Anti-infective and herbicidal activity of N-substituted 2-aminobenzothiazoles

Article abstract of DOI:10.1016/j.bmc.2012.10.007

In this study, a series of N-substituted 2-aminobenzothiazoles was prepared according to a recently developed method. Twelve compounds were tested for their activity related to the inhibition of photosynthetic electron transport (PET) in spinach (Spinacia

Full text of DOI:10.1016/j.bmc.2012.10.007

Bioorganic & Medicinal Chemistry 20 (2012) 7059–7068
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anti-infective and herbicidal activity of N-substituted 2-aminobenzothiazoles
Dagmar Fajkusova , Matus Pesko , Stanislava Keltosova , Jiahui Guo , Zbynek Oktabec a,e,
a,
⇑
b
c
d
f
c
d
a
g
a,h,
⇑
Marcela Vejsova , Peter Kollar , Aidan Coffey , Jozef Csollei , Katarina Kralova , Josef Jampilek
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
Department of Ecosozology and Physiotactics, Faculty of Natural Sciences, Comenius University, Mlynska dolina Ch-2, 842 15 Bratislava, Slovakia
Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland
Department of Toxicology, Institute of Forensic Medicine and Toxicology, 1st Faculty of Medicine, Charles University in Prague, 121 08 Prague 2, Czech Republic
Department of Clinical Microbiology, Charles University Medical School and Teaching Hospital, Sokolska 581, Hradec Kralove 500 05, Czech Republic
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina Ch-2, 842 15 Bratislava, Slovakia
a
b
c
d
e
f
g
h
Research Institute for Pharmacy and Biochemistry, Lidicka 1879/48, 602 00 Brno, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a series of N-substituted 2-aminobenzothiazoles was prepared according to a recently
developed method. Twelve compounds were tested for their activity related to the inhibition of photo-
synthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Primary in vitro screening
of the discussed compounds was also performed against fungal, bacterial and mycobacterial species. The
biological activities of some compounds were comparable or higher than the standards phenoxymethyl-
penicillin or pyrazinamide. The most effective compounds demonstrated insignificant toxicity against the
human monocytic leukemia THP-1 cell line. For all compounds, the structure-activity relationships are
discussed.
Received 18 June 2012
Revised 2 October 2012
Accepted 5 October 2012
Available online 17 October 2012
Keywords:
Substituted 2-aminobenzothiazoles
Lipophilicity
Photosynthetic electron transport inhibition
Spinach chloroplasts
Ó 2012 Elsevier Ltd. All rights reserved.
In vitro antifungal activity
In vitro antimicrobial activity
In vitro antimycobacterial activity
In vitro cytotoxicity
1
6
1
. Introduction
-Aminobenzothiazoles substituted on the endocyclic nitrogen
and serine hydrolases.
neuronal diseases
They showed activity against some
1
7,18
and exhibited significant blood pressure
1
9
2
reduction and increase of myocardial perfusion. Moreover, the
basic 2-substituted-1,3-benzothiazole scaffold is essential for some
herbicides, plant desiccants and defoliant compounds.20 Isopropyl
[(S)-1-[(R)-1-(6-flourobenzothiazolel-2-yl)ethylcarbamoyl]-2-
methylpropyl]-carbamate is a commercially used fungicide which
is effective for controlling the oomycete fungal pathogen Plasmo-
para viticola, which causes downy mildew in grapevines.21 Gener-
ally, benzothiazoles posses a unique and versatile scaffold for
experimental drug design.
The presence of an amide group is characteristic of a number of
herbicidal active compounds
uncouplers of photosynthetic phosphorylation. Over 50% of com-
mercially available herbicides act by reversibly binding to photo-
system II (PS II), a membrane-protein complex in the thylakoid
membranes, which catalyses the oxidation of water and the reduc-
have been studied for their potential biological activities since
the early 1940s when Institute for Chemotherapy in Germany
examined their effects against several infectious diseases. The pri-
mary objective was to compare benzothiazoles with thiazoles
which were already known and used as drugs, especially sulfat-
hiazole being one of the most efficient drugs for various disorders
caused by cocci at that time.1 The heterocyclic system of benzo-
thiazoles might be substituted in many ways and provide com-
pounds with interesting and even unexpected properties. Among
them, the most important is modification of position 2. Diverse
2
2–30
that were also found to be
2
3,4
31
biological activities such as antibacterial, antiparasitic,
fungal,
antioxidant were described. Benzothiazoles are also known as
potent inhibitors of various enzymes, for example phosphodiesterase
anti-
and
5
,6
7–10
11–13
anticancer,
anti-inflammatory/antiallergic
1
4
15
3
2
33–35
tion of plastoquinone, and thereby inhibit photosynthesis.
Both pharmaceuticals and pesticides are designed to target par-
ticular biological functions and, in some cases, these functions
overlap in their molecular target sites or they target similar
⇑
Corresponding authors. Tel.: +420 541562925.
E-mail addresses: adagmar@post.cz (D. Fajkusova), josef.jampilek@gmail.com (J.
Jampilek).
0
968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.007

7
060
D. Fajkusova et al. / Bioorg. Med. Chem. 20 (2012) 7059–7068
processes or molecules. Modern herbicides express low toxicity
against mammals and one of the reasons is that mammals lack
many of the target sites for herbicide action. At present, approxi-
mately 20 mechanisms of action of herbicides are known. It
was determined that inhibitors of protoporphyrinogen oxidase,
sized now to generate a well-organized set (small library)—
Scheme 1. Unsubstituted 2-aminobenzothiazole 3a (R = H) was ob-
tained by basic hydrolysis of the benzoyl derivative 3j.
Attempts to extend the amines series with tert-butyl and cyclo-
hexyl derivatives were unsuccessful. This might be caused by steric
0
4
-hydroxyphenylpyruvate dioxygenase and glutamine synthetase
hindrance on the isothiocyanate carbon that prevented 2,2 -diam-
inhibit these enzymes both in plants and mammals. However,
the consequences of inhibition of the overlapping target site can
be completely different. Therefore, a compound that has lethal ac-
inodiphenyl disulfide from nucleophilic addition. Similarly, in the
set of amides, the yield of tert-butyl 3h, iso-butyl 3g and butyl
derivative 3f was poor, moderate and very good, respectively,
and 2-phenylacetylaminobenzothiazole 3k was obtained in a high-
er yield than 2-cyclohexylcarbonylaminobenzothiazole 3i. The
presence of the carbonyl group further supported the reaction con-
version by its electron withdrawing effects. The best results
exceeding 70% were, therefore, achieved in the amide series
when a substituent providing enough space for the interaction
36
tion on plants may be beneficial for mammals. Such chemicals
are characterized by low toxicity on mammals as a result of quick
metabolism and/or elimination of herbicide from the mammalian
system. Taking into consideration that herbicides may also have
molecular sites of action in mammals, until recently most pharma-
ceutical companies had pesticide divisions, sometimes with a dif-
ferent name. All compounds generated by either division of the
company were evaluated for both pesticide and pharmaceutical
uses. In the past, some leading pesticides have become pharmaceu-
ticals and vice versa. However, little information of this type was
published and must usually be deduced from patent literature.
