European Journal of Medicinal Chemistry 64 (2013) 357e364

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European Journal of Medicinal Chemistry

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

Original article

2-Aminobenzothiazole derivatives: Search for new antifungal agents

Alessia Catalano a, Alessia Carocci a, Ivana Defrenza a, Marilena Muraglia a,
Antonio Carrieri a, Françoise Van Bambeke b, Antonio Rosato a, Filomena Corbo a, *,
Carlo Franchini a
a
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, via E. Orabona n. 4, 70125 Bari, Italy
b
Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: A new series of 6-substituted 2-aminobenzothiazole derivatives were synthesized and screened in vitro
Received 23 October 2012 as potential antimicrobials. Almost all the compounds showed antifungal activity. In particular, com-
Received in revised form pounds 1n,o, designed on the basis of molecular modeling studies, were the best of the series, showing
27 March 2013
MIC values of 4e8 mg/mL against Candida albicans, Candida parapsilosis and Candida tropicalis. None of
Accepted 28 March 2013
Available online 9 April 2013
the two compounds did show any cytotoxicity effect on human THP-1 cells.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Antifungal activity
2-Aminobenzothiazole derivatives
CA-CYP51
Docking
Cytotoxicity

1. Introduction heterocyclic nucleus led to a remarkable antibacterial activity. In a

search for new leads toward potent antimicrobial agents, given the
One of the major problems we are facing today in the context of isosteric relationship existing between SH and NH2 groups,
infectious diseases is the relentless increase and spread of antimi- following a previous work [7], we synthesized a series of 2-amino-
crobial resistance. Thus, studies for the identification of novel tar- 1,3-benzothiazoles (Fig. 1) and tested their in vitro antimicrobial
gets and drugs for the treatment of infectious diseases are at the activity. It was reported, indeed, that several 2-aminobenzothiazole
forefront. In this perspective, for example, we recently determined derivatives, variously substituted, showed antifungal activity, even
the X-ray crystallographic structure of Enterococcus faecalis thy- though much lower than those of the reference antifungal agent
midylate synthase, which should be a potential target for antibac- used [8]. In particular, as it was already done for the series of 2-
terial therapy [1]. Many heterocyclic nuclei, such as 1,3,4- mercapto-1,3-benzothiazole derivatives [6], substitutions at posi-
thiadiazole, benzimidazole, 1,3,5-triazine, and benzothiazole have tion 6 of the aryl moiety was investigated. QSAR and docking
been recently reviewed as antimicrobial agents [2,3]. Our attention studies gave valuable hints assessing lipophilicity and steric hin-
was focused to the benzothiazole nucleus [4]. In fact, benzothiazole drance as main molecular determinants most likely affecting the
derivatives possess a wide spectrum of biological applications such newly synthesized benzothiazole derivatives in their antifungal
as antitumor, schictosomicidal, anti-inflammatory, anticonvulsant, activity.
antidiabetic, antipsychotic, diuretic, and antimicrobial activities [5].
In the past, our research group was interested in the synthesis and
microbiological screening of a series of 2-mercapto-1,3- 2. Results and discussion
benzothiazole derivatives, some of which showed antibacterial
activity against Gram-positive and negative bacterial strains [6]. 2.1. Chemistry
Results showed that the SH moiety at the 2 position of the
Compounds 1a and 1jem were commercially available. Com-
pounds 1bei,n,o (Table 1) were synthesized as depicted in Scheme
* Corresponding author. Tel.: þ39 080 5442746; fax: þ39 080 5442724. 1. Alkyl and aryl alcohols 2eeh,n were reacted with 4-nitrophenol
E-mail address: fi[email protected] (F. Corbo). under Mitsunobu conditions [9e12] to give their nitro derivatives

0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.064
358 A. Catalano et al. / European Journal of Medicinal Chemistry 64 (2013) 357e364

