1,3-Benzothiazoles as Antimicrobial Agents

Article abstract of DOI:10.1002/jhet.2222

Starting from 2-amino-1,3-mercaptobenzothiazoles recently reported (1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h), a series of the corresponding 2-mercapto-1,3-benzothiazole isosters (2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h) were screened for their in vitro antibacterial and antifungal activities. Results underline that the presence of the mercapto moiety at the 2-position of the heterocyclic nucleus is crucial for activity against bacteria. The biological screening against Candida spp. identified commercial 2f as the most promising compound as antifungal against Candida albicans and tropicalis. Molecular modeling studies supported these results. Then, to enlarge structure-activity relationship (SAR) studies on series 1, newly synthesized compounds (1k, 1l, 1m, 1n, 1o, 1p) were reported. All the compounds belonging to this series and bearing a bulky substituent at the 6-position of the aryl moiety showed high antifungal activity.

Full text of DOI:10.1002/jhet.2222

Month 2014
1,3-Benzothiazoles as Antimicrobial Agents
*
*
Ivana Defrenza, Alessia Catalano, Alessia Carocci, Antonio Carrieri, Marilena Muraglia, Antonio Rosato,
Filomena Corbo, and Carlo Franchini
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ‘Aldo Moro’, via E. Orabona n. 4, 70125 Bari, Italy
*
E-mail: alessia.catalano@uniba.it; alessia.carocci@uniba.it
Received November 5, 2013
DOI 10.1002/jhet.2222
Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).
Starting from 2-amino-1,3-mercaptobenzothiazoles recently reported (1a–h), a series of the corresponding
2-mercapto-1,3-benzothiazole isosters (2a–h) were screened for their in vitro antibacterial and antifungal
activities. Results underline that the presence of the mercapto moiety at the 2-position of the heterocyclic nu-
cleus is crucial for activity against bacteria. The biological screening against Candida spp. identified com-
mercial 2f as the most promising compound as antifungal against Candida albicans and tropicalis.
Molecular modeling studies supported these results. Then, to enlarge structure-activity relationship (SAR)
studies on series 1, newly synthesized compounds (1k–p) were reported. All the compounds belonging to this
series and bearing a bulky substituent at the 6-position of the aryl moiety showed high antifungal activity.
J. Heterocyclic Chem., 00, 00 (2014).
INTRODUCTION
The compounds were tested in vitro for their antimicrobial
activities. Results showed that some of these compounds
exerted interesting antifungal activity. The best results were
obtained for compounds bearing a large lipophilic group at
the 6-position of the benzothiazole moiety, such as the
phenoxy and benzyloxy groups. Thus, we decided to investi-
gate the isosteric relationship existing between SH and NH2
moieties and study the corresponding compounds bearing a
2-mercapto-1,3-benzothiazole skeleton (2a–h, Fig. 1). A mo-
lecular modeling study has also been carried out in support of
the results obtained for one of the most active compounds.
Furthermore, in order to improve SAR studies on series 1,
starting from the most active compounds (1i,j) recently
reported [9], six new compounds (1k,p) were synthesized
and tested.
The global diffusion of new antimicrobial infections as
well as the continuously increasing resistance of pathogens
against many of the commonly used antibiotics imposes a
considerable effort to develop alternative therapies to the use
of classical drugs. As part of our ongoing program on antimi-
crobial agents, we focused our attention on the benzothiazole
nucleus, which has been extensively studied and reviewed by
different research groups [1–3]. Benzothiazole derivatives
possess a wide spectrum of biological applications such as
antitumor, schistosomicidal, anti-inflammatory, anticonvul-
sant, antidiabetic, antipsychotic, diuretic, neuroprotective,
and antimicrobial activities [4–6]. Several methods have been
proposed for the synthesis of benzothiazoles [7,8]. For some
years, we are involved in the study of 1,3-benzothiazoles as
antimicrobials [9]. We recently studied two different series
of 1,3-benzothiazoles both as antifungals against Candida
spp. [10] and antibacterials, in particular against Enterococcus
faecalis [11]. In the last year, we determined for the first time
the X-ray crystallographic structure of E. faecalis thymidylate
synthase, which should be an interesting target for antibacte-
rials [12]. This work takes origin from the series of 2-amino-
1,3-benzothiazoles (1a–h, Fig. 1) recently described [10].
RESULTS AND DISCUSSION
Chemistry. Compounds 2b–d were prepared as reported
in Scheme 1. Aniline 4b was commercial, whereas anilines
4c,d were prepared by bromination of anilines 3c,d by
using tetrabutylammonium tribromide (Bu4NBr3) [13].
Anilines 4b–d were then reacted with potassium ethyl
© 2014 HeteroCorporation

I. Defrenza, A. Catalano, A. Carocci, A. Carrieri, M. Muraglia, A. Rosato, F. Corbo, and C. Franchini Vol 000
faecalis. Out of the three new compounds of series 2, only
2b, which bears a small substituent at the 6-position of the
benzothiazole nucleus showed a slight activity against Gram-
positive and in particular against S. aureus. The introduction
of a large lipophilic group, as the phenoxy or benzyloxy
Figure 1. Structures of 6-substituted 1,3-benzothiazole derivatives (1 and 2).
one, as in 2c,d was detrimental for antibacterial activity
and brought to a loss of it in all the bacterial strains tested.
xanthate [9,14] to give the corresponding 6-substituted-1,3-
benzothiazole-2-thiols 2b–d. Compounds 2a,e–h were
prepared as previously described [9]. Compounds 1k,m,o
were prepared as reported in Scheme 2. Benzyl alcohols
5k,m,o were reacted with 4-nitrophenol under Mitsunobu
conditions [15–17] to give their nitro derivatives 6k,m,o,
which were reduced by catalytic hydrogenation [18] to
anilines 7k,m,o. 2-Aminobenzothiazole derivatives 1k,m,o
were prepared via thiocyanation of 7k,m,o [19]. In this
reaction, ammonium thiocyanate and bromine were used to
generate thiocyanogen in situ [20]. 2-Aminobenzothiazole
derivatives 1l,n,p were prepared as reported for 1k,m,o
(Scheme 3). All new synthesized compounds were fully
characterized by routine spectral analyses (IR, NMR, and
mass spectra).