One of the exceptions is fluconazole, a fungicide product discov-
ered by the pharmaceutical sector that is now used both as a phar-
maceutical and also patented as a chemical with applications in
0
with 2,2 -diaminodiphenyl disulfide was introduced (3f and 3k).
Although some difficulties during the preparation (oligomeriza-
tion of isothiocyanates) might occur, this recently developed syn-
thetic approach proved useful, simple and convenient.
2.2. Physico-chemical properties
Lipophilicity is a property that has a major effect on absorption,
distribution, metabolism, excretion and toxicity properties as well
as pharmacological activity, because drugs cross biological mem-
branes through passive transport, which strongly depends on their
lipophilicity. Lipophilicity has been studied and applied as an
important drug property for decades.
Hydrophobicities (logP/ClogP) of compounds 3a–l were calcu-
lated using two commercially available programs (ChemOffice
Ultra 10.0 and ACD/LogP ver. 1.0) and measured by means of
RP-HPLC determination of capacity factors k with subsequent
calculation of logk. The procedure was performed under isocratic
conditions with methanol as an organic modifier in the mobile
phase using an end-capped non-polar C18 stationary RP column.
The results are shown in Table 1 and illustrated in Figure 1.
The results obtained with all the compounds show that the
experimentally-determined lipophilicities (logk) of the discussed
compounds are in accordance with the calculated values of com-
pounds 3a–l as shown in Figure 1. The minimum match of exper-
imental (logk) versus calculated (logP) values was found for ACD/
LogP, see Figure 1B. As expected, unsubstituted 1,3-benzothiazol-
3
6–38
crop production.
As mentioned above, position 2 of benzothiazoles is the most
suitable for affecting physico-chemical properties of these com-
1
6
39
pounds. Following a recently developed method for the synthe-
sis of 2-aminobenzothiazoles, the amino moiety was modified to
obtain biologically effective derivatives and to further explore the
scope of the synthetic approach. A series of twelve compounds
was tested for inhibitory effects on photosynthetic electron trans-
port (PET) in spinach chloroplasts (Spinacia oleracea L.) and also
evaluated against fungal, bacterial and mycobacterial strains. As
many low-molecular-weight drugs cross biological membranes
through passive transport, which strongly depends on their lipo-
philicity, the experimental logk values were determined by high
performance liquid chromatography. The structure–activity rela-
tionships between the chemical structure, physical properties
and in vitro biological activities of all the evaluated compounds
are discussed.
2
2
. Results and discussion
2
-amine 3a showed the lowest lipophilicity (logk), while com-
pounds 3i and 3c exhibited the highest logk values. Generally,
the introduction of a carbonyl moiety (compounds 3c–3k) caused
a decrease in lipophilicity in comparison with amino derivatives,
for example 3e < 3b, 3k < 3d, 3j < 3c. Benzyl-substituted com-
pounds 3d and 3k possessed lower lipophilicity than phenyl deriv-
atives 3c and 3j. Within the series of butyl-substituted compounds
.1. Chemistry
There are many methods for the preparation of 2-aminobenzo-
thiazoles. To avoid working with toxic and inconvenient (2-amino-
thiophenol, diazonium salts) or expensive (transition metal
complexes) starting materials, the procedure reported previously
3
9
3
f–h, the determined logk values increased as follows: iso-Bu
was chosen. Six derivatives 3a–c, 3e, 3j and 3l were, therefore,
(3g) < Bu (3g) < tert-Bu (3h). This experimental data corresponds
available in yields up to 93%, and six new compounds were synthe-
NH2
NH
NH
C
S
R
R
N
S
S
S
a
S
S
+
2 R
N
C
S
2
NH
- 2 H S
R
2
2
S
C
3b-l
NH2
NH
NH
1
R = Me (3b), Ph (3c), Bn (3d),
COMe (3e), COBu (3f), COi-Bu (3g), COt-Bu (3h), COc-Hx (3i), COPh (3j), COBn (3k),
COOMe (3l)
Scheme 1. Synthesis of N-substituted 2-aminobenzothiazoles 3b–l: (a) various conditions, see Section 4.

D. Fajkusova et al. / Bioorg. Med. Chem. 20 (2012) 7059–7068
7061
Table 1
Structure of discussed compounds and comparison of calculated lipophilicities (logP/ClogP) with determined logk values and bulk parameter (MR, reflecting bulkiness)
N
NH
R
S
3
R
Logk
LogP/ClogP ChemOffice
LogP ACD/LogP
MR (cm /mol) Chemoffice
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
g
h
i
H
Me
Ph
Bn
CO-Me
CO-Bu
CO-iBu
CO-tBu
CO-cHx
CO-Ph
CO-Bn
CO-OMe
0.3072
0.3819
0.7301
0.6012
0.3661
0.5780
0.5598
0.5891
0.6880
0.6643
0.5413
0.4251
2.43/1.8230
2.73/2.6266
4.70/4.6056
4.46/4.0746
2.14/1.9914
3.63/3.5784
3.54/3.4484
4.07/3.2284
4.11/4.0224
4.04/3.4804
3.98/3.5294
2.72/2.2765
1.89 ± 0.57
2.23 ± 0.57
3.98 ± 0.57
4.00 ± 0.57
1.84 ± 0.60
3.44 ± 0.60
3.25 ± 0.60
3.07 ± 0.61
3.90 ± 0.60
3.30 ± 0.60
3.62 ± 0.61
2.29 ± 0.61
1.60
6.80
25.28
31.10
11.58
25.53
25.70
25.79
32.77
32.43
36.02
12.89
j
k
l
A
B
5.0
4.0
3.0
2.0
1.0
0.0
0.8
0
0
0
0
0
.7
.6
.5
.4
.3
k
log P [CO] r = 0.936
Clog P r = 0.941
log P [ACD] r = 0.921
0.2
3
a
3e
3b
3l
3k
3g
3f
3h
3d
3j
3i
3c
1.5
2.0
2.5
3.0
log P/Clog P
Clog P [ChemOffice]
3.5
4.0
4.5
5.0
Compounds
Clog P [ChemOffice]
log k
log P [ChemOffice]
log P [ACD/LogP]
log P [ChemOffice]
log P [ACD/LogP]
Figure 1. Comparison of logP data calculated using two programs with experimentally found logk values. Fig. 1A: compounds are arranged in ascending manner according to
experimental logk values; Fig. 1B: match of calculated data with experimentally found logk values.
to only logP calculated by ChemOffice. Generally, it can be as-
sumed that logk values specify lipophilicity within individual ser-
ies of the studied compounds more precisely than calculated logP/
ClogP data.