N benzothiazole moiety of halogens, such as a chlorine and fluorine

atom (1j,k, Fig. 2) and a trifluoromethyl group (1l, Fig. 2) was first
NH2
R = H, Alk, OAlk, OAr investigated. These three compounds were slightly active against
R S all Candida spp. tested. In particular, 1l was the most active of them,
especially against C. albicans. Then, we introduced an alkoxy or
Fig. 1. Structures of 2-amino-1,3-benzothiazole derivatives (1aeo).
aryloxy moiety (1beh) in the same position. Results showed that
antifungal activity enhanced with the increase of steric hindrance
at position 6 of the heterocycle. In fact, compounds 1b and 1c were
3eeh,n, which were reduced by catalytic hydrogenation to give
anilines 6eeh,n. Anilines 6bed,o were commercially available. slightly active while phenoxy and benzyloxy derivatives (1d and 1e,
respectively) were much more potent. Compound 1d was equi-
Aniline 6i was obtained by reducing nitro derivative 4 to aniline 5
and then by submitting the latter to a Mitsunobu reaction with potent to 1c against C. albicans but was much more active against
C. parapsilosis, C. tropicalis and C. krusei (MIC values: 16, 32, 32 vs
phenol. 2-Aminobenzothiazole derivatives 1bei,n,o were prepared
via thiocyanation of 6eei,n,o [13]. In this reaction ammonium 128, 128, 256 mg/mL, respectively). The benzyloxy derivative 1e was
more active than the others against C. albicans and showed high
thiocyanate and bromine were used to generate thiocyanogen in
situ [14]. activity also against C. parapsilosis and C. tropicalis. Thus, we
investigated a possible homologation of the alkyl chain with one to
three methylene moieties obtaining compounds 1f, 1g and 1h,
2.2. Antimicrobial studies respectively. They were generally less active than compound 1e
evidencing that elongation of the alkyl chain was detrimental for
2.2.1. Antibacterial studies activity. In particular, compound 1h was nearly inactive against
According to the Clinical Laboratory Standards Institute (CLSI) fungi. Compound 1i was less active than its position isomer 1e. This
guidelines [15] compounds 1aeo (Table 1) were tested against finding suggests that conceivably moving the oxygen atom is
Gram-positive and Gram-negative bacteria belonging to the ATCC detrimental for activity. Among the series of alkoxy derivatives, we
collection (Staphylococcus aureus 29213, E. faecalis 29212, Escher- also tested the well-known drug riluzole (1m), the only FDA-
ichia coli 25922) using Norfloxacin (NRF) as reference drug. The approved drug to treat amyotrophic lateral sclerosis [17]. It was
results, expressed as MIC (mg/mL), are listed in Table 2. The anti- just slightly active against all the species of Candida. Finally, com-
bacterial screening revealed that the compounds showed very low pounds 1n,o, suggested by modeling studies (see below), were
to no activity against all the bacterial strains tested, thus underlying indeed the most active of the series against C. albicans, C. para-
that isosteric substitution SH/NH2 brought to a loss of the anti- psilosis and C. tropicalis. It is noteworthy that compound 1n was
bacterial activity, as evidenced by comparing 1b,c,jel with their even as potent as the reference on C. tropicalis.
corresponding isosters previously reported [6].
2.3. Molecular modeling studies
2.2.2. Antifungal studies
Compounds 1aeo (Table 1) were screened, according to CLSI A molecular modeling study was carried out to better perceive
guidelines [16], against a panel of fungi strains (Candida albicans and evaluate the biological profile within the series under study.
10231, Candida parapsilosis 22019, Candida tropicalis 750, Candida In the first step we make use of classical 2D-QSAR to gain a sound
krusei 6258) belonging to the ATCC collection. Fluconazole was regression model, showing the dependence of the antifungal ac-
used as reference drug. The results, expressed as MIC (mg/mL), are tivity on lipophilicity. In particular, as proposed by Hansch [18],
listed in Table 3. All the 6-substituted 2-amino-1,3-mercapto pMIC data showed parabolic dependence by hydrophobic che-
benzothiazole derivatives show slight to high antifungal activity motype of benzothiazoles, as scored by the CLogP values (Fig. 2,
against all the Candida spp. tested, while unsubstituted 1a was Table 4). It has to be pointed out that model comprising com-
inactive. The electron-withdrawing effect at the 6-position of the pounds 1bem was indeed statistically significant (r2 ¼ 0.624), but
a valuable increase of the explained variance (r2 ¼ 0.750) was
achieved excluding from the regression model 1d, the lone de-
Table 1 rivative bearing the phenyl ring directly branched to the oxygen at
Structures of compounds 1aeo. position 6 of the aryl moiety. Afterward, the antifungal activity
N was interpreted through docking experiments. Lanosterol 14a-
NH2 demethylase (CYP51) is a member of the cytochrome P450 su-
R S perfamily, which catalyzes the oxidative removal of the 14a-
methyl group of lanosterol to give D14,15-desaturated in-
termediates in ergosterol biosynthesis [19]. The development of
Compd R inhibitors of CYP51 in fungi has provided a rich source of drugs,
1a H such as clotrimazole, ketoconazole, fluconazole, and itraconazole
1b MeeO [20], responsible for the cell growth inhibition due to ergosterol
1c EteO
1d PheO
depletion. The same cytochrome is also targeted by drugs (i.e.
1e BneO benzothiazines and benzoxazines) [20e22] characterized by a
1f Ph(CH2)2eO molecular scaffold similar to the benzothiazoles here presented.
1g Ph(CH2)3eO Moreover, the importance of CYP51 as a plausible target in the
1h Ph(CH2)4eO
treatment of C. albicans infections has been already and success-
1i PhOCH2
1j F fully reported [23e25]. On the basis of this evidence, compounds
1k Cl 1bei were therefore docked into the catalytic site of the homology
1l CF3 based model of C. albicans CYP51 (CA-CYP51) [26,27] which has
1m CF3O already been successfully used in a previous study on antifungal
1n 4-CleBneO
1o 4-ClePheO
agents [7]. Docking binding poses highlighted some interesting
features that might be in charge of the antifungal activity of the
 A. Catalano et al. / European Journal of Medicinal Chemistry 64 (2013) 357e364 359

Scheme 1. Reagents and conditions: (i) 4-nitrophenol (for compounds 3eeh,n) or phenol (for compound 6i), PPh3, DIAD, anhyd THF, rt; (ii) HCOONH4, 10% Pd/C, i-Pr2O/H2O/THF, rt
(for compounds 6feh); H2, 10% Pd/C, MeOH/abs EtOH, rt (for compounds 5 and 6e), H2, 10% Pd/C, abs EtOH, rt (for compounds 6n,o); (iii) Br2, NH4SCN, HCOOH/CH3COOH, 0  C then
rt.

compounds under study. All of them are actually capable to bulkiness and/or lipophilicity, was already highlighted in the 2D-
accommodate the active site mainly anchoring the heterocyclic QSAR study previously mentioned. As long as this evidence was
system nearly perpendicular to the heme group of CA-CYP51, by concerned, the synthesis of novel compounds was perceived and
mean of a coordination bond involving the iron and the nitrogen carried out according to the insights achieved by the modeling
atoms, as it might be perceived from the docking of the most study. Indeed 2D-QSAR highlighted the affect of lipophilicity on
potent antifungal agent 1e depicted in Fig. 3. As a figure of merit, antifungal activity, suggesting thereafter that an increase of this
the same binding topology has also been experimentally observed chemical cliché would have been at least mandatory to challenge
in the X-ray structures of other thiazoles in complex with cyto- the pMIC data. Moreover, the nature of the CA-CYP51 binding cleft
chrome different from the CA-CYP51, but functionally related to was proved from dockings to be most likely characterized by ar-
our target [28]. At the same time, the amino group at position 2 omatic, or at least very low polar, residues. This guided us in the
reinforces the binding engaging Thr311 and Gly307 with design of 1n,o as antifungals with improved activity. As expected
hydrogen-bonding mediated polar interactions. This evidence these analogs were found to be the most potent of the series. The
then facilitates the positioning of the apolar and steric hindered better antimicrobial profile might be ascribed not merely to a
substituents into a most likely hydrophobic dome placed above balancing in lipophilicity, but also to the additional and favorable
the heme group. It is very interesting to note that the benzyl van der Waals interactions occurring between the chlorine atom
moiety of the most potent antifungal agent 1e make extensive pe and the side chain of Val509 (see Fig. 3).
p stackings with Phe228 and Tyr118. Other remarkable indications
have to be ascribed to the reduced accessible surface of the active 2.4. Cytotoxicity assay
site lodge, shaped and delimited by bulky residues, namely
Met508 and Val509. This evidence might justify, at least in part, The most active compounds of the series (1n,o) were tested for
the very low pMIC of derivatives bearing very large and long their cytotoxicity by the measurement of the release of the cytosolic
lipophilic groups hampering a proper docking (i.e. 1h). On the enzyme lactate dehydrogenase (LDH) and by the uptake of trypan
other hand, compounds with a very small group, and lacking the blue in damaged cells. The results obtained on human THP-1 cells
phenyl ring (i.e. 1b,c), might not be capable to establish significant indicate that these compounds show no cytotoxicity. Fig. 4 shows that
interactions, resulting then with a lower antifungal potency. It may 1n caused a significant increase for both markers for concentrations
be noted that the need of a suitable functional group, in terms of 20 mg/mL (4 MIC) and that 1o did not cause any LDH release and
induced a small but significant increase in the number of trypan blue-
Table 2 stained cells when its concentration was 40 mg/mL (10 MIC).
Antibacterial activity results of 2-aminobenzothiazole derivatives 1aeo (MIC, mg/
mL).
Table 3
Microorganism (MIC, mg/mL) Antifungal activity results of 2-aminobenzothiazole derivatives 1aeo (MIC, mg/mL).

