzofuranone analogs or derivatives from Schemes Q-l through Q-11, and the geometry of the double bond is E or Z. In Schemes Q-1 through Q-11, the compounds are of the formula Q-H 2 where the two hydrogens are methylene hydrogens.

In another aspect, the compound has an IC 5 0 of less than 50 micrograms per milliliter against at least one pathogenic strain of Candida or Aspergillus.

In one embodiment, the compound is of formula (III), where each of Y and Z is independently selected from O and S, for example, formulae SO1-S06 and S08-S19 of Scheme P-1. Other embodiments include a compound where: W and W' are selected from H, OH, methoxy, methoxymethyleneoxy, and carboxymethoxy; where Y and Z are O, and at least one of W and W' is OH; where X is a heterocyclic radical, a heteroaryl; where X is C 4 1 0 alkyl, C 4 2 0 alkenyl, or a C 4 2 0 single, Cs 20 bridged, or C 6 20 fused ring moiety containing cycloalkyl, cycloalkenyl, or aryl; where X is a nonaromatic moiety containing cycloalkyl, cycloalkenyl, alkyl, or alkenyl; or where the compound is selected from S12 and S02.

Examples of X include benzyl, WO 99/04789 WO 9904789PCTIUS98/15388 -6- 2,3 -dimethyl-4-methoxyphenyl, 3-benzyloxyphenyl, 3 -phenoxyphenyl, 4-benzyloxy-3-methoxyphenyl, 4-[3-propenoic acid]-phenyl, 2-ethoxy- I -naphthyl, I -(methylthio)ethyl, DL- I -phenylethyl, 4-n-pentyloxyphenyl, I -(phenylsulfonyl)-2-pyrrolyl, 4-(3-dimethylaminopropoxy)phenyl, 3 -phenylpropyl, 2,4-diethoxy-m-tolulyl, 2,6,6-trimethylcyclohexene- 1 -methyl, 2,5-dimethoxy-3-tetrahiydroftiranyl, 4-methyl-S -imidazolyl, 4-n-pentylphenyl, 2-benzyloxy-4,5-dimethoxyphenyl, 1 -pyrenyl, 3,5 -dibenzyloxy-3-methoxyphenyl, 3-methyl -4-methoxyphenyl, 4-n-decyloxyphenyl, 2 ,4-dimethoxy-3-methylphenyl, t-butyl, 3 -(4-t-butylphenoxy)phenyl, 2-n-hexyloxyphenyl, 2-(4-chlorophenylthio)phenyl, cyclopropyl, 2,6-dimethoxy-4-hydroxyphenyl, 4-benzyloxyphenyl, 2-benzyloxyphenyl, 8-hydroxy -1,1 ,7,7-tetramethylj ulolidin-9-yl, 2,3 ,6,7-tetrahydro-8-hydroxyj ulolidin-9-yl, 2-methoxymethyl- 1 -pyrrolidinyl, 5-(2-nitrophenyl)furanyl, 1,1 -dimethyl-2-hydroxyethyl, 5-methylfuranyl, 3-chlorophenyl)furanyl, 2,4-hexadienyl, 5-[3(trifluoromethyl)-phenylfuranyl], 4,5 -dim ethyl-4-pentenyl, imidazolyl, ferrocenyl, 2,6-dimethylhept-5-enyl, -[2-(tri fluoromethyl)-phenyl] furanyl, 5 -(hydroxy-2-nitromethyl)furanyl, 2,4-dimethyl-2,6-heptadienyl, 1 -phenylethyl, 5 2-chlorophenyl)furanyl, benzyl, 5-ethyl-2-furanyl, 5-(4-nitrophenyl)-furanyl, pentamethyiphenyl, 1 -(methyldithio)isopropyl, 4-trifluoromethyiphenyl, 3-fluoro-4-methoxyphenyl, or the X of a compound of Schemes X- I through X- 10, wherein the compounds of Schemes X- 1 through X- 10 have the formulae X-CHO.

The fungal infection can be: an infection of a Candida species, an infection of a fungus resistant to at least one azole antifungal agent where the azole antifungal agent is fluconazole); or an infection of an Aspergillus species.

Examples of pathogen strains include C albi cans, C. glabrata, C krusei, C tropicalis, C parapsilosis, A. fumigatus, and A. niger.

The invention also features aurone derivatives, such as those described in formulae in the Summary section. Examples of these compounds include WO 99/04789 PCT/US98/15388 -7those where X is C3-, 5 alkyl, C 3 8 alkenyl, C 3 cycloalkyl, C 415 cycloalkenyl,

C

5 1 0 bicyclo[a.b.c]alkyl,

C

5 bicyclo[a.b.c]alkenyl, C8- 20 tricyclo[a.b.c.d]alkyl, C8- 20 tricycloalkenyl, C 3 0 heterobicyclo[a.b.c]alkyl, or a combination thereof, where each of a, b, c, and d is independently 0 to 6; X is C 3 5 alkyl, C 3 ,1 alkenyl, C 3 5 cycloalkyl, or C 4 cycloalkenyl; where X is C 5 0 bicyclo[a.b.c.]alkyl,

C

5 0 bicyclo[a.b.c]alkenyl,

C

8 5 tricyclo[a.b.c.d] alkyl, C 8 s- tricycloalkenyl, C3-, 0 heterobicyclo[a.b.c]alkyl, or a combination thereof; where each of W and W' is independently selected from H, hydroxyl, methoxy, hydroxymethyl, and halomethyl; and where W and W' are both hydroxyl; or a combination thereof. The bridges can be ortho-fused or ortho- and peri-fused. The bridge can be alkylene, azo, azimino, biimino, epidioxy, nitrilo, imino, furano, epoxythioxy, epithio, alkanoxy, epoxy, or alkanoxyalkano methanoxymethano). The invention also features additional novel compounds described in the above method of treatment.

In some embodiments, enantiomers of disclosed compounds are separated.

The bridging olefinic bond between Q and X is sometimes preferably E (entgegen) and sometimes preferably Z (zusammen). Depending on the individual embodiment, chiral centers may be or Terms Some terms are defined below, and some terms are defined elsewhere in the disclosure.

Alkyls may be substituted or unsubstituted and may be straight, branched, or cyclic. Preferably, alkyl groups have between 1 and 10 carbon atoms, and more preferably have between 1 and 6 carbon atoms. Examples of alkyls include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, t-pentyl, sec-pentyl, hexyl, cyclohexyl, isohexyl, 2,3-dimethylbutyl, 2,2-dimethylbutyl, 3-ethylpentyl, 3,4-dimethylpentyl, heptyl, octyl, nonyl, decyl, and (2,3,4-trimethylcyclo-hexyl)methyl. An alkylene is a bivalent hydrocarbon, an alkyl group with an additional hydrogen removed, such as WO 99/04789 PCT/US98/15388 -8methylene, propylene, or 1,4-cyclohexylene. Alkoxy groups are alkyl groups linked to the remainder of the molecule, a ring, by an oxygen. Alkoxy groups also include polyethers, such as methoxyethyloxy. Alkyl, alkenyl, alkynyl, aryl, and heterocyclic radicals, whether or not substituting groups, (discussed below) may be linked by alkyl, alkenyl, alkynyl, ether, ester, amide, urea, urethane, amino, thioether, or thioester groups, such as methoxymethyl and alkylthioalkyl.

Alkenyls are alkyl groups with one or more unsaturated carbon-carbon bonds, such as cyclopentenyl, cyclopentadienyl, cyclohexadiene, but-2-enyl, 3,4-dimethylpent-3-enyl, allyl, vinyl, prenyl, isoprenyl, and norbomenyl. Examples of alkenylenes include vinylene and propenylene. Similarly, alkynyl groups have one or more triple bonds, and may also include one or more double bonds.

Aryls include aromatic rings, substituted or unsubstituted, preferably having between 6 and 20 carbon atoms, and more preferably between 6 and 14 carbon atoms, exclusive of substitution on the ring. Examples of aryls include phenyl, naphthyl, indenyl, pentalenyl, anthryl, azulyl, and biphenylyl. Combinations include alkylaryls tolyl, xylyl, mesityl, cumenyl, 2-ethyl-4 methylphenyl) and arylalkyls benzyl, phenylethyl, or arylalkenyls, and divalent arylenes such as 1,4-phenylene.

Haloalkyl (or haloalkenyl or haloalkynyl) includes any alkyl (or alkenyl or alkynyl) group where at least one hydrogen is replaced with a halogen (fluorine, chlorine, bromine, or iodine). Where more than one hydrogen is replaced a dihaloalkyl or a hexahaloalkyl), the halogens are selected independently and may be on the same carbon atom or on different carbon atoms. Amino-substituted, nitro-substituted, or otherwise substituted alkyls (or alkenyls or alkynyl or aryls) are analogous to the above. Halomethyls include perchloromethyl, bromomethyl, and fluorochloromethyl.

Heterocyclic radicals may be aromatic (heteroaryl) or nonaromatic, and substituted or unsubstituted. They have one, two or three rings which are single, fused, bridged rings, or polycyclic. They contain between 2 and 15 carbon atoms in WO 99/04789 PCTUS98/15388 -9the ring, exclusive of substitution. They can be linked to the rest of the molecule through a carbon atom or a heteroatom. Heterocyclic radicals include thienyl, thianthrenyl, furanyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, quinuclidinyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinnolinyl, pteridinyl, 4H-carbazolyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxazinyl, isochromanyl, chromanyl, furazanyl, pyrrolinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrazolinyl, imidazolinyl, piperidyl, piperazinyl, and morpholinyl. Heterocyclic radicals also include benz[h]isoquinolinyl, thieno[2,3-b]furanyl, 2H-furo[3,2-b]pyranyl.

Substituted moieties have one, two, three, or more of the following independently selected substituting moieties (instead of a hydrogen): C.

1 0 o alkyl, C 2 -1 0 alkenyl, C- 10 alkoxy, C2- 10 alkenyloxy, haloalkyl, CI- 1 0 haloalkoxy, aryl, aryloxy, hydroxy, nitro, chloro, fluoro, bromo, and iodo, thiol, cyano, and amino. Substituting moieties also include combinations of the above with carbonyl (acyl), sulfonyl, thionyl thioketone), and carboxyl, such as alkyloxycarbonyl, arylalkyloxy, (N,N-dialkylamino)alkoxy, arylsulfonyl, and carboxylic acids. In some embodiments, substituting moieties have between 1 and 6 carbon atoms, and more preferably have between 1 and 3 carbon atoms. Examples of carbon-containing substituting moieties include chloromethyl, hydroxymethyl, bromoethyl, methoxy, and ethoxy. An alkyl does not have an alkyl or haloalkyl substituent, although, for example, a cycloalkyl may have an alkyl or haloalkyl substituent.

The invention also encompasses compounds identical to any of the disclosed structures formula except that one or more conventional protecting groups are used, such as hydroxyl protecting groups, carboxylate protecting groups, and carbonyl protecting groups. Methods of adding and removing such protecting groups are well known in the art (see, for example, Protective Groups in WO 99/04789 WO 9904789PCTIUS98/15388 Organic Synthesis, by T.W. Greene and P.G.M. Wuts, 2nd ed., 1991, Chapters For example, the following representative hydroxyl protecting groups are provided. There is some overlap between the above-described R moieties and the disclosed hydroxyl protecting groups.

Methyl ethers include methoxymethyl; methyithiomethyl; t-butylthiomethyl; (phenyldimethyldiyl)methoxy-methyl; benzyloxymethyl; p-methoxybenzyloxymethyl; (4-methoxyphenoxy)methyl; gualacolmethyl; t-butoxymethyl; 4pentenyloxymethyl; siloxymethyl; 2-methoxyethoxymethyl; 2,2,2-trichioroethoxymethyl; bis(2-chloroethoxy)methyl; 2-(trimethylsilyl)ethoxymethyl; tetrahydropyran-2-yl; 3-bromotetrahydropyran-2-y!; 1 -methoxycyclohexyl; 4methoxy-tetrahydropyran-2-yl; 4-rnethoxytetrahydrothiopyran-2-yl; 4methoxytetrahydrothio-pyran-2-yl-S,S-dioxido; 1- [(2-chloro-4-methyl)phenyl]-4methoxypiperidin-4-yl; 1 ,4-dioxan-2-yl; tetrahydrofuranyl; tetrahydrothiofuranyl; and 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl.

Ethyl ethers include Il-ethoxyethyl; l-(2-chloroethoxy)ethyl; I1-methyl- Imethoxyethyl; 1-methyl-I -benzyloxy-2-fluoroethyl; 2,2,2-trichioroethyl; 2-trimethylsilylethyl; 2-(phenylselenyl)ethyl; t-butyl; al lyl; p-ch lorophenyl; pmethoxyphenyl; and 2,4-dinitrophenyl.

Benzyl ethers include benzyl; p-methoxybenzyl; 3,4-dimethoxybenzyl; onitrobenzyl; p-nitrobenzyl; p-halobenzyl; 2,6-dichlorobenzyl; p-cyanobenzyl; p-phenylbenzyl; 2- and 4-picolyl; 3-methyl-2-picolyl-N-oxido; diphenylmethyl; p,p'dinitrobenzhydryl; 5-dibenzosuberyl; triphenylmethyl; a-naphthyldiphenylmethyl; pmethoxyphenyldiphenylmethyl; di(p-methoxyphenyl)phenylmethyl; tri(pmethoxyphenyl)methyl; 4-(4'-bromo-phenacyloxy)phenyldiphenylmethyl; tris(4,5-dichlorophthalimidophenyl)methyl; 4 ,4',4"-tnis-(levulinoyloxyphenyl)methyl; 4, 4 ',4"-tris(benzoyloxyphenyl)methyl; 3 -(imidazol-1I-ylmethyl)bis(4',4"dimethoxyphenyl)-methyl; 1,1 -bis(4-methoxyphenyl)-lI'-pyrenylmethyl; 9-anthryl; 9-(9-phenyl)xanthenyl; 9-(9-phenyl-1I0-oxo)anthryl; 1 ,3-benzodithiolan-2yl; and benzisothiazolyl S,S-dioxido.

WO 99/04789 WO 9904789PCT/US98/15388 Silyl ethers include trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; diethylisopropyl-silyl; dimethyithexylsilyl; tbutyldimethylsilyl; t-butyl-diphenylsilyl; tribenzylsilyl; ti-p-xylylsilyl; triphenylsi lyl; diphenylmethylsilyl; and t-butylmethoxyphenylsilyl.

Esters include formate; benzoylformnate; acetate; chioroacetate; trichioroacetate; methoxyacetate; triphenylmethoxyacetate; phenoxyacetate; pchiorophenoxyacetate; p-(phosphate)phenyl acetate; 3-phenyiproprionate; 4oxopentanoate (levulinate); 4,4-(ethylenedithio)pentanoate; pivaloate; adamantoate; crotonate; 4-methoxycrotonate; benzoate; p-phenylbenzoate; and 2,4,6trimethylbenzoate.