Antifungal studies. Compounds 1k–p and 2a–h were
screened, according to CLSI guidelines [22], against a
panel of fungi strains (Candida albicans 10231, Candida
parapsilosis 22019, Candida tropicalis 750, Candida
krusei 6258) belonging to the ATCC collection.
Fluconazole was used as reference drug. The results,
expressed as MIC (μg/mL), are listed in Table 2. Also, in
this case, results for compounds of series 1 (1a–j)
recently reported [10] are listed for comparison. All the
new tested compounds of series 2 showed higher activity
than the corresponding amino isosteres (1a–h) with the
exception of compounds 2c,d, which were inactive.
Conceivably, the presence of a bulky group at 6-position of
the benzothiazole moiety is not well tolerated in the active
site. Among the tested compounds, 2f was the most active,
showing MIC values of 4 and 8 μg/mL against C. albicans
and tropicalis, respectively. All the new tested compounds
of series 1 (1k–p) showed interesting antifungal activity
confirming the importance of a bulky group at the 6-position
of the 2-amino-1,3-benzothiazole nucleus. Position and
electronic properties of the substituent on the aromatic
moiety seems to be not crucial for determining any variation
in activity.
Biological evaluation.
Antibacterial studies. According to the Clinical Laboratory
Standard Institutes (CLSI) guidelines [21], the newly
synthesized compounds 1k–p and 2b–d were tested against
Gram-positive and Gram-negative bacteria belonging to the
ATCC collection (Staphylococcus aureus 29213, E. faecalis
29212, Escherichia coli 25922) using oxacillin and
norfloxacin as reference drugs. The results, expressed as
Minimum Inhibitory Concentration (MIC) (μg/mL), are
reported in Table 1. As a comparison, results for 1a–j and
2a,e–h previously reported [9,10] are listed too. None of the
Molecular modeling studies.
A molecular modeling
study was carried out to better perceive and evaluate the
biological profile as antifungal of the most active
compound of the series under study (2f). To fulfill this
goal, the cytochrome P450-dependent lanosterol 14α-
demethylase (CYP51) of C. albicans was elicited as the
newly synthesized compounds of series
1 showed
improvement in antibacterial activity with respect to those
previously reported, with the only exception of compounds
1k,m,o, which showed a MIC value of 64 μg/mL against E.
Scheme 1. Reagents and conditions: (i) Bu4NBr3, CH2Cl2/MeOH, rt; (ii) potassium ethyl xanthate, DMF, reflux.
Scheme 2. Synthesis of 2-amino-1,3-benzothiazole derivatives 1k,m,o.
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Scheme 3. Synthesis of 2-amino-1,3-benzothiazole derivatives 1l,n,p.
congeners. The heterocyclic system of the most potent
antifungal agent is indeed anchored nearly perpendicular
to the heme group of CA-CYP51, by means of a
coordination bond involving the iron atom and the sulfur
in its thione tautomeric form, as it might be perceived
from the docking 2f depicted in Figure 2. As a figure of
merit, a similar type of coordination has been proved by
studies carried out on the basis of analytical, spectral (IR
and VIS), and magnetic data [27]. At the same time, the
chlorine atom at 6-position of the benzothiazole moiety
reinforces the binding with favorable Van der Waals
interactions engaged with the residues at the boundary of
a reduced accessible surface of the active site lodge, shaped
and delimited by residues, namely, Tyr118, Leu121,
Phe228, His310, Met508, and Val509. Thus, this evidence
facilitates then the positioning of the apolar and less steric
hindered substituent into this most likely hydrophobic
dome placed above the heme group. The same kind of
favorable interactions pattern might also be ascribed to
compounds with very small groups (i.e., 2e) but not to
primary target capable to explain the antifungal activity of
our derivatives through docking experiments. This
cytochrome is in fact targeted by antifungals, such as
azoles (i.e., fluconazole and miconazole), and also by
drugs (i.e., benzothiazines and benzoxazine) responsible
for the cell growth inhibition due to ergosterol depletion
determined by CYP51 blockade [23–25]. As long as this
evidence is concerned, compound 2f was therefore
docked into the catalytic site of the homology-based
model of C. albicans CYP51, which was successfully
used in a similar study on structurally related antifungal
agents [10,26]. Dockings binding poses some
highlighted, interesting features that might be in charge
of the antifungal activity of 2f and also of the synthesized
Table 1
Antibacterial activity results (MIC, μg/mL).
Microorganism (MIC, μg/mL)a
Gram-positive
Gram-negative
E.c. 25922
>512
512
Compound
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
2a
X
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
S
R
S.a. 29213
>512
256
>512
>512
256
>512
>512
128
512
256
256
>512
64
E.f. 29212
>512
512
H
CF3O
Ph–O
Bn–O
CF3
Cl
>512
>512
256
>512
>512
128
512
256
64
256
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
50
F
Me-O
4-Cl-Bn-O
4-Cl-Ph-O
2-Cl-Bn-O
2–Cl–Ph–O
4–Me–Bn–O
4–Me–Ph–O
2–Me–Bn–O
2–Me–Ph–O
H
64
256
64
256
>512
256
>512
50
100
2b
2c
2d
2e
2f
2g
2h
S
S
S
S
S
S
S
CF3O
Ph–O
Bn–O
CF3
Cl
F
Me–O
64
128
>512
>512
>512
>512
>512
>512
100
>512
>512
3.12
12.5
25
>512
>512
100
100
>512
50
50
OXA
NRF
—
—
—
—
0.25
0.5
16
4
>512
0.03
Data for compounds 1a–j were taken from ref. [10]; data for compounds 2a,e–h were taken from ref. [9]
S.a., Staphylococcus aureus; E.f., Enterococcus faecalis; E.c., Escherichia coli; OXA, oxacillin; NRF, norfloxacin.
aAntibacterial activity was estimated by using CLSI assay [21].
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I. Defrenza, A. Catalano, A. Carocci, A. Carrieri, M. Muraglia, A. Rosato, F. Corbo, and C. Franchini Vol 000
Table 2
Antifungal activity results (MIC, μg/mL).