Gram-positive Gram-negative Microorganism (MIC, mg/mL)

S.a. 29213 E.f. 29212 E.c. 25922 C.a. 10231 C.p. 22019 C.t. 750 C.k. 6258
1a >512 >512 >512 1a >512 >512 >512 >512
1b 128 128 >512 1b 256 256 256 512
1c 512 512 >512 1c 128 128 128 256
1d >512 >512 >512 1d 128 16 32 32
1e >512 >512 >512 1e 8 8 8 64
1f >512 128 >512 1f 8 16 32 16
1g 512 128 >512 1g 32 16 16 16
1h 512 256 >512 1h 512 128 512 >512
1i 256 128 >512 1i 32 16 16 64
1j >512 >512 >512 1j 256 128 256 256
1k >512 >512 >512 1k 64 64 64 128
1l 256 256 >512 1l 16 64 64 64
1m 256 512 512 1m 64 64 128 128
1n 256 256 >512 1n 4 8 4 64
1o 512 512 >512 1o 8 4 8 32
NRF 0.5 4 0.03 FCN 2 2 4 32

Antibacterial activity was estimated by using CLSI assay [15]. Abbreviations: S.a.: Antifungal activity was estimated by using CLSI assay [16]. Abbreviations: C.a.:
S. aureus; E.f.: E. faecalis; E.c.: E. coli; NRF: norfloxacin. C. albicans; C.p.: C. parapsilosis; C.t.: C. tropicalis; C.k.: C. krusei; FCN: fluconazole.
360 A. Catalano et al. / European Journal of Medicinal Chemistry 64 (2013) 357e364

Fig. 2. Plot of pMIC vs CLogP data. Data not included in the regression model are
represented by square. Fig. 3. Docking poses for 1e (left) and 1n (right) in the active site of CA-CYP51. Ligands
and iron atom are displayed as ball and stick to help interpretation.

3. Conclusion potent than the reference compound fluconazole against C. krusei.

In conclusion, the presence of the substitution at position 2 of the
A new series of 2-amino-1,3-benzothiazole derivatives were heterocyclic moiety is crucial for benzothiazole derivatives activity:
synthesized, characterized by mass, 1H NMR and IR spectra, and C, in particular, 2-mercapto derivatives were generally more active
H, N, analyses. Comparison of these compounds with the series of against bacteria, while the 2-amino ones were more potent against
2-mercapto-1,3-benzothiazole derivatives previously reported [6] fungi. Moreover, as also suggested by the molecular modeling
showed that the isosteric substitution SH/NH2 at position 2 studies, data suggests the importance of the substitution with
brought to the loss of activity against bacteria but to the appearance bulky groups at the 6-position of the 2-aminobenzothiazole moiety
of an interesting activity toward fungi strains. This result confirms for enhancing antifungal activity, being the 6-benzyloxy derivative
our previous hypothesis that the SH moiety at the 2 position of the 1e the best of the series against most of the fungal strains used.
heterocyclic nucleus is crucial for antibacterial activity. In this work, Homologation of the alkyl chain brought to a reduction of anti-
substitutions at the 6-position of the heterocyclic moiety were fungal activity, except for C. krusei, against which the most active of
investigated. All the newly synthesized compounds (1bei,n,o), the series were compounds 1f and 1g. The best compounds of the
together with commercial 1a,jem, were investigated for their series, 1n,o, were also screened for their cytotoxicity and did not
in vitro antimicrobial activities. Among the screened samples, no show any toxic effects for human THP-1 cells when concentrations
compound showed interesting antibacterial activity, as expected. are maintained below 4 (1n) or 10 (1o) their MIC. The current
On the contrary, most of the compounds were interesting for their study therefore suggests that 2-amino-1,3-benzothiazoles 1n,o are
antifungal activity. In particular, compound 1n showed the highest promising scaffolds for the development of novel antifungal agents
antifungal activity against C. albicans (MIC: 4 mg/mL), followed by against Candida spp.
compounds 1e,f,o (MIC: 8 mg/mL). Compound 1n was also very
active against C. tropicalis, showing the same MIC value as the
4. Experimental
reference (4 mg/mL), followed by compounds 1n,o (MIC: 8 mg/mL).
Compound 1o was the best of the series against C. parapsilosis (MIC:
4.1. Chemistry
4 mg/mL) followed by compounds 1e,n (MIC: 8 mg/mL). It is worthy
of note that both compounds 1f and 1g were indeed two-fold more
Chemicals were purchased from SigmaeAldrich or Lancaster.
Yields refer to purified products and were not optimized. The
Table 4 structures of the compounds were confirmed by routine spectro-
Lipohilicity, binding energy and antifungal activity of derivatives 1beo.
metric and spectroscopic analyses. Only spectra for compounds not
pMICa CLogPb FEB (kcal/mol)c Rankd Populatione previously described are given. Alcohols 2eeh,n and 4 and anilines
1b 2.85 1.81 5.00 1 68 6bed,o were commercially available. Melting points were deter-
1c 3.18 2.34 5.07 4 2 mined on a Gallenkamp apparatus in open glass capillary tubes and
1d 3.28 3.88 6.64 1 129
are uncorrected. Infrared spectra were recorded on a PerkineElmer
1e 4.51 3.46 6.95 1 13
1f 4.53 3.69 6.67 2 12 (Norwalk, CT) Spectrum One FT spectrophotometer and band po-
1g 3.95 4.37 7.07 2 15 sitions are given in reciprocal centimeters (cm1). 1H NMR and 13C
1h 2.77 4.85 ndf e e NMR spectra were recorded on a Varian VX Mercury spectrometer
1i 3.90 3.46 7.16 1 53 operating at 300 and 75 MHz for 1H and 13C, respectively, using
1j 2.82 1.94 4.87 1 88
1k 3.46 2.49 5.42 1 171
CDCl3 and DMSO-d6 as solvents. Chemical shifts are reported in
1l 4.13 2.46 5.08 1 110 parts per million (ppm) relative to the residual non-deuterated
1m 3.56 2.84 4.59 2 31 solvent resonance: CDCl3, d 7.26 (1H NMR) and d 77.3 (13C NMR);
1n 4.86 4.06 6.49 1 70 DMSO-d6, d 2.48 (1H NMR) and d 40.1 (13C NMR). J values are given
1o 4.54 4.48 6.19 5 3
in Hz. Gas chromatography (GC)/mass spectroscopy (MS) was
a
Expressed as Log ¼ 1/MIC. performed on a HewlettePackard 6890e5973 MSD at low resolu-
b
Calculated using Advanced Chemistry Development (ACD) Software. tion. Liquid chromatography (LC)/mass spectroscopy (MS) was
c
Free Energy of Binding.
d
Pose rank according to the FEB scoring.
performed on a spectrometer Agilent 1100 series LC-MSD Trap
e
Number of similar docked conformations found in the same cluster. System VL. Elemental analyses were performed on a Eurovector
f
No docking poses (see text). Euro EA 3000 analyzer. Chromatographic separations were
 A. Catalano et al. / European Journal of Medicinal Chemistry 64 (2013) 357e364 361