Experience has shown that a parameter representing the vol-
ume of substituents on each compound relative to other members
of the same series may often be correlated with the biological data.
Of the various bulk parameters proposed, molar refraction reflect-
ing bulkiness (MR) was chosen. These values are not dependent on
the position of the substituent and can be calculated by various
chemical software, similarly as logP. In Table 1 there are the MR
values calculated by ChemOffice Ultra 10.0, and the correlation be-
tween experimental logk and calculated MR is shown in Figure 2
and the following equation:
log k ¼ 0:304 ðꢀ0:048Þ þ 0:010ðꢀ0:002ÞMR
r ¼ 0:861; s ¼ 0:073; F ¼ 28:55; n ¼ 12
0
0
0
0
0
0
0
.8
.7
.6
.5
.4
.3
.2
3
c
3i
3
j
3
g
h
3d
3f
3k
3
k
r = 0.861
3
b
3l
3e
3a
0
.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
3
MR [cm /mol]
Figure 2. Match of calculated bulk parameters expressed as MR (reflecting bulkiness) data with experimentally found logk values. (rhombs = amines, squares = amides,
triangle = carbamate).

7
062
D. Fajkusova et al. / Bioorg. Med. Chem. 20 (2012) 7059–7068
2
.3. Biological activities
OMe (3l). The carbamate moiety (Group 3) seems to be the most
advantageous as was described recently.
2
7,29
The compounds under investigation could be divided into three
groups based on their chemical structure: Group 1 includes amines
a–d; Group 2 containes carboxamides 3e–k; and Group 3 in-
It can be stated that the dependence of PET-inhibiting activity
on the lipophilicity was linear up to logk = 0.54 and with the fur-
ther increase of logk it gradually decreased. The most active com-
3
cludes carbamate 3l. Compounds within Group 1 and Group 2
can also be divided according to whether they contain an aromatic
or a non-aromatic substituent. The compounds showed a wide
range of biological activities, and some interesting structure-activ-
ity relationships were observed. All the results are summarized in
Table 2.
pound phenylacetamide 3k has a lipophilicity optimum at
log k = 0.54. Within the series of 2-substituted benzothiazoles the
length and branching of the chain seem to be also important. It
is evident that PET inhibition increases with the chain length (–
COC
4
H
9
, 3f) or mono-b-branched (–COCH
2
C
6
H
5
, 3k) substituent,
-branched
while in the case of short chain (compounds 3a,b,e) or
a
substituent as tert-butyl 3h, cyclohexyl 3i or phenyl 3c,j PET inhi-
bition dramatically decreases. It is evident that the bulkiness of the
N-substituents influences PET-inhibiting activity. Excellent PET
inhibition by benzyl substituted amides has also been recently de-
2
.3.1. Inhibition of photosynthetic electron transport (PET) in
spinach chloroplasts
The activity of all the evaluated derivatives related to inhibition
of photosynthetic electron transport (PET) in spinach (Spinacia
oleracea L.) chloroplasts was moderate or rather low relative to
the standard, see Table 2. Only 2-phenylacetylaminobenzothiazole
2
7,30
scribed by Otevrel et al. and Gonec et al.
Several series of alkyl substituted benzothiazole derivatives
were also recently investigated for their photosynthesis-inhibiting
4
0–42
3
k expressed high PET-inhibiting activity (IC50 = 1.11
l
mol/L)
activity.
PET inhibition in spinach chloroplasts was also ob-
43
which is higher than DCMU. Despite the relatively low inhibitory
activity of the rest of the studied compounds, the structure-activity
relationships can be discussed. Generally, Group 1 (amines 3a–d)
showed lower PET-inhibiting activity than the carboxamides or
the carbamate, that is –NH-Me (3b) < –NHCO-Me (3e) < –NHCO-
served for 2-(6-acetamidobenzothiazolethio)acetic acid esters.
The dependence of PET-inhibiting activity on the lipophilicity of
the derivatives with R = n-alkyl and allyl showed a quasiparabolic
course, the most active compound was the hexyl derivative
(IC50 = 47 lmol/L). The effect of 2-(alkoxycarbonylmethylthio)-6-
Table 2
IC50 values related to PET inhibition in spinach chloroplasts of N-substituted 2-aminobenzothiazoles in comparison with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)
standard; in vitro antifungal activity (IC80/IC50) of compounds compared to fluconazole (FLU) standard; in vitro antibacterial activity (IC80) of compounds compared to
phenoxymethylpenicillin (PMP) standard; in vitro antimycobacterial activity (IC90) of compounds in comparison with isoniazid (INH) and pyrazinamide (PZA) standards; and
in vitro cytotoxicity assay (LD50) of choice compounds
[lmol/L]
PET IC50
MIC/IC80a/IC50b
MIC/IC80
MIC/IC90
MK
LD50
CAa
CKa
TAa
TMb
MRSA
SE
2
4
4 h
8 h
24 h
48 h
24 h
48 h
72 h
120 h
24 h
48 h
24 h
48 h
5 days
3
a
1722
>500
500
125
50
ND
>125
>500
>500
500
>500
ND
>125
>125
250
>500
125
>125
>125
>125
500
>500
>125
>125
500
>500
62.50
125
>250
>250
—
>500
>500
125
>500
ND
125
>125
250
>500
62.50
>125
125
>125
>500
>500
125
>125
250
>500
31.25
62.25
250
>500
>500
62.50
62.50
ND
>125
>125
250
>500
>500
500
500
ND
>500
>500
125
125
ND
>125
>125
250
>500
31.25
62.50
>125
>125
125
>1997
>1827
—
—
>
3
b
364.4
2
3
3
c
d
57.7
ND
ND
ND
—
—
>125
>125
250
500
31.25
125
125
125
250
500
>125
>125
125
500
31.25
62.25
250
250
—
>
125
62.50
25
31.25
1.25
125
25
62.50
50
>125
125
125
25
3.90
.81
125
50
3
3
3
3
3
3
3
3
e
86.4
21.0
34.1
74.6
113.2
77.3
1.11
24.2
1300
1087
480
>30
>30
>30
ND
1
250
f
31.25
62.50
62.50
62.50
62.50
250
>125
>125
62.25
125
15.62
125
125
125
—
3
g
h
i
1
1040
960
2
500
>125
>125
>500
>500
125
500
125
500
—
>30
>30
>30
27.5
>
j
533
1
k
l
256
7
533
2
—
0.06
500
—
0.24
0.48
—
DCMU
FLU
1.9
—
—
—
—
—
3.91
15.62
—
1.95
3.91
—
—
—
0.12
PMP
—
—
250
62.50
250
—
—
—
500
INH
PZA
—
—
—
—
—
—
—
—
—
—
—
—
<72.9
>812
—
—
—
ND = not determined due to precipitation during the experiment or interaction with 2,6-dichlorophenol-indophenol (DCPIP).
