Naphthalimido derivatives as antifolate thymidylate synthase inhibitors

1011

Eur J Med Che,t 11996) 31, 1011-1016 © Elsevier, Paris

Naphthalimido

Short communication

d e r i v a t i v e s as a n t i f o l a t e t h y m i d y l a t e

synthase inhibitors

MP Costil*, D Tondil, M Rinaldi~, D Barlocc02*, G Cignarella 2, DV SantP. C Musiua, I Pudu 4, G Vacca 4, P La Colla 4 II)il~arttmento Science Farma~eutiche. Vta Campi 183. 41100 Modena: 2L~'tituto Chtmwa Farma~ eutk a e Tosstcologica, Viale Abruzzi 42. 20131 Milan, ltah: 3Department o[ Biochemt,st O' and Bu)ph3stcs, Universttv o[- Californta, San Francis~ o, CA 9£t143, USA; 4Dipartimento Biologia Stwrmwntale, Sez Microbiologia, Vtale Regtna Margherita 45, 09124 Cagliari, Italy (Recmved 22 February 1996: accepted 24 June 1996)

S u m m a r y - - A new series of N-(subsntuted)benzyl-1,8-naphthahmides 4, structurally related to the prevtously reported thymidylate synthase (TS) inhibitor naphthalems 3, were synthesized and compounds tested for their inhibition o f several specms of TS. Moreover, their in v~tro cvtotoxicitv together with antimycotic and antibacterial properties were assayed. While no activity was detected in the antibacterlal tests, the m-nitro ~4ae) and the p-nitro (4af) derivatives were found able to partially inhibit TS at low micromolar concentrations. Introduction of nitro or (substitutedI-ammo groups in p o s m o n 4 of the naphthahc ring always led to le~s active compounds

thymidylate synthase / inhibition / naphthalimido derivatives

Introduction

Thymidylate synthase (TS) is an interesting enzyme target in cancer chemotherapy since its inhibition blocks the de novo synthesis of 2'-deoxythymidine-5'monophosphate (dTMP): DNA is no longer synthesized and the cell replication stops I1]. A double substrate enzyme can be inhibited by compounds competing with either 2'-deoxyuridine-5'-monophosphate {dUMP) or a cofactor function. The antifolates are a very interesting tool because of their potential high structural specificity. In fact, selected modifications in the basic pteridinic ring give compounds with either antimicrobial or antitumor action [2]. TSinhibiting agents are usually structurally related to the cofactor and follow a similar metabolic pathway. However, though favouring their accumulation in the cells and their affinity towards TS binding sites, this similarity is also the source of resistance for the best known folate analogues such as CB3717 [3]. Recently, some compounds non-structurally related to folate but with good affinity towards folate binding site of TS have been synthesized with the aim of overcoining the above-mentioned problems [4, 5].

We have previously described several classes of phthalido derivatives (1-3) provided with general competitive inhibition pattern and apparent inhibition constant in the low micromolar range against Lactobacillus casei TS ILcTS}. While their activity was reported to slightly increase passing from 1 to 3, the specific interactions with the enzyme are common

"

O

-

O

l

0... ~0~ / ~

3

For I, 2, 3

*Correspondence and reprints

~

Rj ~ R2 - (substnuled)phenol RI = OH R2 - (substituted)phenol

2

O

R I

4

For 4 R = (substltuted)benzyl

1012 to these ligands and involve the two phenolic hydroxy groups and the lactonic carbonyl [6, 7]. However, it should be noted that some derivatives with only one phenolic ring still retain LcTS inhibitory activity (Costi, unpublished results). On this basis, to further assess the real importance of these requirements, we have now modified the lactonic moiety of structure 3 to the imidic derivatives 4. Besides giving more information on the essential requirements for these TS inhibitors, this substitution should increase their stability with respect to the models 1-3 [8, 9]. This paper describes the synthesis of compounds 4 and their ability to inhibit different species" TS. Moreover, several in vitro assays have been performed; cytotoxicity has been evaluated on MT-4 and C8166 cells together with their capability to stop the cell multiplication of various fungi and bacteria.