Fig. 4. Toxicity of 1o (left) and 1n (right) for THP-1 monocytes, as assessed by measuring LDH release (left axis) or trypan blue staining (right axis). Data are mean  SD of 3 independent
determinations (when non visible, SD are smaller than the symbol). Statistical analysis: one-way ANOVA with Dunnet’s post-hoc test vs control: *: p < 0.05; **: p < 0.01.

performed on silica gel columns by column chromatography on (%) 271 (Mþ, 13), 91 (100); 1H NMR (CDCl3): d 1.75e1.95 (m, 4H,
silica gel (Kieselgel 60, 0.040e0.063 mm, Merck, Darmstadt, Ger- CH2CH2CH2CH2), 2.71 (t, J ¼ 6.9 Hz, 2H, CH2eAr), 4.06 (t, J ¼ 5.6 Hz,
many) as described elsewhere [29e31]. TLC analyses were per- 2H, CH2eOAr), 6.92 (d, J ¼ 9.1 Hz, 2H, Ar), 7.15e7.35 (m, 5H, Ar), 8.19
formed on precoated silica gel on aluminum sheets (Kieselgel 60 (d, J ¼ 9.1 Hz, 2H, Ar); 13C NMR (CDCl3): d 27.9 (1C), 28.8 (1C), 35.7
F254, Merck). (1C), 68.9 (1C), 114.6 (2C), 126.1 (2C), 126.2 (1C), 128.6 (4C), 141.6
(1C), 142.1 (1C), 164.4 (1C).

4.2. General procedure for the synthesis of 4-substituted

4.2.4. Synthesis of 1-chloro-4-[(4-nitrophenoxy)methyl]benzene
nitrobenzene derivatives (3eeh,n) and 4-(phenoxymethyl)aniline
(3n)
(6i)
Prepared as reported above for 3e starting from 2n and 4-
nitrophenol. Yield: 47%; off-white crystals: mp 114e115  C
The method adopted for the synthesis of 1-benzyloxy-4-
(EtOAc/petroleum ether); IR (KBr): 1505, 1337 (NO2) cm1; GC/MS
nitrobenzene (3e) is described. A solution of diisopropyl azodi-
(70 eV) m/z (%) 263 (Mþ, 2), 125 (100); 1H NMR (CDCl3): d 5.13 (br s,
carboxylate (DIAD, 5.60 g, 27.8 mmol) in dry THF (60 mL) was
exch D2O, 2H, NH2), 7.02 (d, J ¼ 9.1 Hz, 2H, Ar), 7.32e7.44 (m, 4H,
added dropwise to a solution of benzyl alcohol (2e) (2.0 g,
Ar), 8.21 (d, J ¼ 9.3 Hz, 2H, Ar); 13C NMR (CDCl3): d 70.1 (1C), 115.1
18.5 mmol), 4-nitrophenol (3.86 g, 27.8 mmol), and triphenyl-
(2C), 126.2 (2C), 129.0 (2C), 129.2 (2C), 134.2 (1C), 134.7 (1C), 142.1
phosphine (7.28 g, 27.8 mmol) in dry THF (100 mL) under N2 at-
(1C), 163.6 (1C).
mosphere at room temperature. The reaction mixture was stirred
overnight and then concentrated in vacuo. Et2O was added to the
4.2.5. 4-(Phenoxymethyl)aniline (6i)
residue and the solid filtered off. The filtrate was evaporated and
Prepared as reported above for 3e starting from 5 and phenol.
the residue was purified by column chromatography on silica gel
Yield: 59%; gold crystals: mp 177e178  C (CHCl3/hexane); IR (KBr):
(EtOAc/petroleum ether 1:9) to give a white solid which was
3398 (NH2) cm1; GC/MS (70 eV) m/z (%) 199 (Mþ, 3), 106 (100); 1H
recrystallized from EtOAc/petroleum ether to give 3.33 g (79%) of
NMR (CDCl3): d 3.70 (br s, exch D2O, 2H, NH2), 4.93 (s, 2H, CH2), 6.68
white crystals: mp 107e108  C; IR (KBr): 1509, 1347 (NO2) cm1;
(d, J ¼ 8.8 Hz, 2H, Ar), 6.95e7.05 (m, 3H, Ar), 7.20e7.35 (m, 3H, Ar);
GC/MS (70 eV) m/z (%) 229 (Mþ, 3), 91 (100). Other spectroscopic 13
C NMR (DMSO-d6): d 60.4 (1C), 112.8 (2C), 115.4 (2C), 123.8 (1C),
data were in agreement with the literature [32].
127.4 (1C), 128.8 (2C), 130.1 (2C), 148.4 (1C), 171.0 (1C).