The MIC determination was performed according to the CLSI reference protocol:
a
M27-A2 for yeasts (IC80 value).
b
M38-A for moulds (IC50 value); CA = Candida albicans ATCC 44859, CK = C. krusei E28, TA = Trichosporon asahii 1188, TM = Trichophyton mentagrophytes 445,
MRSA = methicillin-resistant Staphylococcus aureus H 5996/08, SE = S. epidermidis H 6966/08, MK = Mycobacterium kansasii CIT11/06.

D. Fajkusova et al. / Bioorg. Med. Chem. 20 (2012) 7059–7068
7063
A 160
B 120
100
120
3
j
8
6
4
2
0
0
0
0
8
4
0
0
0
3
g
3k
600
650
700
λ [nm]
750
800
0.0
0.2
0.4
0.6
c [mmol/L]
0.8
1.0
1.2
Figure 3. Emission fluorescence spectra of chlorophyll a in suspension of spinach chloroplasts without and with 0.23, 0.45, 0.67 and 1.00 mmol/L of N-1,3-benzothiazol-2-yl-
-phenylacetamide 3k (curves from top to bottom) (A), and dependence of intensity of chlorophyll a fluorescence emission band at 686 nm in suspension of spinach
chloroplasts on concentration of studied compounds 3k (square), 3g (ring) and 3j (triangle) (B) (ex = 436 nm).
2
aminobenzothiazoles on photosynthetic apparatus was similar to
-alkylthio-6-R-benzothiazoles, where R = 6-formamido-, 6-acet-
advantageous, in the case of anti-TM activity 2-penta-
noylaminobenzothiazole (3f) with lipophilicity logk = 0.58 was the
most efficient. In this case a longer (C ) chain (3f) that can be mono-
branched in b-position (3g) or unsaturated (3k), the radical analogue
of 3g, increases anti-TM activity. A short chain or -branched (tert-
2
amido- and 6-benzoylamino–, and as a probable site of their action
4
the oxygen evolving complex was suggested.44
Because the studied compounds inhibited Hill reaction, they
can be considered to be PS II herbicides. Upon addition of diphe-
nylcarbazide (DPC, an artificial electron donor with the known site
a
butyl, cyclohexyl, phenyl) chain caused a decrease in activity.
4
5
of action in the intermediate Z/D on the donor side of PS II ) to
chloroplasts, activity of which was inhibited by 3k, PET was prac-
tically completely restored. Thus, it can be assumed this benzothi-
azole derivatives did not damage PET between photosynthetic
centres PS II (P 680) and PS I (P 700), and their site of action is sit-
uated at the donor side of PS II. The site of action situated at the
donor side of PS II was found also for 2-alkylthio-6-R-
2.3.3. In vitro antibacterial susceptibility testing
All compounds were tested for their in vitro antibacterial activ-
ity against two Gram positive bacterial strains methicillin-resistant
Staphylococcus aureus H 5996/08 (MRSA) and Staphylococcus epide-
rmidis H6966/08 (SE). The results are shown in Table 2. Generally,
all the compounds expressed only moderate antibacterial activity,
apart from 2-phenylacetylaminobenzothiazole (3k) and 2-penta-
noylaminobenzothiazole (3f), where the antibacterial activity is
comparable with or higher than the standard phenoxymethylpen-
icillin against methicillin-resistant S. aureus. Similarly as men-
tioned above, it can be concluded that the amines (Group 1) did
not possess any antibacterial activity in comparison with the
amides (Group 2) or the carbamate (Group 3).
The dependences of the antibacterial activity of compounds
3b,e–h,j–l against methicillin-resistant S. aureus on lipophilicity
(logk) was performed (see Fig. 4). Based on the results obtained
it is possible to declare that lipophilicity (optimum at logk = 0.54)
is the most important parameter. Similarly, based on Table 2, it can
be assumed that antibacterial activity against S. aureus is also af-
fected by bulk parameters expressed as MR of R substituents, when
4
1
benzothiazoles.
The effects of studied compounds on the photosynthetic centres
of chloroplasts were investigated by studying chlorophyll a fluo-
rescence. Figure 3A shows how the chlorophyll a fluorescence
emission band at 686 nm is influenced in the presence of N-1,3-
benzothiazol-2-yl-2-phenylacetamide. The decreased intensity of
this emission band belonging to the chlorophyll-protein complexes
mainly in photosystem (PS) II46 suggested PS II as the site of action
of the studied inhibitor. As shown in Figure 3B, the intensity of the
chlorophyll a fluorescence emission band at 686 nm in the suspen-
sion of spinach chloroplasts decreased with increasing concentra-
tion of the studied compounds and the rate of fluorescence
decline correlated with the effectiveness of PET inhibition. The
greatest decrease was observed with the most effective PET inhib-
double-branching on the a-position of a substituent (i.e. tert-butyl
3
3
itor 3k (R = CO-Bn, IC50 = 1.1
R = CO-iBu, IC50 = 34.1 mol/L) the fluorescence intensity was af-
fected to a lesser extent, and even less effect was observed with
lmol/L), in the presence of 3g
(MR = 25.79 cm /mol) 3h and phenyl (MR = 32.43 cm /mol) 3j)
caused an expressive decrease in activity. The character of the
dependence of anti-MRSA activity on logk is the same as the
dependence of PET inhibition on lipophilicity, and phenylacet-
amide 3k is the most effective compound with logk = 0.54.
(
l
3
j (R = CO-Ph, IC50 = 77.3 lmol/L).
2
.3.2. In vitro antifungal susceptibility testing
The evaluation of in vitro antifungal activity of the synthesized
2.3.4. In vitro antimycobacterial evaluation
compounds was performed against Candida albicans ATCC 44859
CA), Candida krusei E28 (CK), Trichosporon asahii 1188 (TA) and
Although all the compounds were evaluated for their in vitro
antimycobacterial activity against Mycobacterium kansasii CIT11/
06 (MK), most compounds did not show any activity. Only 2-phen-
ylacetylaminobenzothiazole (3k) expressed high activity against
M. kansasii. Though the antimycobacterial activity of 3k is less than
that of isoniazid, it is threefold higher than the activity of pyrazin-
amide. Similar trends, as mentioned above, that is lipophilicity
(optimum at logk = 0.54) as well as the bulkiness of the N-substit-
uents within the amide series, influence antimycobacterial activity.