Microorganism (MIC, μg/mL)a
Compound
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
2a
2b
2c
2d
2e
2f
2g
2h
FCN
X
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
S
S
S
S
S
S
S
S
—
R
C.a. 10231
C.p. 22019
C.t. 750
>512
128
32
C.k. 6258
>512
128
32
H
>512
64
128
8
16
64
256
256
4
>512
64
16
8
64
64
128
256
8
CF3O
Ph–O
Bn–O
CF3
Cl
8
64
64
256
256
4
8
8
8
8
8
16
16
32
16
64
64
128
256
512
64
32
16
4
16
F
Me–O
4–Cl–Bn–O
4–Cl–Ph–O
2–Cl–Bn–O
2–Cl–Ph–O
4–Me–Bn–O
4–Me–Ph–O
2–Me–Bn–O
2–Me–Ph–O
H
8
8
4
16
8
4
8
8
4
8
16
8
8
16
8
32
16
4
32
32
>512
>512
16
4
32
32
>512
>512
64
32
64
64
2
CF3O
Ph–O
Bn–O
CF3
Cl
F
Me–O
—
32
>512
>512
16
>512
>512
32
8
64
64
4
16
32
64
32
32
32
2
Data for compounds 1a–j were taken from ref. [10].
C.a., Candida albicans; C.p., Candida parapsilosis; C.t., Candida tropicalis; C.k., Candida krusei; FCN, fluconazole.
aAntifungal activity was estimated by using CLSI assay [22].
those bearing bulky pendants (i.e., 2c,d) not being capable to
straightforwardly explore the limited hydrophobic dome,
resulting then with a lower antifungal potency. As
previously assessed, also, dockings highlight the need of
tiny substituents as mandatory chemical cliché for
enhanced antifungal activity in this series of compounds.
the contrary, in series 2, small groups at the same position
(2e,f) are preferable to bulky ones (2c,d), and molecular
modeling study furnished a plausible interpretation of this
evidence. It is noteworthy that, among all the compounds
tested, 2-mercapto compound 2f was particularly interesting
as antifungal, showing MIC values of 4 and 8 μg/mL against
C. albicans and tropicalis, respectively.
CONCLUSIONS
EXPERIMENTAL
Chemistry.
A number of 2-amino-1,3-benzothiazole (1a–p) and 2-
mercapto-1,3-benzothiazole derivatives (2a–h) were screened
for their in vitro antibacterial and antifungal activities. All the
new compounds described in this paper were completely
characterized. Results showed that antibacterial activity, in
particular against S. aureus, is peculiar for compounds
belonging to series 2, thus corroborating the finding that
SH group is crucial for antibacterial activity [9]. Both series
of compounds 1 and 2 showed different levels of antifungal
activity, depending on the substitution at position 6 of the
benzothiazole moiety: in series 1, the presence of bulky
substituents confers higher antifungal activity (1d,i–p); on
General.
Chemicals were purchased from Sigma-Aldrich
(Italy) or Lancaster (Lancaster Synthesis Ltd., White Lund
Industrial Estate, Lancashire, UK). Yields refer to purified
products and were not optimized. The structures of the
compounds were confirmed by routine spectrometric and
spectroscopic analyses. Only spectra for compounds not
previously described are given. Anilines 3c,d, 4b, 1l,n,p, and
benzothiazoles 2a,e–h were commercial or prepared as
previously described [9,10]. Melting points were determined on a
Gallenkamp apparatus (Weiss-Gallenkamp, London, UK) in open
glass capillary tubes and are uncorrected. Infrared spectra (IR)
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residue was purified by column chromatography (EtOAc/petroleum
ether 1:9) to give 0.43 g (61%) of a brown oil: IR (neat): 3464,
1
3375 cmÀ1; H NMR: δ 3.97 (br s, exch. D2O, 2H, NH2), 6.75 (d,
J= 8.8 Hz, 1H, Ar), 6.86 (dd, J= 8.7, 2.6 Hz, overlapped to d at
6.94, J= 7.7 Hz, 3H, Ar), 7.05 (t, J= 7.4 Hz, 1H, Ar), 7.16 (d,
J= 2.5 Hz, 1H, Ar), 7.30 (t, J= 8.0 Hz, 2H, Ar); 13C NMR:
δ = 109.4 (1C), 116.5 (1C), 117.7 (2C), 120.6 (1C), 122.8 (1C),
124.3 (1C), 129.9 (2C), 140.8 (1C), 148.8 (1C), 158.6 (1C); GC-
MS (70 eV electron impact) m/z (%) 263 (M+, 100).
4-(Benzyloxy)-2-bromoaniline (4d).
Yield 26%; brown
1H NMR: δ
solid: mp 62–63°C; IR (KBr): 3416, 3310 cmÀ1
;
3.80 (br s, 2H, NH2), 4.97 (s, 2H, CH2), 6.72 (d, J = 8.8 Hz, 1H,
Ar), 6.80 (dd, J = 8.8, 2.7 Hz, 1H, Ar), 7.10 (d, J = 2.7 Hz, 1H,
Ar), 7.28–7.44 (m, 5H, Ar); 13C NMR: δ 71.2 (1C), 109.8 (1C),
116.3 (1C), 116.8 (1C), 119.2 (1C), 127.7 (2C), 128.2 (1C),
128.8 (2C), 137.2 (1C), 138.5 (1C), 152.1 (1C); GC-MS (70 eV
electron impact) m/z (%) 277 (M+, 20), 91 (100).
General procedure for the synthesis of 6-substituted-1,3-
benzothiazole-2-thiols (2b–d).