4.2.1. 1-Nitro-4-(2-phenylethoxy)benzene (3f)

Prepared as reported above for 3e starting from 2f and 4- 4.3. General procedure for the synthesis of 4-substituted anilines
nitrophenol. Yield: 72%; white solid: mp 59e60  C (EtOAc/petro- (6feh) [34,35]
leum ether); IR (KBr): 1510, 1338 (NO2) cm1; GC/MS (70 eV) m/z
(%) 243 (Mþ, 14), 105 (100). Other spectroscopic data were in The method adopted for the synthesis of 4-(2-phenylethoxy)
agreement with the literature [33]. aniline (6f) is described. A stirring suspension of 10% Pd/C (0.17 g) in
2-propanol (10 mL) and water (1 mL) was brought to reflux. Next,
4.2.2. 1-Nitro-4-(3-phenylpropoxy)benzene (3g) nitro derivative 3f (0.40 g, 1.64 mmol) dissolved in THF (4 mL) and
Prepared as reported above for 3e starting from 2g and 4- solid ammonium formate (0.41 g, 6.58 mmol) were added. After
nitrophenol. Yield: 25%; slightly yellowish crystals: mp 82e83  C 1.5 h the catalyst was removed by filtration and the solvent was
(EtOAc/hexane); IR (KBr): 1514, 1332 (NO2) cm1; GC/MS (70 eV) m/ evaporated in vacuo to give a crude residue that was taken up with
z (%) 257 (Mþ, 49), 91 (100); 1H NMR (CDCl3): d 2.15 (quintet, EtOAc and washed with water. Then the solvent was removed in
J ¼ 6.9 Hz, 2H, CH2CH2CH2), 2.83 (t, J ¼ 7.4 Hz, 2H, CH2eAr), 4.05 (t, vacuo giving 0.28 g (80%) of a dark brown oil: GC/MS (70 eV) m/z (%)
J ¼ 6.3 Hz, 2H, CH2eOAr), 6.93 (d, J ¼ 9.3 Hz, 2H, Ar), 7.15e7.35 (m, 213 (Mþ, 100). Other spectroscopic data were in agreement with
5H, Ar), 8.19 (d, J ¼ 9.3 Hz, 2H, Ar); 13C NMR (CDCl3): d 30.7 (1C), the literature [24].
32.2 (1C), 67.9 (1C), 114.6 (2C), 126.1 (2C), 126.4 (1C), 128.7 (4C),
141.1 (1C), 142.0 (1C), 164.3 (1C). 4.3.1. 4-(3-Phenylpropoxy)aniline (6g)
Prepared as reported above for 6f starting from 3g. Yield: 72%;
4.2.3. 1-Nitro-4-(4-phenylbutoxy)benzene (3h) brown solid: mp 60e61  C; IR (KBr): 3403 (NH2) cm1; GC/MS
Prepared as reported above for 3e starting from 2h and 4- (70 eV) m/z (%) 227 (Mþ, 94), 109 (100); 1H NMR (CDCl3): d 2.06
nitrophenol. Yield: 78%; white crystals: mp 54e55  C (EtOAc/pe- (quintet, J ¼ 7.0 Hz, 2H, CH2CH2CH2), 2.80 (t, J ¼ 7.6 Hz, 2H, CH2eAr),
troleum ether); IR (KBr): 1498, 1331 (NO2) cm1; GC/MS (70 eV) m/z 3.42 (br s, exch D2O, 2H, NH2), 3.89 (t, J ¼ 6.3 Hz, 2H, CH2eOAr), 6.64
362 A. Catalano et al. / European Journal of Medicinal Chemistry 64 (2013) 357e364