(
Trichophyton mentagrophytes 445 (TM), and the results are shown
in Table 2. All the compounds expressed only moderate antifungal
activity, except N-1,3-benzothiazol-2-yl-2-phenylacetamide (3k).
Generally, it can be concluded that Group 1 did not possess any
antifungal activity in comparison with the amides (Group 2) or
the carbamate (Group 3). It can be stated that the dependence of
in vitro antifungal activity on the lipophilicity seems to be impor-
tant; nevertheless, as discussed above, substituent bulkiness also
plays a significant role. Although in the case of anti-CA activity it
is evident that amide substituted by benzyl moiety (compound
2.3.5. In vitro cytotoxicity assay
Compounds 3e–l were tested for their in vitro cytotoxicity using
a human monocytic leukemia THP-1 cell line. The cytotoxicity was
3
k with a lipophilicity optimum at logk = 0.54) is the most

7
064
D. Fajkusova et al. / Bioorg. Med. Chem. 20 (2012) 7059–7068
4.2
4.0
3.8
3.6
3.4
3.2
3.0
2.8
3
k
3
f
3g
3l
3e
3
j
3h
3b
0
.30
0.36
0.42
0.48
0.54
0.60
0.66
0.72
log
k
Figure 4. Dependence of in vitro antibacterial activity against methicillin-resistant S. aureus (log 1/MIC [mol/L]) on lipophilicity (logk) of studied compounds 3a–l.
rhombs = amines, squares = amides, triangle = carbamate).
(
5
4
3
2
1
5.0
5.0
5.0
5.0
5.0
52.91
35.84
1
8.74
9
.24
7
.20
5
.0
-5.0
0
.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
Concentration [µmol/L]
3
e
3f
3g
3h
3i
3j
3k
3l
Figure 5. Relative cytotoxicity of compounds 3e–3l in leukemia THP-1 cell line.
evaluated as the LD50 value (LD50—lethal dose to 50% of the cell
population). The results are presented in Table 2. Compound 3h
compounds were tested for their ability to inhibit photosynthetic
electron transport (PET) in spinach chloroplasts (Spinacia oleracea
L.) and for their in vitro antifungal, antibacterial and antimycobac-
terial activity. 2-Phenylacetylaminobenzothiazole (3k) showed
excellent PET inhibition, higher than the standard DCMU. This
compound also expressed relatively high antimycobacterial
activity against M. kansasii. Two compounds, namely 2-phen-
ylacetylaminobenzothiazole (3k) and 2-pentanoylaminobenzo-
thiazole (3f), expressed high activity against C. albicans and
methicillin-resistant S. aureus. Both exhibited activity comparable
with or higher than the standard phenoxymethylpenicillin. Gener-
ally, amines 3a–d showed lower biological activity than carboxam-
ides 3e–3k or carbamate 3l indicating that amide/carbamate
groups are essential for biological effect. Furthermore, it could be
concluded that lipophilicity is fundamental for the biological activ-
ities of all compounds in all assays. It can be stated that the depen-
dence of activity on the lipophilicity was parabolic or bilinear. Also,
the substitution of an amide moiety by a bulky group or a long al-
kyl chain is important for high herbicidal or anti-infective activity.
The most effective biological compounds 3e–g and 3i–l were
was not soluble in concentrations 15 and 30
lmol/L, the highest
concentration prepared in DMSO for the toxicity assay was
7
.5
lmol/L. The LD50 value for all tested compounds 3e–g and
3
i–k was determined to be >30
lmol/L. A significant change in via-
bility was found only for compound 3l with the value of
LD50 = 27.5 mol/L for the cell line used in the assay, see Figure 5.
l
It could be derived from the data that none of the compounds
tested were cytotoxic in the range of concentrations used in these
cytotoxicity assays (e.g. LD50 of oxaliplatin 1.7 ± 6.4
camptothecin 0.16 ± 0.07 mol/L assessed in this line formerly
showed much lower values). The value of LD50 for all tested com-
pounds was more than 25 mol/L.
lmol/L and
l
l
3
. Conclusions
Six new N-substituted 2-aminobenzothiazoles were success-
fully prepared according to the recently developed procedure,
which proved to be useful, simple and convenient. In total, twelve

D. Fajkusova et al. / Bioorg. Med. Chem. 20 (2012) 7059–7068
7065
tested for their in vitro cytotoxicity against the human monocytic
leukemia THP-1 cells. None of the compounds with LD50 >25 mol/
4.2.3. 2-Pentanoylaminobenzothiazole (3f)
l
Valeroyl chloride (1.6 g, 13.4 mmol), potassium thiocyanate
L were cytotoxic in the range of concentrations used in these
cytotoxicity assays.
(1.4 g, 14.8 mmol), 1 (1.0 g, 4.0 mmol). Yield: 1.4 g (74%). IR
ꢁ1
(cm ): 3054 (
m
, Ar-H), 2951, 2929 (
, C@O), 1600 ( , C–N), 1548 (d, NH), 1446, 1379 (d
272, 1244, 758 ( , Ar), 724 (
m
as, CH
2
), 2867 (
m
s
, CH
, CH
3
),
),
1
1
695 (
m
m
s
3
1
c
c, CH
2
); H NMR (CDCl
3
), d: 11.48
4
4
. Experimental
(
br s, 1H, NH), 7.90–7.80 (m, 1H, Ar), 7.80–7.72 (m, 1H, Ar),
.52–7.40 (m, 1H, Ar), 7.40–7.28 (m, 1H, Ar), 2.47 (t, 2H,
J = 6.7 Hz, NHCOCH CH CH CH ), 1.69 (pent, 2H, J = 7.6 Hz, NHC-
OCH CH CH CH ), 1.31 (sext, 2H, J = 7.4 Hz, NHCOCH CH CH CH ),
.86 (t, 3H, J = 7.4 Hz, NHCOCH ); C NMR (CDCl ), d:
71.87, 159.57, 147.84, 131.99, 126.29, 123.99, 121.61, 120.39,
7
.1. Chemistry
2
2
2
3
All reagents and solvents used are commercially available.
2
2
2
3
2
2
2
3
1
3
0
1
3
2
2
CH
CH CH
2 2 3
3
Reaction conversions were monitored by TLC on alumina-backed
silica gel 40 F254 plates (Merck, Darmstadt, Germany) in diethyl
ether and evaluated under UV (254 nm). Infrared (IR) spectra were
recorded on a Smart MIRacle™ ATR ZnSe for Nicolet™ Impact 6700
FT–IR spectrometer (Thermo Scientific, USA). The spectra were ob-
15 2
6.26, 27.01, 22.18, 13.60; HR-MS: for C12H N
OS [M+H]+ calcd
35.0827 m/z, found 235.0896 m/z.