The method adopted for the
synthesis of 6-(trifluoromethoxy)-1,3-benzothiazole-2-thiol (2b) is
described. To a solution of aniline 4b (0.84 g, 3.3 mmol) in DMF
(12 mL), potassium ethyl xanthate (1.57 g, 9.8 mmol) was added
and brought to reflux. After 1 h, the solvent was evaporated and the
residue was taken up with EtOAc and extracted with 6 N HCl. The
organic layer was washed with water, then dried, and the solvent
evaporated to give an orange solid in quantitative yield: mp 224–
1
225°C; IR (KBr): 3107, 3055, 2937, 2868, 1493, 1476 cmÀ1; H
NMR (DMSO-d6): δ 7.30–7.45 (m, 2H, Ar), 7.82 (s, 1H, Ar), 13.8
(br s, 1H, SH); 13C NMR (DMSO-d6): δ 113.9 (1C), 115.8 (1C),
120.7 (br q, JCF = 234 Hz, 1C), 121.3 (1C), 131.5 (1C), 141.1 (1C),
145.4 (1C), 191.5 (1C); GC-MS (70 eV electron impact) m/z (%)
251 (M+, 100). Anal. Calcd. for C8H4F3NOS2 (251.25): C, 38.24;
H, 1.60; N, 5.57. Found: C, 38.13; H, 1.99; N, 5.39.
Figure 2. Docking poses for 2f in the active site of CA-CYP51. Ligand and
iron atom are displayed as ball and stick to help interpretation. [Color figure can
be viewed in the online issue, which is available at wileyonlinelibrary.com.]
were recorded on a Perkin-Elmer (Norwalk, CT) Spectrum One FT
spectrophotometer and band positions are given in reciprocal
centimeters (cmÀ1). 1H NMR and 13C NMR spectra were
recorded on a Varian VX Mercury spectrometer (Varian Inc.,
Palo Alto, CA) operating at 300 and 75MHz for 1H and 13C,
respectively, using CDCl3 and DMSO-d6 (where indicated) as
solvents. Chemical shifts are reported in parts per million (ppm)
relative to the residual non-deuterated solvent resonance: CDCl3, δ
7.26 (1H NMR) and δ 77.3 (13C NMR); DMSO-d6, δ 2.48 (1H
NMR) and δ 40.1 (13C NMR). J values are given in hertz. GC-MS
6-Phenoxy-1,3-benzothiazole-2-thiol (2c). Yield 45%; off-
white solid: mp 119–120°C (EtOAc/petroleum ether); IR (KBr):
1
3100, 3051, 2917, 2850, 1487, 1469 cm–1; H NMR (DMSO-d6):
δ 6.95–7.18 (m, 4H, Ar), 7.25–7.48 (m, 4H, Ar), 13.7 (br s, 1H,
SH); 13C NMR (DMSO-d6): δ 112.9 (1C), 119.0 (2C), 119.4
(1C), 124.2 (2C), 130.8 (2C), 131.7 (1C), 154.1 (2C), 157.6
(2C); GC-MS (70eV electron impact) m/z (%) 259 (M+, 100).
Anal. Calcd. for C13H9NOS.20.17H2O (259.35): C, 59.52; H,
3.59; N, 5.34. Found: C, 59.78; H, 3.55; N, 5.47.
was performed on
a Hewlett–Packard 6890–5973 MSD
6-(Benzyloxy)-1,3-benzothiazole-2-thiol (2d). Yield 29%;
light brown solid: mp 197–198°C; IR (KBr): 3098, 3027, 2928,
(Hewlett-Packard, Palo Alto, CA) at low resolution. Liquid
chromatography/mass spectroscopy (LC-MS) was performed on a
spectrometer Agilent 1100 series LC-MSD Trap System VL (VL
Work station, Agilent, Palo Alto, CA). Elemental analyses were
1
2873, 1483 cmÀ1; H NMR (DMSO-d6): δ 5.08 (s, 2H, CH2), 7.05
(dd, J= 8.8, 2.5 Hz, 1H, Ar), 7.15–7.50 (m, 7H, Ar); GC-MS (70 eV
electron impact) m/z (%): 273 (M+, 2), 91 (100); LC-MS m/z (%)
272 (M+ – 1). Anal. Calcd. for C14H11NOS2. 0.17H2O (276.37):
C, 60.84; H, 4.13; N, 5.07. Found: C, 60.97; H, 4.14; N 5.12.
General procedure for the synthesis of 4-nitrophenyl ethers
(6k,m,o). The method adopted for the synthesis of 2-chlorobenzyl-
4-nitrophenyl ether (6k) is described. A solution of DIAD (6.40 g,
31.6 mmol) in dry THF (80 mL) was added dropwise to a solution of
benzyl alcohol (5k) (3.0 g, 21.0 mmol), 4-nitrophenol (4.39 g,
31.6 mmol), and triphenylphosphine (8.27 g, 31.6 mmol) in dry THF
(70 mL) under N2 atmosphere at room temperature. The reaction
mixture was stirred overnight and then concentrated in vacuo. Et2O
was added to the residue and the solid filtered off. The filtrate was
evaporated and the residue was purified by column chromatography
on silica gel (EtOAc/petroleum ether, 2:8) to give 1.5 g of an off-white
solid, which was recrystallized from EtOAc/petroleum ether to give
performed on
a
Eurovector Euro EA 3000 analyzer.
Chromatographic separations were performed on silica gel
columns by column chromatography on silica gel (Kieselgel 60,
0.040–0.063mm, Merck, Darmstadt, Germany) as previously
described [28–31]. TLC analyses were performed on precoated
silica gel on aluminum sheets (Kieselgel 60F254, Merck).
General procedure for the synthesis of 2-bromo-4-aryloxyanilines
(4c,d).
The method adopted for the synthesis of 2-bromo-4-
phenoxyaniline (4c) is described. To a solution of aniline 3c (0.5 g,
2.7 mmol) in CH2Cl2 (13 mL) and MeOH (7 mL) was added
Bu4NBr3 (1.2 g, 2.5 mmol), and the mixture was stirred at room
temperature for 5 h. Et2O (8 mL) and Na2SO3 saturated solution
(15 ml) were added and the phases were separated. The organic
phase was washed with water, dried (Na2SO4), and filtered. The
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mixture was allowed to warm to room temperature overnight.
Sodium hydroxide pellets and ice were added with stirring until
pH 11 was attained, and the mixture was extracted with EtOAc.