(d, J ¼ 8.5 Hz, 2H, Ar), 6.75 (d, J ¼ 9.1 Hz, 2H, Ar), 7.15e7.35 (m, 5H, ether 1:1) to give 0.93 g (65%) of an orange solid: mp 163e164  C;
Ar); 13C NMR (CDCl3): d 31.2 (1C), 32.4 (1C), 67.9 (1C), 116.0 (2C), IR (KBr): 3436 (NH2) cm1; GC/MS (70 eV) m/z (%) 194 (Mþ, 81),
116.9 (2C), 126.1 (1C), 128.7 (4C), 139.8 (1C), 141.9 (1C), 152.6 (1C). 165 (100); 1H NMR (DMSO-d6): d 1.28 (t, J ¼ 6.9 Hz, 3H, CH3), 3.34
(br s, exch D2O, 2H, NH2), 3.96 (quartet, J ¼ 7.0 Hz, 2H, CH2), 6.64e
4.3.2. 4-(4-Phenylbutoxy)aniline (6h) 6.80 (m, 1H, Ar), 7.16e7.28 (m, 2H, Ar); 13C NMR (DMSO-d6): d 15.5
Prepared as reported above for 6f starting from 3h. Yield: 90%; (1C), 64.1 (1C), 106.8 (1C), 114.0 (1C), 118.7 (1C), 132.5 (1C), 147.4
brown oil; IR (neat): 3361 (NH2) cm1; GC/MS (70 eV) m/z (%) 241 (1C), 154.1 (1C), 165.4 (1C). Anal. calcd. for C9H10N2OS$0.17H2O
(Mþ, 53), 109 (100); 1H NMR (CDCl3): d 1.72e1.88 (m, 4H, (197.25) %: C, 54.80; H 5.28; N 14.20. Found: C, 55.02; H 5.13; N
CH2CH2CH2CH2), 2.62e2.75 (m, 2H, CH2eAr), 3.12 (br s, 2H, NH2), 13.92.
3.85e3.95 (m, 2H, CH2eOAr), 6.60e6.68 (m, 2H, Ar), 6.70e6.78 (m,
2H, Ar), 7.15e7.35 (m, 5H, Ar); 13C NMR (CDCl3): d 28.1 (1C), 29.3 4.5.1. 6-Methoxy-1,3-benzothiazol-2-amine (1b)
(1C), 35.9 (1C), 68.7 (1C), 116.0 (2C), 116.7 (2C), 126.0 (1C), 128.5 Prepared as reported above for 1c starting from 6b. Yield: 26%;
(2C), 128.7 (2C), 140.0 (1C), 142.5 (1C), 152.6 (1C). beige crystals: mp: 162e163  C (CHCl3/hexane); IR (KBr): 3388
(NH2) cm1; GC/MS (70 eV) m/z (%) 180 (Mþ, 81), 165 (100); 1H
4.4. General procedure for the synthesis of 4-substituted anilines (5 NMR (DMSO-d6): d 3.32 (br s, exch D2O, 2H, NH2), 3.71 (s, 3H, CH3),
and 6e,n) 6.75e6.85 (m, 1H, Ar), 7.15e7.30 (m, 2H, Ar); 13C NMR (CDCl3):
d 56.1 (1C), 105.6 (1C), 113.9 (1C), 119.9 (1C), 132.9 (1C), 146.4 (1C),
The method adopted for the synthesis of 4- 155.9 (1C), 164.3 (1C). Anal. calcd. for C8H8N2OS$0.50H2O (189.24)
aminophenylmethanol (5) is described. Catalytic hydrogenation of %: C, 50.78; H 4.79; N 14.80. Found: C, 50.82; H 4.21; N 14.43.
4 (1.50 g, 9.8 mmol) in 30 mL of a mixture of MeOH and absolute
EtOH (2/1) was conducted at room temperature for 24 h in the 4.5.2. 6-Phenoxy-1,3-benzothiazol-2-amine (1d)
presence of 10% palladium on carbon at 10 bar. The catalyst was Prepared as reported above for 1c starting from 6d. Yield: 65%;
removed by filtration and the residue taken up with EtOAc and brown crystals: mp: 171e172  C (EtOAc/petroleum ether); GC/MS
washed with water. The solvent was removed to give 1.03 g (85%) of (70 eV) m/z (%) 242 (Mþ, 100). Anal. calcd. for C13H10N2OS$0.20H2O
a yellow solid which was recrystallized from EtOAc/petroleum (245.90) %: C, 63.50; H 4.26; N 11.39. Found: C, 63.80; H 3.89; N
ether to give 0.65 g of yellowish crystals: mp 65e66  C; GC/MS 11.41. Other spectroscopic data were in agreement with the litera-
(70 eV) m/z (%) 123 (Mþ, 100). Other spectroscopic data were in ture [13].
agreement with the literature [36].
4.5.3. 6-(Benzyloxy)-1,3-benzothiazol-2-amine (1e)
4.4.1. 4-Benzyloxyaniline (6e) Prepared as reported above for 1c starting from 6e. Yield: 20%;
Prepared as reported above for 5 starting from 3e. Yield: 90%; brown crystals: mp: 152e153  C (EtOAc/petroleum ether); IR (KBr):
dark green solid: mp 68e69  C; IR (KBr): 3355 (NH2) cm1; GC/MS 3436 (NH2) cm1; GC/MS (70 eV) m/z (%) 256 (Mþ, 15), 165 (100); 1H
(70 eV) m/z (%) 199 (Mþ, 19), 108 (100); LC/MS m/z (%): 200 NMR (CDCl3): d 5.09 (s, 2H, CH2), 5.21 (br s, exch D2O, 2H, NH2), 6.98
(Mþ þ H). Other spectroscopic data were in agreement with the (dd, J ¼ 8.8, 2.5 Hz, 1H, Ar benzothiazole), 7.20 (d, J ¼ 2.5 Hz, 1H, Ar
literature [24]. benzothiazole), 7.28e7.48 (m, 6H, Ar); 13C NMR (CDCl3): d 71.1 (1C),
106.9 (1C), 114.8 (1C), 120.0 (1C), 127.7 (2C), 128.2 (1C), 128.8 (2C),
4.4.2. Synthesis of 4-[(4-chlorobenzyl)oxy]aniline (6n) 132.9 (1C), 137.2 (1C), 146.6 (1C), 155.1 (1C), 164.2 (1C). Anal. calcd.
Prepared as reported above for 5 starting from 3n. This reaction for C14H12N2OS$0.33H2O (262.32) %: C, 64.10; H 4.87; N 10.68.
was carried out at room temperature for 20 min in the presence of Found: C, 64.27; H 4.75; N 10.32.
10% palladium on carbon at 3 bar. Yield: 49%; brown crystals: mp
108e109  C (EtOAc/hexane); IR (KBr): 3366 (NH2) cm1; GC/MS 4.5.4. 6-(2-Phenylethoxy)-1,3-benzothiazol-2-amine (1f)
(70 eV) m/z (%) 233 (Mþ, 14), 108 (100); 1H NMR (CDCl3): d 3.44 (br Prepared as reported above for 1c starting from 6f. Prepared as
s, exch D2O, 2H, NH2), 4.95 (s, 2H, CH2), 6.58e6.67 (m, 2H, Ar), reported above for 1c starting from 6f. Yield: 59%; brown crystals:
6.75e6.85 (m, 2H, Ar), 7.34 (s, 4H, Ar). mp 116e117  C (EtOAc/hexane); IR (KBr): 3432 (NH2) cm1; GC/MS
(70 eV) m/z (%) 270 (Mþ, 100); 1H NMR (CDCl3): d 3.10 (t, J ¼ 7.1 Hz,
4.5. General procedure for the synthesis of 6-substituted-2-amino- 2H, CH2eAr), 4.18 (t, J ¼ 7.0 Hz, 2H, CH2eOAr), 5.19 (br s, exch D2O,
1,3-benzothiazoles (1bei) 2H, NH2), 6.90 (dd, J ¼ 8.8, 2.5 Hz, 1H, Ar), 7.12 (d, J ¼ 2.5 Hz, 1H, Ar),
7.20e7.37 (m, 5H, Ar), 7.43 (d, J ¼ 8.8 Hz, 1H, Ar); 13C NMR (CDCl3):
The synthesis of 6-ethoxy-1,3-benzothiazol-2-amine (1c), ob- d 36.1 (1C), 69.8 (1C), 106.6 (1C), 114.6 (1C), 119.9 (1C), 126.7 (1C),
tained following a general procedure for the preparation of ami- 128.7 (2C), 129.2 (2C), 132.8 (1C), 138.4 (1C), 146.3 (1C), 155.1 (1C),
nobenzothiazoles described in the literature [13,14] is described. 164.2 (1C). Anal. calcd. for C15H14N2OS (270.35) %: C, 66.64; H 5.22;
Aniline 6c (1.0 g, 7.36 mmol) and NH4SCN (1.6 g, 21.9 mmol) were N 10.36. Found: C, 66.27; H 5.22; N 10.26.
dissolved in a 20% formic acideglacial acetic acid mixture
(100 mL) and cooled to 3  C with stirring, under N2. With the 4.5.5. 6-(3-Phenylpropoxy)-1,3-benzothiazol-2-amine (1g)
exclusion of light from the reaction mixture, bromine (0.30 mL Prepared as reported above for 1c starting from 6g. Yield: 41%;
dissolved in 20 mL of glacial acetic acid) was added dropwise, gold crystals: mp 126e127  C (EtOAc/hexane); IR (KBr): 3419
while the reaction temperature was kept between 3  C and 0  C. (NH2) cm1; GC/MS (70 eV) m/z (%) 284 (Mþ, 81), 166 (100); 1H
The light shield was removed and the mixture was allowed to NMR (CDCl3): d 2.05e2.20 (m, 2H, CH2CH2CH2), 2.82 (t, J ¼ 7.6 Hz,
warm to room temperature overnight. Sodium hydroxide pellets 2H, CH2eAr), 3.96 (t, J ¼ 6.3 Hz, 2H, CH2eOAr), 5.18 (br s, exch D2O,
and ice were added with stirring until pH 11 was attained, and the 2H, NH2), 6.91 (dd, J ¼ 8.5, 2.6 Hz, 1H, Ar), 7.11 (d, J ¼ 2.5 Hz, 1H, Ar),
mixture was extracted with EtOAc. The organic layer was sepa- 7.15e7.35 (m, 5H, Ar), 7.44 (d, J ¼ 8.5 Hz, 1H, Ar); 13C NMR (CDCl3):
rated and filtered through celite to remove polythiocyanogen d 31.1 (1C), 32.4 (1C), 67.9 (1C), 106.4 (1C), 114.6 (1C), 119.9 (1C),
(SCN)n. The organic layer was then washed with water, saturated 126.2 (1C), 128.8 (4C), 132.8 (1C), 141.7 (1C), 146.3 (1C), 155.3 (1C),
NaHCO3 and brine; then, the solvent was evaporated in vacuo. The 164.1 (1C). Anal. calcd. for C16H16N2OS (284.38) %: C, 67.58; H 5.67;
residue was purified by flash chromatography (EtOAc/petroleum N 9.85. Found: C, 67.52; H 5.60; N 9.81.
 A. Catalano et al. / European Journal of Medicinal Chemistry 64 (2013) 357e364 363