ꢁ1
4
.2.4. 2-(3-Methyl)butanoylaminobenzothiazole (3g)
tained by accumulation of 256 scans with 2 cm resolution in the
region of 4000–600 cm . All 1H and C NMR spectra were re-
ꢁ
1
13
Isovaleryl chloride (1.6 g, 13.4 mmol), potassium thiocyanate
(
(
CH
1
1
1.4 g, 14.8 mmol), 1 (1.0 g, 4.0 mmol). Yield: 0.9 g (47%). IR
corded on a Bruker Avance III 400 MHz FT-NMR spectrometer
ꢁ1
1
13
cm ): 3055 (
), 1697 ( , C@O), 1599 (
248, 752 ( , Ar), 728 ( , CH
H, NH), 7.89–7.82 (m, 1H, Ar), 7.82–7.74 (m, 1H, Ar), 7.52–7.40
3 2 s
m, Ar-H), 2962 (mas, CH ), 2929 (mas, CH ), 2868 (m ,
(
400 MHz for H and 100 MHz for C, Bruker Comp., Karlsruhe,
Germany). Chemicals shifts are reported in ppm (d) using internal
Si(CH as the reference with diffuse, easily exchangeable signals
3
m
c
m
, C–N), 1538 (d, NH), 1440, 1268,
1
c
2 3
); H NMR (CDCl ), d: 10.67 (br s,
3 4
)
being omitted. Mass spectra were measured using a LTQ Orbitrap
Hybrid Mass Spectrometer (Thermo Electron Corporation, USA)
with direct injection into an APCI source (400 °C) in the positive
mode.
(
m, 1H, Ar), 7.40–7.28 (m, 1H, Ar), 2.34 (d, 2H, J = 6.7 Hz,
NHCOCH CH(CH ), 2.16 (non, 1H, J = 6.7 Hz, NHCOCH CH(CH ),
.92 (d, 6H, J = 6.7 Hz, NHCOCH ); C NMR (CDCl ), d:
71.06, 159.11, 147.89, 132.02, 126.26, 124.01, 121.57, 120.58,
2
3
)
2
2
3 2
)
1
3
0
1
4
2
2
3
CH(CH )
2
3
Compounds 3a–c, 3e, 3j and 3l were described and published
12 15 2
C H N
OS [M+H]+ calcd
recently by Fajkusova.39
5.55, 25.96, 22.26; HR-MS: for
35.0827 m/z, found 235.0896 m/z.
4
.2.1. 2-Benzylaminobenzothiazole (3d)
0
4.2.5. 2-(2,2-Dimethyl)propanoylaminobenzothiazole (3h)
Trimethylacetyl chloride (1.6 g, 13.4 mmol), potassium thiocya-
nate (1.4 g, 14.8 mmol), 1 (1.0 g, 4.0 mmol). Yield: 0.2 g (9%). IR
A
suspension of 2,2 -diaminodiphenyl disulfide (1) (1.5 g,
6
.1 mmol) in toluene (30 mL) was treated with benzyl isothiocya-
nate (1.8 g, 12.1 mmol) and stirred at room temperature. A clear
solution was quickly formed; colourless crystals precipitated with-
in 48 h. After filtration the solution was partially evaporated to
provide another solid. Both crops were washed with toluene and
ꢁ1
(
cm ): 3061 (
C@O), 1601 ( , C–N), 1537 (d, NH), 1446, 1298, 1278, 762 (
H NMR (CDCl ), d: 8.89 (br s, 1H, NH), 7.85–7.79 (m, 1H, Ar),
.79–7.73 (m, 1H, Ar), 7.50–7.38 (m, 1H, Ar), 7.38–7.27 (m, 1H,
m
, Ar-H), 2959 (
m
as, CH
3
), 2868 (
m
s
, CH
3
), 1686 (
m,
m
c, Ar);
1
3
ꢁ1
7
dried well in the air to give pure 3d (1.6 g, 55%). IR (cm ): 3060
, Ar-H), 3030 ( , Ar-H), 2897 ( , CH), 2853 ( , CH ), 1614 (
C–N), 1572 (d, NH), 1453, 1269, 1256, 1235, 753 ( , Ar), 742 ( , Ar),
), d: 7.56 (ddd, 1H, J = 0.6 Hz,
1
3
Ar), 1.37(s, 9H, NHCOC(CH
3
)
3
);
3
C NMR (CDCl ), d: 176.46,
(m
m
m
m
c
s
2
m,
1
2
2
59.91, 147.96, 132.01, 126.24, 123.93, 121.43, 120.88, 39.35,
7.22; HR-MS: for C12H N OS [M+H] calcd 235.0827 m/z, found
15 2
35.0896 m/z.
c
+
1
6
98
(c,
Ar);
H NMR (CDCl
3
J = 1.3 Hz, J = 7.9 Hz, Ar), 7.47 (ddd, 1H, J = 0.6 Hz, J = 1.3 Hz,
J = 8.1 Hz, Ar), 7.44–7.7.18(m, 6H, Ar), 6.15 (br s, 1H, NH), 4.63 (s,
1
3
4
.2.6. 2-Cyclohexylcarbonylaminobenzothiazole (3i)
Cyclohexanecarbonyl chloride (1.6 g, 10.8 mmol), potassium
3
1
1
H, CH
3 3
); C NMR (CDCl ), d: 167.42. 152.31, 137.46, 130.49,
28.91, 128.81, 127.85, 127.66, 127.51, 125.96, 121.61, 120.81,
18.96, 49.38. HR-MS: for C14H N S [M+H] calcd 241.07212 m/
13 2
+
thiocyanate (1.2 g, 11.8 mmol), 1 (0.8 g, 3.2 mmol). Yield: 0.9 g
ꢁ1
(
53%). IR (cm ): 3056 (
m
, Ar-H), 2927 (
2 s 2
mas, CH ), 2850 (m , CH ),
z, found 241.0791 m/z.
