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European Journal of Medicinal Chemistry 53 (2012) 246e253

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Synthesis, anti-Candida activity, and cytotoxicity of new(4-(4-iodophenyl)thiazol-2-yl)hydrazine derivatives

Daniela Secci a, Bruna Bizzarri a,*, Adriana Bolasco a, Simone Carradori a, Melissa D’Ascenzio a,Daniela Rivanera b, Emanuela Mari c, Lucia Polletta c, Alessandra Zicari c

aDipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, ItalybDipartimento Sanità Pubblica e Malattie Infettive, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, ItalycDipartimento di Medicina Sperimentale, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy

a r t i c l e i n f o

Article history:Received 16 November 2011Received in revised form6 March 2012Accepted 6 April 2012Available online 14 April 2012

Keywords:Antifungal drugsCandida spp.CytotoxicityHydrazoneHantzsch reactionThiazole

* Corresponding author. Tel./fax: þ39 06 49913772E-mail address: bruna.bizzarri@uniroma1.it (B. Biz

0223-5234/$ e see front matter � 2012 Elsevier Masdoi:10.1016/j.ejmech.2012.04.006

a b s t r a c t

Novel (4-(4-iodophenyl)-thiazol-2-yl)hydrazine derivatives were assayed for their in vitro anti-Candidaactivity, compared to topical and systemic antifungal drugs, against twenty-seven clinical isolates. Thepresence of aliphatic chains or specific heteroaromatic rings on hydrazone moiety at position C2 and a 4-iodophenyl at C4 of the thiazole ring gave a promising inhibitory activity especially against Candidaalbicans and Candida krusei. The most active compounds have been also evaluated for their cytotoxicityand in association with clotrimazole for anti-Candida activity.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Candida species are the most common opportunistic fungalpathogens in humans, with Candida albicans being the most prev-alent microbial population in mucosal and systemic infections.Candidiasis encompasses infections that range from superficial,such as oral thrush and vaginitis, to systemic and potentially life-threatening diseases [1,2]. Candida infections are usually confinedto severely immunocompromised people, cancer, transplants aswell as surgery patients [3]. C. albicans and C. glabrata account forapproximately 80% of Candida spp. recovered from patients withcandidemia or invasive candidiasis [4]. Candida glabrata hasrecently become very important because of its increasing incidenceworldwide, its association with fluconazole resistance in up to 20%of clinical specimens, and its overall decreased susceptibility toother azoles and polyenes [5]. Candida parapsilosis is also animportant species to consider in hospitalized patients because it isespecially common in infections associated with vascular cathetersand prosthetic devices [6]. Finally, Candida tropicalis has frequentlybeen considered an important cause of candidemia in patients with

.zarri).

son SAS. All rights reserved.

leukemia and in those who have undergone transplantation [7].Epidemiologic studies suggest that the problem is not undercontrol and, in fact, the incidence of drug resistance to antifungalagents shows there is an urgent need for new chemical entitieswith alternative mode of action.

From an overview of the most recent literature, it emerged that(4-aryl-thiazol-2-yl)hydrazines have been investigated for theirsignificant antimicrobial activity against a large number of clinicallyrelevant fungal [8e10] and (myco)bacterial [11] strains. Thesestructures are endowed with a different mechanism of action inrelation to azoles and amphotericin B (inhibition of the fungalbiofilm growth, low toxicity profile) [12], and an encouraginginhibitory activity against Escherichia coli FabH enzyme. Thus, the(thiazol-2-yl)hydrazine nucleus has attracted much interest in thedevelopment of pharmacologically active compounds. Keeping thisin view and in continuation of our research on biologically potentmolecules in the field [13e16], in this paper we report on thesynthesis and the biological evaluation (in vitro antifungal andantibacterial activity, association with clotrimazole, and cytotox-icity) of a new series of 2,4-disubstituted-1,3-thiazoles bearingdifferent moieties on double bond C]N and a 4-iodophenyl inposition C2 and C4 of thiazole nucleus, respectively (Table 1). Ourchoice of an iodine atom derived from the good results obtained

Table 1Structures and minimal inhibitory concentration (MIC) of compounds 1e25, clotrimazole (C), fluconazole (F), and griseofulvin (G) against twenty-seven clinical strains ofCandida spp.

Aliphatic and cycloaliphatic derivatives

R NHN

S

N

ITested fungi (MIC values mg/mL)

C. albicans(10 strains)

C. krusei(3 strains)

C. sakè(2 strains)

C. glabrata(4 strains)

C. tropicalis(4 strains)

C. parapsilosis(4 strains)

R1 Pentan-2-yl 0.25e2 4 32 2e8 0.50e2 2e82 Pentan-3-yl 0.25e2 256 256 2e8 0.50e2 2e83 Heptan-2-yl 0.25e2 2 32 2e8 0.50e2 2e84 Cyclopentyl 2e4 2 32 2e4 0.50e2 2e45 2-CH3ecyclopentyl 0.25e2 2 32 2e8 0.50e2 2e86 3-CH3ecyclopentyl 0.25e2 2 4 2e8 0.50e2 2e87 Cyclohexyl 0.50e2 2 16 2e4 2e4 2e48 2-CH3ecyclohexyl 0.25e2 128 128 2e8 0.50e2 2e89 3-CH3ecyclohexyl 2e4 128 128 4e8 2e4 2e410 4-CH3ecyclohexyl 0.50e2 64 64 2e4 2e8 2e411 Cycloheptyl 0.25e2 64 64 2e8 0.50e2 2e8