Carbonates include methyl carbonate; 9-fluorenyl-methylcarbonate; ethyl carbonate; 2,2,2-trichioroethyl carbonate; 2-(trimethylsilyl)ethyl carbonate; 2-(phenylsulfonyl)ethyl carbonate; 2-(triphenylphosphono)ethyl carbonate; isobutyl carbonate; vinyl carbonate; allyl carbonate; p-nitrophenyl carbonate; beuzyl carbonate; pmethoxybenzyl carbonate; 3,4-dimethoxybenzyl carbonate; o-nitrobenzyl carbonate; p-nitrobenzyl carbonate; S-benzyl thiocarbonate; 4-ethoxy-1-naphthyl carbonate; and methyl dithiocarbonate.

Protecting groups with assisted cleavage include 2-iodobenzoate; 4-azidobutyrate; 4-nitro-4-methylpentanoate; o-(dibromomethyl)benzoate; 2-formylbenzenesulfonate; 2-(methylthiomethoxy)ethyl carbonate; 4-(methylthiomethoxy)-butyrate; and 2-(methylthiomethoxymethyl) benzoate.

Miscellaneous esters include 2,6-dichloro-4-methylphenoxyacetate; 2,6dichloro-4-( 1,1,3 ,3-tetramethyl-butyl)phenoxyacetate; 2,4-bis( 1,1-dimethylpropyl)phenoxy-acetate; chlorodiphenylacetate; isobutyrate; monosuccinoate; (E)-2-methyl- 2-butenoate (tigloate); o-(methoxycarbonyl)benzoate; p-benzoate; a-naphthoate; nitrate; alkyl N,N,N',N'-tetramethylphosphorodiamidate; N-phenylcarbamate; borate; dimethylphosphinothioyl; and 2,4-dinitrophenyl-sulfenate.

Sulfonates include methanesulfonate (mesylate); benzylsulfonate; and tosylate.

WO 99/04789 PCT/US98/15388 -12- Cyclic acetals and ketals include methylene; ethylidene; 1-tbutylethylidene; 1-phenylethylidene; 4-methoxyphenylethylidene; 2,2,2trichloroethylidene; acetonide (isopropylidene); cyclopentylidene; cyclohexylidene; cycloheptylidene; benzylidene; p-methoxybenzylidene; 2,4-dimethoxybenzylidene; 3,4-dimethoxybenzylidene; and or 4-nitrobenzylidene.

Cyclic ortho esters include methoxymethylene; ethoxymethylene; dimethoxymethylene; 1-methoxyethylidene; 1-ethoxyethylidine; 1,2-dimethoxyethylidene; a-methoxybenzylidene; 1-(N,N-dimethylamino)ethylidene derivative; a-(N,N-dimethylamino)benzylidene derivative; and 2-oxacyclo-pentylidene.

Note that these cyclic ortho esters, like the bivalent organic moieties recited above for adjacent pairs of substituents R, and R 2 in formula may react with non-adjacent hydroxyl moieties. For example, a bivalent organic moiety recited in the preceding paragraph or recited above for adjacent pairs of substituents may be selected for two nonadjacent substituents on the same molecule or for any two substituents on two separate molecules. The two separate molecules can be the same or different, and are selected from compounds disclosed herein.

Silyl derivatives include di-t-butylsilylene group; 1,3-(1,1,3,3tetraisopropyldisiloxanylidene) derivative; tetra-t-butoxydisiloxane-1,3-diylidene derivative; cyclic carbonates; cyclic boronates; ethyl boronate; and phenyl boronate.

Preferred protecting groups for catechols include cyclic acetals and ketals such as methylene, acetonide, cyclohexylidene, and diphenylmethylene; and cyclic esters such as cyclic borate and cyclic carbonate.

The invention encompasses other Cilo hydroxyl protecting groups not individually identified above which are pharmaceutically acceptable, and are optionally metabolized cleaved or modified) to form one of the compounds disclosed herein. In other words, the invention encompasses metabolic precursors of the disclosed compounds and metabolites of the disclosed compounds having antimicrobial activity.

WO 99/04789 PCT/US98/15388 -13- The invention also encompasses amides, amine salts, and other organic salts of the disclosed compounds. Amides may be formed by reacting a disclosed compound or activated derivative thereof with any naturally-occurring amino acid, an oligopeptide having up to 10 4, 3, or 2) residues, a peptidomimetic having a molecular weight less than 300, or any C 1 20 organic moiety having an amino group that is not already described above. The term "naturally occurring amino acid" is meant to include the 20 common a-amino acids (Gly, Ala, Val, Leu, Ile, Ser, Thr, Asp, Asn, Lys, Glu, Gin, Arg, His, Phe, Cys, Trp, Tyr, Met and Pro), and other amino acids that are natural products, such as norleucine, ethylglycine, orithine, methylbutenylmethylthreonine, and phenylglycine. Examples of amino acid side chains include H (glycine), methyl (alanine), -CH 2

-(C=O)-NH

2 (asparagine), -CH,-SH (cysteine), and -CH(OH)CH 3 (threonine).

Subjects or patients of the disclosed methods may be any living animal, plant, or plant product grain or feed). Animals include mammals, particularly humans. Animals also include domestic animals bred for food or as pets, such as horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals. Plants include trees, crops, grasses, and flowering plants.

WO 99/04789 PCTIUS98/15388 -14- Cn Sclicaiic Q-2 WO 99/04789 PCTIUS98/15388 -16- WO 99/04789 PCT/US98/15388 -17- WO 99/04789 PCT/US98/15388 -18- WO 99/04789 PCT[US98/15388 -19- WO 99/04789 WO 9904789PCTIUS98115388 0z 0 0L 0 oz 0 0 0 0.

z 0 0 0 0 0 r.

z -n 0 0 Iz 0 C) SCiJCf ic Q-8

H

3C yN 0 0 0

S

5 -k

CH

2 0

N

NII

Q( 0

S

Ks

N-NH

2 0 cil .0 lCl HO 0 61ol 0

C(CH

3 3

N

N

H

3

C/

C(CH

3 3

N-

N

H 0 Sclictlic Q-9 0

NH

O N 0

H

00 N N

H

0 Bi0§) cc =o 11 3

CO

OH

000

NH

I0 SclICnTIC Q- 1) .1 3 coH- 2 CO 0

CH

2

OC

2 0Cfi 3

H

3

COH

2 CO -0

IH

3

CI

H

3 C 0co( 0 0 OG 2

OCH-

3 G1H 2 i

H

3

COH

2 C0 0 0 0 0OGHO 2

CH

3

CH

2 -1-l 0

HJO

0 0H 2 CO 0

K.

0 0 OCI 013 C11 3

H

3 C0H 2 C0 0 ~0 V1 3 C OCH 2 0C1 3

H

3 00H 2 C0 0

OCH

2 00CH 3

OH

3

FH

3 COI-1 2 00 0 00H1 2 0CH 3

UH

2 V1 3 CO11 2

G

OGI 1 2 0oC9 I., 0-

H

3

C

of Jac I-k- 0 Scliciuic Q- I I

H

3

COH

2

CI

H

3 00D- 2 C0 0 01

OHOCH

2

CH

3

H

3

COH

2 CO A0 0 OCI-1 2 0CH 3

CH

2

H

3 COI 12CO A0 HO

OCH

2

OCH

3

CH

3 0

H

3 COI H 3

COH

2 CO A0 0 2

OCH'CCI

3

OCH

2 0CH 3

H

3

GOH

2 C A 0

'C)N

I'13G

OCH

2 0CH 3

OCH

2 0CH 3 PSS057 WO 99/04789 WO 9904789PCTIUS98/15388 Scheme X- I \i

C

C

C

C

C

RC

Z

C C C WO 99/04789 WO 9904789PCTIUS98/15388 -26- F- 0 COaCH, 3 Scheme X-2 Ph-< I 'Ph

H

3

C

Fi 3 c 0

H

N

NH

H

H

3 C CHO

P%.

Ph f Ph HO0 0 HO0

OH

OH

HO0

OCH

3

OH

HO0

OH

HO0

OH

OH

3 H 0

OH

PN

Ph IP Ho 0 WO 99/04789 WO 9904789PCTfUS98/15388 -27- Scheme X-3

C

1'-a

-I

0 C

C

C

C

0-

C

C

z

C

C o: cHC

CH)

WO 99/04789 WO 9904789PCTIUS98/15388 -28- '7 040

"I

-:1

C

Scheme X-4 WO 99/04789 WO 9904789PCT/US98/15388 -29- Schcn'ie X-'

N'-

z

N

I

~~Lc z 0 0 z

C

z

'LY"

C

C

C

C Z 0 0

Q

0

C

C',

In 0

C

b

C

C

C

Os

C

C-)

C

WO 99/04789 PCTIUS98/15388 Schmc!"e X-6 _1 /4: z

-Z

C

r, I r h h

=X

C)

C-

C)

iI?3

CZ

Ck= zz c z Ph z C8 cl z o 11 o -r!

I-

=-n Ca

ON

C

Cra HOOC 0 cbjffAH

H

-r 0~ 0 NC N

H

0

H

0

H

H3C-..N

N

U",

3 0

H

F

3

C"

H Y ol 0N 0 H 3c 0 0

H

H

N~ 0 0 2

NJD

3 WO 99/04789 PCTIUS98/15388 -32- Schcme X-S

C

r)

ZC

&0 c

C"

C

o

C

2

C

oC c C-)i c

C

CQ

WO 99/04789 PCTIUS98/15388 -33- Scheme X-9 -ti r) c 8-Q~o r! 0

-N

0~~M o n z

C;

0 r, kai c c d n

C'

-x -0 0 xc r-)

C

0 0 -0 0o

IC'

WO 99/04789 WO 9904789PCTIUS98/15388 -34- Scheme X- 1

A

J~c

C

I

0' CG--r 1:

/CT

-0

C

C)

CA(

-an C C r) C WO 99/04789 PCT/US98/15388 Matrix Synthesis Over 3000 aurones were synthesized by matrix methodology and screened for antimicrobial activity. Numerous benzofuranones (Schemes Q-l through Q-11) and aldehydes (Schemes X-1 through X-10) were obtained commercially or synthesized. One-hundred and sixty aldehydes were purchased.

The aldehydes were divided into two sets or plates of 80 aldehydes. Each set was reacted with a given benzofuranone. Phenolic hydroxyl groups were protected, for example, as methoxymethyl ethers. In general, the alicyclic or aliphatic aldehydes were less reactive than the aromatic aldehydes and thus required more vigorous conditions, such as higher temperatures. Even so, yields were generally lower than the aromatic aldehydes. Portions of the adducts were screened for antimicrobial activity, and the remainder deprotected with trimethylsilyl chloride in methanol to yield the unprotected aurone, which was also screened for antimicrobial activity.

Specifically, a solution of benzofuranone in methanol, (1 M, 10 ul) was added to a 2 ml polypropylene tube containing 100 /l of methanol. After a solution of methanolic sodium methoxide (0.5 M, 22 was added, the reaction was shaken for 1 minute. A solution of the aldehyde in methanol (1 M, 10 was added and the reaction was left shaking for 5 minutes. After partitioning between ethyl acetate (1 ml) and water (0.5 ml), the organic layer was collected and transferred to another 2 ml polypropylene tube and allowed to dry. The dried sample was redissolved in 1 ml methanol and divided into 2 equal portions, one of which was dried and tested for antimicrobial activity. To the second portion was added trimethylsilyl chloride After standing at room temperature for 8 hours, ethyl acetate (1 ml) and saturated sodium bicarbonate (500 p1) were added. The organic layer was collected and dried.

The dried sample was tested for antimicrobial activity. The compounds were tested for antimicrobial activity using, for example, methods described in WO 97/26873.

WO 99/04789 PCT/US98/15388 -36- Antifungal Activity Thirty-eight 96-well plates were prepared with the above samples and tested against both C. albicans and A. fumigatus at either 8 mg/ml or 12.5 pg/ml.

Plates with high activity were retested at the same concentration to confirm activity.

Based on the above, preferred compounds have at least one phenolic hydroxyl group on the benzofuranone portion of the aurone, preferably at position in formulae I-IV). Compounds with 2,3-dihydroxyphenyl or 2,3,4-trihydroxyphenyl (derived from the aldehyde reagent) exhibited good inhibition against Candida, yet exhibited little inhibition of Aspergillus. Some preferred anti-Candida compounds have polar substituents on the aldehyde portion of the aurone. Turning to anti-Aspergillus compounds, aurones including 3,5-di-t-butyl-4-hydroxyphenyl and 2,4-difluorophenyl (again derived from the aldehyde reagent) generally exhibited good inhibition. Less polar substituents tend to improve inhibition of Aspergillus.

Inhibition can be measured in terms of an IC 5 0 an MIC, or a percent inhibition relative to control (absence of test compound) at a given concentration, such as 8 pg/ml or 12.5 Mg/ml. In general, a percent inhibition of at least 30% at 8 ,ug/ml or 12.5 zg/ml is preferred at least 40%, at least 50%, at least 65%, and least 70%, and at least Preferred compounds of formula (II) exhibit a percent inhibition of at least 70%, including two as high as 87%. These compounds have formulae where W' is OH, V is 3',4'-(1,1-dimethylpropyloxy) to form a dihydropyran series, and X is selected from 3,4-dimethoxyphenyl, 4-t-butylphenyl, 2(prop-2-enyloxy)phenyl, 3-phenoxyphenyl, 3-ethoxy-4-hydroxyphenyl, 6-cyclohexenyl, and norbom-4-yl.

The inhibition values of 60 compounds are shown in Tables 1 through 3 on the next three pages.

WO 99/04789 WO 9904789PCTIUS98/15388 -37- HO HO H 0 H 0 H 0 N IOH

NOH

OH

H OH O0 o OH OH CA 49% AF 40% 12.5gjg/mI CA 92% AF 0% CAO0% AF 0% 12.5jig/mI CA 94% AF 31% CA 89% AF 0% 12.5gg/mI CA 0% CA 95% CA 57% CA 79% CA 94% AF 43% AF 36% AF 48% AF 27% AF 44% 12.5pgg/mI 12.5pLg/mI 12.5 tg/mI 12.5pgg/mI 12.5gg/mI CA 67% AF 94% 8pRg/ml CA 87% AF 0% 8lig/mI CA 80% AF 76% 8pg/ml CA 96% AF 52% 8jtg/mI CAO0% AF 69% 8pgg/mI 4 4 4- CA 53% AF 96% 8pgg/mI CAO0% AFO0% 8gg/mI CA 52% AF 68% 8jig/mI CA 45% AFO0% 8pgg/mI CA 69% AF 8gig/ml L SUBSTITUTE SHEET (RULE 26) WO 99/04789 WO 9904789PCTIUS98/15388 -38- SUBSTITUTE SHEET (RULE 26) H 0 H 0 H H 0 H 0 H 0 H 0 O I HCO H,O H C 3

H

3

H

3 H 3 1 i1; 1 BU

CH

3 E I T CH 3 HC CH 3 HO 0 CA 7% CA N% CA 11% CA 18% CA 31% CA O% CA 51% CA 26%

H

3 C OHAFO% AF13% AFO% AF18% AF41% AFO% AF53% AF13% C 12.5 tg/ml @12.5gg/ml @12.5g/ml @12.5gg/ml @12.5pgg/ml @12.5jig/ml @12.5gig/ml @12.51gg/ml

CH

3 m HO N CA O% CA O% CA O% CA O% CA O% CA O% CA 6% CA 4% 2 AF O% AF 13% AF O% AF O% AF O% AF O% AF 48% AF 17% (0 OH0 012.5R.g/mi 12.5ptg/mI 12.5gg/md 12.5pgg/mI 12.5pRg/niI @12.5jig/mI 12.5jig/mI 12.54ig/ml 0 N CA 82% CA 87% CA 13% CA 58% CA 73% CA 76% CA O% CAO% AF 97% AF 94% AF 34% AF 93% AF 68% AF 68% AF 28% AF 11% O 8jg/mI 8jig/ml a 8jig/ml 8jg/ml 8pgg/mI Bjig/mI 8g~g/ml 8gg/mi OH 0 HO N CA 7% CA 56% CA 4% CA 10% CA 16% CA 6% CA 16% CA 42% AF 49% AF 69% AF 11% AF 47% AF 47% AF 22% AF 7% AF 81% H OH CO 8pg/mI 8jig/ml 8jig/ml 8pig/mI 84g/ml 8gg/ml 8gg/ml 8gg/mI

H

00 00 WO 99/04789 PCT[US98/15388 Antibacterial Activity Compounds described herein also have activity against bacteria. The compounds shown in the table below were tested using assays known in the art and were found to have activity against Staphylococcus aureus.

WO 99/04789 WO 9904789PCTIUS98/15388 -41- STRUCTUREb INHIBITION OF GROWTH AT 1 Qpg/mI OH 0 0O% 010 0 3

H

OH 0 b B 100 Br OH 0 OH 100 HOO 1- CI ~OH ~98.6 I -V %HO O, 0v H 2

C

0 Fc76.5 SUBSTITUTE SHEET (RULE 26) WO 99/04789 PCT/US98/15388 -42- Use Compositions including one or more disclosed compounds are useful for inhibiting microbial infections, or combinations of infections. The invention features a method for inhibiting a microbial infection in a subject, which method includes administering a pharmaceutically effective amount of such a composition. One aspect is a method for inhibiting a fungal infection which is resistant or sensitive to known therapies, such as fluconazole or other azoles. Examples of fluconazole-resistant or fluconazole-sensitive strains include C. glabrata, C. kefyr, and C. tropicalis.

Formulation and Administration A disclosed composition contains from about 0.1 to 90% by weight (such as about 0.1 to 20%, or about 0.5 to 10%) of active compound(s). Disclosed compositions can be formulated as solids or liquids for oral administration, or as liquids or semi-solids (ointments, creams) for topical administration. The compositions can also be formulated for administration by nebulization or inhalation, or administration by intravenous, intramuscular, or intraperitoneal injection.

Formulations for controlled release, including implantable or biodegradable or biocompatible matrices, are also contemplated. Controlled release includes continuous and intermittent release. Methods of formulation, including pharmaceutical carriers, are well-known to those in the art. The effective amount of active compound(s) used to practice the present invention for therapeutic or prophylactic treatment of conditions caused by or contributed to by a microbial infection varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

There now follow particular examples that describe the preparation of compounds of the invention, and the methods of the invention. These examples are WO 99/04789 PCTIUS98/15388 -43provided for the purpose of illustrating the invention, and should not be construed as limiting.

Example 1: 2-Amino-4.6-dimethoxy-a-chloroacetophenone Boron trichloride (1M in CH 2 Cl 2 6.5 ml) was added to a dry N 2 flushed flask through a septum and cooled on ice. 3,5-Dimethoxyaniline (1.0 g, 6.5 mmol), dissolved in dry CH 2 C1 2 was added, followed by dropwise addition of chloroacetonitrile (0.500 ml, 7.8 mmol). The mixture was stirred under N 2 for minutes, and ZnCl 2 (0.98 g, 7.2 mmol) was added. The green mixture was refluxed for 1 hour and stirred 18 hours at room temperature. Hydrochloric acid (2N, 5 ml) was added; the mixture was refluxed for 30 minutes, allowed to cool to 25 and an excess ofNaOH (2N) was added. After two extractions with CH 2 C1 2 the organic phases were dried (MgSO 4 filtered, and concentrated in vacuo to afford the crude product (1.013 g, Purification by column chromatography (eluent:

CH

2

CI

2 /MeOH: 99/1) gave (834 mg, HPLC-MS: M' (230). 'H-NMR (CDC1,, 250 MHz): 6 6.5 (broad s, 2 5.65 2 4.70 2 3.79 3 H); 3.72 3 H).

Example 2: 4.6-Dimethoxy-3-indolinone (6) 2-Amino-4,6-dimethoxy-a-chloroacetophenone (670 mg, 2.9 mmol) was dissolved in dry acetone (10 ml). K 2

CO

3 (604 mg, 4.4 mmol) and a little KI was added, and the mixture was refluxed for 4 hours, then stirred at room temperature for 2 days until the starting material had disappeared. The solvent was evaporated in vacuo, and water (20 ml) was added to the compound. Extraction with CH 2

CI

2 drying with MgSO 4 filtration, and evaporation of the solvent in vacuo afforded the crude product. Purification by column chromatography (eluent: CH 2

CI

2 /MeOH: 99/1) gave (394 mg, 70%) as green crystals. 'H-NMR (CDC1 3 250 MHz): 6 6.5 (broad s, 2 5.65 (dd, 2 4.39 2 4.39 2 3.82 3 3.72 3 H).

WO 99/04789 PCT/US98/15388 -44- Example 3: 2-Amino-4.5,6-trimethox-a-chloroacetophenone (8) The target compound was synthesized in the manner described for the synthesis of(5) using 3,4,5-trimethoxyaniline (2.5 g, 13.6 mmol), BCl1 (1 M in CHzCl 2 13.6 ml), ZnCl 2 (2.04 g, 15 mmol), and chloroacetonitrile (1.03 ml, 16.3 mmol) in dry CH 2 CI2. Purification by column chromatography as described for afforded (1.06 g, 30%) as a cystalline product. 'H-NMR (CDC1 3 250 MHz): 6 6.5-5.5 (broad s, 2 5.9 1 4.70 2 3.95 3 3.80 3 3.65 3

H).

Example 4: 4.5.6-Trimethoxv-3-indolinone (9) Compound was synthesized from in the same manner as described for the synthesis of using 2-amino-4,5,6-trimethoxy-a-chloroacetophenone (1.05 g, 4.0 mmol), K 2

CO

3 (838 mg, 6.1 mmol), a little KI, and dry acetone (50 ml).

Purification by column chromatography (eluent: CH 2

CI

2 /MeOH: 9/1) afforded (9) (190 mg, 21%) as red crystals.

Example 5: Preparation of 6-methoxv-3-benzofuranone Chloroacetonitrile (3.5 ml, 55.2 mmol) was added dropwise to a stirred solution containing 3-methoxyphenol (5 ml, 46 mmol) and zinc chloride (6.9 g, 50.6 mmol) in anhydrous dioxane (30 ml) at room temperature. The resulting solution was saturated with dry hydrogen chloride gas. After stirring at room temperature overnight, the yellow precipitate was filtered and washed with anhydrous ether (100 ml). The collected precipitate was dissolved in water (80 ml) and heated to reflux for 1 hour. The solution was allowed to cool to approximately 40'C, and aqueous sodium hydroxide (20% w/v) (7.5 ml) was added. After stirring at that temperature for 30 minutes a pale yellow precipitate had formed. A heterogeneous system was then taken to pH 7 by addition of hydrochloric acid (1 The precipitate was filtered, washed with water, and recrystallized from acetone to give the desired compound as a light yellow powder (4.14 g, 'H NMR: 6 (ppm): 3.85 (OMe); WO 99/04789 PCT/US98/15388 4.9 (CH2); 6.50-6.46 (2 H-Phenyl); 7.70-7.66 (1 H-Phenyl).

Example 6: Preparation of 6 -methoxv-3-benzothiofuranone or 4-methoxy-3-benzothiofuranone Chloroacetonitrile (0.62 ml, 9.8 mmol) was added dropwise to a stirred solution containing 3-methoxythiophenol (1 ml, 8.1 mmol) and aluminum chloride (1.19 g, 8.9 mmol) in anhydrous ether (10 ml) at room temperature. The resulting solution was saturated with dry hydrogen chloride gas. After stirring at room temperature overnight, the pale yellow precipitate was filtered and washed with anhydrous ether (30 ml). The collected precipitate was dissolved in water (25 ml) and heated to reflux for 1 hour. After cooling to approximately 40 0 C, aqueous sodium hydroxide (20% w/v) (2.6 ml) was added. After stirring at that temperature for minutes the solution was then adjusted to pH 7 by the addition of hydrochloric acid (1 The resulting solution was extracted with ethyl acetate (2 X 50 ml). The combined organic extracts were washed with brine, dried over magnesium sulfate, and concentrated in vacuo to afford an orange oil. Column chromatography, using dichloromethane with 1% methanol, gave a light yellow solid (770 mg, a single compound by TLC and HPLC.

Example 7: Preparation of 4 6 -dimethoxv-3-benzofuranone Benzofuranone (3 g, 18.1 mmol) was dissolved in DMF (100 ml). To this was added Li 2

CO

3 (5.4 g, 72.4 mmol) and methyl iodide (3.5 ml, 54.3 mmol) in one portion. A nitrogen atmosphere was maintained, and the reaction was stirred for 18 hours at 70C. The mixture was filtered, and water was added to the solution. The DMF/water phase was extracted with dichloromethane (3 X 100 ml) and the organic phase was washed with a saturated NaHCO 3 solution (2 X 100 ml). After drying with MgSO 4 and concentrating the solution, solid yellow crystals formed. These were washed with cold ethanol to give a single compound by HPLC. Yield: 2.2 g WO 99/04789 PCT/US98/15388 -46- Example 8: Preparation of 4.6-dimethoxv-3-benzofuranthione This reaction was performed under nitrogen and anhydrous conditions.

4,6-Dimethoxy-3-benzofuranone (1.5 g, 6.6 mmol) was dissolved in dry toluene ml), and Lawesson's reagent (1.6 g, 4 mmol) was added. The mixture was refluxed with stirring for 18 hours. The mixture was cooled to room temperature and purified by chromatography with 1:1 ether:petroleum ether. Concentration of the fractions yielded yellow/orange crystals. The crystals were washed with the eluent and clean yellow crystals were produced. 'H NMR showed at least two compounds, probably the thioketone and the thiol. Yield: 400 mg NMR: 6(ppm): 3.86, 3.88, 3.94, 3.96 (OMe, 2 from thioketone and 2 from thiol), 6 (ppm): 4.04 6 (ppm): 6.40 6.52 (aromatic).

Example 9: 4-Methoxv-3-benzofuranone and 6-methoxv-3-benzofuranone (3) These compounds were prepared from 3-methoxyphenol (6 ml, mmol), ZnCI 2 (8.2 g, 60 mmol), and chloroacetonitrile (4.2 g, 66 mmol) in dry ether (100 ml). Purification of the crude product by column chromatography (eluent: CHC1 2 /MeOH: 99/1) gave (1.147 g, a single compound by HPLC (98% pure, recrystallized from EtOH). 'H-NMR (Acetone-d 6 400 MHz): 6( 7.45 (1 H, d); 6.64 (2 H, 4.62 2 3.95 3 H) and the more polar compound (1.48 g, HPLC (84% purity). 'H-NMR (Acetone -d 6 6(400 MHz): values corresponded to previous synthesis of(3).

Example 10: 4.6-Bismethoxymethoxvbenzofuranone To two grams (12 mmol) benzofuranone dissolved in 100 ml DMF was added 6 ml triethylamine. After adding 2.8 ml (36 mmol) methoxymethyl chloride (MOM-C1) dropwise over 15 minutes, the solution was stirred for 18 hours at room temperature. Water (100 ml) was added to quench the excess MOM-CI. The resultant mixture was extracted with 100 ml brine, dried with MgSO 4 and concentrated to give a brown oil which contained some DMF. Chromatographic WO 99/04789 PCTJLS98/15388 -47purification (3:1 ethyl acetate:hexane with 2% diisopropylethylamine) gave the di-MOM protected benzofuranone as an oil. Recrystallization from water gave the product as fine, light brown needles (2.6 g, Example 11: 4.6-Bismethoxvmethoxvbenzofuranone thioketone This reaction was performed under nitrogen and anhydrous conditions. To a solution of the product of Example 12 (0.3 g, 1.35 mmol) dissolved in 15 ml dry toluene was added Lawesson's reagent (0.36 g, 0.9 mmol). The reaction was refluxed overnight until the ketone was consumed, by TLC. Chromatographic purification (3:1 dichloromethane: petroleum ether and 2% diisopropylethylamine; or 3:1 ether:petroleum ether and 2% diisopropylethylamine) gave a slightly yellow clear oil.

TLC showed a major and minor product.

Example 12: Inhibition of Fungal Growth Each test compound was tested against nine isolates in an eight-point dose response assay ranging from 50 /g/ml to 0.39 g/ml. Aspergillus MIC's (minimum inhibitory concentrations) were scored visually after 48 and 72 hours at 37 0 C. All Candida MIC's were scored visually after a 24 hour incubation at 35 0

C.

Amphotericin B (2.5 /g/ml) and 5-flucytosine (2.0 gg/ml) were standard controls for each antifungal assay. In each case, total inhibition was observed for all assays relative to amphotericin B and 5-flucytosine. The results are shown below in Table 4, MIC values in btg/ml after 72 hours. HFF toxicity was analyzed after a 24 hour incubation at 37°C CO2 MTS/PMS was added, and the sample absorbance was read at 450 nm.

WO 99/04789 WO 9904789PCTIUS98/15388 TABLE 4 Minimum Inhibitory Concentrations Pathogen a A. fumigatis ATCC8001 (XI) a A. fumigatus ATCC8001 (X2) A. fumigatus 94-2766 CA. niger C albicans ATCC9002 8 C. tropicalis ATCC750 C krusei ATCC625 8 d C. glabrata (Fluconazole resistant) C parapsilosis ATCC900I18 S02 12.5 S12 12.5 S17 6.25 6.25 (>50) 6.25 (>50) 6.25 (50) 6.25 (50) >50 50 (50) 12.5 12.5 6.25 0.39 6.25 12.5 >50 6.25 0.39 6.25 6.25 6.25 12.5 3.125 6.25 0.39 12.5 a Reference strain from Chrisope Technologies b Clinical isolate from J. R. Graybill c Clinical isolate from A. Sugar d Clinical isolate from M. Rinaldi Scheme P- I shows several compounds of the invention.

WO 99/04789 -9 Scheme P-1 OH 0

OH

HH

OH 9- H PCTIUS98/ 15388 S02 6 S06 WO 99/04789 PCT/US98/15388 Scheme P- I

HO'

S08 OMe SlO

OCH

3 S12 WO 99/04789 PCT[US98/15388 -51- Scheme P- I 0

H

_0 HOJ S14

OCH

3 S16 S17 S18 1 S19 04/06 2002 10:15 FAX 61 3 92438333 GRIFFITH HACK [M009 52 All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

Other Embodiments From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word ."comprises" has a corresponding meaning.

What is claimed is: S.:"4 if\ruzsfl~t'KOSVP8peCi\8SS7699S.1 SPECI.doc 3/06/02

Claims (29)

1. A method for treating a microbial infection, said method comprising administering to a patient a pharmaceutical composition containing a compound of formula (IA): Y R R R (IA) wherein each R is independently H, OH, Br, Cl, I, amino, thiol, nitro, C,_ 4 alkoxy, C 4 alkenyloxy, C2- alkoxyalkyleneoxy, C. 4 alkylthio, C 3 .18 alkyl, or C 3 alkenyl; or two adjacent Rs, taken together, are a C 2 1 8 bivalent moiety containing at least one oxgen atom, substituted or disubstituted with A or B, or both, A being H, OH, Br, Cl, I, amino, or thiol, and B being H, C,-o 1 alkyl, C2 18alkenyl, or C 6 .g aryl; provided that at least two Rs are not H; further provided that when each of two Rs is one of OH, C,4 alkoxy, C1. 4 alkenyloxy, or C 2 6 alkoxyalkyleneoxy, and X is phenyl substituted with two substituents independently selected from OH, alkoxy, and alkenyloxy, the remaining R cannot be prenyl; further provided that when each of two Rs is one of OH, C. 4 alkoxy, C. 4 alkenyloxy, or C2 6 alkoxyalkyleneoxy, and X is phenyl substituted with three substituents independently selected from OH, alkoxy, and alkenyloxy, the remaining R cannot be prenyl; further provided that when each of two Rs is one of OH, C 4 alkoxy, C.1 4 alkenyloxy, or C 2 6 alkoxyalkyleneoxy, and X is phenyl substituted with a prenyl substituent and with two additional substituents independently selected from OH, alkoxy, and alkenyloxy, the remaining R cannot be H or OH; further provided that when each of two Rs is one of OH, C,. 4 alkoxy, C,. 4 alkenyloxy, or C. 6 alkoxyalkyleneoxy, and X is phenyl substituted with a substituent containing three rings and with two additional substituents independently selected WO 99/04789 WO 9904789PCTIUS98/15388 -54- from OH, alkoxy, and alkenyloxy, the remaining R cannot be prenyl; X is C 4 1 0 alkyl, C 4 20 alkenyl, or a C 420 single, C 6 20 bridged, or C 6 20 fused ring moiety containing cycloalkyl, cycloalkenyl, aryl, heterocycle, or heteroaryl, wherein X is substituted with H, OH, Cl, Br, 1, amino, cyano, nitro, alkyl, alkoxy, alkenyl, or alkenyloxy; provided that if X is a heteroaryl or heterocyclic moiety where two Rs are each OH and meta to each other, then the remaining R is H and ortho to each of the two hydroxyls, Y and Z are each 0, and a ring atom of X is linked directly to the Sp 2 carbon atom adjacent to X, and substituted with H, OH, Cl, Br, 1, amino, cyano, alkyl, alkoxy, alkenyl, or alkenyloxy; and each of Y and Z is independently selected from 0, S, and NH; or a pharmaceutically acceptable salt or ester thereof.

2. The method of claim 1, wherein X is selected from benzyl, -dimethoxyphenyl, 2,3-dimethyl-4-methoxyphenyl,

3-benzyloxyphenyl, 3-phenoxyphenyl, 4-benzyloxy-3-methoxyphenyl, 4-[3-propenoic acid]-phenyl, 2-ethoxy- I -naphthyl, I -(methylthio)ethyl, DL- I -phenylethyl, 4-n-pentyloxyphenyl, 1 -(phenylsulfonyl)-2-pyrrolyl, 4-(3-dimethylaminopropoxy)phenyl, 3-phenylpropyl, 2,4-diethoxy-m-tolulyl, 2,6,6-trimethylcyclohexene- 1 -methyl, -dimethoxy-3-tetrahydrofuranyl,

4-methyl-5-imidazolyl, 4-n-pentylphenyl, 2-benzyloxy-4,5 -dimethoxyphenyl, 1 -pyrenyl, 3,5 -dibenzyloxy-3-methoxyphenyl, 3-methyl-4-methoxyphenyl, 4-n-decyloxyphenyl, 2,4-dimethoxy-3-methylphenyl, t-butyl, 3-(4-t-butylphenoxy)phenyl, 2-n-hexyloxyphenyl, 2-(4-chlorophenylthio)phenyl, cyclopropyl, 2,6-dimethoxy-4-hydroxyphenyl, 4-benzyloxyphenyl, 2-benzyloxyphenyl, 8-hydroxy -1,1 ,7,7-tetramethylj ulolidin-9-yl, 2,3 ,6,7-tetrahydro-8-hydroxyjulolidin-9-yl, 2-methoxymethyl-l1-pyrrolidinyl,

5-(2-nitrophenyl)fuiranyl, 1,1 -dimethyl-2-hydroxyethyl, 5-(3-chlorophenyl)furanyl, 2,4-hexadienyl, 5-[3(trifluoromethyl)phenylfuranyl], 4,5-dimethyl-4-pentenyl, imidazolyl, ferrocenyl, 2,6-dimethylhept-5-enyl, 5-[2-(trifluoromethyl)phenyl]fiiranyl, 5-(hydroxy-2-nitromethyl)furanyl, 04/06 2002 10:15 FAX 61 3 92438333 GRIFFITH HACK ao010 55 2,4-dimethyl-2,6-heptadienyl, 1-phenylethyl, 5-(2- chorophenyl)furanyl, benzyl, 5-ethyl-2-furanyl, 5-(4- nitrophenyl)furanyl, pentamethylphenyl, 1- (methyldithio)isopropyl, 4-trifluoromethylphenyl, 3- fluoro-4-methoxyphenyl, or the X of a compound of Schemes X-1 through X-10, wherein the compounds of Schemes x-1 through X-10 have the formulae X-CHO. 3. The method of claim 1 or claim 2, wherein said compound is selected from formulae (III), and (IV): Y W' Y X X 15 x I II Rb y Ra X Rc X w z w z III IV S. 25 wherein V is bivalent C2-1i moiety containing at least one oxygen atom and substituted with A, B, or both; each of W and W' is independently selected from the values for A, cyano, nitro, C0-d alkoxy, C1-4 alkenyloxy, C2-6 alkyloxyalkyleneoxy, C2-7 carboxyalkyloxy, C7-15 arylalkoxy, and C 1 4 alkylthio; Ra is H, C3-ia alkyl, C3-18 alkenyl, C5-18 cyclohexenyl, or C6-18 aryl; and each of Rb and Re is independently selected from H and C1- 4 alkyl. 4. The method of any one of claims 1 to 3, wherein H:\nuClanet\Kiap\Speci\B5876-9t.1 SPECI.doc 3/Oti02 04/06 2002 10:15 FAX 61 3 92438333 GRIFFITH HACK [aOil 56 said compound is of the formu selected from the Qs of the cc through Q-11, wherein the coml Q-1 have the formula Q-H 2 The method of any o said compound has an ICso of 1 millilitre against at least oi Candida or Aspergillus.

6. The method of any oi V contains between 1 and 3 ril

7. The method of any o] 15 V is selected from the follow: .a Q=(CHX), wherein Q is mpounds of Schemes Q-1 )ounds of Schemes Q-1 through ie of claims 1 to 4, wherein ess than 50 micrograms per 1e pathogenic strain of ie of igs. claims 3 to 5, wherein t. ooc- A BT ie of claims 3 to 6, wherein .ng formulae: O0 xe of claims 3 to 5, wherein -ng formulae:

8. The method of any o V is selected from the follow A sq

9. The method of any o Ra is selected from H, prop-2- 2-enyl, but-2-enyl, 3-methylb 2,6-dienyl, (cyclohexenyl)met 2,6,10-trienyl, and benzyl. The method of any o said compound is of formula ie of claims 3 to 8, wherein enyl, cinnamyl, 2-methylprop- it-2-enyl, 3,7-dimethylocta- lyl, 3,7,11-trimethyldodeca- ie of claims 3 to 9, wherein and each of Y an Z is iI:\Su.anOt\Ko \p peci\5866-98.l SCPECI.dLc 3/06/02 04/06 2002 10:15 FAX 61 3 92438333 GRIFFITH HACK M012 57 independently selected from O aId S.

11. The method of any one of claims 1 to 10, wherein said compound is selected from S01, 502, S03, 504, S06, S08, SO9, 810, S11, 512, S13, S14, 515, 516, S17, S18, and S19.

12. The method of any one of claims 3 to 11, wherein each of W and W' is independently selected from H, OH, methoxy, methoxymethyleneoxy, and carboxymethoxy.

13. The method of claim 12, wherein Y and Z are 0, and at least one of W and W' is OH. 15 14. The method of any one of claims 1 to 13, wherein *5*q X is a heterocyclic or heteroaryl moiety- The method of any one of claims 1 to 13, wherein X is selected from C4-10 alkyl, C4-20 alkenyl, and a C4-20 single, C6- 20 bridged, or C6-20 fused ring moiety containing cycloalkyl, cycloalkenyl, or aryl.

16. The method of claim 15, wherein X is a nonaromatic moiety containing cycloalkyl, cycloalkenyl, 25 alkyl, or alkenyl.

17. The method of any one of claims 1 to 16, wherein said compound is selected from S17 and 519.

18. The method of claim 15, wherein X is an aryl moiety.

19. The method of any one of claims 1 to 18, wherein said compound is selected from S12 and S02. The method of any one of claims 1 to 19, wherein said microbial infection is a fungal infection. H:\u'nnue2t\Keep\Speci\85676-98.1 fSPECI..oc 3/06/02 04/06 2002 10:16 FAX 61 3 92438333 GRIFFITH HACK @1013 58

21. The method of claim 20, wherein said fungal infection is an infection of a Candida species.

22. The method of claim 20, wherein said fungal infection is an infection of a fungus resistant to at least one azole antifungal agent.

23. The method of claim 22, wherein said azole antifungal agent is fluconazole.

24. The method of claim 20, wherein said fungal infection is an infection of an Aspergillus species.

25. The method of claim 20, wherein said fungal infection is selected from an infection due to C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, A. fumigatus, or A. niger.

26. The method of any one of claims 1 to 19, wherein said microbial infection is a bacterial infection.

27. A compound selected from formulae (IV) below: 25 Y X X V1 w z z I II Y Rb y Ra X R X W W III IV wherein: H:\%usannet\cKeep\Sp.i\SB76-9B.1 BPECI.doc 3/06/02 04/06 2002 10:16 FAX 61 3 92438333 GRIFFITH HACK [a014 59 V is a bivalent C2-18 moiety containing at least one oxygen atom and substituted with A, B, or both wherein A and B are as defined in claim 1; each of W and W' is independently selected from the values for A, cyano, nitro, 01-4 alkoxy, C1-4 alkenyloxy, C2..6 alkyloxyalkyleneoxy, C2-7 carboxyalkyloxy, C7-15 arylalkoxy, and C1-4 alkyithio; Ra is H, C 3 18 alkyl, C3-16 alkenyl, Cs-18 cyclohexenyl, or Ce-le aryl; each of Rb and R, is independently selected from H and C1-.4 alkyl; X is substituted or unsubstituted C3-15s alkyl, C3. ,a alkenyl, C3-15 cycloalkyl, C4-15 cycloalkenyl, C4-20 .bicyclo[a.b.c]alkyl, CS-20 bicyclo[a.b.clalkenyl, tricyclo[a.b.c.d]alkyl, C8- 20 tricycloalkenyl, C2-20 heterobicyclo[a.b.c]alkyl, or a combination thereof, S.wherein each of a, b, c, and d is independently 0 to and each of Y and Z is independently selected from O and S.

28. The compound of claim 27, wherein X is C 3 -1 alkyl, C3-le alkenyl, C3-15 cycloalkyl, C4-15 cycloalkenyl, Cs- bicyclo[a.b.c]alkyl, Cs-10 bicyclo[a.b.c]lalkenyl, tricyclo[a.b.c.d]alkyl, Ce- 20 tricycloalkenyl, C3-10 heterobicyclo[a.b.c]alkyl, or a combination thereof, wherein each of a, b, c, and d is independently 0 to 6. S29. The compound of claim 27 or claim 28, wherein X is C3-15 alkyl, C 3 -1.1 alkenyl, C3- 1 5 s cycloalkyl, or C4-15 cycloalkenyl. The compound of claim 27 or claim 28, wherein X is C5-10o bicyclo[a.b.c]alkyl, Cs-10 bicyclo[a.b.c]alkenyl, C8- is tricyclo[a.b.c.d]alkyl, CE-15s tricycloalkenyl, or C3-10 heterobicyclo[a.b.cJalkyl, or a combination thereof. :\su:annetC\Keep\$pecL\8S976-9a.1 BPECI.doc 3/06/02 04/06 2002 10:16 FAX 61 3 92438333 GRIFFITH HACK 0015 60

31. The compound of any one of claims 27 to wherein each of W and W' is independently selected from H, hydroxyl, methoxy, hydroxymethyl, and halomethyl.

32. The compound of claim 31, wherein W and W' are both hydroxyl.

33. Use of a compound of the formula (IA) as defined in claim 1 for the manufacture of a medicament for treating a microbial infection.

34. A pharmaceutical composition comprising an effective amount of a compound of formulae (IV) as defined in any one of claims 27 to 32 together with a pharmaceutically acceptable carrier. Methods for treating a microbial infection, See substantially as hereinbefore described with reference to the examples.

36. Compounds of formulae processes for their preparation or pharmaceutical compositions containing them, substantially as hereinbefore described with reference to the examples. Dated this 3rd day of June 2002 PHYTERA INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\2u.anne \Kee\Speci\65876-98,1 $ilCI.doc 3/06/02