1693 (
1243, 760 (
1H, NH), 7.93–7.66 (m, 1H, Ar), 7.82–7.74 (m, 1H, Ar), 7.54–7.40
m
, C@O), 1599 (
m, C–N), 1543 (d, NH), 1443, 1272, 1262,
c, CH ); H NMR (CDCl ), d: 11.12 (br s,
2 3
1
c
, Ar), 733 (
4
.2.2. General procedure for synthesis of 2-aminobenzothiazole
derivatives 3f–i and 3k
The corresponding acyl chloride was added dropwise to a solu-
tion of potassium thiocyanate in dry acetone (20 mL for 3f–h,
(
m, 1H, Ar), 7.40–7.28 (m, 1H, Ar), 2.44–2.20 (m, 1H, CH of cyclo-
1
3
hexyl), 2.00–0.83 (m, 10H, cyclohexyl);
3
C NMR (CDCl ), d:
1
4
2
74.90, 159.81, 147.84, 132.01, 126.26, 123.99, 121.59, 120.42,
1
5 mL for 3i and 3k) under stirring at room temperature. Potas-
17 2
4.96, 29.18, 25.38, 25.13; HR-MS: for C14H N
OS [M+H]+ calcd
sium chloride precipitated immediately and approx. after 10 min
was filtered off and washed with dry acetone. The solution con-
taining the corresponding acyl isothiocyanate was evaporated to
dryness. This residue was then dissolved in chloroform (15 mL
for 3–h, 10 mL for 3i and 3k), a solid rest was removed and a clear
61.0983 m/z, found 261.1054 m/z.
4
.2.7. 2-Phenylacetylaminobenzothiazole (3k)
Phenylacetyl chloride (1.7 g, 10.8 mmol), potassium thiocya-
nate (1.2 g, 11.8 mmol), 1 (0.8 g, 3.2 mmol). Yield: 1.2 g (71%). IR
0
solution added dropwise to a solution of 2,2 -diaminodiphenyl
ꢁ1
(
cm ): 3063 (
C–N), 1544 (d, NH), 1453, 1263, 1239, 757 (
NMR (CDCl ), d: 9.96 (br s, 1H, NH), 7.87–7.77 (m, 1H, Ar), 7.77–
.68 (m, 1H, Ar), 7.47–7.38 (m, 1H, Ar), 7.38–7.27 (m, 4H, Ar),
m, Ar-H), 3038 (
m
, Ar-H), 1697 (
m
, C@O), 1597 (
m,
H
disulfide (1) in chloroform (20 mL for 3f–h, 15 mL for 3i and 3k)
under stirring at room temperature. Within 2 days the yellow reac-
tion mixture turned pale; hydrogen sulfide evolved. Precipitated
crystals were filtered off, and a second crop was obtained by partial
evaporation of the solution (ethanol was added as needed). Both
portions were washed with chloroform (or ethanol) and dried in
the air to provide pure 3f–i and 3k. The studied compounds are
presented in Table 1.
c
, Ar), 693 (
c
, Ar); 1
3
7
7
1
3
2 3
.27–7.16 (m, 2H, Ar), 3.87 (s, 2H, NHCOCH Ph); C NMR (CDCl ),
d: 169.38, 158.49, 148.01, 132.69, 132.12, 129.40, 129.28, 128.00,
26.35, 124.10, 121.52, 120.69, 43.49; HR-MS: for C15 OS
1
13 2
H N
+
[
M+H] calcd 269.3336 m/z, found 269.0741 m/z.

7
066
D. Fajkusova et al. / Bioorg. Med. Chem. 20 (2012) 7059–7068
4
.2. Lipophilicity determination using HPLC (capacity factor k/
dark for10 min prior to the measurement. Due to low aqueous sol-
calculated logk)
ubility the compounds were added to a chloroplast suspension in
DMSO solution. The DMSO concentration in all samples was the
same as in the control (10%).
A Waters Alliance 2695 XE HPLC separation module and a
Waters Photodiode Array Detector 2996 (Waters Corp., Milford,
MA, USA) were used. A SymmetryÒ
C
5
l
m, 4.6 ꢂ 250 mm, Part
4.5. In vitro antifungal susceptibility testing
18
No. WAT054275 (Waters Corp., Milford, MA, USA) chromato-
graphic column was used. The HPLC separation process was mon-
itored by Empower™ 2 Chromatography Data Software, Waters
The broth microdilution test48,49 was used for the assessment of
in vitro antifungal activity of the synthesized compounds against
Candida albicans ATCC 44859, C. krusei E28, Trichosporon asahii
1188 and Trichophyton mentagrophytes 445. Fluconazole (FLU)
was used as a standard, since it is a clinically used antimycotic
drug. The procedure was performed with a twofold dilution of
the compounds in RPMI 1640 (Sevapharma a.s., Prague, Czech
Republic) buffered to pH 7.0 with 0.165 mol of 3-morpholino-pro-
pane-1-sulfonic acid (MOPS, Sigma, Germany). The final concentra-
2
009 (Waters Corp., Milford, MA, USA). A mixture of MeOH p.a.
70%) and H O-HPLC—Mili-Q Grade (30%) was used as a mobile
phase. The total flow of the column was 1.0 mL/min, injection vol-
ume, 30 L, column temperature, 30 °C, and sample temperature,
0 °C. The detection wavelength of 210 nm was chosen. The KI
methanolic solution was used for the dead time (t ) determination.
Retention times (t ) were measured in minutes. The capacity fac-
tors k were calculated using the Empower™ 2 Chromatography
Data Software according to formula k = (t –t )/t , where t is the
retention time of the solute, whereas t denotes the dead time ob-
(
2
l
1
D
R
tions of the compounds ranged from 500 to 0.975 lmol. Drug-free
R
D
D
R
controls were included. The MIC determination was performed
according to the CLSI reference protocol M27-A2 for yeasts (IC80
value) and M38-A for moulds (IC50 value). IC50 values were defined
as a 50% reduction of growth in comparison with the control, and
IC80 values were defined as an 80% reduction of growth in compar-
ison with the control. The trays were incubated at 35 °C, and MICs
were read visually for filamentous fungi and photometrically for
yeasts as absorbance at 540 nm after 24 h and 48 h. The MIC values
for the dermatophytic strain (T. mentagrophytes) were determined
after 72 h and 120 h. MICs were determined twice and in duplicate.
The results are summarized in Table 2.
D
tained using an unretained analyte. Logk, calculated from the
capacity factor k, is used as the lipophilicity index converted to
logP scale. The logk values of the individual compounds are shown
in Table 1.
4
.3. Lipophilicity calculations
LogP, that is the logarithm of the partition coefficient for n-oct-
anol/water, was calculated using the programs CS ChemOffice Ul-
tra ver. 10.0 (CambridgeSoft, Cambridge, MA, USA) and ACD/LogP
ver. 1.0 (Advanced Chemistry Development Inc., Toronto, Canada).
ClogP values (the logarithm of n-octanol/water partition coeffi-
cient based on established chemical interactions) were generated
by means of CS ChemOffice Ultra ver. 10.0 (CambridgeSoft, Cam-
bridge, MA, USA) software. The results are shown in Table 1.