(Hetero)aryl derivatives

NHN

S

N

I

R1

Cy

Cy R1

12 Fur-2-yl H 0.50e2 64 64 4e8 2e8 2e413 Fur-2-yl CH3 0.25e2 128 128 2e8 0.50e2 2e814 Thiophen-2-yl H 2e8 128 128 4e8 2e4 2e415 Thiophen-2-yl CH3 2e4 2 4 4e8 2e4 0.50e216 Pyridin-2-yl CH3 0.25e2 64 64 2e8 0.50e2 2e817 Pyridin-3-yl H 2e4 64 64 4e8 2e4 2e418 Pyridin-3-yl CH3 0.25e2 64 64 2e8 0.50e2 2e819 Pyridin-4-yl H 0.25e2 128 128 2e8 0.50e2 2e820 Pyridin-4-yl CH3 0.25e2 2 4 2e8 0.50e2 2e821 1H-Indol-3-yl H 0.50e2 4 4 4e8 2e4 0.50e222 Benzodioxol-5-yl H 2e4 2 4 4e8 2e4 2e423 Naphthalen-1-yl H 0.25e2 64 64 2e8 0.50e2 2e824 Naphthalen-2-yl CH3 2e4 64 64 4e8 2e4 2e425 Coumarin-3-yl CH3 2e8 2 4 4e8 2e4 2e4C 2e4 2 2 0.25e2 1e4 0.50e2F 4e64 4e16 4e16 4e16 4e16 4e16G 8 8 8 8 8 8

D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253 247

with other halogens (F, Cl, Br) and the possibility to increase thelipophilicity of this scaffold. According to the established guidelinesof Clinical and Laboratory Standards Institute (CLSI) and the Euro-pean Committee for Antimicrobial Susceptibility Testing (EUCAST),we assayed the susceptibility of twenty-seven clinical Candida spp.and bacterial strains to our compounds by determining theirminimum inhibitory concentration (MIC) [17].

2. Chemistry

The new derivatives were synthesized as reported in ourprevious communication [16]. Different carbonyl compounds werereacted directly with thiosemicarbazide with catalytic amounts ofacetic acid in ethanol. These thiosemicarbazones were subse-quently condensed with newly synthesized a-bromo-4-iodo-ace-tophenone (obtained by bromination of 4-iodo-acetophenone withBr2 in CHCl3) to 2,4-disubstituted-1,3-thiazoles (Hantzsch reaction)as shown in Scheme 1. All synthesized compounds were fullycharacterized by analytical and spectral data. In general in the IR

spectrum, they showed strong bands at about w3200 andw1595 cm�1 due to the presence of NH and C]N group, respec-tively. In MS spectra, the fragment peaks which correspond to lossof eI (M � 127) from the molecular ion are consistent with thepostulated structure. Furthermore, because of the hindered rota-tion of the double bond around the C]N group, the synthesizedproducts could exist as a couple of conformers E/Z, each interactingin different way with the biological target. Diastereoselectivedynamic HPLC and theoretical studies about the E/Z interconver-sion and the configurational stability of structurally similar thio-semicarbazone derivatives showed that E-form dominated on Z-form [18].

3. Pharmacology

First, derivatives 1e25, dissolved in dimethylsulfoxide (DMSO),were evaluated for their antibacterial activity (Table S1, SupportingInformation). Organisms from routine clinical Gram-positive(Staphylococcus aureus, Staphylococcus warneri, Streptococcus

Oi

N

HN

ii

NHN

S

N

INH2

S

H2N

HN

NH2

S

H3CO

I

O

I

Br

iii

1-25

Scheme 1. Synthesis of compounds 1e25. Reagents: (i) thiosemicarbazide, EtOH, acetic acid (cat.), rt; (ii) Br2, CHCl3; (iii) a-bromo-4-iodo-acetophenone, EtOH, rt.

D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253248

pyogenes, Enterococcus faecalis) and Gram-negative isolates (E. coli,Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris)from the respiratory tract were collected from specimens ofpatients at the Hospital ‘Azienda Policlinico Umberto I�’ (SapienzaUniversity of Rome). The isolates were subcultured on a qualifiedmedium to ensure purity. The isolates were identified by conven-tional methodologies; all isolates were subcultured to ensureoptimal growth. The in vitro antibacterial activities of thecompounds were determined by the broth microdilution method,as recommended by the National Committee for Clinical LaboratoryStandards (NCCLS) [19] with Mueller-Hinton II broth (BBL Micro-biology Systems, Cockeysville, MD).

Then, the antifungal activity was evaluated against twenty-seven clinical fungal isolates of Candida spp. (C. albicans, C. krusei,C. parapsilosis, C. tropicalis, C. sakè, and C. glabrata) and comparedwith topical and systemic reference drugs clotrimazole, flucona-zole, and griseofulvin (Table 1). The used clinical isolates wererecently collected from specimens of patients at the ‘AziendaPoliclinico Umberto I�’ (Sapienza University of Rome) and wereobtained from haematology/oncology and surgery departments,which also included an intensive care unit. In particular, thesamples were isolated from the upper and lower respiratory tract,blood, and indwelling venous catheters; the isolates were identi-fied by conventional methodologies. Prior to testing, each isolatewas subcultured on a qualified medium to ensure purity andoptimal growth. All derivatives were dissolved in DMSO. Thein vitro antifungal activities were determined by the broth micro-dilution method with Sabouraud dextrose broth (BBL MicrobiologySystems, Cockeysville, MD) as recommended by the NCCLS [20].

Furthermore, the most active and representative compounds1, 2, 3, 5, 6, 20, and 23 (concentration range 0.5e4 mg/mL) weretested against a number of clinical isolates of C. albicans andC. krusei in combination with clotrimazole (concentration range

Table 2Effects of the association between the most active compounds (2 mg/mL) and clotrimazo

Comp. 18 h, MIC (mg/mL) 24 h, MIC (mg/mL)

C. albicans (4 strains) C. krusei (2 strains) C. albicans (4 strain

C 2 4 2C þ 1 0.5 2 1C þ 2 1 4 2C þ 3 0.5 2 1C þ 5 0.5 2 1C þ 6 1 2 2C þ 20 1 2 1C þ 23 0.5 4 0.5

a C. albicans strains isolated from bronchial washing of transplant patient and from blb C. krusei strains isolated from blood culture of coronary unit’s patient and from wou

0.125e256 mg/mL), to evaluate a possible association (additive/synergistic effect) between the substances on the fungal strains’growth [20] and to have in this way a preliminary insight into thepossible mechanism of action of this scaffold (Table 2).