The organic layer was separated and filtered through celite to
remove polythiocyanogen (SCN)n. The organic layer was then
washed with water, saturated NaHCO3 aqueus solution and brine;
then, the solvent was evaporated in vacuo. The residue was purified
by flash chromatography (EtOAc/petroleum ether, 1:1) to give
0.41 g (66%) of dark brown solid, which was recrystallized from
THF/petroleum ether to give 0.12 g of dark brown crystals: mp
163–164°C; IR (KBr): 3457, 3440, 3071, 1635, 1544, 1471, 1451,
1264, 1227 cmÀ1; 1H NMR (DMSO-d6): δ 5.10 (s, 2H, CH2), 6.88
(dd, J= 8.8, 2.2 Hz, 1H, Ar), 7.16–7.28 (m, 3H, exch D2O, 2H,
NH2, Ar), 7.30–7.42 (m, 3H, Ar), 7.44–7.52 (m, 1H, Ar), 7.54–
7.64 (m, 1H, Ar); 13C NMR (DMSO-d6): δ 68.2 (1C), 107.5 (1C),
114.3 (1C), 118.8 (1C), 128.0 (1C), 130.1 (1C), 130.4 (1C), 130.7
(1C), 132.6 (1C), 133.2 (1C), 135.2 (1C), 148.0 (1C), 153.8 (1C),
165.7 (1C); GC-MS (70 eV electron impact) m/z (%) 290 (M+, 15),
165 (100). Anal. Calcd. for C14H11ClN2OS (290.77): C, 57.83; H,
3.81; N, 9.63. Found: C, 57.52; H, 3.87; N, 9.56.
1.24 of off-white crystals: mp 101–102°C (EtOAc/petroleum ether); IR
(KBr): 1588, 1505, 1342, 1259 cmÀ1; 1H NMR (CDCl3): δ 5.26 (s, 2H,
CH2), 7.05 (d, J= 9.1 Hz, 2H, Ar), 7.26–7.36 (m, 2H, Ar), 7.38–7.54
(m, 2H, Ar), 8.22 (d, J= 9.3 Hz, 2H, Ar); GC-MS (70 eV electron
impact) m/z (%) 263 (M+, 5), 125 (100).
4-Methylbenzyl 4-nitrophenyl ether (6m).
Yield 60%;
off-white crystals: mp 108–109°C (EtOAc/petroleum ether); IR
1
(KBr): 1591, 1505, 1339, 1255 cmÀ1; H NMR (CDCl3): δ 2.37
(s, 3H, CH3), 5.12 (s, 2H, CH2), 7.02 (d, J = 9.3 Hz, 2H, Ar),
7.15–7.38 (m, 4H, Ar), 8.19 (d, J = 9.3 Hz, 2H, Ar); GC-MS
(70 eV electron impact) m/z (%) 243 (M+, <1), 105 (100).
2-Methylbenzyl 4-nitrophenyl ether (6o). Yield 83%; off-
white crystals: mp 92–93°C (EtOAc/petroleum ether); IR (KBr):
1
1594, 1505, 1339 1255 cm–1; H NMR (CDCl3): δ 2.38 (s, 3H,
CH3), 5.14 (s, 2H, CH2), 7.05 (d, J = 9.3 Hz, 2H, Ar), 7.20–7.35
(m, 3H, Ar), 7.38 (d, J= 7.4 Hz, 1H, Ar), 8.22 (d, J =9.3Hz, 2H,
Ar); GC-MS (70 eV electron impact) m/z (%) 243 (M+, 1), 105 (100).
General procedure for the synthesis of 4-substituted
benzyloxyanilines (7k,m,o).
The method adopted for the
synthesis of 4-[(2-chlorobenzyl)oxy]aniline (7k) is described.
Catalytic hydrogenation of 6k (0.05 g, 0.19 mmol) in 4 mL of
mixture of MeOH and absolute EtOH (3/1) was conducted at
room temperature for 45 min in the presence of 10% palladium
on carbon at 10 bar. The catalyst was removed by filtration and
the residue was taken up with EtOAc and washed with water.
The solvent was removed to give 0.04 g (90%) of a dark brown
6-[(4-Methylbenzyl)oxy]-1,3-benzothiazol-2-amine (1m).
Yield 80%; slightly yellowish crystals: mp 180–181°C (CH2Cl2);
IR (KBr): 3439, 3288, 3050, 1638, 1603, 1541, 1516, 1457,
1
1274, 1204 cmÀ1; H NMR (CDCl3): δ 2.36 (s, 3H, CH3), 5.02
(s, 2H, CH2), 5.22 (br s, exch D2O, 2H, NH2), 6.97 (dd, J = 8.8,
2.5 Hz, 1H, Ar), 7.15–7.24 (m, 3H, Ar), 7.32 (d, J = 8.0 Hz, 2H,
Ar), 7.44 (d, J = 8.8, 1H, Ar); 13C NMR (CDCl3): δ 21.4 (1C),
71.0 (1C), 106.9 (1C), 114.8 (1C), 119.9 (1C), 127.9 (2C), 129.5
(2C), 132.7 (1C), 134.1 (1C), 138.0 (1C), 146.4 (1C), 155.1
(1C), 164.3 (1C); GC-MS (70 eV electron impact) m/z (%) 270
(M+, 20), 105 (100). Anal. Calcd. for C15H14N2OS (270.35): C,
66.64; H, 5.22; N, 10.36. Found: C, 66.18; H, 5.18; N, 10.26.
6-[(2-Methylbenzyl)oxy]-1,3-benzothiazol-2-amine (1o). Yield
72%; beige crystals: mp 154–155°C (THF/petroleum ether); IR
(KBr): 3444, 3294, 3077, 1644, 1598, 1538, 1463, 1456, 1225,
oil: IR (neat): 3432, 3358, 3219, 1509, 1233 cmÀ1 1H NMR
;
(CDCl3): δ 3.38 (br s, exch D2O, 2H, NH2), 5.10 (s, 2H, CH2),
6.64–6.72 (m, 2H, Ar), 6.78–6.88 (m, 2H, Ar), 7.22–7.32 (m,
3H, Ar), 7.34–7.42 (m, 1H, Ar), 7.52–7.58 (m, 1H, Ar); GC-
MS (70 eV electron impact) m/z (%) 233 (M+, 12), 108 (100).