H

for R--NI~

NttCORa ,I

4

R=H, NH2

¢

CI

a}

~o-~-~,

CICI"Fz--~

RI / Nail /

DMF

b) (RzCO)20/pvndm¢/60 °C c)

C I ~ O C 1

,Nail/DMF

CI

5

Chemistry Compounds 4 were prepared following two alternatives pathways depicted in schemes 1 and 2. According to Method a (scheme 1), the appropriate 1,8-naphthalimide was condensed with the required benzyl chloride in the presence of sodium hydride, using DMF as the solvent. The desired 4 were isolated by suction after pouring the mixture onto ice/water. The acyl-substimted derivatives were easily obtained by condensing the corresponding amino substituted with acetic or butyric anhydride in pyridine at 60 °C. According to Method b (scheme 2), a solution of 4-nitro-l,8-naphthalic anhydride was refluxed overnight with the required benzylamino derivative in ethanol. All compounds were finally purified by trimration with hot ethanol or by silica-gel chromatography, eluting with dichloromethane/methanol (see table I for data).

4

R

Rx

4aa 4ab 4ac 4ad

H

H

H H

4ae 4af 4ag

H H H NH2

3-OCH3 4-OCH3 4-F 3-NO2 4-N02 3-NO:,4-C1 H

4ba

H

R2

The compounds were tested for their inhibitory activity against LcTS. The most interesting derivatives were also screened against Cryptococcus neoformans TS (CnTS) and human TS. The inhibition assays were performed spectrophotometrically by standard methods (see table II). Moreover, they were evaluated in vitro for cytotoxicity against MT-4 and C8166 cell lines. Finally, the capability of compounds to inhibit the multiplication of various human pathogenic fungi (such as Candida albicans, C parapsilosis, C paratropicalis, Trycophyton mentagrophytes, Microsporum canis, and Aspergillus fumigatus) and bacteria (Staphylococcus aureus, group D Streptococcus,

R

Rt

112

4bb. 4b¢ 4bd 4be

NFIz

3-OCH3 4-OCH3 4-F 3-NO2 4-NO2 3-NO2 4-NO2 4-NO2

CH3 CH3 (CH2)2CH~

4bf

NH2 N]-I2 NIt2 ,'NH~

4de 4dr 4ef

Scheme 1. Method a.

cH2 +

Biological assays

4

H2N--CH2--~

R~

NO2

NO2 Rh=H 4ca =NO2 4cf a) EtOH 1 70 °C / 5h

Scheme 2. Method b.

Pseudomonas aeruginosa and Escherichia coli) was tested. Econazole and streptomycin were used as reference compounds for antimycotic and antibacterial assays, respectively.

1013 Table I. Physicochemical properties of compounds 4.

Compound

R

RI

Yield (%) Mp (° C)

Formula

~H-NMR 8(ppm)

4aa

H

H

72

195-197

C19HI3NO2 5.4 (s, 2H), 7.3-7.4 (m, 5H), 7.9 (t, 2H, J = 7.7 Hz), 8.5 (dd, 4H, J = 7.3 and 8.2 Hz)

4ab

H

3-OCH~

70

146-147

C2oHjsNO3

3.8 (s, 3HI, 5.3 Is, 2H), 6.8-6.9 (m, 1H), 7.1-7.2 (m, 3H), 7.7 (t, 2H, J = 7.8 Hz), 8.2 (d, 2H, J = 8.2 Hz), 8.6 (d, 2H, J = 7.2 Hz)

4ac

H

4-OCH~

50

185-186

C2~IHI~NO3

3.7 (s, 3H), 5.3 (s, 2H), 6.7 (d, 2H, J = 8.4 Hz), 7.3 (d, 2H, J = 8.4 Hz), 7.7 (t, 2H, J = 7.7 Hz), 8.5 (rid, 4H, J = 7.7 and 9,0 Hz)

4ad

H

4-F

55

181-183

C19HI2FNOz

5,4 (s, 2H), 7.1 (t, 2H, J = 8.7 Hz), 7.3-7.4 (m, 2H), 7,9 (t, 2H, J = 7.7 Hz), 8.5 (dd, 4H, J = 6.9 and 7.7 Hz)

4ae

H

3-NO,

53

207-209

CI9HI2N204

5.4 (s, 2H), 7.7 (dd, lH, J = 7.9 and 8.0 Hz), 7.9 (dd, 3H~ J = 7.7 and 8.0 Hz), 8.0-8.1 (m, lH), 8.3 (s, IH), 8.6 (dd, 4H, J = 7.0 and 7.9 Hz)

4af

H

4-NO.