4.5.6. 6-(4-Phenylbutoxy)-1,3-benzothiazol-2-amine (1h) each bacterial strain were prepared in Cation Adjusted Muellere
Prepared as reported above for 1c starting from 6h. Yield: 62%; Hinton broth (CAMHB) and incubated at 37  C until the growth
beige solid: mp 98e99  C; IR (KBr): 3428 (NH2) cm1; GC/MS ceased. The turbidity of bacterial cell suspension was calibrated to
(70 eV) m/z (%) 298 (Mþ, 28), 166 (100); 1H NMR (CDCl3): d 1.70e 0.5 McFarland Standard by spectrophotometric method (625 nm,
1.90 (m, 4H, CH2CH2CH2CH2), 2.65e2.75 (m, 2H, CH2eAr), 3.90e range 0.08e0.10), and further the standardized suspension was
4.05 (m, 2H, CH2eOAr), 5.13 (br s, exch D2O, 2H, NH2), 6.89 (dd, diluted 1:100 with CAMHB to have 1e2  106 CFU/mL. All wells were
J ¼ 8.8, 2.5 Hz, 1H, Ar), 7.11 (d, J ¼ 2.5 Hz, 1H, Ar), 7.14e7.34 (m, 5H, seeded with 100 mL of inoculum. A number of wells containing only
Ar), 7.43 (d, J ¼ 8.8 Hz, 1H, Ar); 13C NMR (CDCl3): d 28.1 (1C), 29.1 inoculated broth as control growth were prepared. The plates were
(1C), 35.8 (1C), 68.7 (1C), 106.3 (1C), 114.5 (1C), 119.9 (1C), 126.0 incubated at 37  C for 24 h, and the MIC values were recorded as the
(1C), 128.6 (2C), 128.7 (2C), 132.9 (1C), 142.4 (1C), 146.3 (1C), 155.3 last well containing no bacterial growth. The MICs were determined
(1C), 164.1 (1C). Anal. calcd. for C17H18N2OS$0.2H2O (302.00) %: C, by using an antibacterial assay repeated twice in triplicate. Nor-
67.61; H 6.14; N 9.28. Found: C, 67.84; H 6.02; N 9.22. floxacin was used as reference drug.