1H NMR (CDCl3 + D2O): δ 5.09 (s, 2H, CH2), 6.60–6.70 (m,
2H, Ar), 6.78–6.88 (m, 2H, Ar), 7.20–7.30 (m, 3H, Ar), 7.35–
7.42 (m, 1H, Ar), 7.50–7.60 (m, 1H, Ar); GC-MS (70 eV
electron impact) m/z (%) 233 (M+, 12), 108 (100).
1
1210 cmÀ1; H NMR (CDCl3): δ 2.38 (s, 3H, CH3), 5.03 (s, 2H,
4-[(4-Methylbenzyl)oxy]aniline (7m).
The reaction was
CH2), 5.30 (br s, exch D2O, 2H, NH2), 6.95–7.05 (m, 1H, Ar),
7.15–7.30 (m, 4H, Ar), 7.35–7.50 (m, 2H, Ar); 13C NMR (CDCl3):
δ 19.1 (1C), 69.7 (1C), 106.8 (1C), 114.7 (1C), 119.8 (1C), 126.3
(1C), 128.6 (1C), 128.9 (1C), 130.7 (1C), 132.6 (1C), 135.0 (1C),
136.9 (1C), 146.3 (1C), 155.2 (1C), 164.5 (1C); GC-MS (70 eV
electron impact) m/z (%) 270 (M+, 44), 105 (100). Anal. Calcd. for
C15H14N2OS.0.50H2O (279.36): C, 66.49; H, 5.41; N, 10.03.
Found: C, 64.49; H, 5.20; N, 10.05.
carried out at room temperature for 30 min in the presence of
5% palladium on carbon at 5 bar. Yield: quantitative; dark
brown crystals: mp 106–107°C; IR (KBr): 3448, 3360 1509,
1
1229, 1217 cmÀ1; H NMR (CDCl3): δ 2.36 (s, 3H, CH3), 3.50
(br s, exch D2O, 2H, NH2), 4.95 (s, 2H, CH2), 6.60–6.70 (m,
2H, Ar), 6.75–6.90 (m, 2H, Ar), 7.18 (d, J = 7.7 Hz, 2H, Ar),
7.31 (d, J = 8.0Hz, 2H, Ar); GC-MS (70 eV electron impact) m/z
(%) 213 (M+, <1), 105 (100).
6-(2-Chlorophenoxy)-1,3-benzothiazol-2-amine (1l). Yield
57%; slightly brown crystals: mp 172–173°C (EtOAc/petroleum
ether); IR (KBr): 3420, 3249, 3056, 1622, 1584, 1549, 1530,
4-[(2-Methylbenzyl)oxy]aniline (7o).
The reaction was
carried out at room temperature for 30 min in the presence of
10% palladium on carbon at 5 bar. Yield 91%; dark brown oil;
1456, 1252, 1230cmÀ1 1H NMR (CDCl3): δ 5.41 (br s, exch
;
1
IR (neat): 3685, 3621, 3020, 2400, 1511, 1215 cmÀ1; H NMR
D2O, 2H, NH2), 6.90–7.10 (m, 3H, Ar), 7.15–7.25 (m, 2H, Ar),
7.40–7.55 (m, 2H, Ar); 13C NMR (CDCl3): δ 111.2 (1C), 117.7
(1C), 120.0 (1C), 120.1 (1C), 124.5 (1C), 125.4 (1C), 128.1 (1C),
131.0 (1C), 132.7 (1C), 148.4 (1C), 152.4 (1C), 153.5 (1C), 165.5
(1C); GC-MS (70eV electron impact) m/z (%) 276 (M+, 100).
Anal. Calcd. for C13H9ClN2OS (276.74): C, 56.42; H, 3.28; N,
10.12. Found: C, 56.28; H, 3.26; N, 10.04.
(CDCl3): δ 2.37 (s, 3H, CH3), 3.23 (br s, exch D2O, 2H, NH2),
4.96 (s, 2H, CH2), 6.62–6.74 (m, 2H, Ar), 6.75–6.90 (m, 2H,
Ar), 7.15–7.30 (m, 3H, Ar), 7.39 (d, J = 7.1 Hz, 1H, Ar); GC-
MS (70 eV electron impact) m/z (%) 213 (M+, 34), 108 (100).
General procedure for the synthesis of (1k–p).
The
synthesis of 6-[(2-chlorobenzyl)oxy]-1,3-benzothiazol-2-amine
(1k) is described. Aniline 7k (0.50 g, 2.15 mmol) and NH4SCN
(0.57 g, 7.5 mmol) was dissolved in a 20% formic acid/glacial
acetic acid mixture (50 mL) and cooled to À3°C with stirring,
under N2. With the exclusion of light from the reaction mixture,
bromine (0.17 mL dissolved in 10 mL of glacial acetic acid) was
added dropwise, whereas the reaction temperature was kept
between À3°C and 0°C. The light shield was removed and the
6-(4-Methylphenoxy)-1,3-benzothiazol-2-amine (1n). Yield
38%; beige crystals: mp 166–167°C (EtOAc/petroleum ether); IR
(KBr): 3423, 3284, 3079, 1640, 1599, 1541, 1505, 1455, 1259,
1
1216, 1206 cmÀ1; H NMR (CDCl3): δ 2.33 (s, 3H, CH3), 5.35
(br s, exch D2O, 2H, NH2), 6.85–6.95 (m, 2H, Ar), 7.00 (dd,
J = 8.8, 2.5 Hz, 1H, Ar), 7.12 (d, J = 8.8 Hz, 2H, Ar), 7.20 (d,
J = 2.5Hz, 1H, Ar), 7.47 (d, J = 8.8 Hz, 1H, Ar); 13C NMR
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2014
Search for New Antimicrobials
(CDCl3): δ 20.9 (1C), 111.4 (1C), 118.1 (1C), 118.7 (2C), 119.9
(1C), 130.5 (2C), 132.6 (1C), 132.9 (1C), 147.9 (1C), 153.3 (1C),
155.8 (1C), 165.3 (1C); GC-MS (70 eV electron impact) m/z (%)
256 (M+, 100), 165 (31). Anal. Calcd. for C14H12N2OS.0.2H2O
(259.93): C, 64.69; H, 4.81; N, 10.78. Found: C, 64.73; H, 4.70;
N, 10.77.
plates were incubated at 37°C for 24–48 h, and the MIC values
were recorded as the last well containing no fungal growth. The
MIC was determined by using an antifungal assay repeated
twice in triplicates. Fluconazole was used as reference drug.