83

268-269

CI9HI2N~O4 5.4 (s, 2H), 7.7 Id, 2H, J = 8.4 Hz), 7.9 (t, 2H, J = 7.7 Hz), 8.2 (d, 2H~ J = 8.4 Hz), 8.6-8.7 (m, 4H)

4ag

H

3-NO2,4-C1

81

186-188

CI~HIICIN204 5.4 Is, 2H), 7.5 (d, 1H, J -- 8.5 Hz), 7.7 (d, IH, J = 7.4 Hz), 7.9 (t, 2H, J = 7.8 Hz), 8.3 (d, 1H, J = 0.5 Hz), 8.5 (d, 4H, J = 7.7 Hz)

4ba

NH 2

H

60

240-241

C19H14N2O2

4bb

NH2

3-OCH~

54

214-216

C2oH16N203 3.7 (s, 3H), 5.3 (s, 2H), 6.8-7.0 (m, 4H), 7.3 (dd, 1H, J = 7.6 and 8.0 Hz), 7.5 (s, 2H, exch with D20), 7.7 (dd, IH, J = 7.6 and 8.0 Hz), 8.3 (d, 1H, J = 8.0 Hz), 8.5 (d, 1H, J = 7.0 Hz), 8.7 (d, IH, J = 8.6 Hz)

4be

NH2

4-OCH)

73

186-189

C2oHj~N203

4bd

NH~

4-F

90

>260

5.4 (s, 2H), 6.9 (d, 1H, J = 8.0 Hz), 7.2-7.3 (m, 5H), 7.4 (br s, 2H, exch with D20), 7 7 It, IH, J = 7.2 Hz), 8.3 (d, 1H, J = 8.0 Hz), 8.4 (d, 1H, J = 7.2 Hz), 8.7 (d, IH, J = 8.0 Hz)

3.7 (s, 3H), 5.3 (s, 2H), 6.7 (d, 3H, J = 8.0 Hz), 7.3 (d, 2H, J = 7.8 Hz), 7.5 (s, 2H, exch with D20), 7.7 (t, 1H, J = 6.9 Hz), 8.3 (d, 1H, J = 8.6 HzL 8.5 (d, 1H, J = 6.9 Hz), 8.7 (d, 1H, J = 8.6 Hz)

CI~Ht3FN202 5.3 (s, 2H), 6.9 (d, 1H, J = 8.6 Hz), 7.2 (t, 3H, J= 8.1 Hz), 7.3-7.4 (m, 2H), 7.5 (s, 2H, exch with D20), 7.7 (t, IH, J = 8.1 Hz), 8.3 (d, 1H, J = 8.7 Hz), 8.5 (d, 1H, J = 7 . 1 H z ) , 8.7 (d, 1H, J = 8.6 Hz)

1014 Table I. Continued. Cot,wound 4be

R

Rj

Deht(%)

M p ( ° C)

NH~

3-NO,

95

268-269

f f orti, llA[[i

IH-NMR ~ (l)pm)

CI,HI3N~O4 5.3 (s, 2H), 6.9 (d, IH, J = 8.2 Hz), 7.6-7.8 (m, 5H), 8.2-8.3 (m, 3HI, 8.5 (d, 1H, J = 7.2 Hz), 8.7 (d, IH, J = 8.2 Hz) CI~HI~N~O4

5.3 (s, 2HL 6.9 (d, 1H, J = 9.3 Hz), 7.6-7.7 (m, 5H), 8.2-8.3 (m, 3H), 8 5 (d, 1H, J = 7 3 Hz), 8.7 (d, 1H, J = 8.3 Hz)

CI,jHleN20 a

5.3 (s, 2H), 7.2-7.4 (m, 5H), 8.1 (t, IH, J = 8.1 Hz), 8.6-8.8 (m, 4HI

260-262

C~H~N)O,

5.4 Is, 2H), 7.7 ld, 2H, J = 8.1 Hz), 8.0-8.1 (m, 3H), 8.6-8.8 (m, 4H)

46

186-188

CeIH~sN~O5

2.3 (s, 3H), 5.4 (s, 2H), 7.7 (t, 1H, J = 7.7 Hz), 7 . 9 8.0 (m, 2H), 8.2 (d, 1H. J = 7.7 Hz), 8.4-8.5 (m, 2H), 8.6 (t, 2H, J = 7.7 Hz), 8.8 (d, 1H, J = 7.7 Hz), 10.5 (s, 1H, exch with D=O)

4-NO~

45

>280

C=tHIsN~O~

2.3 (s, 3H), 5.4 (s, 2HI, 7.7 (d, 2H, J = 8.6 Hz), 8.0 (t, IH, J = 8.1 Hz), 8.2 (d, 2H, J = 8.6 Hz), 8.4 (d, IH, J = 8.1 Hz), 8.7 (dd, 2H, J = 7.7 and 8.6 Hz), 8.8 ld, 1H. J = 8.6 Hz), 10.5 (s, IH, exch with D_,O)