4.6. In vitro antibacterial susceptibility testing
The synthesized compounds were evaluated for in vitro anti-
bacterial activity against methicillin-resistant Staphylococcus aur-
eus
H
5996/08 and Staphylococcus epidermidis H 6966/08.
Phenoxymethylpenicillin (PMP) was used as the standard since it
is a clinically used antibacterial drug. All strains were sub-cultured
on nutrient agar (HiMedia) and maintained on the same medium at
4
.4. Study of inhibition photosynthetic electron transport (PET)
in spinach chloroplasts
4
°C. Prior to testing, each strain was passaged onto nutrient agar,
Chloroplasts were prepared from spinach (Spinacia oleracea L.)
according to Masarovicova and Kralova.47 The inhibition of photo-
synthetic electron transport (PET) in spinach chloroplasts was
determined spectrophotometrically (Genesys 6, Thermo Scientific,
USA), using an artificial electron acceptor 2,6-dichlorophenol-indo-
phenol (DCIPP) according to Kralova et al.,40 and the rate of photo-
synthetic electron transport was monitored as photoreduction of
DCPIP. The measurements were carried out in phosphate buffer
and bacterial inocula were prepared by suspending a small portion
of bacterial colony in sterile 0.85% saline. The cell density was ad-
justed to 0.5 McFarland units using a densitometer (Densi-La-Me-
ter, PLIVA Lachema Diagnostika, Czech Republic). The final
inoculum was made by 1:20 dilution of the suspension with the
test medium (Mueller-Hinton broth). The compounds were dis-
solved in DMSO, and the anti-bacterial activity was determined
using Mueller-Hinton broth (MH broth, HiMedia, pH 7.0 ± 0.2).
Controls consisted of MH broth and DMSO alone. The final concen-
tration of DMSO in the MH broth did not exceed 1% (v/v) of the to-
tal solution composition. The activity of the studied compounds
was determined as the minimal inhibition concentration (MIC)
according to NCCLS guidelines using broth microdilution test.
The MICs were defined as 90% inhibition of bacterial growth com-
pared to the control and were determined after 24 and 48 h of sta-
tic incubation at 37 °C. After incubation MICs were read visually as
an absorbance at 540 nm. The results are shown in Table 2.
(
(
0.02 mol/L, pH 7.2) containing sucrose (0.4 mol/L), MgCl
0.005 mol/L) and NaCl (0.015 mol/L). The chlorophyll content
2
was 30 mg/L in these experiments, and the samples were irradi-
2
ated (ꢃ100 W/m with 10 cm distance) with a halogen lamp
5
0
(
250 W) using a 4 cm water filter to prevent warming of the sam-
ples (suspension temperature 22 °C). The studied compounds were
dissolved in DMSO due to their limited water solubility. The ap-
plied DMSO concentration (up to 4%) did not affect the photochem-
ical activity in spinach chloroplasts. The inhibitory efficiency of the
studied compounds was expressed by IC50 values, that is, by molar
concentration of the compounds causing 50% decrease in the oxy-
gen evolution rate relative to the untreated control. The compara-
4.7. In vitro antimycobacterial evaluation
ble IC50 value for a selective herbicide 3-(3,4-dichlorophenyl)-1,1-
Mycobacterium kansasii CIT11/06 were grown in Middlebrook
broth (MB), supplemented with Oleic-Albumin-Dextrose-Catalase
supplement (OADC, Becton Dickinson, UK). Identification of these
isolates was performed using biochemical and molecular protocols.
At log phase growth, culture (10 mL) was centrifuged at
15,000 rpm/20 min using a bench top centrifuge (Model CR 4-12,
Jouan Inc., UK). Following removal of the supernatant, the pellet
was washed in fresh Middlebrook 7H9GC broth and re-suspended
Ò
dimethylurea, DCMU (Diurone ) was about 1.9
are summarized in Table 2.
lmol/L. The results
The emission fluorescence spectra were recorded on a fluores-
cence spectrophotometer F-2000 (Hitachi, Tokyo, Japan) at room
temperature (24 °C). The samples of chloroplast suspension
(
10 mg chlorophyll/L) with and without the studied inhibitor were
excited at 436 nm using a slit width of 10 nm and were kept in the

D. Fajkusova et al. / Bioorg. Med. Chem. 20 (2012) 7059–7068
7067
in fresh supplemented MB (10 mL). The turbidity was adjusted to
damaged cells results in an increase of LDH enzyme activity in the
culture supernatant. This increase in the amount of enzyme activ-
ity in the supernatant directly correlates to the amount of forma-
zan formed during a limited time period. The formazan dye
formed is water soluble and shows a broad absorption maximum
at approximately 500 nm.
8
match McFarland standard No. 1 (3 ꢂ 10 cfu) with MB broth. A
further 1:20 dilution of the culture was then performed in MB
broth.
The antimicrobial susceptibility of all four mycobacterial spe-
cies was investigated in a 96-well plate format. In these experi-
ments, sterile deionised water (300 lL) was added to all outer-
perimeter wells of the plates to minimize evaporation of the med-
Acknowledgments
ium in the test wells during incubation. Each evaluated compound
(
(
100
100
l
l
L) was incubated with each of the mycobacterial species
L). Dilutions of each compound were prepared in duplicate.
This study was supported by the Slovak Grant Agency VEGA,
Grant No. 1/0612/11, by Sanofi-Aventis Pharma Slovakia and by
the Irish Department of Agriculture Fisheries and Food
08RDCIT601.
For all synthesized compounds, final concentrations ranged from
000 g/mL to 8 g/mL. All compounds were prepared in DMSO,
1
l
l
and subsequent dilutions were made in supplemented MB. The
plates were sealed with parafilm and incubated at 37 °C for 5 days.
Following incubation, a 10% addition of alamarBlue (AbD Serotec)
was mixed into each well and readings at 570 nm and 600 nm
were taken, initially for background subtraction and subsequently
after 24 h re-incubation. The background subtraction is necessary
for strongly coloured compounds, where the colour may interfere
with the interpretation of any colour change. For non-interfering
compounds, a blue colour in the well was interpreted as an ab-
sence of growth and a pink colour was scored as growth. The
MIC was initially defined as the lowest concentration, which pre-
vented a visual colour change from blue to pink. Isoniazid (INH)
and pyrazinamide (PZA) were used as the standards as they are
clinically used antimycobacterial drugs. The MIC for mycobacteria
was defined as a 90% or greater (IC90) reduction of growth in com-
parison with the control. The MIC/IC90 value is routinely and
widely used in bacterial assays and is a standard detection limit
according to the Clinical and Laboratory Standards Institute (CLSI,
www.clsi.org). The results are summarized in Table 2.
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