Molecular modeling studies.
Semi-rigid dockings were
carried out by means of AutoDock ver. 4.2 [32] on the recently
published homology models [33]. Blind dockings were initially
carried out to explore the accessibility of the target and also to
prove the hypothesis of an inhibition of CA-CYP51 activity by
the coordination of the iron atom elicited by the benzothiazole
moiety of the studied compound. Calculating the affinity maps
on the entire protein surface, the most favorable binding pose
was, in all the instances, exactly inside the active site lodge
surrounding the heme group. Afterwards, the cytochrome
binding site was defined as a 50 × 50 × 50 cubic box, 0.374 Å
spaced, comprising the residues of the active site lodge above
the heme plane (see Results and Discussion). AMBER and
OPLS005 charges were used for protein and 2f, respectively.
For each compound, 200 runs were carried out, exploring the
conformational space with the Lamarckian genetic algorithm.
To increase the docking performance, the population size and
the number of energy evaluations were raised to 300 and
5,000,000 in that order, and a unique pose endowing a free
energy of binding of À4.68 kcal/mol was detected.
6-(2-Methylphenoxy)-1,3-benzothiazol-2-amine (1p). Yield
92%; yellow crystals: mp 146–147°C (CHCl3/hexane); IR (KBr):
3430, 3256, 3055, 1626, 1584, 1553, 1551, 1457, 1248, 1224,
1
1194cmÀ1; H NMR (CDCl3): δ 2.27 (s, 3H, CH3), 5.36 (br s,
exch D2O, 2H, NH2), 6.86 (d, J = 8.0Hz, 1H, Ar), 6.90–7.0 (m,
1H, Ar), 7.05 (t, J = 7.4Hz, 1H, Ar), 7.10–7.20 (m, 2H, Ar), 7.25
(d, J = 7.4 Hz, 1H, Ar), 7.46 (d, J = 8.8 Hz, 1H, Ar); 13C NMR
(CDCl3): δ 16.5 (1C), 110.2 (1C), 117.2 (1C), 119.2 (1C), 119.9
(1C), 123.9 (1C), 127.4 (1C), 129.8 (1C), 131.7 (1C), 132.7 (1C),
147.6 (1C), 153.6 (1C), 155.4 (1C), 165.2 (1C); GC-MS (70eV
electron impact) m/z (%) 256 (M+, 100). Anal. Calcd. for
C14H12N2OS.0.33H2O (262.33): C, 64.10; H, 4.87; N, 10.68.
Found: C, 64.31; H, 4.58; N, 10.74.
Antibacterial studies.
The in vitro minimum inhibitory
concentrations (MICs, μg/mL) were assessed by the broth
microdilution method, using 96-well plates, according to CLSI
guidelines [21]. Stock solutions of the tested compounds were
obtained in DMSO. Stock solutions of lower concentrations were
prepared for those substances, which did not dissolve well. Then
twofold serial dilutions in the suitable test medium between 512 and
0.5 μg/mL were plated. To be sure that the solvent had no adverse
effect on bacterial growth, a control test was carried out by using
DMSO at its maximum concentration along with the medium.
Bacteria strains available as freeze-dried discs, belonging to the
ATCC collection, were used, Gram-positive strains 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 the
reproducibility, a series of criovials of all microbial strains in
glycerolic medium were set up and stored at À80°C. Pre-cultures of
each bacterial strain were prepared in Cation Adjusted Mueller-
Hinton broth (CAMHB) and incubated at 37°C until the growth
ceased. The turbidity of bacterial cell suspension was calibrated to
0.5 McFarland Standard by spectrophotometric method (625 nm,
range 0.08–0.10), and further, the standardized suspension was
diluted 1:100 with CAMHB to have 1–2×106 CFU/mL. All wells
were seeded with 100 μL of inoculum. A number of wells containing
only inoculated broth as control growth were prepared. The plates
were incubated at 37°C for 24 h, and the MIC values were recorded
as the last well containing no bacterial growth. The MIC was
determined by using an antibacterial assayed repeated twice in
triplicate. Oxacillin and norfloxacin were used as reference drugs.
Acknowledgments. This work was accomplished, thanks to
the financial support of the Ministero dell’Istruzione,
dell’Università e della Ricerca (MIUR). The authors would like
to acknowledge Prof. Antonio Macchiarulo for supplying the
CA-CYP51 homology model.
Conflict of interest. The authors did not report any conflict
of interest.
REFERENCES AND NOTES
[1] Sharma, P.C.; Sinhmar, A.; Sharma, A.; Rajak, H.; Pathak, D. P.
J Enz Inhib Med Chem 2013, 28, 240.
[2] Chaudhary, P.; Sharma, P. K.; Sharma, A.; Varshney, J. Int J
Curr Pharm Res 2010, 2, 5.
[3] Ahmed, K.; Yellamelli Valli Venkata, S.; Mohammed, N. A.;
Sultana, F.; Methuku, K. R. Expert Opin Investig Drugs 2012, 21, 619.
[4] Weekes, A. A.; Westwell, A. D. Curr Med Chem 2009, 16,
2430.
[5] Yadav, P. S.; Devprakash, D.; Senthilkumar, G. P. Intern J
Pharm Sci Drug Res 2011, 3, 1.
[6] Bruno, C.; Carocci, A.; Catalano, A.; Cavalluzzi, M. M.; Corbo,
F.; Franchini, C.; Lentini, G.; Tortorella, V. Chirality 2006, 18, 227.
[7] Khokra, S. L.; Arora, K.; Mehta, H.; Aggarwal, A.; Yadav, M.
Int J Pharm Sci Res 2011, 2, 1356.
Antifungal studies.