4.5.7. 6-(Phenoxymethyl)-1,3-benzothiazol-2-amine (1i) 4.7. Antifungal studies

Prepared as reported above for 1c starting from 6i. Yield: 46%;
yellow crystals: mp 168e169  C (EtOAc/petroleum ether); IR (KBr): Antifungal studies [16] were carried out against C. albicans
3393 (NH2) cm1; GC/MS (70 eV) m/z (%) 256 (Mþ, 1), 163 (100); 1H 10231, C. parapsilosis 22019, C. tropicalis 750, C. krusei 6258,
NMR (CDCl3): d 5.10 (s, 2H, CH2), 5.27 (br s, exch D2O, 2H, NH2), belonging to the ATCC collection. Preparation of stock solutions and
6.88e7.08 (m, 3H, Ar), 7.22e7.40 (m, 3H, Ar), 7.55 (d, J ¼ 8.3 Hz, 1H, purity of cultures preservation were obtained as above described
Ar), 7.69 (s, 1H, Ar); 13C NMR (CDCl3): d 70.2 (1C), 115.1 (2C), 119.5 for antibacterial studies. Pre-cultures of each yeast strain were
(1C), 120.4 (1C), 121.2 (1C), 126.0 (1C), 129.7 (2C), 131.6 (1C), 132.3 prepared in Sabouraud broth 2% glucose (SAB), and incubated at
(1C), 152.2 (1C), 159.0 (1C), 166.2 (1C). Anal. calcd. for 37  C until the growth ceased. The turbidity of yeast stock sus-
C14H12N2OS$0.5H2O (265.32) %: C, 63.37; H 4.94; N 10.56. Found: C, pension was calibrated to 0.5 McFarland Standard by spectropho-
63.11; H 4.66; N 10.49. tometric method (530 nm, range 0.12e0.15), and further the
standardized suspension was diluted first 1:50 with SAB and then
4.5.8. 6-[(4-Chlorobenzyl)oxy]-1,3-benzothiazol-2-amine (1n) 1:20 in the same medium to have 1e5  106 CFU/mL. All wells were
Prepared as reported above for 1c starting from 6n. Yield: 20%; seeded with 100 mL of inoculum. A number of wells containing only
light pink crystals: mp 199e200  C (EtOAc/petroleum ether); IR inoculated broth as control growth were prepared. The plates were
(KBr): 3439 (NH2) cm1; GC/MS (70 eV) m/z (%) 290 (Mþ, 18), 165 incubated at 37  C for 24e48 h, and the MIC values were recorded
(100); 1H NMR (CDCl3): d 5.03 (s, 2H, CH2), 5.07 (br s, exch D2O, 2H, as the last well containing no fungal growth. The MICs were
NH2), 6.95 (dd, J ¼ 8.8, 2.7 Hz, 1H, Ar), 7.17 (d, J ¼ 2.5 Hz, 1H, Ar), 7.36 determined by using an antifungal assay repeated twice in tripli-
(s, 4H, Ar), 7.45 (d, J ¼ 8.8 Hz, 1H, Ar); 13C NMR (CDCl3): d 69.7 (1C), cates. Fluconazole was used as reference drug.
107.5 (1C), 114.4 (1C), 118.7 (1C), 129.1 (2C), 130.1 (2C), 132.5 (1C),
133.0 (1C), 137.0 (1C), 147.8 (1C), 153.7 (1C), 165.6 (1C). Anal. calcd. 4.8. Molecular modeling studies
for C14H11ClN2OS (290.77) %: C, 57.83; H 3.81; N 9.63. Found: C,
58.16; H 3.77; N 9.70. CLogP were calculated with ACD/ChemSketch v.12.01. Semi-rigid
dockings were carried out by means of AutoDock ver. 4.2 [37] on
4.5.9. 6-(4-Chlorophenoxy)-1,3-benzothiazol-2-amine (1o) the recently published homology models [26]. Blind dockings were
Prepared as reported above for 1c starting from 6o. Yield: 87%; initially carried out to explore the accessibility of the target, and also
slightly green powder: mp 168e169  C (CHCl3/hexane); IR (KBr): to prove the hypothesis of an inhibition of CA-CYP51 activity by the
3377 (NH2) cm1; GC/MS (70 eV) m/z (%) 276 (Mþ, 100); 1H NMR coordination of the iron atom elicited by the benzothiazole moiety of
(CDCl3): d 5.27 (br s, exch D2O, 2H, NH2), 6.91 (d, J ¼ 8.8 Hz, 2H, Ar), the studies compounds. Calculating the affinity maps on the entire
7.0 (dd, J ¼ 8.5, 2.5 Hz, 1H, Ar), 7.15e7.35 (m, 3H, Ar), 7.50 (d, protein surface the most favorable binding pose was, in all the in-
J ¼ 8.5 Hz, 1H, Ar); 13C NMR (CDCl3): d 112.1 (1C), 118.5 (1C), 119.5 stances, exactly inside the active site lodge surrounding the heme
(2C), 120.0 (1C), 128.0 (1C), 129.9 (2C), 132.8 (1C), 148.8 (1C), 152.0 group. Afterward the cytochrome binding site was then defined as a
(1C), 157.1 (1C), 165.7 (1C). Anal. calcd. for C13H9ClN2OS (276.74) %: 60  60  60 cubic box, 0.374  A spaced, centered on the iron atom of
C, 56.42; H 3.28; N 10.12. Found: C, 56.25; H 3.38; N 9.85. the heme group of CA-CYP51. Amber and AM1 charges were used for
protein and ligands respectively. For each compound 200 runs were
4.6. Antibacterial studies carried out, exploring the conformational space with the Lamarckian
Genetic Algorithm (LGA). To increase the docking performance the
The in vitro minimum inhibitory concentrations (MICs, mg/mL) population size and the number of energy evaluations were raised to
were assessed by the broth microdilution method, using 96-well 300 and 5,000,000 in that order, and for the whole docked molecules
plates, according to CLSI guidelines [15]. Stock solutions of the tested the best free energy of binding (FEB) poses were selected. To save
compounds were obtained in DMSO. Stock solutions of lower con- computational time, the lone high active compound 1e was submit-
centrations were prepared for those substances which did not ted to flexible docking carried out exploring the side chains flexibility
dissolve well. Then two-fold serial dilutions in the suitable test of the seven residues comprising the binding site proximity, namely
medium between 512 and 0.5 mg/mL were plated. To be sure that the Tyr118, Leu121, Phe228, His310, Thr311, Leu376 and Met508. The 200
solvent had no adverse effect on bacterial growth, a control test was samplings carried in this way gave results similar to the previous 200
carried out by using DMSO at its maximum concentration along rigid runs, with a RMSD between the selected poses of 0.619.
with the medium. Bacteria strains available as freeze-dried discs,
belonging to the ATCC collection, were used: Gram-positive strains 4.9. Toxicity for eukaryotic cells
such as S. aureus 29213, E. faecalis 29212, and Gram-negative one
such as E. coli 25922. To preserve the purity of cultures and to allow Toxicity was assessed toward THP-1 cells (ATCC TIB-202), a hu-
the reproducibility, a series of criovials of all microbial strains in man myelomonocytic cell line displaying macrophage-like activity
glycerolic medium were set up and stored at 80  C. Pre-cultures of [38]. These cells were maintained as a loose suspension in RPMI 1640
364 A. Catalano et al. / European Journal of Medicinal Chemistry 64 (2013) 357e364

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[11,40]. LDH release was measured with the Roche LDH kit (cyto- [16] CLSI, Reference Method for Broth Dilution Antifungal Susceptibility Testing of
Yeast. Approved standard, third ed., Clinical and Laboratory Standards Insti-
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18427.
the Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR). [29] M.M. Cavalluzzi, C. Bruno, G. Lentini, A. Lovece, A. Catalano, A. Carocci,
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