4-NO,

86

>280

C>Hj,N~05

1.1 (t, 3H, J = 7 Hz), 1.6-1.8 (m, 2H), 2.6 (s. 2H), 5.4 (s, 2H), 7.7 Id, 2H, J = 8 Hz), 7.9 (t, 1H, J = 7 7 Hz), 8.2 (d. 2H, J = 8.0 Hz), 8.4 (d, I H, J = 7.7 Hz), 8.6 (t, 2H, J = 7.7 Hz), 8 8 (d, IH, J = 7.7 HzL 10.3 (s, IH, exch with D,O)

4bf

NH,

4-NO~

91

4ca

NO~

H

89

4cf

NO~

4-N()~

77

4de

NHCOCH 3

3-NO~

4df

NHCOCHa

4ef NHCO(CH_,):CHx

280-282

Results and discussion

TS inhibition T h e c o m p o u n d s w e r e e v a l u a t e d for t h e i r d i r e c t i n h i b i tory p r o p e r t i e s a g a i n s t L c T S . S i n c e t h e i r l o w w a t e r s o l u b i l i t y p r e v e n t e d us f r o m m e a s u r i n g d e t a i l e d s t e a d y - s t a t e k i n e t i c p a r a r n e t e r s , w e d e t e r m i n e d the p e r c e n t a g e o f i n h i b i t i o n at the h i g h e s t c o n c e n t r a t i o n c o m p a t i b l e with t h e i r s o l u b i l i t y ( t a b l e II). T h o u g h the m a j o r i t y o f the test c o m p o u n d s was f o u n d i n a c t i v e in this assay, the m e t a ( 4 a e ) a n d p a r a ( 4 a f t nitro d e r i v a t i v e s w e r e a b l e to i n h i b i t the e n z y m e b y a v a l u e o f a b o u t 3 5 % at 5 jaM. T h e p r e s e n c e o f a c h l o r i n e a t o m

in the papzl p o s i t i o n o f 4 a e ( c o m p o u n d 4 a g ) b r o u g h t a b o u t a t e n f o l d loss o f activity. M o r e o v e r , it s h o u l d be n o t e d that the i n t r o d u c t i o n o f a nitro (4el), a m i n o ( 4 b e , 4 b f ) o r a m i d o ( 4 d e , 4 d r , 4ef) g r o u p in p o s i t i o n 4 o f the n a p h t h a l i c r i n g a l w a y s l o w e r e d the i n h i b i t o r y activity, t h o u g h b y d i f f e r e n t d e g r e e s . To v e r i f y the p r e s e n c e o f a p o s s i b l e specificity, the m o s t i n t e r e s t i n g c o m p o u n d s w e r e also t e s t e d l b r their i n h i b i t o r y p r o p e r t i e s a g a i n s t o t h e r s p e c i e s o f TS, eg, C n T S a n d h u m a n TS. T h e i r p e r c e n t a g e i n h i b i t i o n s (table II) i n d i cate that no s i g n i f i c a n t s p e c i f i c i t y c o u l d b e o b s e r v e d . H o w e v e r , c o m p o u n d 4af, w h i c h s h o w s a l m o s t the same inhibitory activity against LcTS and CnTS, did not i n h i b i t h u m a n TS.

1015

Table II. TS inhibition. Compound

lnhtbition a LeTS

Human TS

CnTS

4ae

35 (5 gM)

NI (5 jaMl

4 (5 IM)

4af

33 (5 gM)

NI (5 btM)

35 (5 ,tM)

4ag

30 (50 jaM)

Nd

35 (50 ,tM)

4be

21 (50 jaM)

Nd

N1 (50 aM)

4bf

26 (50 JaM)

Nd

N1 (50 aM)

4cf

17 (5 jaM)

Nd

NI (5 .tM)

4de

16 (50 btM)

Nd

NI (50 LtM)

4dr

18 (50 jaM)

Nd

NI (50 aM}

4el

31 (50 btM)

Nd

Nd

o f c o m p o u n d s 4 will b e a n c h o r e d at the top o f the f o l a t e a c t i v e site as s e e n for the p h e n o l r i n g o f the r e f e r e n c e c o m p o u n d s 17], the n a p h t h a l e n e r i n g o f 4 c o u l d a r r a n g e in d i f f e r e n t w a y s with r e s p e c t to the m o d e l in the large s p a c e o f the b i n d i n g p o c k e t on the enzyme.

Experimental protocols

aPercentage inhibition at a given concentration (in parentheses); NI: no inhibition: Nd: not determined.

Cell replication inhibition

N o n e o f the test c o m p o u n d s s h o w e d a n y s i g n i f i c a n t a c t i v i t y in these a s s a y s up to a 300 btM c o n c e n t r a t i o n . The apparent inconsistency between these results and T S i n h i b i t i o n d a t a for the m o s t a c t i v e c o m p o u n d s c o u l d p o s s i b l y be e x p l a i n e d a s s u m i n g a b a d cell m e m b r a n e p e n e t r a t i o n , due to t h e i r low w a t e r s o l u b i lity. T h e r e f o r e , f u r t h e r efforts will b e d i r e c t e d to i m p r o v e this p a r a m e t e r .

Conclusion In c o n c l u s i o n , the a b o v e r e s u l t s for this c l a s s i n d i c a t e m u c h l o w e r a c t i v i t y w i t h r e s p e c t to the m o d e l in the e n z y m a t i c a s s a y s a n d a b s e n c e o f a c t i v i t y in the o t h e r in v i t r o a s s a y s . T h e b e t t e r p r o f i l e s h o w n by the nitros u b s t i t u t e d 4ae and 4af s e e m s to s u g g e s t that the p r e s e n c e o f a s t r o n g e l e c t r o n - a t t r a c t i n g g r o u p on the phenyl ring which could induce a significant polarization o f the a r o m a t i c ring, is an e s s e n t i a l r e q u i r e m e n t f o r T S - i n h i b i t i n g p r o p e r t i e s o f this class, thus c o n f i r m i n g w h a t has a l r e a d y b e e n seen in the m o d e l [5-7]. H o w e v e r , c o n t r a r y to the m o d e l , s u b s t i t u t i o n o f the n a p h t h a l e n e r i n g l e d to less a c t i v e (4et') or v e r y w e a k l y a c t i v e ( 4 d e , 4 d f and 4 e l ) c o m p o u n d s . In this r e g a r d , it s h o u l d be n o t e d that the m e t h y l e n e b r i d g e c o n f e r s m u c h m o r e c o n f o r m a t i o n a l f r e e d o m to the n a p h t h a l i c ring, w i t h r e s p e c t to the q u a t e r n a r y c a r b o n o f the m o d e l 3. T h u s , a s s u m i n g that the b e n z y l i c ring

Chemistry

Melting points were determined on a Buchi 510 capillary melting points apparatus and are uncorrected Analyses indicated by the symbols were within _+0 4r~ of the theoretical values. IH-NMR spectra were recorded on a Bruker AC200 spectrometer: chemtcal shifts are reported as 8 (ppm) relative to tetramethylsilane as internal standard and coupling constants (J) are in Hz, Sphttmg patterns are designated as follows: s. smglet: br s, broad smglet; d, doublet: dd, doublet of doublets: t, triplet: q, quartet, m, multiplet. DMSO-d,, was used as the solvent, unless othervdse noted. TLC on sihca gel plates was used to check product purity Silica gel 60 IMerck: 70-230 mesh) was used for column chromatography The structures of all compounds were consistent w~th their analyncal and spectroscopic data. N-( Substmaed)benzyl- 1,8-naphthalintides 4. Method a To a solution of the reqmred 1,8-naphthalimide (0.01 moll in DMF (40 mL), Nail (0.02 moll was added and the mixture stirred for 30 rain at rt. The appropriate benzyl chloride wa~ then added and the mixture stirred for further 3 h at ft. Water was added under coohng, the so-formed precipitate hltered by suction and thoroughly washed with ethanol (see table I for data). N-( Substituted)benzyl-4-nitro- 1,8-naphthuhmides 4. Method b To a solution of 4-mtro-1,8-naphthahc anhydride (0.02 moll in toluene (30 mL) a solution of the properly substituted benzylamine (0.02 moll in ethanol (30 mL~ was added dropwise and the mixture refluxed for 5 h. After evaporation of the solvent, the desired 4 were purified by column chromatography, eluting with CH,CL/MeOH 9:1 (see table I for data) N-gNttrobenzyl)-4-acyl- l,8-nctphthahnmle~s 4. General method A solution of the reqmred 4 (0.01 moll and the appropriate anhydride (0.05 moll in pyridme (30 mL) was stirred overnight at 60 °C. After coohng, the mixture was poured onto water (30 mL) and extracted w'lth CH,CI, (3 × 20 mL). The organic layer was washed in succession with 6 N HCI and water After evaporation of the solvent, the desired 4 were purified by column chromatography, eluting with CH~CIJMeOH 9:1 (see table I for data) N-( 3,4-Diehlorobenzo 3 I )~1,8-nat~hthaltmJde 5 To a solution of 1,8-naphthalimide (0 2 g, 0.001 moll in DMF (6 mL), Nail (0.033 g, 0.001 moll wa~ added in ~mall portions and the mixture stirred for 30 rain at ft. Equimolar 3,4-dichlorobenzoylchloride was then added and the mixture snrred tbr further 3 h at rt. Water 13 mL) was caunously added and the soformed precipitate filtered by suction to gwe 0.27 g (72%) of 5 IH-NMR 8 7.8-8.0 (m, 4H), 8.5 (d, 5H, J = 7 7 Hz). Anal CI,.HoCI,NO~ (C, H, N)

1016

Biology Enzyme assays

Plasmids that express LcTS in E c o l i strain X2913 have been described previously [10]. The enzyme was purified by column chromatography method using phosphocellulose (PI 1, Biorad) and hydroxyapatite (HAP, Bmrad) resin, with phosphate buffer as eluent [11]. CnTS and human TS have been purified as reported [12, 13]. The enzyme preparations were >95% homogeneous as shown by SDS-polyacrylamide gel electrophoresis. The purified enzyme have been stored at - 8 0 °C in l0 m M phosphate buffer, pH 7.0, 0.1 m M EDTA until use. TS activity was monitored spectrophotometrically at 340 nm using a Perkin-Elmer X15 spectrophotometer as previously described [14]. Assays were performed at 20 °C in the standard assay buffer, which contained 50 m M N-tris-(hydroxymethyl2-aminoethane)sulfonic acid (TES), pH 7.4, 25 m M MgClz, 6.5 mM formaldehyde, 1 mM EDTA, 75 mM ~-mercaptoethanol; d U M P concentration was 120 ~ M and N s , N m - m e t h y l e n e t e t r a hydrofolate 82 btM. The inhibitory properties of the compounds were determined as percent lnhlbmon at a given concentration. In vitro assays Compounds. The compounds were dissolved in D M S O at an initial concentration of 300 g M and they were serially diluted in culture medium. Cells. MT-4 and C8166 cells were grown in RPMI-1640 containing 10% fetal calf serum (FCS), 100 | U / m L penicillin G and 100 btg/mL streptomycin. Cell culture were checked periodically for the absence o f mycoplasma contamination with a MycoTect Kit (Gibco). Bacterial and fungal strains were obtained either from American Type Culture Collection (ATCC) or from Clinica Dermoslfilopatica, Universlt?a dl Cagliari. Cytotoxicto'. This was evaluated against the above cells and was based on the viability of Mock-infected cells, as momtored by MTT method [15]. assays. Yeast blastospores were obtained from a 30-h-old shaken culture incubated at 30 °C In Sabouraud dextrose broth, whereas the dermatophyte inoculum was scraped aseptically with a spatula from a 7-day-old agar culture. The macerate was then finely suspended in Sabouraud dextrose broth using a glass homogenizer. Glycerol, final concentration 10%, was added as a cryoprotective agent to both the yeast and the dermatophyte suspension, allquots of which

Antimycotw

were then stored in liquid nitrogen. Test tubes were inoculated with 103 blastospores or colony forming units (cfu)/mL. The minimal inhibitory concentration (MIC) was determined by serial dilutions using Sabouraud dextrose broth (pH 5.7) and incubating at 37 °C. Microorganism growth was determined after 1 day (yeast) or 3 days (dermatophytes). The MIC was defined as the c o m p o u n d concentration at which no microscopic signs of fungal growth were detectable. Fungicidal activity was tested by subactivations of negative test tubes in Sabouraud dextrose agar. Antibacterial assays. S a u r e u s , group D S t r e p t o c o c c u s . E c o l i and P a e r u g i n o s a were recent clinical isolates. Test were

carried out in nutrient broth, pH 7.2. The lnoculum consisted in 103 cells. MICs were determined after 18 h of incubation at 37 °C in the presence o f different compounds concentrations

Acknowledgments Thanks are due to B Sholchet (University of Chicago) for providing the X-ray crystal structure o f the binary complex LcTS-phenolphthalein and to F Soragm (Universit/a di Modena) for her excellent technical assistance.

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