h and 13c nmr spectra were recorded at ambient temperature in cdcl3 and dmso on a bruker amc instrument operating at 300.12 and 75.47 mhz , respectively or a bruker drx instrument operating at 500 , respectively 125 mhz . si gel for chromatography was silica gel 60 ( 0.20.5 mm merck ) for analytical work and for preparative work tlc silica gel 60 gf 254 merck . haliclona cymaeformis ( esper , 1794 ) order haplosclerida , family chalinidae , was collected by scuba diving in the gulf of thailand near koh chang ( 2 meter depth ) , trad province in march 2004 . haliclona baeri ( wilson , 1952 ) was also collected by scuba diving in the in the coastal area of chonburi province ( 3 meter depth ) . sumaitt putchakarn and the vouchers of the specimens , registered as bims - i 953 and bims - i 1766 , respectively were deposited at bangsaen institute of marine science ( bims ) , burapha university , bangsaen , chonburi , thailand . the materials were frozen immediately at 20c prior to extraction . the sample ( 1.3 kg net weight ) was thawed , homogenized with etoh ( 3 l ) , allowed to stand for 24 h in a dark chamber and filtered . the residue on the filter paper was again extracted with etoh ( 2  3l ) , the aqueous alcoholic extracts were combined , evaporated at reduced pressure to ca . the etoac extracts were combined and concentrated at reduced pressure to give the crude etoac extract . this ( 10 g ) was chromatographed over a si gel column ( 120 g ) and eluted with petrol - chcl3 and chcl3-me2o , 350 ml fractions being eluted as follows . frs 162 ( petrol - chcl3 , 1:1 ) , 6378 ( petrol - chcl3 , 3:7 ) , 7995 ( petrol - chcl3 , 1:9 ) , 96182 ( chcl3-me2o , 4:1 ) , 183206 ( chcl3- me2o , 3:2 ) . frs 101114 were combined ( 107.8 mg ) and purified by tlc ( si gel , chcl3-me2o frs 125198 were combined ( 185.6 mg ) and purified by tlc ( si gel , chcl3-meoh - hco2h , 93:7:1 ) to give 13.1 mg 3,4-dihydroxybenzoic acid ( 2 ) and 8.0 mg of tetillapyrone ( 5and 6 ) . the sample of haliclona cymaeformis ( 7.6 kg net weight ) was thawed , homogenized with etoh ( 10l ) , allowed to stand for 24 h in the dark chamber and filtered . the residue on the filter paper was again extracted with etoh ( 2  10l ) , the aqueous alcoholic extracts were combined , evaporated at reduced pressure to give ca . the etoac extracts were combined and concentrated at reduced pressure to give a crude etoac extract ( 21 g ) . this extract was applied to flash chromatography column of si gel ( 150 g ) and eluted with chcl3 ( frs . 15 ; 5  200 ml ) , chcl3-meoh , , 49:1 ( frs 615 ; 10  200 ml ) , chcl3-meoh , , 19:1 ( frs1620 ; 5  200 ml ) , chcl3-meoh , , 9:1 ( frs 2122 ; 2  500 ml ) , etoac ( frs 2324 ; 2  500ml ) and meoh ( frs 2526 ; 2  500ml ) . frs 1824 were combined ( 3.1 g ) , applied on the flash chromatography column of ods - a gel ( 35 g ) and eluted with mixture of meoh - h2o ; 7:3 ( sfr 1 ; 200ml ) , meoh - h2o ; 4:1 ( sfr 2 ; 200ml ) , meoh - h2o ; 9:1 ( sfr 3 ; 2  100ml ) , meoh ( sfr 4 ; 400ml ) , chcl3-meoh - h2o ; 14:6:1 ( sfr 5 , 400ml ) . sfr 1 ( 156 mg ) was purified by tlc si gel , chcl3-meoh - hco2h , 19:1:0.1 ) to give 3.5 mg of p - hydroxybenzaldehyde ( 3 ) and 15 mg of tetillapyrone ( 5 and 6 ) . sfr 5 ( 103 mg ) was purified by tlc ( si gel , chcl3-meoh - hco2h , 19:1:0.1 ) to give 7 mg of phenylacetic acid ( 4 ) and 17 mg of nortetillapyrone ( 7 and 8) . maleimide-5-oxime ( 1 ) : yellow viscous mass ; ei hrms 122.02723 ( m),cald for c4h4n2o2 , 122.02728.hnmr ( 300 mhz , dmso ) : 11.03 brs ( nh ) , 10.86 brs ( = n - oh ) , 7.40d ( j=7.6 hz , h-4 ) , 5.45d ( j=7.6 hz , h-3 ) , cnmr ( 75.47 mhz , dmso ) : 164.37 ( co-2 ) , 151.54 ( c-5 ) , 142.22 ( ch-4 ) , 100.24 ( ch-3 ) . the effect of compounds on the growth of human tumor cell lines were evaluated according to the procedure adopted by national cancer institute ( nci , usa ) for the in vitro anticancer drug discovery screen which uses the protein - binding dye sulforhodamine b ( srb ) to assess cell growth inhibition [ 13 , 14 ] and according to the method described by kijjoa et al . . antifungal activity of maleimide-5-oxime ( 1 ) , p - hydroxybenzaldehyde ( 3 ) , phenylacetic acid ( 4 ) , tetillapyrone ( 5 and 6 ) and nortetillapyrone ( 7 and 8) was evaluated against candida , aspergillus and dermatophyte strains : three yeasts clinical strains , one of cryptococcus neoformans ph1 , one of candida dubliniensis ph10 and one of c. glabrata d10r , as well as , four american type culture collection ( atcc ) type strains ( c. albicans attc 10231 , c. tropicalis atcc 13803 , c. krusei atcc 6258 and c. parapsilosis atcc 90018 ) ; one aspergillus clinical strain ( a. flavus f44 ) , as well as , two atcc type strains ( a. niger atcc 16404 , and a. fumigatus atcc 46645 ) ; five dermatophyte clinical strains ( microsporum canis ff1 , m. gypseum ff3 , trichophyton rubrum ff5 , t. mentagrophytes ff7 and epidermophyton floccosum ff9 ) . the fungal isolates were identified by standard microbiology methods and stored on sabouraud dextrose broth with glycerol at 70c and subcultured in sabouraud - chloramphenicol agar . minimal inhibitory concentration ( mic ) was determined by a microdilution method according to the clsi reference method m27-a2 for yeasts , and m38-a for filamentous fungi . the test was performed in sterile 96 flat - bottom well microtitration plates and the medium used was mops - buffered rpmi 1640 . briefly , mics were determined for all yeasts strains with the adjusted final inoculum of 14  10 cfu / ml , prepared from a 2448 hours culture . with filamentous fungi ( except for epidermophyton floccosum ) , the final inoculum used was 48  10cfu / ml , prepared from a sporulated culture . for epidermophyton floccosum , the final inoculum was 8  10 to 1.6  10 cfu / ml . two fold serial dilutions of compounds in dmso , ranging from 250 to 15.125 g / ml , were tested . in addition , reference antifungal compounds , fluconazole ( pfizer , united kingdom ) for yeasts and dermatophytes or amphotericin b ( sigma ) for aspergillus , were used as the standard antifungal drugs . twofold serial dilutions ranging from 128 to 0.25 g / ml for fluconazole and 16 to 0.063 g / ml for amphotericin b were used . plates were incubated at 30c for 48 hours for candida species , 72 hours for cryptococcus neoformans , 45 days for aspergillus species and 8 days for dermatophytes . quality control determinations of the mic of fluconazole were ensured by testing c. parapsilosis atcc 90018 . h and 13c nmr spectra were recorded at ambient temperature in cdcl3 and dmso on a bruker amc instrument operating at 300.12 and 75.47 mhz , respectively or a bruker drx instrument operating at 500 , respectively 125 mhz . si gel for chromatography was silica gel 60 ( 0.20.5 mm merck ) for analytical work and for preparative work tlc silica gel 60 gf 254 merck . haliclona cymaeformis ( esper , 1794 ) order haplosclerida , family chalinidae , was collected by scuba diving in the gulf of thailand near koh chang ( 2 meter depth ) , trad province in march 2004 . haliclona baeri ( wilson , 1952 ) was also collected by scuba diving in the in the coastal area of chonburi province ( 3 meter depth ) . sumaitt putchakarn and the vouchers of the specimens , registered as bims - i 953 and bims - i 1766 , respectively were deposited at bangsaen institute of marine science ( bims ) , burapha university , bangsaen , chonburi , thailand . the sample ( 1.3 kg net weight ) was thawed , homogenized with etoh ( 3 l ) , allowed to stand for 24 h in a dark chamber and filtered . the residue on the filter paper was again extracted with etoh ( 2  3l ) , the aqueous alcoholic extracts were combined , evaporated at reduced pressure to ca . the etoac extracts were combined and concentrated at reduced pressure to give the crude etoac extract . this ( 10 g ) was chromatographed over a si gel column ( 120 g ) and eluted with petrol - chcl3 and chcl3-me2o , 350 ml fractions being eluted as follows . frs 162 ( petrol - chcl3 , 1:1 ) , 6378 ( petrol - chcl3 , 3:7 ) , 7995 ( petrol - chcl3 , 1:9 ) , 96182 ( chcl3-me2o , 4:1 ) , 183206 ( chcl3- me2o , 3:2 ) . frs 101114 were combined ( 107.8 mg ) and purified by tlc ( si gel , chcl3-me2o frs 125198 were combined ( 185.6 mg ) and purified by tlc ( si gel , chcl3-meoh - hco2h , 93:7:1 ) to give 13.1 mg 3,4-dihydroxybenzoic acid ( 2 ) and 8.0 mg of tetillapyrone ( 5and 6 ) . the sample of haliclona cymaeformis ( 7.6 kg net weight ) was thawed , homogenized with etoh ( 10l ) , allowed to stand for 24 h in the dark chamber and filtered . the residue on the filter paper was again extracted with etoh ( 2  10l ) , the aqueous alcoholic extracts were combined , evaporated at reduced pressure to give ca . the etoac extracts were combined and concentrated at reduced pressure to give a crude etoac extract ( 21 g ) . this extract was applied to flash chromatography column of si gel ( 150 g ) and eluted with chcl3 ( frs . 15 ; 5  200 ml ) , chcl3-meoh , , 49:1 ( frs 615 ; 10  200 ml ) , chcl3-meoh , , 19:1 ( frs1620 ; 5  200 ml ) , chcl3-meoh , , 9:1 ( frs 2122 ; 2  500 ml ) , etoac ( frs 2324 ; 2  500ml ) and meoh ( frs 2526 ; 2  500ml ) . frs 1824 were combined ( 3.1 g ) , applied on the flash chromatography column of ods - a gel ( 35 g ) and eluted with mixture of meoh - h2o ; 7:3 ( sfr 1 ; 200ml ) , meoh - h2o ; 4:1 ( sfr 2 ; 200ml ) , meoh - h2o ; 9:1 ( sfr 3 ; 2  100ml ) , meoh ( sfr 4 ; 400ml ) , chcl3-meoh - h2o ; 14:6:1 ( sfr 5 , 400ml ) . sfr 1 ( 156 mg ) was purified by tlc si gel , chcl3-meoh - hco2h , 19:1:0.1 ) to give 3.5 mg of p - hydroxybenzaldehyde ( 3 ) and 15 mg of tetillapyrone ( 5 and 6 ) . sfr 5 ( 103 mg ) was purified by tlc ( si gel , chcl3-meoh - hco2h , 19:1:0.1 ) to give 7 mg of phenylacetic acid ( 4 ) and 17 mg of nortetillapyrone ( 7 and 8) . maleimide-5-oxime ( 1 ) : yellow viscous mass ; ei hrms 122.02723 ( m),cald for c4h4n2o2 , 122.02728.hnmr ( 300 mhz , dmso ) : 11.03 brs ( nh ) , 10.86 brs ( = n - oh ) , 7.40d ( j=7.6 hz , h-4 ) , 5.45d ( j=7.6 hz , h-3 ) , cnmr ( 75.47 mhz , dmso ) : 164.37 ( co-2 ) , the effect of compounds on the growth of human tumor cell lines were evaluated according to the procedure adopted by national cancer institute ( nci , usa ) for the in vitro anticancer drug discovery screen which uses the protein - binding dye sulforhodamine b ( srb ) to assess cell growth inhibition [ 13 , 14 ] and according to the method described by kijjoa et al . . antifungal activity of maleimide-5-oxime ( 1 ) , p - hydroxybenzaldehyde ( 3 ) , phenylacetic acid ( 4 ) , tetillapyrone ( 5 and 6 ) and nortetillapyrone ( 7 and 8) was evaluated against candida , aspergillus and dermatophyte strains : three yeasts clinical strains , one of cryptococcus neoformans ph1 , one of candida dubliniensis ph10 and one of c. glabrata d10r , as well as , four american type culture collection ( atcc ) type strains ( c. albicans attc 10231 , c. tropicalis atcc 13803 , c. krusei atcc 6258 and c. parapsilosis atcc 90018 ) ; one aspergillus clinical strain ( a. flavus f44 ) , as well as , two atcc type strains ( a. niger atcc 16404 , and a. fumigatus atcc 46645 ) ; five dermatophyte clinical strains ( microsporum canis ff1 , m. gypseum ff3 , trichophyton rubrum ff5 , t. mentagrophytes ff7 and epidermophyton floccosum ff9 ) . the fungal isolates were identified by standard microbiology methods and stored on sabouraud dextrose broth with glycerol at 70c and subcultured in sabouraud - chloramphenicol agar . minimal inhibitory concentration ( mic ) was determined by a microdilution method according to the clsi reference method m27-a2 for yeasts , and m38-a for filamentous fungi . the test was performed in sterile 96 flat - bottom well microtitration plates and the medium used was mops - buffered rpmi 1640 . briefly , mics were determined for all yeasts strains with the adjusted final inoculum of 14  10 cfu / ml , prepared from a 2448 hours culture . with filamentous fungi ( except for epidermophyton floccosum ) , the final inoculum used was 48  10cfu / ml , prepared from a sporulated culture . for epidermophyton floccosum , two fold serial dilutions of compounds in dmso , ranging from 250 to 15.125 g / ml , were tested . in addition , reference antifungal compounds , fluconazole ( pfizer , united kingdom ) for yeasts and dermatophytes or amphotericin b ( sigma ) for aspergillus , were used as the standard antifungal drugs . twofold serial dilutions ranging from 128 to 0.25 g / ml for fluconazole and 16 to 0.063 g / ml for amphotericin b were used . plates were incubated at 30c for 48 hours for candida species , 72 hours for cryptococcus neoformans , 45 days for aspergillus species and 8 days for dermatophytes . quality control determinations of the mic of fluconazole were ensured by testing c. parapsilosis atcc 90018 . the effect of compounds on the growth of human tumor cell lines were evaluated according to the procedure adopted by national cancer institute ( nci , usa ) for the in vitro anticancer drug discovery screen which uses the protein - binding dye sulforhodamine b ( srb ) to assess cell growth inhibition [ 13 , 14 ] and according to the method described by kijjoa et al . . antifungal activity of maleimide-5-oxime ( 1 ) , p - hydroxybenzaldehyde ( 3 ) , phenylacetic acid ( 4 ) , tetillapyrone ( 5 and 6 ) and nortetillapyrone ( 7 and 8) was evaluated against candida , aspergillus and dermatophyte strains : three yeasts clinical strains , one of cryptococcus neoformans ph1 , one of candida dubliniensis ph10 and one of c. glabrata d10r , as well as , four american type culture collection ( atcc ) type strains ( c. albicans attc 10231 , c. tropicalis atcc 13803 , c. krusei atcc 6258 and c. parapsilosis atcc 90018 ) ; one aspergillus clinical strain ( a. flavus f44 ) , as well as , two atcc type strains ( a. niger atcc 16404 , and a. fumigatus atcc 46645 ) ; five dermatophyte clinical strains ( microsporum canis ff1 , m. gypseum ff3 , trichophyton rubrum ff5 , t. mentagrophytes ff7 and epidermophyton floccosum ff9 ) . the fungal isolates were identified by standard microbiology methods and stored on sabouraud dextrose broth with glycerol at 70c and subcultured in sabouraud - chloramphenicol agar . minimal inhibitory concentration ( mic ) was determined by a microdilution method according to the clsi reference method m27-a2 for yeasts , and m38-a for filamentous fungi . the test was performed in sterile 96 flat - bottom well microtitration plates and the medium used was mops - buffered rpmi 1640 . briefly , mics were determined for all yeasts strains with the adjusted final inoculum of 14  10 cfu / ml , prepared from a 2448 hours culture . with filamentous fungi ( except for epidermophyton floccosum ) , the final inoculum used was 48  10cfu / ml , prepared from a sporulated culture . for epidermophyton floccosum , two fold serial dilutions of compounds in dmso , ranging from 250 to 15.125 g / ml , were tested . in addition , reference antifungal compounds , fluconazole ( pfizer , united kingdom ) for yeasts and dermatophytes or amphotericin b ( sigma ) for aspergillus , were used as the standard antifungal drugs . twofold serial dilutions ranging from 128 to 0.25 g / ml for fluconazole and 16 to 0.063 g / ml for amphotericin b were used . plates were incubated at 30c for 48 hours for candida species , 72 hours for cryptococcus neoformans , 45 days for aspergillus species and 8 days for dermatophytes . quality control determinations of the mic of fluconazole were ensured by testing c. parapsilosis atcc 90018 .