4. Cell toxicity

Compounds that have been demonstrated to achieve thestrongest anti-Candida activity were also analyzed in two steps toestimate their ability to induce cytotoxic effects, at concentrationsranging from 0.05 to 100 mg/mL (Table S2, Supporting Information),on a cultured cell line derived from a human laryngeal epidermoidcarcinoma of the larynx (Hep2). Cell viability (EC50) was firstexamined using Trypan Blue exclusion test (Table 3), as previouslydescribed [21], followed by optical microscopy examination (Fig. 1).Afterwards, MTT Cell Proliferation Assay was performed in thesame conditions used for the cytotoxicity tests to analyze variationin cell proliferation rate and conversely, when metabolic eventslead to apoptosis or necrosis, the reduction in cell viability (Table 3).

5. Results and discussion

Derivatives 1e25 displayed a good inhibitory activity against allclinical fungal isolates, with a selectivity oriented towards C. albi-cans and C. tropicalis (MIC values lower than ones obtained aftertreatment with reference drugs) (Table 1). A discrete antifungalactivity was also reported against other Candida spp. (MIC valuescomparable with those obtained with reference drugs). Withrespect to the other series of (thiazol-2-yl)hydrazines previouslysynthesized by us [13�16], the presence of a 4-iodophenyl at C4improved the antimicrobial activity of these 2,4-disubstituted-1,3-thiazoles. The introduction of this halogen increased theClog P values of the newly synthesized molecules (Supporting

le (C) (2 mg/mL) on C. albicansa and C. krusei.b

48 h, MIC (mg/mL)

s) C. krusei (2 strains) C. albicans (4 strains) C. krusei (2 strains)

4 2 42 2 24 2 42 2 22 2 24 2 42 2 24 2 4

ood culture of surgery unit’s patient.nd’s swab of surgery unit’s patient.

Table 3Anti-proliferative and cytotoxic effect (EC50) of themost active compounds tested onHep2 cells after 24 h of incubation at 37 �C.a

Comp. MTT Cell Proliferationassay EC50 (mg/mL)

Trypan Blue exclusiontest EC50 (mg/mL)

1 45 nd2 50 nd3 42 625 30 306 50 5120 25 3023 35 40Clotrimazole 48 40

nd ¼ not determined.a Data represent arithmetic means � SD of at least three independent

experiments.

D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253 249

information) and confirmed an interesting correlation betweenClog P values >5.34 (i.e. 7.11 for compound 23) and a strongerantifungal activity in this scaffold. In addition, some of the mostactive and representative compounds (1e3, 5, 6, 20, and 23) weretested in association with clotrimazole to evaluate a potentialsynergistic/additive effect with the azoles class (Table 2). The bestconcentrations of the selected compounds and clotrimazole resul-ted in the lowest MIC values reported in Table 1. In particular,compounds 5, 23 and 20 were the most potent in inhibitingC. albicans with MIC values of 0.5 mg/mL (2-fold reduction forcompounds 5 and 23) and 1 mg/mL (1-fold reduction for compound20) in comparison with clotrimazole alone (MIC ¼ 2 mg/mL) at 18 hand 24 h, while compounds 1e3 and 6 showed a similar behavioronly at 18 h. When the MIC end points were determined following48 h of incubation, the observed anti-Candida effect was slightlylower. As regards C. krusei strains, the combined treatment of clo-trimazole plus selected derivatives, after 18e48 h, gave a slightreduction of the MIC values (2e4 mg/mL) with respect to those ofthe control (MIC ¼ 4 mg/mL). In general, MIC values obtained fromthis drugs association may suggest the possibility of using them incombination, in order to lower their single dosage and so to limittheir corresponding toxicity.

Considering this, the same selected derivatives (1e3, 5, 6, 20,and 23) and clotrimazole were investigated for their cytotoxiceffects (EC50) by Trypan blue exclusion test andMTTassay (Table 3).The results suggested that both the newly synthesized compounds

Fig. 1. Morphological changes of Hep2 cells after treatment with compound 23. Cells have beincubated for 24 h with culture medium alone represents the control. Cells are all adherentare well evident in each cell. Cells are strictly near one to each other because of cell to cell cowith 50 mg/mL of compound 23. The number of cells is significantly reduced and cell shape ithe flask surface. Cells show various dimensions and shapes, plasma membranes are not intaevident (red arrows). C) Hep2 cells after incubation with 100 mg/mL of compound 23. Cells aare largely evident as well as the presence of intra-cytoplasmatic vacuoles (red arrows). Nuclcolour in this figure legend, the reader is referred to the web version of this article.)

and the reference drug clotrimazole (a well-known drug widelyused in therapy) exert the same cytotoxicity trend. For a bettercomprehension of this behavior, cells after treatment withcompound 23 (50 and 100 mg/mL) have been analyzed by opticalmicroscopy (40X) to assess the biological effects on cell structuralelements. As reported in Fig. 1, at concentrations higher (50 and100 mg/mL) than its best MIC value (0.25 mg/mL), compound 23exerts a cytotoxic effect and induces cellular modificationsinvolving the changing of cell shape and volume, presence ofapoptotic blebs and of intra-cytoplasmatic vacuoles, and inductionof nuclear fragmentation. The reported data suggested that thecombined treatment could lead to an encouraging synergic effecton the fungal strains’ growth which might be useful to limit theinduction of resistance to clotrimazole and its toxicity.

In this study we investigated the synthesis and in vitro antimi-crobial activity of new thiazolylhydrazine derivatives by deter-mining the minimum inhibitory concentrations with the EUCASTbroth microdilution method. The substitution of the nucleus linkedto the hydrazone moiety had a deep impact on the antifungalactivity on this scaffold. Based on the reported results, derivatives1e3, 5, 6, 20, and 23 could be also used in association with azolederivatives to enhance their antifungal activity. Moreover, dis-playing a better activity and a lower cytotoxicity compared to thedrug reference clotrimazole, they represent good templates for thedevelopment of novel broad-spectrum anti-Candida spp. agents.

6. Experimental protocols

The chemicals, solvents for synthesis, and spectral gradesolvents were purchased from Aldrich (Italy) and used withoutfurther purification. Melting points (uncorrected) were determinedautomatically on an FP62 apparatus (Mettler-Toledo). 1H NMRspectra were recorded at 400 MHz on a Bruker spectrometer usingCDCl3 or DMSO-d6 as solvent. Chemical shifts are expressed asd units (parts per millions) relative to the solvent peak. Couplingconstants J are valued in Hertz (Hz). IR spectra were registered ona Perkin Elmer FT-IR Spectrometer Spectrum 1000 in anhydrouspotassium bromide (KBr). Elemental analyses for C, H, and N wererecorded on a PerkineElmer 240 Bmicroanalyzer and the analyticalresults were within �0.4% of the theoretical values for allcompounds. All reactions were monitored by TLC performed on0.2 mm thick silica gel plates (60 F254 Merck). The GC/MS unit

en analyzed after 24 h of incubation at 37 �C by optical microscopy (40X): A) Hep2 cellsto the flask, plasma membranes are intact, cell shape is regular, and one or more nucleintacts. The black arrows indicate cells in mitosis. B) Hep2 cells after incubation for 24 hs completely changed. Cells are losing their cell to cell contacts and getting detached byct and blebs are present (black arrow). In several cells intra-cytoplasmatic vacuoles arere all detached by the flask surface and are floating in the supernatant. Apoptotic blebsei are reduced in small fragments (black arrow). (For interpretation of the references to

D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253250

consisted of a PerkineElmer Autosystem XL gas chromatograph.The samples were introduced directly into the ion source. Electronionization (EI) mass spectra were obtained by a Faisons QMD 1000mass spectrometer (70 eV, 200 mA, ion source temperature of200 �C, carrier gas helium).

6.1. General procedure for the synthesis of derivatives 1e25

The appropriate carbonyl compound (50 mmol) was dissolvedin 50 mL of ethanol and magnetically stirred with an equimolarquantity of thiosemicarbazide for 24 h at room temperature withcatalytic amounts of acetic acid. The desired thiosemicarbazoneprecipitated from reaction mixture, was filtered, crystallized fromsuitable solvent, and dried. Equimolar quantities of 4-iodo-aceto-phenone and bromine, both dissolved in chloroform, were stirredfor 4 h at room temperature until the presence of HBr disappeared.The solution was evaporated under vacuum and the obtained paleyellow solid was washed with petroleum ether to give a-bromo-4-iodo-acetophenone in good yield (94%). Equimolar amounts of theprepared thiosemicarbazone (50 mmol) and a-bromo-4-iodo-ace-tophenone (50 mmol), both suspended in 50 mL of ethanol, werereacted at room temperature under magnetic stirring for 10 h. Theprecipitate was filtered and purified by chromatography to givecompounds 1e25 in high yield.

6.1.1. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(pentan-2-ylidene)hydrazine (1)

Yield 89%, mp 210e211 �C; 1H NMR (CDCl3, 400 MHz) d (ppm):0.95e0.99 (t, J¼ 7.3 Hz, 3H, CH3),1.60e1.66 (m, 2H, CH2), 2.19 (s, 3H,CH3C]N), 2.34e2.37 (m, 2H, CH2), 6.73 (s, 1H, C5Hethiazole),7.45e7.47 (d, J ¼ 7.7 Hz, 2H, Ar), 7.82e7.84 (d, J ¼ 7.7 Hz, 2H, Ar),12.23 (bs, 1H, NH, D2O exch.).

6.1.2. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(pentan-3-ylidene)hydrazine (2)

Yield 77%, mp 184e185 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 1.01e1.06 (m, 6H, 2 � CH3), 2.37e2.41 (m, 4H, 2 � CH2),6.78 (s, 1H, C5Hethiazole), 7.62e7.64 (m, 2H, Ar), 7.78e7.80 (m, 2H,Ar), 11.01 (bs, 1H, NH, D2O exch.).

6.1.3. 1-(Heptan-2-ylidene)-2-(4-(4-iodophenyl)thiazol-2-yl)hydrazine (3)

Yield 92%, mp 212e213 �C; 1H NMR (CDCl3, 400 MHz) d (ppm):0.90e0.93 (t, J ¼ 7.1 Hz, 3H, CH3), 1.33e1.38 (m, 4H, 2 � CH2),1.57e1.61 (m, 2H, CH2), 2.18 (s, 3H, CH3), 2.35e2.38 (t, J¼ 7.1 Hz, 2H,CH2), 6.73 (s, 1H, C5Hethiazole), 7.45e7.47 (d, J ¼ 7.9 Hz, 2H, Ar),7.82e7.84 (d, J ¼ 7.9 Hz, 2H, Ar), 12.21 (bs, 1H, NH, D2O exch.).

6.1.4. 1-Cyclopentylidene-2-(4-(4-iodophenyl)thiazol-2-yl)hydrazine (4)

Yield 86%, mp 213e215 �C; 1H NMR (CDCl3, 400 MHz) d (ppm):1.84e1.87 (m, 2H, CH2ecyclopentyl), 1.93e1.96 (m, 2H,CH2ecyclopentyl), 2.51e2.55 (m, 2H, CH2ecyclopentyl), 2.62e2.66(m, 2H, CH2ecyclopentyl), 6.72 (s, 1H, C5Hethiazole), 7.45e7.47 (d,J ¼ 8.7 Hz, 2H, Ar), 7.82e7.84 (d, J ¼ 8.7 Hz, 2H, Ar), 12.07 (bs, 1H,NH, D2O exch.).

6.1.5. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(2-methylcyclopentylidene)hydrazine (5)

Yield 87%, mp 148e150 �C; 1H NMR (CDCl3, 400 MHz)d (ppm): 1.21e1.28 (m, 3H, CH3ecyclopentyl), 1.73e1.78 (m, 1H,CHecyclopentyl), 1.99e2.11 (m, 2H, CH2ecyclopentyl), 2.55e2.65(m, 2H, CH2ecyclopentyl), 3.70e3.76 (m, 2H, CH2ecyclopentyl),6.78 (s, 1H, C5Hethiazole), 7.48e7.50 (d, J ¼ 8.6 Hz, 2H, Ar),7.76e7.78 (d, J ¼ 8.6 Hz, 2H, Ar), 12.14 (bs, 1H, NH, D2O exch.).

6.1.6. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(3-methylcyclopentylidene)hydrazine (6)

Yield 88%, mp 177e179 �C; 1H NMR (CDCl3, 400 MHz)d (ppm): 1.09e1.14 (m, 3H, CH3ecyclopentyl), 1.48e1.53 (m, 1H,CHecyclopentyl), 2.10e2.21 (m, 2H, CH2ecyclopentyl), 2.54e2.66(m, 2H, CH2ecyclopentyl), 2.82e2.90 (m, 2H, CH2ecyclopentyl),6.72 (s, 1H, C5Hethiazole), 7.44e7.46 (d, J ¼ 8.7 Hz, 2H, Ar),7.81e7.83 (d, J ¼ 8.7 Hz, 2H, Ar), 12.11 (bs, 1H, NH, D2O exch.).

6.1.7. 1-Cyclohexylidene-2-(4-(4-iodophenyl)thiazol-2-yl)hydrazine (7)

Yield 84%, mp 186e188 �C; 1H NMR (CDCl3, 400 MHz) d (ppm):1.62e1.69 (m, 4H, 2 � CH2ecyclohexyl), 1.71e1.78 (m, 2H,CH2ecyclohexyl), 2.48e2.54 (m, 2H, CH2ecyclohexyl), 2.63e6.70(m, 2H, CH2ecyclohexyl), 6.72 (s, 1H, C5Hethiazole), 7.45e7.47 (d,J ¼ 8.6 Hz, 2H, Ar), 7.81e7.83 (d, J ¼ 8.6 Hz, 2H, Ar), 12.03 (bs, 1H,NH, D2O exch.).

6.1.8. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(2-methylcyclohexylidene)hydrazine (8)

Yield 96%, mp 157e159 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 1.06e1.08 (m, 3H, CH3ecyclohexyl), 1.12e1.17 (m, 1H,CHecyclohexyl), 1.43e1.52 (m, 2H, CH2ecyclohexyl), 1.69e1.76(m, 2H, CH2ecyclohexyl), 1.78e1.85 (m, 2H, CH2ecyclohexyl),2.37e2.39 (m, 1H, CHecyclohexyl), 2.97e2.99 (m, 1H,CHecyclohexyl), 7.29 (s, 1H, C5Hethiazole), 7.62e7.64 (d, J¼ 7.8 Hz,2H, Ar), 7.73e7.75 (d, J ¼ 7.8 Hz, 2H, Ar), 10.98 (bs, 1H, NH, D2Oexch.).

6.1.9. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(3-methylcyclohexylidene)hydrazine (9)

Yield 99%, mp 168e170 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 1.01e1.03 (m, 3H, CH3ecyclohexyl), 1.23e1.26 (m, 1H,CHecyclohexyl), 1.78e1.84 (m, 2H, CH2ecyclohexyl), 1.92e1.97 (m,2H, CH2ecyclohexyl), 2.13e2.19 (m, 2H, CH2ecyclohexyl),2.49e2.51 (m, 1H, CHecyclohexyl), 3.02e3.04 (m, 1H,CHecyclohexyl), 6.73 (s, 1H, C5Hethiazole), 7.43e7.47 (m, 2H, Ar),7.80e7.83 (m, 2H, Ar), 12.44 (bs, 1H, NH, D2O exch.).

6.1.10. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(4-methylcyclohexylidene)hydrazine (10)

Yield 95%, mp 151e153 �C; 1H NMR (CDCl3, 400 MHz)d (ppm): 0.97e0.99 (m, 3H, CH3ecyclohexyl), 1.19e1.22 (m,2H, 2 � CHecyclohexyl), 1.73e1.75 (m, 1H, CHecyclohexyl),1.97e2.00 (m, 2H, 2 � CHecyclohexyl), 2.18e2.21 (m, 2H,2� CHecyclohexyl), 2.51e2.54 (m, 2H, 2� CHecyclohexyl), 6.71 (s,1H, C5Hethiazole), 7.45e7.47 (d, J ¼ 8.2 Hz, 2H, Ar), 7.82e74 (d,J ¼ 8.2 Hz, 2H, Ar), 12.36 (bs, 1H, NH, D2O exch.).

6.1.11. 1-Cycloheptylidene-2-(4-(4-iodophenyl)thiazol-2-yl)hydrazine (11)

Yield 80%, mp 200e201 �C; 1H NMR (CDCl3, 400 MHz) d (ppm):1.61e1.69 (m, 6H, 3 � CH2ecycloheptyl), 1.82e1.86 (m, 2H,CH2ecycloheptyl), 2.55e2.58 (m, 2H, CH2ecycloheptyl), 2.68e2.71(m, 2H, CH2ecycloheptyl), 6.72 (s, 1H, C5Hethiazole), 7.46e7.48 (d,J ¼ 8.6 Hz, 2H, Ar), 7.82e7.84 (d, J ¼ 8.6 Hz, 2H, Ar), 12.23 (bs, 1H,NH, D2O exch.).

6.1.12. 1-(Furan-2-ylmethylene)-2-(4-(4-iodophenyl)thiazol-2-yl)hydrazine (12)

Yield 84%, mp 271e272 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 6.53 (m, 1H, C4Hefuran), 6.87 (s, 1H, C5Hethiazole),7.33 (m, 1H, C5Hefuran), 7.62e7.64 (m, 2H, Ar), 7.68e7.70 (m,1H, C3Hefuran), 7.79e7.81 (m, 4H, Ar), 11.15 (bs, 1H, NH, D2Oexch.).

D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253 251

6.1.13. 1-(1-(Furan-2-yl)ethylidene)-2-(4-(4-iodophenyl)thiazol-2-yl)hydrazine (13)

Yield 84%, mp 272e273 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 2.24 (s, 3H, CH3), 6.57 (m, 1H, C4Hefuran), 6.83 (s, 1H,C5Hethiazole), 7.37 (m, 1H, C5Hefuran), 7.65e7.67 (d, J ¼ 8.0 Hz,2H, Ar), 7.71e7.73 (m, 1H, C3Hefuran), 7.75e7.77 (d, J ¼ 8.0 Hz, 2H,Ar), 11.04 (bs, 1H, NH, D2O exch.).

6.1.14. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(thiophen-2-ylmethylene)hydrazine (14)

Yield 93%, mp 190e192 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 7.08e7.10 (m, 1H, C4Hethiophene), 7.35e7.36 (m, 1H,C3Hethiophene), 7.37 (s, 1H, C5Hethiazole), 7.57e7.58 (m,C5Hethiophene), 7.63e7.65 (d, J ¼ 8.5 Hz, 2H, Ar), 7.74e7.76 (d,J ¼ 8.5 Hz, 2H, Ar), 8.20 (s, 1H, HeC]N), 12.23 (bs, 1H, NH, D2Oexch.).

6.1.15. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(1-(thiophen-2-yl)ethylidene)hydrazine (15)

Yield 95%, mp 263e265 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 2.32 (s, 3H, CH3C]N), 7.06e7.07 (m, 1H, C4Hethiophene),7.34e7.35 (m, 1H, C3Hethiophene), 7.37 (s, 1H, C5Hethiazole),7.50e7.52 (m, 1H, C5Hethiophene), 7.65e7.67 (d, J ¼ 8.1 Hz, 2H,Ar), 7.75e7.77 (d, J ¼ 8.1 Hz, 2H, Ar), 12.25 (bs, 1H, NH, D2O exch.).

6.1.16. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(1-(pyridin-2-yl)ethylidene)hydrazine (16)

Yield 99%, mp 168e170 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 2.42 (s, 3H, CH3C]N), 7.50 (s, 1H, C5Hethiazole),7.54e7.57 (m, 1H, C5Hepyridine), 7.67e7.69 (d, J ¼ 8.5 Hz, 2H,Ar), 7.77e7.69 (d, J ¼ 8.5 Hz, 2H, Ar), 8.13e8.15 (m, 2H, C3He andC4Hepyridine), 8.67e8.68 (m,1H, C6Hepyridine), 11.75 (bs, 1H, NH,D2O exch.).

6.1.17. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(pyridin-3-ylmethylene)hydrazine (17)

Yield 88%, mp 221e223 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 7.47 (s,1H, C5Hethiazole), 7.64e7.67 (m, 2H, Ar), 7.76e7.78(m, 2H, Ar) 7.79e7.81 (m, 1H, C5Hepyridine), 8.11e8.14 (m, 1H,C4Hepyridine), 8.37e8.39 (m, 1H, C6Hepyridine), 8.69e8.71 (m,1H, C2Hepyridine), 8.96 (s, 1H, HeC]N), 12.56 (bs, 1H, NH, D2Oexch.).

6.1.18. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(1-(pyridin-3-yl)ethylidene)hydrazine (18)

Yield 70%, mp 228e230 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 2.39 (s, 3H, CH3C]N), 7.53 (s, 1H, C5Hethiazole),7.69e7.71 (m, 2H, Ar), 7.74e7.76 (m, 2H, Ar), 7.83e7.85 (m, 1H,C5Hepyridine), 8.16e8.18 (m,1H, C4Hepyridine), 8.42e8.44 (m,1H,C6Hepyridine), 8.77e8.79 (m,1H, C2Hepyridine), 12.16 (bs, 1H, NH,D2O exch.).

6.1.19. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(pyridin-4-ylmethylene)hydrazine (19)

Yield 99%, mp >290 �C; 1H NMR (DMSO-d6, 400 MHz) d (ppm):7.58 (s, 1H, C5Hethiazole), 7.65e7.67 (d, J ¼ 7.9 Hz, 2H, Ar),7.77e7.79 (d, J ¼ 7.9 Hz, 2H, Ar), 8.08e8.09 (m, 2H, C3He andC5Hepyridine), 8.13 (s, 1H, HeC]N), 8.80e8.81 (m, 2H, C2He andC6Hepyridine), 13.11 (bs, 1H, NH, D2O exch.).

6.1.20. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(1-(pyridin-4-yl)ethylidene)hydrazine (20)

Yield 92%, mp >290 �C; 1H NMR (DMSO-d6, 400 MHz) d (ppm):2.36 (s, 3H, CH3C]N), 7.56 (s, 1H, C5Hethiazole), 7.67e7.69 (d,J ¼ 8.3 Hz, 2H, Ar), 7.78e7.80 (d, J ¼ 8.3 Hz, 2H, Ar), 8.14e8.16 (d,

J ¼ 7.2 Hz, 2H, C3He and C5Hepyridine), 8.82e8.84 (d, J ¼ 7.2 Hz,2H, C3He and C5Hepyridine), 12.09 (bs, 1H, NH, D2O exch.).

6.1.21. 1-((1H-Indol-3-yl)methylene)-2-(4-(4-iodophenyl)thiazol-2-yl)hydrazine (21)

Yield 90%, mp 230e231 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 7.22e7.25 (m, 3H, indole), 7.34 (s, 1H, C5Hethiazole),7.48e7.50 (m, 1H, indole), 7.66e7.68 (m, 2H, Ar), 7.75e7.77 (m,2H, Ar), 8.23e8.25 (m, 1H, indole), 8.27 (s, 1H, HeC]N), 11.56 (bs,1H, NH, D2O exch.), 11.88 (bs, 1H, NH-indole, D2O exch.).

6.1.22. 1-(Benzo[d][1,3]dioxol-5-ylmethylene)-2-(4-(4-iodophenyl)thiazol-2-yl)hydrazine (22)

Yield 80%, mp 218e220 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 6.07 (s, 2H, OCH2O), 6.98e7.01 (m, 1H,C7Hebenzodioxole), 7.11e7.12 (m, 1H, C4H-benzodioxole),7.24e7.27 (m, 1H, C6Hebenzodioxole), 7.36 (s, 1H, C5Hethiazole),7.65e7.67 (d, J ¼ 7.9 Hz, 2H, Ar), 7.74e7.76 (d, J ¼ 7.9 Hz, 2H, Ar),7.96 (s, 1H, HeC]N), 12.05 (bs, 1H, NH, D2O exch.).

6.1.23. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(naphthalen-1-ylmethylene)hydrazine (23)

Yield 84%, mp 191e193 �C; 1H NMR (DMSO-d6, 400 MHz)d (ppm): 7.43 (s, 1H, C5Hethiazole), 7.62e7.64 (m, 2H, Ar),7.65e7.67 (m, 3H, Ar), 7.70e7.72 (m, 2H, Ar), 7.76e7.80 (m, 4H, Ar),8.01 (s, 1H, HeC]N), 10.19 (bs, 1H, NH, D2O exch.).

6.1.24. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(1-(naphthalen-2-yl)ethylidene)hydrazine (24)

Yield 75%, mp 228e230 �C; 1H NMR (CDCl3, 400 MHz) d (ppm):2.40 (s, 3H, CH3C]N), 6.95 (s, 1H, C5Hethiazole), 7.52e7.55 (m, 2H,Ar), 7.56e7.58 (d, J¼ 6.7 Hz, 2H, Ar), 7.72e7.74 (d, J¼ 6.7 Hz, 2H, Ar),7.94e7.97 (m, 3H, Ar), 8.04e8.05 (m, 1H, Ar), 8.12e8.14 (m, 2H, Ar),11.57 (bs, 1H, NH, D2O exch.).

6.1.25. 1-(2H-Chromen-2-oxo-3-ylethylidene)-2-(4-(4-iodophenyl)thiazol-2-yl)hydrazine (25)

Yield 77%, mp 153e155 �C; 1H NMR (CDCl3, 400 MHz) d (ppm):2.38 (s, 3H, CH3C]N), 6.91 (s, 1H, C5Hethiazole), 7.31e7.33 (m, 1H,Ar), 7.55e7.57 (d, J ¼ 8.3 Hz, 2H, Ar), 7.60e7.63 (m, 2H, Ar),7.74e7.76 (d, J ¼ 8.3 Hz, 2H, Ar), 8.06 (s, 1H, Ar), 12.52 (bs, 1H, NH,D2O exch.).

6.2. Antibacterial activity

Microtiter plates containing serial dilutions of each compoundranging from 128 to 0.5 mg/mL were inoculated with each organismto yield the appropriate density (105/mL) in a 100 mL final volume;each plate included positive controls (bacteria withouta compound), and a negative control (medium only). The plateswere incubated for 18e22 h at 35 �C. The Minimal InhibitoryConcentration (MIC) for all isolates was defined as the lowestconcentration of antibacterial agent that completely inhibited thegrowth of the organism, as detected by the unaided eye.

6.3. Antifungal activity

Microtiter plates containing serial dilutions of each compoundwere inoculated with each organism to yield the appropriatedensity (103/mL) in a 100 mL final volume; each plate includedpositive controls (fungi without a compound) and a negativecontrol (medium only). The plates were incubated for 24 h at 37 �C.The MIC for all isolates was defined as the lowest concentration ofantifungal agents that completely inhibited the growth of theorganism, as detected by unaided eye.

D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253252

6.4. Broth microdilution susceptibility test method

The yeast inoculum was adjusted to a concentration of0.5e2.5 � 103 CFU/mL in RPMI medium, and an aliquot of0.1 mL was added to each well of the microdilution plate. Theplates were incubated at 35 �C, the MIC end points were readvisually following 18, 24, and 48 h of incubation and weredefined as the lowest concentration that produced a prominentdecrease in turbidity compared with that of the drug-free growthcontrol.

6.5. Cytotoxicity assay

The Hep2 cell line, delivered from a human laryngealepidermoid carcinoma of the larynx and purchased froma Korean Cell Line Bank (KCLB No. 10023), was a kind gift by Prof.R. Misasi (Department of Experimental Medicine e SapienzaUniversity of Rome). Cells were cultured in Dulbecco’s modifiedeagle media (DMEM), supplemented with 10% fetal bovineserum (FBS; Gibco, USA) and 1% streptomycinepenicillin. Celllines were maintained as adherent type cultures under humid-ified atmosphere in 5% CO2 at 37 �C in Dulbecco’s modifiedEagle’s culture medium. Experiments were performed on cellsgrown to 60e70% confluency. The stock solutions of the inves-tigated compounds were prepared in sterile DMSO and succes-sive dilutions were made in culture medium; the DMSO percentpresent in culture medium never exceeded 0.5%. Hep2 cells inthe exponential phase of growth (1 � 105/mL) were seeded into24-well microplate and incubated for 24 h with six differentconcentrations of the compounds (100e0.05 mg/mL). Someplates containing cells alone or cells and DMSO representedthe negative controls, whereas cells incubated with 1 mMnatrium nitroprusside represented the positive one. After theincubation period, cells were mechanically scraped off from theplates and an aliquot was diluted (1:1) with a solution 0.4%Trypan blue stain. After few minutes at room temperature, cellswere counted under an optical microscope in a Thoma hemo-cytometer chamber by two different operators. On the basis thatTrypan blue is a vital dye [22] and can enter and interact withthe cells unless the plasmatic membrane is damaged, bluestained cells were considered as having died. Values areexpressed as % of viable cells. Cell viability in control sampleswas always 97e98%.

6.6. MTT proliferation assay

Hep2 proliferation rate was evaluated using a commercial MTTproliferation assay kit (ATCC, Manassas VA 20108). Cells wereseeded in triplicate in 96-wells plate at the density of 1 � 105/wellin culture mediumwithout fetal calf serum (FCS) and incubated for24 h at 37 �C in atmosphere of air and 5% CO2 with the compoundsunder investigation at the same concentrations used for the TrypanBlue test. Cells incubated with culture medium alone representedthe controls, wells containing medium alone represented theblanks. At the end of incubation time, 10 mL of MTT Reagent wasadded to each well and plates were incubated at 37 �C for 2 h. Cellswere periodically examined under an inverted microscope toevaluate the presence of intracellular punctuate purple precipitate:when it was clearly visible, 100 mL of Detergent Reagent was addedto each well and the plate was incubated with the cover, fora further 2 h at room temperature in the dark. The absorbance wasmeasured in each well at 570 nm in a plate reader (Digital andAnalog Reader, UniMed Scien) using a test wavelength of 550 nmand a reference wavelength of 650 nm. The optical density at

650 nm (OD650) was subtracted from the OD550 to eliminatenonspecific background.

6.7. Optical microscopy

Hep2 cells, at the end of the incubation time with the differentcompounds, have been detached from the flasks using a manualscraper, washed a couple of time with phosphate buffer saline(PBS) and fixed overnight at þ4 �C with a solution of 2.5% glutar-aldehyde in 0.1 M PBS (pH 7.3). Cells were then fixed with 2%osmium tetroxide in the same buffer for 2 h at room temperature.After that, cells were dehydrated atþ4 �C in graded steps of ethanol(30% up to 100%) 100 at step, treated with toluene and embedded48 h at 60 �C in epossidic resin (Epon 812). Thin sections (30 mm),obtained using an ultra microtome (Ultra Cut Leica), were pro-cessed for Azure II staining plus Fuchsin and analyzed with anoptical microscopy equipped with a camera to acquire images(NIKON E-1000) [23].

Acknowledgments

This work was supported by grants from MURST (2008e2009).

Appendix A. Supplementary material

Supplementary material associated with this article canbe found, in the online version, at doi:10.1016/j.ejmech.2012.04.006.

References

[1] F.C. Odds, Future Microbiol. 5 (1) (2010) 67e79.[2] M.A. Pfaller, D.J. Diekema, Clin. Microbiol. Rev. 20 (1) (2007) 133e163.[3] (a) D.K. Morales, D.A. Hogan, PLoS Pathog. 6 (4) (2010) e1000886;

(b) S.K. Pitman, R.H. Drew, J.R. Perfect, Expert Opin. Emerg. Drugs 16 (3)(2011) 559e586.

[4] E.G. Playford, J. Lipman, T.C. Sorrell, Drugs 70 (7) (2010) 823e839.[5] L. Li, S. Redding, A. Dongari-Bagtzoglou, J. Dent. Res. 86 (3) (2007)

204e215.[6] (a) E.C. van Asbeck, K.V. Clemons, D.A. Stevens, Crit. Rev. Microbiol. 35 (4)

(2009) 283e309;(b) D. Trofa, A. Gácser, J.D. Nosanchuk, Clin. Microbiol. Rev. 21 (4) (2008)606e625.

[7] R.J. Kothavade, M.M. Kura, A.G. Valand, M.H. Panthaki, J. Med. Microbiol. 59(2010) 873e880.

[8] T.V. Kochikyan, E.V. Arutyunyan, M.A. Samvelyan, V.S. Arutyunyan,A.A. Avetisyan, R.V. Paronikyan, G.M. Stepanyan, Pharm. Chem. J. 43 (2009)144e147.

[9] S.K. Bharti, G. Nath, R. Tilak, S.K. Singh, Eur. J. Med. Chem. 45 (2010)651e660.

[10] A. Arshad, H. Osman, M.C. Bagley, C.K. Lam, S. Mohamad, A.S.M. Zahariluddin,Eur. J. Med. Chem. 46 (9) (2011) 3788e3794.

[11] (a) P.-C. Lv, K.-R. Wang, Y. Yang, W.-J. Mao, J. Chen, J. Xiong, H.-L. Zhu, Bioorg.Med. Chem. Lett. 19 (2009) 6750e6754;(b) A.M. Vijesh, A.M. Isloor, V. Prabhu, S. Ahmad, S. Malladi, Eur. J. Med. Chem.45 (2010) 5460e5464.

[12] A. De Logu, M. Saddi, M.C. Cardia, R. Borgna, C. Sanna, B. Saddi, E. Maccioni,J. Antimicrob. Chemother. 55 (2005) 692e698.

[13] F. Chimenti, B. Bizzarri, D. Secci, A. Bolasco, R. Fioravanti, P. Chimenti,A. Granese, S. Carradori, D. Rivanera, D. Lilli, A. Zicari, S. Distinto, Bioorg. Med.Chem. Lett. 17 (2007) 4635e4640.

[14] F. Chimenti, S. Carradori, D. Secci, A. Bolasco, P. Chimenti, A. Granese,B. Bizzarri, J. Heterocycl. Chem. 3 (2009) 575e578.

[15] F. Chimenti, B. Bizzarri, A. Bolasco, D. Secci, P. Chimenti, A. Granese,S. Carradori, M. D’Ascenzio, M.M. Scaltrito, F. Sisto, J. Heterocycl. Chem. 47 (6)(2010) 1269e1274.

[16] (a) F. Chimenti, B. Bizzarri, A. Bolasco, D. Secci, P. Chimenti, A. Granese,S. Carradori, D. Lilli, D. Rivanera, Eur. J. Med. Chem. 46 (2011) 378e382;(b) F. Chimenti, D. Secci, A. Bolasco, P. Chimenti, A. Granese, S. Carradori,M. Yáñez, F. Orallo, M.L. Sanna, B. Gallinella, R. Cirilli, J. Med. Chem. 53 (2010)6516e6520;(c) F. Chimenti, D. Secci, A. Bolasco, P. Chimenti, A. Granese, S. Carradori,M. Yáñez, F. Orallo, F. Ortuso, S. Alcaro, Bioorg. Med. Chem. 18 (2010)5715e5723.

D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253 253

[17] National Committee for Clinical Laboratory Standards, PerformanceStandards for Antimicrobial Susceptibility Testing. Supplement M100-S19,vol. 29 n 3, National Committee for Clinical Laboratory Standards, Wayne,PA, 2009.

[18] R. Cirilli, R. Ferretti, F. La Torre, D. Secci, A. Bolasco, S. Carradori, M. Pierini,J. Chromatogr. A 1172 (2007) 160e169.

[19] National Committee for Clinical Laboratory Standards Approvedstandard M7-A8, in: , eighth ed., Methods for Dilution AntimicrobialSusceptibility Tests for Bacteria that Grow Aerobically, vol. 29 n 2National Committee for Clinical Laboratory Standards, Wayne, PA,2009.

[20] National Committee for Clinical Laboratory Standard. Reference Method forBroth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standardthird ed. DM27-A3 vol. 28 n 14 National Committee for Clinical LaboratoryStandard, Wayne, Pa.

[21] F. Chimenti, B. Bizzarri, A. Bolasco, D. Secci, P. Chimenti, A. Granese,S. Carradori, D. Rivanera, A. Zicari, M.M. Scaltrito, F. Sisto, Bioorg. Med. Chem.Lett. 20 (2010) 4922e4926.

[22] R. Freshney, Culture of Animal Cells: A Manual of Basic Technique, A.R. LissInc., New York, 1987.

[23] C. Chimenti, E. Morgante, G. Tanzilli, E. Mangieri, G. Critelli, C. Gaudio,M.A. Russo, A. Frustaci, Circ. Heart Fail. 1 (2008) 161e169.