Antifungal studies [22] were carried
out against C. albicans 10231, C. parapsilosis 22019, C.
tropicalis 750, and C krusei 6258 belonging to the ATCC
collection. Preparation of stock solutions and purity of cultures
preservation was obtained as described earlier for antibacterial
studies. Pre-cultures of each fungi strain were prepared in
Sabouraud broth 2% glucose (SAB) and incubated at 37°C until
the growth ceased. The turbidity of fungi stock suspension was
calibrated to 0.5 McFarland Standard by spectrophotometric
method (530 nm, range 0.12–0.15), and further, the standardized
suspension was diluted first 1:50 with SAB and then 1:20 in the
same medium to have 1–5 × 106 CFU/mL. All wells were
seeded with 100 μL of inoculum. A number of wells containing
only inoculated broth as control growth were prepared. The
[8] Gupta, A.; Rawat, S. J Curr Pharm Res 2010, 3, 13.
[9] Franchini, C.; Muraglia, M.; Corbo, F.; Florio, M.; Di Mola, A.;
Rosato, A.; Matucci, R.; Nesi, M.; Van Bambeke, F.; Vitali, C. Arch Pharm
Chem Life Sci 2009, 342, 605.
[10] Catalano, A.; Carocci, A.; Defrenza, I.; Muraglia, M.; Carrieri, A.;
Van Bambeke, F.; Rosato, A.; Corbo, F.; Franchini, C. Eur J Med Chem
2013, 64, 357.
[11] Armenise, D.; Carocci, A.; Catalano, A.; Muraglia, M.;
Defrenza, I.; De Laurentis, N.; Rosato, A.; Corbo, F.; Franchini, C.
J Chem 2013. Article number 181758, DOI: http://dx.doi.org/10.1155/
2013/181758.
[12] Pozzi, C.; Ferrari, S.; Cortesi, D.; Luciani, R.; Stroud R. M.;
Catalano, A.; Costi, M. P.; Mangani, S. Acta Crystallogr 2012, D68,
1232.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

I. Defrenza, A. Catalano, A. Carocci, A. Carrieri, M. Muraglia, A. Rosato, F. Corbo, and C. Franchini Vol 000
[13] Ferreira, I. C. F. R.; Queiroz, M. J. R. P.; Kirsch, G. Tetrahedron
2003, 59, 3737.
[23] Sheehan, D. J.; Hitchcock, C. A.; Sibley, C. M. Clin Microbiol
Rev 1999, 12, 40.
[14] Handte, R.; Willms, L.; Blume, E. U.S. Patent 239445,
Chem. Abstr. 1984, 14, 4431813.
[15] Carocci, A.; Catalano, A.; Lovece, A.; Lentini, G.; Duranti, A.;
Lucini, V.; Pannacci, M.; Scaglione, F.; Franchini, C. Bioorg Med Chem
2010, 18, 6496.
[16] Cavalluzzi, M. M.; Catalano, A.; Bruno, C.; Lovece, A.;
Carocci, A.; Corbo, F.; Franchini, C.; Lentini, G.; Tortorella, V. Tetrahedron:
Asymmetry 2007, 18, 2409.
[17] Cavalluzzi, M. M.; Bruno, C.; Lentini, G.; Lovece, A.;
Catalano, A.; Carocci, A.; Franchini, C. Tetrahedron: Asymmetry 2009,
20, 1984.
[18] Bruno, C.; Cavalluzzi, M. M.; Carocci, A.; Catalano, A.;
Franchini, C.; Lentini, G. Drug Metab Lett 2013, 7, 52.
[19] Baell, J. B.; Duggan, P. J.; Forsyth, S. A.; Lewis, R. J.; Lok, Y. P.;
Schroeder, C. I. Bioorg Med Chem 2004, 12, 4025.
[20] Nagarajan, K.; Hendi, S. B.; Goud, A. N., Sen, H. G.; Deb, B. N.
Ind J Pharm Sci 1991, 48, 85.
[21] CLSI. Methods for dilution antimicrobial susceptibility tests
for bacteria that grow aerobically. Approved Standard, 8th Ed. M7-A8,
Clinical and Laboratory Standards Institute, Wayne, PA 2009.
[22] CLSI. Reference method for broth dilution antifungal suscepti-
bility testing of yeast. Approved standard, 3rd Ed. M27-A3, CLSI, Clini-
cal and Laboratory Standards Institute, Wayne, PA 2008.
[24] Lamb, D.; Kelly, D.; Kelly, S. Drug Res Updates 1999, 2, 390.
[25] Lepesheva, G. I.; Waterman, M. R. Biochim Biophys Acta
2007, 1770, 467.
[26] Armenise, D.; Muraglia, M.; Florio, M.A.; De Laurentis, N.;
Rosato, A.; Carrieri, A.; Corbo, F.; Franchini, C. Arch Pharm Chem Life
Sci 2012, 345, 407.
[27] Khullar, I. P.; Agarwala, U. Can J Chem 1975, 53, 1165.
[28] Catalano, A.; Budriesi, R.; Bruno, C.; Di Mola, A.; Defrenza, I.;
Cavalluzzi, M. M.; Micucci, M.; Carocci, A.; Franchini, C.; Lentini, G. Eur J
Med Chem 2013, 65, 511.
[29] Catalano, A.; Desaphy, J.-F.; Lentini, G.; Carocci, A.;
Di Mola, A.; Bruno, C.; Carbonara, R.; De Palma, A.; Budriesi R.;
Ghelardini, C.; Perrone, M. G.; Colabufo, N. A.; Conte Camerino, D.;
Franchini, C. J Med Chem 2012, 55, 1418.
[30] Catalano, A.; Carocci, A.; Corbo, F.; Franchini, C.; Muraglia, M.;
Scilimati, A.; De Bellis, M.; De Luca, A.; Conte Camerino, D.; Sinicropi, M. S.;
Tortorella, V.; Franchini, C. Eur J Med Chem 2008, 43, 2535.
[31] Catalano, A.; Carocci, A.; Lentini, G.; Defrenza, I.; Bruno, C.;
Franchini, C. Drug Metab Lett 2012, 6, 182.
[32] Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.;
Goodsell, D. S.; Olson, A. J. J Comput Chem 2009, 16, 2785.
[33] Macchiarulo, A.; Costantino, G.; Fringuelli, D.; Vecchierelli, A.;
Schiaffella, F.; Fringuelli, R. Bioorg Med Chem 2002, 10, 3415.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet