A note to the biological activity of benzoxazine derivatives containing the thioxo group

Article abstract of DOI:10.1016/j.ejmech.2010.02.037

New 3-benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-benzyl-2H-1,3-benzoxazine-2,4(3H)-dithiones were synthesized. The compounds were tested for in vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium kansasii and Mycobacterium avium. The replacement of the carbonyl group by the thiocarbonyl group increased the antimycobacterial activity. The most active derivatives were more active than isonicotinhydrazide (INH). The cytotoxicity and the antiproliferative activity were studied as well.

Full text of DOI:10.1016/j.ejmech.2010.02.037

European Journal of Medicinal Chemistry 45 (2010) 2719e2725
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A note to the biological activity of benzoxazine derivatives containing the
thioxo group
,
a
a
a
a
Karel Waisser a , Eva Petrlíková , Milan Perina , Vera Klimesová , Jirí Kunes , Karel Palát Jr. ,
ꢀa
*
ꢀ
ꢀ
ꢀ
ꢀ
Jarmila Kaustová b, Hans-Martin Dahse c, Ute Möllmann c
a Department of Inorganic and Organic Chemistry, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Heyrovský-Str. 1203, 50005 Hradec Králové, Czech Republic
b
ꢀ
Department for Diagnostics of Mycobacteria, Regional Institute of Public Health in Ostrava, Partyzánské námestí 7, 70200 Ostrava, Czech Republic
c Leibniz Institute for Natural Product Research and Infection Biology e Hans Knoell Institute, Beutenbergstr. 11a, D-07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
New 3-benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-benzyl-2H-1,3-benzoxazine-2,4(3H)-
dithiones were synthesized. The compounds were tested for in vitro antimycobacterial activity against
Mycobacterium tuberculosis, Mycobacterium kansasii and Mycobacterium avium. The replacement of the
carbonyl group by the thiocarbonyl group increased the antimycobacterial activity. The most active
derivatives were more active than isonicotinhydrazide (INH). The cytotoxicity and the antiproliferative
activity were studied as well.
Received 11 July 2008
Received in revised form
12 February 2010
Accepted 15 February 2010
Available online 20 February 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Benzoxazine
Thioxo group
Antimycobacterial activity
Cytotoxicity
Antiproliferative activity
1. Introduction
benzylsulfanyl benzoxazole [4]. We suppose that the mechanism of
their antimycobacterial action is different from the mechanisms of
In the period dating from 1985 there has been an increase in the
occurrence of tuberculosis in both Europe and North America. In the
developing countries, due to the insufficient medical care and
hygienic standards, a number of mycobacterial strains become
resistant to antituberculotic drugs used (i.e. multidrug resistant
strains). Due to the contemporary migration of the population with
this infection, itisoftentransferredtoEuropeandNorthAmerica. The
multidrugresistantstrains ofMycobacterium tuberculosis are brought
to Central Europe mainly from the countries of the former Soviet
Union. In addition, this unfavorable state is also being influenced by
an increase in the occurrence of AIDS which is often accompanied by
mycobacterial diseases. Our aim is the development of drugs that
show the activity against M. tuberculosis and against the potential
pathogenic strains of mycobacterium (Mycobacterium avium and
Mycobacterium kansasii). The purpose of our research is the devel-
opment of new antituberculotic agents with high activity. We have
recently studied some hybrid molecules of estrone [1], many isosters
of salicylanilides and salicylamides [2,3] and some derivatives of
recent commercial drugs [5,6]. The most active compounds show
better activity than INH. The goal is to study the antimycobacterial
activity of some compounds with the thioxo group, i.e. substituted
3-benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones, 3-benzyl-2H-
1,3-benzoxazine-2,4(3H)-dithiones prepared from N-benzylsalicy-
lamides. The compounds in this study are the derivatives of ben-
zoxazinediones in which one or both oxo groups were replaced by
the thioxo group. We focused both on their cytotoxicity and
antiproliferative activity. Many antituberculous agents containing
the thioxo group were important in the history of antituberculotics,
e.g. p-acetamidobenzaldehyde thiosemicarbazone (Thiacetazone)
[7], 4, 40-bis(isopentoxy)thiocarbanilide (Isoxyl) [8] and 2-ethyl-
pyridine-4-carbotioamide (Ethionamide) [9]. The replacement of
the oxo group by the thioxo group generally increases anti-
mycobacterial activity. The compounds with the thioxo group are
usually the inhibitors of mycolic acid biosynthesis [8,9]. Our study
comes out from a previous QSAR calculation [10].
2. Chemistry
The synthesis of starting 3-benzyl-2H-1,3-benzoxazine-2,4(3H)-
diones has already been described [11]. Initially we studied the
* Corresponding author. Tel.: þ420 495067276; fax: þ420 465067166.
E-mail address: waisser@faf.cuni.cz (K. Waisser).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.037

2720
K. Waisser et al. / European Journal of Medicinal Chemistry 45 (2010) 2719e2725
H37RV and ATCC 27294), M. kansasii CNCTC My 235/80 (identical
with ATCC 12 478, resistant to INH), M. avium CNCTC My 330/88
(identical with ATCC 25291, resistant to INH) and M. kansasii 6509/
96 using the dilution-micromethod for the determination of
minimum inhibitory concentrations (MIC). All these strains were
obtained from the Czech National Collection of Type Cultures
(CNCTC), National Institute of Public Health, Prague, with the
exception of M. kansasii 6509/96. This strain was clinically isolated,
because of the fact that INH-resistant strains of M. kansasii have not
been found in the Czech Republic.
All prepared compounds were also tested for their cytotoxicity
(cell death) and antiproliferative activity (retardation of cell
proliferation). HeLa (ATCC CRL-1730, human cervical cancer cells)
were used for the cytotoxic assay, while HUVEC (ATCC CRL-1730,
human umbilical vein endothelial cells) and K-562 (DSM ACC 10,
human immortalised myelogenous leukaemia cells) served for the
evaluation of antiproliferative activity. For these studies, we used
different colorimetric assays to determine the number of viable
cells. Under our experimental conditions, the optical density
measured from the CellTiter-BlueÒ reagent and methylene blue
assay is proportional to the number of viable cells.
Fig. 1. 2D NMR: a correlation between the hydrogen in position 5 and the thiocarbonyl
group.
conditions of the synthesis of 3-benzyl-4-thioxo-2H-1,3-benzox-
azine-2(3H)-ones and 3-benzyl-2H-1,3-benzoxazine-2,4(3H)-dithiones.
The compounds prepared by the reaction of 3-benzyl-2H-1,3-
benzoxazine-2,4(3H)-diones with P4S10 form a chromatographic
swimmingly separable mixture of sulfur analogues. These products
differ in their colour, 3-benzyl-4-thioxo-2H-1,3-benzoxazine-2
(3H)-ones crystallise as yellow or orange solids, while 3-benzyl-2H-
1,3-benzoxazine-2,4(3H)-dithiones form red coloured crystals. The
fractions of pure compounds were collected and crystallized from
ethanol. The mother liquor was evaporated. The residue contained
the pure compound (controlled by TLC). Additionally we crystallized
the residue from ethanol. Two methods were developed for the
conversion of the oxo group to the thioxo group in 3-benzyl-2H-1,3-
benzoxazine-2,4(3H)-diones. The conditions of the first method (A)
were very mild (lower temperature, an equivalent amount of the
reagents and a shorter time of reaction). 3-Benzyl-4-thioxo-2H-1,3-
benzoxazine-2(3H)-ones were the main products. The position of
sulfur was established by 2D NMR. The position of the thiocarbonyl
group was determined via gHMQC correlation of hydrogen in posi-
tion 5 with the carbon atom of the thiocarbonyl group in position 4
over three bonds. A cross-peak of the hydrogen in position 5 and the
thiocarbonyl group is displayed in Fig. 1.
4. Results and discussion
We prepared two groups of benzoxazine derivatives with the
thioxo group: 3-benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones
(2ae2i)
and
3-benzyl-2H-1,3-benzoxazine-2,4(3H)-dithiones
(3ae3i). The mixture of these sulfur analogues was obtained by the
reaction of previously prepared 3-benzyl- 2H-1,3-benzoxazine-2,4
(3H)-diones (1ae1i) with phosphorus pentasulfide. We used two
different methods for this preparation. The first method (A) was
mild, the reaction temperature was 180e190 ꢀC, the equivalent
molecular amount of the reagent was used and the melting time
was 20 min. The increase in temperature, the duration of the
An increase in the reagents, temperature and reaction time was
used in the method (B). The reaction yielded a mixture of both
products. The synthetic pathway is illustrated in Scheme 1. The first
two steps have already been published [11,12].
reaction and the excess of phosphorus pentasulfide (P4S10
)
3. Biology
provided both products, increasing the yield (method B). The
temperature of the reaction was 200e210 ꢀC, a three times larger
amount of phosphorus pentasulfide was used and the duration of
melting was 40 min. The products were separated by column
chromatography. Only 3-benzyl-4-thioxo-2H-1,3-benzoxazine-2
(3H)-ones were obtained by method A in some cases (2ee2i). The
frequencies in the region (1735e1758 cmꢁ1) within the infrared
spectra are characteristic of 3-benzyl-4-thioxo-2H-1,3-benzox-
azine-2(3H)-ones. The second method resulted in the production of
both products. The location of sulfur for 3-benzyl-4-thioxo-2H-1,3-
benzoxazine-2(3H)-ones was elaborated by gHMQC experiments.
The correlation of the chemical shifts of the carbon in the thi-
ocarbonyl moiety with the chemical shifts of H-5 was observed (see
Fig. 1). The structure of 3-benzyl-2H-1,3-benzoxazine-2,4(3H)-
dithiones was also confirmed by NMR. The modifications of mole-
cules were made by various substituents.
In vitro antimycobacterial activity of the compounds against
M. tuberculosis CNCTC My 331/88, M. kansasii CNCTC My 235/80,
M. avium CNCTC My 330/88 and M. kansasii 6509/96 are summa-
rized in Table 1. To compare the MIC (minimum inhibition
concentration) values, the previously described 3-benzyl-2H-1,3-
benzoxazine-2,4(3H)-diones (1ae1i) [9] were inserted in Table 1.
Thus the replacement of the oxo group by the thioxo group
increases the antimycobacterial activity. All studied compounds are
active against INH-resistant strains M. kansasii CNCTC My 235/80
and M. avium CNCTC My 330/88. The antimycobacterial activity
against M. tuberculosis of compounds 2d, 2e, 2g and 3a is compa-
rable with the activity of INH but the activity against M. kansasii
6509/96 is higher. All strains are active to rifampicin but the activity
Scheme 1. Synthesis of 3-benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-
benzyl-2H-1,3-benzoxazine-2,4(3H)-dithiones. a Ref [12], b Ref. [11]. Substituents are
described in Table 1.
3-Benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones, 3-benzyl-2H-
1,3-benzoxazine-2,4(3H)-dithiones and corresponding 3-benzyl-2H-
1,3-benzoxazine-2,4(3H)-diones were used for the biological evaluations.
The in vitro antimycobacterial activity of these compounds was
evaluated against M. tuberculosis CNCTC My 331/88 (identical with

K. Waisser et al. / European Journal of Medicinal Chemistry 45 (2010) 2719e2725
2721
Table 1
Minimum inhibitory activity of 3-benzyl-2H-1,3-benzoxazine-2,4(3H)-diones 3-benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-benzyl-2H-1,3-benzoxazine-2,4(3H)-
dithiones.
O
O
O
S
O
S
O
O
S
N
N
N
R2
R1
2
R2
R2
R1
R1
3
1
Compounds
MIC (mmol/L) Incubation time 14 d/21 d
R1
R2
M. tuberculosis My 331/88
M. avium My 330/88
M. kansasii My 235/80
M. kansasii 6509/96
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
2e
2f
2g
2h
2i
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
H
H
H
32/32
62.5/62.5
62.5/125
32/62.5
32/32
62.5/125
32/32
32/32
16/16
1/2
1/2
1/2
1/1
1/1
2/2
1/1
2/4
32/32
1/1
32/62.5
62.5/62.5
32/62.5
32/62.5
32/32
125/125
32/32
32/32
16/32
8/8
8/8
4/8
2/2
4/4
4/4
1/1
8/8
62.5/62.5
1/1
8/8
n
n
8/16
62.5/62.5
0.5/0.5
16/16
16/32
62.5/62.5
62.5/125
32/62.5
62.5/62.5
62.5/62.5
125/125
62.5/62.5
62.5/62.5
62.5/62.5
4/8
8/8
8/8
1/2
4/8
16/16
2/4
16/16
62.5/62.5
2/2
8/8
n
n
8/16
62.5/62.5
4/4
16/32
32/32
62.5/62.5
62.5/125
125/125
62.5/125
a/125
125/125
62.5/62.5
62.5/62.5
62.5/62.5
4/8
4/8
4/4
1/2
4/4
16/16
2/4
16/16
62.5/62.5
1/2
4/8
n
n
8/16
62.5/62.5
4/4
16/32
32/32
6-Br
6-Cl
6-CH3
6-Cl
6,8-Cl2
H
6-Br
6-Cl
H
6-Br
6-Cl
6-CH3
6-Cl
6,8-Cl2
H
6-Br
6-Cl
H
4-F
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
H
H
H
H
4-F
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
H
H
H
H
4-F
6-Br
6-Cl
6-CH3
6-Cl
6,8-Cl2
H
1/2
n
n
1/2
32/32
1/2
4/8
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
6-Br
6-Cl
4/8
INH
1/1
>250/>250
>250/>250
8/8
Rifampicin
0.25/0.5
16/65
0.25/0.25
0.125/0.25
Antimycobacterial activity of 3-benzyl-2H-1,3-benzoxazine-2,4(3H)-diones was taken over from the Ref. [11]. n, The test was not evaluated due to the low solubility of the
compound.
of rifampicin against M. avium CNCTC My 330/88 is low. Thus,
rifampicin has no medical importance in this case.
Above all, the compounds in the study possess excellent anti-
mycobacterial activity. It was also necessary to study their side
effects, cytotoxicity and antiproliferative activity. The results are
shown in Figs. 2e4.
The antimycobacterial activities of sulfur analogs of 3-benzyl-
2H-1,3-benzoxazine-2,4(3H)-diones, especially 3-benzyl-4-thioxo-
2H-1,3-benzoxazine-2(3H)-ones, are strong, but due to their
cytotoxicity they seemed not to be prospective as antituberculous
drugs.
the mobile phase. Crystallization of products was carried out from
ethanol. The 1H NMR and 13C NMR spectra of new compounds were
recorded in CDCl3 or DMSO-d6 solutions at ambient temperature on
a Varian Mercury-Vx BB 300 spectrometer operating at 300 MHz for
1H NMR, and 75 MHz for 13C NMR. Chemical shifts were recorded as
d
values in parts per million (ppm) and were indirectly referenced to
tetramethylsilane via the solvent signal (2.49 for 1H or 39.7 for 13C).
The position of the thiocarbonyl group was determined via gHMQC
correlation of hydrogen in position 5 with the carbon atom of the
thiocarbonyl group in position 4 over three bonds. The starting 3-
benzyl-2H-1,3-benzoxazine-2,4(3H)-diones were prepared in our
previous paper [11] (see Scheme 1).
5. Experimental protocols
5.1.1. Chemistry, synthetic procedures for the preparation of
3-benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-benzyl-
2H-1,3-benzoxazine-2,4(3H)-dithiones
5.1. Chemistry, general information
The melting points were determined on a Kofler apparatus. The
samples for the analyses and antimycobacterial tests were dried over
P2O5 at 61 ꢀC and 66 Pa for 24 h. Elemental analyses (C, H, N) were
performed on a CHNS-O CE elemental analyzer (Fisions EA 1110,
Milan) and were within ꢂ0.4% of the theoreticalvalues. The IR spectra
were measured in KBr pellets on a Nicolet Impact 400 apparatus; the
wavenumbers are given in cmꢁ1. TLC was performed on silica gel
plates precoated with a fluorescent indicator Silufol UV 254 þ 366
(Kavalier Votice, Czech Republic), hexaneeacetone (3:1) was used as
A) 3-Benzyl-2H-1,3-benzoxazine-2,4(3H)-diones prepared in the
previous paper [10] (3.8 mmol) were melted with P4S10
(3.8 mmol) for 20 min (180e190 ꢀC). After cooling to the room
temperature, a 10% potassium carbonate solution (60 mL) was
poured intothe reaction mixture; the crude product was filtered
off, and dissolved in toluene (p.a., 40 mL). Column chromatog-
raphyon silica gel gave 3-benzyl-4-thioxo-2H-1,3-benzoxazine-
2(3H)-ones (2ae2d) and 3-benzyl-2H-1,3-benzoxazine-2,4

2722
K. Waisser et al. / European Journal of Medicinal Chemistry 45 (2010) 2719e2725
Fig. 2. Cytotoxicity (HeLa): 1) 3-benzyl-2H-1,3-benzoxazine-2,4(3H)-diones, 2) 3-
benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones, 3) 3-benzyl-2H-1,3-benzoxazine-2,4
(3H)-dithiones.
Fig. 4. Antiproliferative activity (HUVEC): 1) 3-benzyl-2H-1,3-benzoxazine-2,4(3H)-
diones, 2) 3-benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones, 3) 3-benzyl-2H-1,3-
benzoxazine-2,4(3H)-dithiones.
(3H)-dithiones (3ae3d). In other cases only 3-benzyl-4-thioxo-
2H-1,3-benzoxazine-2(3H)-ones (2ee2i) were isolated.
5.1.3. 3-Benzyl-6-bromo-4-thioxo-2H-1,3-benzoxazine-2(3H)-one
(2b)
B) 3-Benzyl-2H-1,3-benzoxazine-2,4(3H)-diones prepared in the
previous paper [10] (3.8 mmol) were melted with of P4S10
(10.1 mmol) for 40 min (200e210 ꢀC). After cooling to the room
temperature, a 10% potassium carbonate solution (60 mL) was
poured into the reaction mixture; the crude product was
filtered off, and dissolved in toluene (p.a., at the most 40 mL).
Column chromatography on silica gel gave 3-benzyl-4-thioxo-
2H-1,3-benzoxazine-2(3H)-ones (2ee2i) and 3-benzyl-2H-1,3-
benzoxazine-2,4(3H)-dithiones (3ee3i).
3-Benzyl-6-bromo-4-thioxo-2H-1,3-benzoxazine-2(3H)-one (2b)
was prepared using procedure A from 3-benzyl-6-bromo-2H-1,3-
benzoxazine-2,4(3H)-dione (1.26 g, 3.8 mmol) to yield 21% (0.277 g,
0.796 mmol) of the title compound as a crystalline yellow solid. Mp.
130e133 ꢀC, IR nC]O 1758 cmꢁ1
.
For C15H10BrNO2S (348.2) calculated: 51.72% C, 4.02% N, 2.87% H,
9.20% S; found: 51.64% C, 3.97% N, 2.94% H, 9.06% S.
1H NMR (300 MHz, DMSO-d6)
d
8.34 (d, 1H, J ¼ 2.4 Hz, H5), 7.98
(dd, 1H, J ¼ 8.1 Hz, J ¼ 2.4 Hz, H7), 7.43 (d, 1H, J ¼ 8.1 Hz, H8),
7.37e7.23 (m, 5H, H20, H30, H40, H50, H60), 5.64 (s, 2H, CH2). 13C NMR
(75 MHz, DMSO-d6)
127.4, 127.3, 122.1, 119.4, 117.6, 52.0.
d 190.2, 148.8, 144.9, 138.7, 135.1, 133.1, 128.4,
5.1.2. 3-Benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-one (2a)
3-Benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-one (2a) was
synthesized using procedure A from 3-benzyl-2H-1,3-benzoxazine-
2,4(3H)-dione (0.96 g, 3.8 mmol) to yield 20% (0.204 g,
0.758 mmol) of the title compound as a crystalline yellow solid. Mp.
5.1.4. 3-Benzyl-6-chloro-4-thioxo-2H-1,3-benzoxazine-2(3H)-one
(2c)
115e116 ꢀC, IR nC]O 1735 cmꢁ1
.
3-Benzyl-6-chloro-4-thioxo-2H-1,3-benzoxazine-2(3H)-one (2c)
was prepared using procedure A from 3-benzyl-6-chloro-2H-1,3-
benzoxazine-2,4(3H)-dione (1.09 g, 3.8 mmol) to yield 26% (0.3 g,
0.988 mmol) of the title compound as a crystalline yellow solid. Mp.
For C15H11NO2S (269.3 g/mol) calculated: 66.91% C, 5.20% N,
4.09% H, 11.90% S; found: 66.77% C, 5.15% N, 4.14% H, 11.68% S.
1H NMR (300 MHz, DMSO-d6)
d
8.30 (dd, 1H, J ¼ 8.4 Hz,
120e122 ꢀC, IR nC]O 1762 cmꢁ1
.
J ¼ 2.7 Hz, H5), 7.86e7.80 (m, 1H, H7), 7.45e7.41 (m, 2H, H6, H8),
7.37e7.21 (m, 5H, H20, H30, H40, H50, H60), 5.66 (s, 2H, CH2). 13C NMR
For C15H10ClNO2S (303.8) calculated: 59.31% C, 4.61% N, 3.29% H,
10.54% S; found: 59.49% C, 4.57% N, 3.34% H, 10.45% S.
(75 MHz, DMSO-d6)
d 91.7, 149.6, 145.3, 136.6, 135.4, 131.7, 128.5,
1H NMR (300 MHz, DMSO-d6)
d
8.21 (d, 1H, J ¼ 2.7 Hz, H5), 7.88
127.4, 127.3, 125.9, 120.5, 116.7, 51.7.
(dd, 1H, J ¼ 8.7 Hz, J ¼ 2.7 Hz, H7), 7.50 (d, 1H, J ¼ 8.7 Hz, H8),
7.38e7.21 (m, 5H, H20, H30, H40, H50, H60), 5.64 (s, 2H, CH2). 13C NMR
(75 MHz, DMSO-d6)
d 190.3, 148.4, 144.9, 135.9, 135.1, 130.1, 129.9,
128.5, 127.4, 127.3, 121.7, 119.2, 52.0.
5.1.5. 3-Benzyl-6-methyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-one
(2d)
3-Benzyl-6-methyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-one (2d)
was prepared using procedure A from 3-benzyl-6-methyl-2H-1,3-
benzoxazine-2,4(3H)-dione (1.02 g, 3.8 mmol) to yield 22% (0.25 g,
0.872 mmol) of the title compound as a crystalline yellow solid. Mp.
132e135 ꢀC, IR nC]O 1746 cmꢁ1
.
For C16H13NO2S (283.4) calculated: 67.82% C, 4.62% H, 4.94% N,
11.32% S; found: 67.51% C, 4.61% H, 4.90% N, 11.00% S.
1H NMR (300 MHz, DMSO-d6)
d
8.08 (d, 1H, J ¼ 2.1 Hz, H5), 7.63
(dd, 1H, J ¼ 8.1 Hz, J ¼ 2.1 Hz, H7), 7.35e7.20 (m, 6H, H8, H20, H30,
H40, H50, H60), 5.66 (s, 2H, CH2), 2.39 (s, 3H, CH3). 13C NMR (75 MHz,
Fig. 3. Antiproliferative activity (K-562): 1) 3-benzyl-2H-1,3-benzoxazine-2,4(3H)-
diones, 2) 3-benzyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones, 3) 3-benzyl-2H-1,3-
benzoxazine-2,4(3H)-dithiones.
DMSO-d6)
120.1, 116.6, 51.6, 20.5.
d 191.7, 147.7, 145.4, 137.4, 135.4, 131.2, 128.5, 127.4, 127.3,

K. Waisser et al. / European Journal of Medicinal Chemistry 45 (2010) 2719e2725
2723
5.1.6. 6-Chloro-3-(4-fluorobenzyl)-4-thioxo-2H-1,3-benzoxazine-2
(3H)-one (2e)
5.1.10. 6-Chloro-3-(3,4-dichlorobenzyl)-4-thioxo-2H-1,3-
benzoxazin-2(3H)-one (2i)
6-Chloro-3-(4-fluorobenzyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-
one (2e) was prepared using procedure A from 6-chloro-3-(4-flu-
orobenzyl)-2H-1,3-benzoxazine-2(3H)-dione (1.16 g, 3.8 mmol) to
yield 24% (0.29 g, 0.911 mmol) of the title compound and using
procedure B to yield 34% (0.42 g,1.290 mmol) of a crystalline yellow
6-Chloro-3-(3,4-dichlorobenzyl)-4-thioxo-2H-1,3-benzoxazin-2
(3H)-one (2i) was prepared using procedure A from 6-chloro-
3-(3,4-dichlorobenzyl)-2H-1,3-benzoxazin-2(3H)-dione (1.36 g,
3.8 mmol) to yield 21% (0.30 g, 0.833 mmol) of the title compound
and using procedure B to yield 27% (0.38 g, 1.025 mmol) of a crys-
solid. Mp. 127e129 ꢀC, IR nC]O 1751 cmꢁ1
.
talline yellow solid. Mp. 157e160 ꢀC, IR nC]O 1756 cmꢁ1
.
For C15H9ClFNO2S (321.8) calculated: 55.99% C, 2.82% H, 4.35% N,
9.94% S; found: 55.94% C, 4.26% N, 2.91% H, 9.92% S.
For C15H8Cl3NO2S (372.7) calculated: 48.32% C, 32.16% H, 3.76%
N, 8.60% S; found: 48.42% C, 2.05% H, 3.39% N, 8.29% S.
1H NMR (300 MHz, DMSO-d6)
d
8.19 (d, 1H, J ¼ 2.4 Hz, H5), 7.87
1H NMR (300 MHz, DMSO-d6)
d
8.20 (d, 1H, J ¼ 3.0 Hz, H5), 8.00
(dd, 1H, J ¼ 8.7 Hz, J ¼ 2.4 Hz, H7), 7.49 (d, 1H, J ¼ 8.7 Hz, H8),
(dd, 1H, J ¼ 9.0 Hz, J ¼ 3.0 Hz, H7), 7.71 (d, 1H, J ¼ 2.1 Hz, H20), 7.56
(d, 1H, J ¼ 8.4 Hz, H50), 7.52 (d, 1H, J ¼ 9.0 Hz, H8), 7.39 (dd, 1H,
J ¼ 8.4 Hz, J ¼ 2.1 Hz, H60), 5.59 (s, 2H, CH2). 13C NMR (75 MHz,
7.46e7.40 (m, AA0, BB0, 2H, H20, H6), 7.16e7.08 (m, AA0, BB0, 2H, H30,
H50), 5.60 (s, 2H, CH2). 13C NMR (75 MHz, DMSO-d6)
d 190.3, 161.6
(d, J ¼ 241.6 Hz), 148.4, 145.0, 135.9, 131.3 (d, J ¼ 3.1 Hz), 129.9 (d,
DMSO-d6) d 190.5, 148.6, 145.1, 136.4, 135.9, 131.2, 130.5, 130.0,
J ¼ 18.8 Hz), 129.8, 129.7, 121.8, 119.2, 115.4 (d, J ¼ 21.1 Hz), 51.4.
129.9, 129.8, 129.3, 127.9, 121.9, 119.2, 51.3.
5.1.7. 6,8-Dichloro-3-(3,4-dichlorobenzyl)-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2f)
5.1.11. 3-Benzyl-2H-1,3-benzoxazine-2,4(3H)-dithione (3a)
3-Benzyl-2H-1,3-benzoxazine-2,4(3H)-dithione (3a) was
prepared using procedure A from 3-benzyl-2H-1,3-benzoxazine-
2,4(3H)-dione (0.96 g, 3.8 mmol) to yield 22% (0.24 g, 0.835 mmol)
of the title compound as a crystalline red solid. Mp. 147e150 ꢀC.
For C15H11NOS2 (285.0) calculated: 63.13% C, 4.91% N, 3.89% H,
22.47% S; found: 62.88% C, 4.85% N, 3.95% H, 22.38% S.
6,8-Dichloro-3-(3,4-dichlorobenzyl)-4-thioxo-2H-1,3-benzox-
azine-2(3H)-one (2f) was prepared using procedure A from 6,8-
dichloro-3-(3,4-dichlorobenzyl)-2H-1,3-benzoxazine-2(3H)-dione
(1.49 g, 3.8 mmol) to yield 24% (0.37 g, 0.911 mmol) of the title
compound and using procedure B to yield 34% (0.53 g, 1.292 mmol)
of a crystalline yellow solid. Mp. 169e170 ꢀC, IR nC]O 1739 cmꢁ1
.
1H NMR (300 MHz, DMSO-d6)
d
8.22 (dd, 1H, J ¼ 8.1 Hz,
For C15H7Cl4NO2S (407.1) calculated: 44.26% C, 1.73% H, 3.44% N,
7.88% S; found: 44.03% C, 1.83% H, 3.52% N, 7.72% S.
J ¼ 1.2 Hz, H5), 7.90e7.84 (m, 1H, H7), 7.53e7.44 (m, 2H, H6, H8),
7.29e7.18 (m, 5H, H20, H30, H40, H50, H60), 6.20 (s, 2H, CH2). 13C NMR
1H NMR (300 MHz, DMSO-d6)
d
8.23 (d, 1H, J ¼ 2.4 Hz, H5), 8.15
(75 MHz, DMSO-d6) d 187.1, 178.4, 149.6, 136.9, 134.3, 131.8, 128.5,
127.2, 127.1, 126.6, 121.9, 116.4, 57.
(d, 1H, J ¼ 2.4 Hz, H7), 7.71 (d, 1H, J ¼ 2.1 Hz, H20), 7.57 (d, 1H,
J ¼ 8.4 Hz, H50), 7.40 (dd, 1H, J ¼ 8.4 Hz, J ¼ 2.1 Hz, H60), 5.58 (s, 2H,
CH2). 13C NMR (75 MHz, DMSO-d6)
d
189.6, 145.2, 144.5,136.1, 135.0,
5.1.12. 3-Benzyl-6-bromo-2H-1,3-benzoxazine-2,4(3H)-dithione
(3b)
131.2, 130.5, 129.9, 129.4, 129.1, 129.1, 127.8, 122.9, 121.8, 51.6.
3-Benzyl-6-bromo-2H-1,3-benzoxazine-2,4(3H)-dithione (3b)
was prepared using procedure A from 3-benzyl-6-bromo-2H-1,3-
benzoxazine-2,4(3H)-dione (1.26 g, 3.8 mmol) to yield 21% (0.29 g,
0.795 mmol) of the title compound as a crystalline red solid. Mp.
157e160 ꢀC.
5.1.8. 3-(3,4-Dichlorobenzyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-
one (2g)
3-(3,4-Dichlorobenzyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-one
(2g) was prepared using procedure A from 3-(3,4-dichlorobenzyl)-
2H-1,3-benzoxazine-2(3H)-dione (1.22 g, 3.8 mmol) to yield 24%
(0.31 g, 0.911 mmol) of the title compound and using procedure B
to yield 52% (0.67 g, 1.976 mmol) of a crystalline yellow solid. Mp.
For C15H10BrNOS2 (364.9) calculated: 49.46% C, 3.85% N, 2.75% H,
17.61% S; found: 49.32% C, 3.61% N, 2.82% H, 17.75% S.
1H NMR (300 MHz, DMSO-d6)
d
8.26 (d, 1H, J ¼ 2.4 Hz, H5), 7.54
169e170 ꢀC, IR nC]O 1739 cmꢁ1
.
(dd, 1H, J ¼ 8.4 Hz, J ¼ 2.4 Hz, H7), 7.53 (d, 1H, J ¼ 8.4 Hz, H8),
For C15H9Cl2NO2S (338.2) calculated: 53.27% C, 2.69% H, 4.14% N,
9.48% S; found: 53.08% C, 2.67% H, 4.10% N, 9.55% S.
7.29e7.17 (m, 5H, H20, H30, H40, H50, H60), 6.16 (s, 2H, CH2). 13C NMR
(75 MHz, DMSO-d6)
d 185.6, 177.9, 148.9, 139.1, 134.1, 133.2, 128.5,
1H NMR (300 MHz, DMSO-d6)
d
8.29 (dd, 1H, J ¼ 8.4 Hz,
127.1, 126.6, 123.5, 119.1, 118.8, 57.9.
J ¼ 2.40 Hz, H5), 7.86e7.80 (m, 1H, H7), 7.71 (d, 1H, J ¼ 2.1 Hz, H20),
7.55 (d, 1H, J ¼ 8.1 Hz, H50), 7.45e7.37 (m, 3H, H6, H8, H60), 5.61 (s,
5.1.13. 3-Benzyl-6-chloro-2H-1,3-benzoxazine-2,4(3H)-dithione
(3c)
2H, CH2). 13C NMR (75 MHz, DMSO-d6)
d 191.9, 149.8, 145.4, 136.7,
136.5, 131.5, 131.2, 130.5, 129.9, 129.4, 127.8, 125.9, 120.7, 116.7, 51.0.
3-Benzyl-6-chloro-2H-1,3-benzoxazine-2,4(3H)-dithione (3c)
was prepared using procedure A from 3-benzyl-6-chloro-2H-1,3-
benzoxazine-2,4(3H)-dione (1.09 g, 3.8 mmol) to yield 16% (0.20 g,
0.638 mmol) of the title compound as a crystalline red solid. Mp.
148e150 ꢀC.
5.1.9. 6-Bromo-3-(3,4-dichlorobenzyl)-4-thioxo-2H-1,3-
benzoxazin-2(3H)-one (2h)
6-Bromo-3-(3,4-dichlorobenzyl)-4-thioxo-2H-1,3-benzoxazin-2
(3H)-one (2h) was prepared using procedure A from 6-bromo-3-
(3,4-dichlorobenzyl)-2H-1,3-benzoxazin-2(3H)-dione (1.52 g, 3.8 mmol)
to yield 23% (0.37 g, 0.875 mmol) of the title compound and using
procedure B to yield 30% (0.48 g, 1.141 mmol) of a crystalline yellow
For C15H10ClNOS2 (319.0) calculated: 56.33% C, 4.38% N, 3.15% H,
20.05% S; found: 56.28% C, 4.52% N, 3.08% H, 19.95% S.
1H NMR (300 MHz, DMSO-d6)
d
8.13 (d, 1H, J ¼ 2.7 Hz, H5), 7.93
(dd, 1H, J ¼ 8.7 Hz, J ¼ 2.7 Hz, H7), 7.60 (d, 1H, J ¼ 8.7 Hz, H8),
solid. Mp. 180e183 ꢀC, IR nC]O 1739 cmꢁ1
.
7.34e7.18 (m, 5H, H20, H30, H40, H50, H60), 6.17 (s, 2H, CH2). 13C NMR
For C15H8BrCl2NO2S (417.1) calculated: 43.16% C, 1.93% H, 3.36%
N, 7.69% S; found: 43.33% C, 2.00% H, 3.31% N, 7.58% S.
(75 MHz, DMSO-d6) d 185.8, 178.0, 148.5, 136.3, 134.1, 130.9, 130.2,
128.5, 127.2, 126.6, 123.1, 118.9, 58.0.
1H NMR (300 MHz, DMSO-d6)
d
8.33 (d, 1H, J ¼ 2.1 Hz, H5), 8.00
(dd, 1H, J ¼ 8.7 Hz, J ¼ 2.1 Hz, H7), 7.71 (d, 1H, J ¼ 2.1 Hz, H20), 7.57
(d, 1H, J ¼ 8.1 Hz, H50), 7.44 (d, 1H, J ¼ 8.7 Hz, H8), 7.39 (dd, 1H,
J ¼ 8.1 Hz, J ¼ 2.1 Hz, H60), 5.59 (s, 2H, CH2). 13C NMR (75 MHz,
5.1.14. 3-Benzyl-6-methyl-2H-1,3-benzoxazine-2,4(3H)-dithione
(3d)
3-Benzyl-6-methyl-2H-1,3-benzoxazine-2,4(3H)-dithione (3d)
was prepared using procedure A from 3-benzyl-6-methyl-2H-1,3-
benzoxazine-2,4(3H)-dione (1.02 g, 3.8 mmol) to yield 15% (0.17 g,
DMSO-d6)
d 190.4, 149.0, 145.1, 138.7, 136.4, 133.0, 131.2, 130.5,
129.9, 129.3, 127.9, 122.3, 119.4, 117.5, 51.3.

2724
K. Waisser et al. / European Journal of Medicinal Chemistry 45 (2010) 2719e2725
0.571 mmol) of the title compound as a crystalline red solid. Mp.
118e119 ꢀC.
1H NMR (300 MHz, DMSO-d6)
d
8.25 (d, 1H, J ¼ 2.5 Hz, H5), 8.03
(dd, 1H, J ¼ 8.8 Hz, J ¼ 2.5 Hz, H7), 7.68 (d, 1H, J ¼ 1.9 Hz, H20), 7.54
(d overlapped, 1H, J ¼ 8.2 Hz, H50), 7.52 (d overlapped, 1H,
J ¼ 8.8 Hz, H8), 7.35 (dd 1H, J ¼ 8.2 Hz, J ¼ 1.9 Hz, H60), 6.10 (s, 2H,
For C16H13NOS2 (299.0) calculated: 64.18% C, 4.68% N, 4.38% H,
21.42% S; found: 64.32% C, 4.63% N, 4.23% H, 21.60% S.
1H NMR (300 MHz, DMSO-d6)
d
8.00 (d, 1H, J ¼ 2.1 Hz, H5), 7.69
CH2). 13C NMR (75 MHz, DMSO-d6)
d 185.9, 178.2, 149.1, 139.0, 135.6,
133.0, 131.2, 130.5, 129.7, 128.6, 127.2, 123.7, 119.0, 118.7, 57.4.
(dd, 1H, J ¼ 8.4 Hz, J ¼ 2.1 Hz, H7), 7.43 (d, 1H, J ¼ 8.4 Hz, H8),
7.31e7.19 (m, 5H, H20, H30, H40, H50, H60), 6.20 (s, 2H, CH2) 2.39 (s,
3H, CH3). 13C NMR (75 MHz, DMSO-d6)
d
187.0, 178.3, 147.9, 137.8,
5.1.19. 6-Chloro-3-(3,4-dichlorobenzyl)-2H-1,3-benzoxazin-2(3H)-
dithione (3i)
136.8, 134.4, 131.1, 128.4, 127.1, 126.5, 121.6, 116.2, 57.6, 20.6.
6-Chloro-3-(3,4-dichlorobenzyl)-2H-1,3-benzoxazin-2(3H)-
dithione (3i) was prepared using procedure B from 6-chloro-3-(3,4-
dichlorobenzyl)-2H-1,3-benzoxazin-2(3H)-dione (1.36 g, 3.8 mmol)
to yield 42% (0.62 g, 1.595 mmol) of the title compound as a crys-
talline red solid. Mp. 183e185 ꢀC.
5.1.15. 6-Chloro-3-(4-fluorobenzyl)-2H-1,3-benzoxazine-2(3H)-
dithione (3e)
6-Chloro-3-(4-fluorobenzyl)-2H-1,3-benzoxazine-2(3H)-dithione
(3e) was prepared using procedure B from 6-chloro-3-(4-fluo-
robenzyl)-2H-1,3-benzoxazine-2(3H)-dione (1.16 g, 3.8 mmol) to
yield 30% (0.39 g, 1.139 mmol) of the title compound as a crystalline
red solid. Mp. 147e150 ꢀC.
For C15H8Cl3NOS2 (388.7) calculated: 46.35% C, 3.60% N, 2.07% H,
16.50% S; found 46.09% C, 3.35% N, 1.97% H, 16.49% S.
1H NMR (300 MHz, DMSO-d6)
d
8.11 (d, 1H, J ¼ 2.5 Hz, H5), 7.91
For C15H6 ClFNOS2 (337.8) calculated: 53.33% C, 4.15% N, 2.69% H,
18.98% S; found: 53.52% C, 3.86% N, 2.80% H, 18.99% S.
(dd, 1H, J ¼ 8.8 Hz, J ¼ 2.5 Hz, H7), 7.67 (d, 1H, J ¼ 1.4 Hz, H20), 7.59
(d, 1H, J ¼ 8.8 Hz, H8), 7.542 (d, 1H, J ¼ 8.8 Hz, H50), 7.35 (dd 1H,
J ¼ 8.2 Hz, J ¼ 1.4 Hz, H60), 6.10 (s, 2H, CH2). 13C NMR (75 MHz,
1H NMR (300 MHz, DMSO-d6)
d
8.12 (d, 1H, J ¼ 2.6 Hz, H5), 7.91
(dd, 1H, J ¼ 8.8 Hz, J ¼ 2.6 Hz, H7), 7.59 (d, 1H, J ¼ 8.8 Hz, H8),
DMSO-d6) d 186.0, 178.2, 148.7, 136.2, 135.5, 131.2, 130.8, 130.5,
7.42e7.32 (2H, m, H20, H60), 7.16e7.06 (m, 2H, H30, H50), 6.13 (s, 2H,
130.0, 129.7, 128.6, 127.2, 123.3, 118.8, 57.4.
CH2). 13C NMR (75 MHz, DMSO-d6)
d
8, 178.0, 161.4 (d, J ¼ 242.8 Hz),
5.2. Biology
148.5, 136.3, 130.9, 130.2 (d, J ¼ 3.2 Hz), 130.1, 128.8 (d, J ¼ 8.0 Hz),
123.2, 118.9, 115.2 (d, J ¼ 21.5 Hz), 57.4.
5.2.1. Antimycobacterial assay
5.2.1.1. Antimycobacterial susceptibility testing. For the in vitro
evaluation of the antimycobacterial activity of the substances, the
following strains were used: M. tuberculosis CNCTC My 331/88
(identical with H37RV and ATCC 27294), M. kansasii CNCTC My
235/80 (identical with ATCC 12 478), M. avium CNCTC My 330/88
(identical with ATCC 25291), obtained from the Czech National
Collection of Type Cultures (CNCTC), National Institute of Public
Health, Prague, and a clinical isolate of M. kansasii 6509/96. The
antimycobacterial activity of the compounds was determined in
5.1.16. 6,8-Dichloro-3-(3,4-dichlorobenzyl)-2H-1,3-benzoxazine-2
(3H)-dithione (3f)
6,8-Dichloro-3-(3,4-dichlorobenzyl)-2H-1,3-benzoxazine-2(3H)-
dithione (3f) was prepared using procedure B from 6,8-dichloro-3-
(3,4-dichlorobenzyl)-2H-1,3-benzoxazine-2(3H)-dione (1.49 g,
3.8 mmol) to yield 51% (0.82 g, 1.938 mmol) of the title compound
as a crystalline red solid. Mp. 178e181 ꢀC.
For C15H7Cl4NOS2 (420.9) calculated: 42.57% C, 3.31% N, 1.67% H,
15.16% S; found 42.31% C, 3.05% N, 1.68% H, 15.40% S.
1H NMR (300 MHz, DMSO-d6)
d
8.26 (d, 1H, J ¼ 2.5 Hz, H5), 8.06
ꢀ
the Sula semisynthetic medium (SEVAC, Prague). This medium
(d, 1H, J ¼ 2.5 Hz, H7), 7.68 (d, 1H, J ¼ 2.2 Hz, H20), 7.55 (d, 1H,
(with bovine serum) is routinely used in the Czech Republic. Each
strain was simultaneously inoculated into a Petri dish containing
the LöwensteineJensen medium for the control of sterility of the
inoculum and its growth. The compounds were added to the
medium in DMSO solutions. The final concentrations were 1000,
J ¼ 8.2 Hz, H50), 7.37 (dd, 1H, J ¼ 8.2 Hz, J ¼ 2.2 Hz, H60), 6.09 (s, 2H,
CH2). 13C NMR (75 MHz, DMSO-d6)
d 185.2, 177.2, 145.3, 135.3, 135.2,
131.2, 130.5, 130.4, 129.7, 129.1, 128.5, 127.2, 124.3, 121.3, 57.6.
500, 250, 125, 62.5, 32, 16, 8, 4, 2, 1 and 0.5 mmol/L. The MICs were
5.1.17. 3-(3,4-Dichlorobenzyl)-2H-1,3-benzoxazine-2(3H)-dithione
(3g)
determined after incubation at 37 ꢀC for 14 days and 21 days. The
MIC was the lowest concentration of the antimycobacterially
effective substance (on the above concentration scale) at which
the inhibition of the growth of mycobacteria occurred. The eval-
uation was repeated three times and the values of the MIC were
the same.
3-(3,4-Dichlorobenzyl)-2H-1,3-benzoxazine-2(3H)-dithione (3g)
was prepared using procedure B from 3-(3,4-dichlorobenzyl)-2H-
1,3-benzoxazine-2(3H)-dione (1.22 g, 3.8 mmol) to yield 42%
(0.565 g, 1.595 mmol) of the title compound as a crystalline red
solid. Mp. 170e172 ꢀC.
For C15H9Cl2NOS2 (354.3) calculated: 50.85% C, 3.95% N, 2.56% H,
18.10% S; found 50.56% C, 3.85% N, 2.49% H, 18.14% S.
5.2.2. Antiproliferative and cytotoxic assay
1H NMR (300 MHz, DMSO-d6)
d 8.25e8.17 (m,1H, H5), 7.91e7.83
5.2.2.1. Cells and culture conditions. Cell proliferation and cytotox-
icity assays using various cancer cell line models are among the
basic assays used to assess the efficacy and potency of different
anti-cancer compounds. Therefore, we used the following cell
lines:
(m, 1H, H7), 7.67 (s, 1H, H20), 7.56e7.42 (m, 3H, H6, H8, H50),
7.37e7.30 (m, 1H, H60), 6.13 (s, 2H, CH2). 13C NMR (75 MHz, DMSO-
d6) d 187.5, 178.6, 149.8, 136.9, 135.8, 131.6, 131.2, 130.5, 129.6, 128.7,
127.1, 127.0, 122.2, 116.3, 57.2.
5.1.18. 6-Bromo-3-(3,4-dichlorobenzyl)-2H-1,3-benzoxazin-2(3H)-
dithione (3h)
Cells
Cell culture medium
DMEM (CAMBREX 12-614F)
ꢃ HUVEC (ATCC CRL-1730)
6-Bromo-3-(3,4-dichlorobenzyl)-2H-1,3-benzoxazin-2(3H)-dithione
(3h) was prepared using procedure B from 6-bromo-3-(3,4-
dichlorobenzyl)-2H-1,3-benzoxazin-2(3H)-dione (1.52 g, 3.8 mmol)
to yield 43% (0.71 g, 1.634 mmol) of the title compound as a crys-
talline red solid. Mp. 168e171 ꢀC.
RPMI 1640 (CAMBREX 12-167F)
RPMI 1640 (CAMBREX 12-167F).
ꢃ K-562 (DSM ACC 10)
ꢃ HeLa (DSM ACC 57)
Cells were grown in the appropriate cell culture medium supple-
mented with 10 mL/L ultraglutamine 1 (CAMBREX 17-605E/U1),
500 mL/L gentamicin sulfate (CAMBREX 17-518Z), and 10% heat
For C15H8BrCl2NOS2 (430.8) calculated: 41.59% C, 3.23% N, 1.86%
H, 14.81% S; found 41.37% C, 2.87% N, 1.98% H, 15.10% S.

K. Waisser et al. / European Journal of Medicinal Chemistry 45 (2010) 2719e2725
2725
inactivated fetal bovine serum (PAA A15-144) at 37 ꢀC in high
density polyethylene flasks (NUNC 156340).
The adherent HUVEC and HeLa cells were fixed by glutaralde-
hyde (MERCK 1.04239.0250) and stained with a 0.05% solution of
methylene blue (SERVA 29198) for 15 min. After gently washing,
the stain was eluted with 0.2 mL of 0.33 N HCl in the wells. The
optical densities were measured at 660 nm in a SUNRISE micro-
plate reader (TECAN).
The GI50 and CC50 values were defined as being where the dose
response curve intersected the 50% line, compared to the untreated
control. These comparisons of the different values were performed
with the software Magellan (TECAN). The GI50 and CC50 values were
defined as being where the dose response curve intersected the 50%
line, compared to the untreated control. These comparisons of the
different values were performed using the software Magellan (TECAN).
5.2.3. Antiproliferative assay
The test substances were dissolved in DMSO before being
diluted in DMEM. The adherent cells were harvested at the loga-
rithmic growth phase after soft trypsinization, using 0.25% trypsin
in PBS containing 0.02% EDTA (Biochrom KG L2163). For each
experiment approximately 10,000 cells were seeded with 0.1 mL
culture medium per well of the 96-well microplates (HUVEC: NUNC
167008, K-562: NUNC 163320). To test the antiproliferative effect of
compounds on HUVEC and K-562, the cells were incubated for 72 h
in plates prepared with control and different dilutions of test
substances.
Acknowledgements
5.2.4. Cytotoxic assay
For the cytotoxicity assay, HeLa cells were preincubated for 48 h
without the test substances. To test the cytotoxic effect of
compounds on HeLa, the dilutions of the compounds were carried
out carefully on the subconfluent monolayers of HeLa cells after the
preincubation time. After the incubation time, the cytolytic effects
of compounds were analysed in comparison with the negative
control.
This work is a part of the research project No. MSM0021620822
of the Ministry of Education of the Czech Republic.
References
ꢀ
[1] J. Adamec, R. Beckert, D. Weiss, V. Klimesová, K. Waisser, U. Möllmann,
J. Kaustová, V. Buchta, Bioorg. Med. Chem. 15 (2007) 2898e2906.
ꢀ
ꢀ
ꢀ
[2] K. Waisser, J. Matyk, H. Divisová, P. Husáková, J. Kunes, V. Klimesová,
J. Kaustová, U. Möllmann, H.M. Dahse, M. Miko, Arch. Pharm. Chem. Life Sci.
339 (2006) 616e620.
5.2.5. Condition of incubation
ꢀ
[3] R. Dolezal, S. Van Damme, P. Bultinck, K. Waisser, Eur. J. Med. Chem. 43 (2008)
The cells were incubated with dilutions of the test substances in
microplates for 72 h at 37 ꢀC in a humidified atmosphere and 5%
CO2. This incubation was found to be an optimum time for the
evaluation of the cytotoxicity and the inhibition of cell proliferation
by finding out the number of viable cells stained with the CellTiter-
BlueÒ reagent or methylene blue.
869e876.
ꢀ
ꢀ
[4] V. Klimesová, J. Kocí, K. Waisser, J. Kaustová, U. Möllmann, Eur. J. Med. Chem.
44 (2009) 2286e2293.
[5] D.J. Hlasta, J.P. Demers, B.D. Foleno, S.A. Fraga-Spano, J. Guan, J.J. Hilliard, M.
J. Macielag, K.A. Ohemeng, C.M. Sheppard, Z. Sui, G.C. Webb, M.A. Weidner-
Wells, H. Werblood, J.F. Barrett, Bioorg. Med. Chem. Lett. 8 (1998) 1923e1928.
[6] M.J. Macielag, J.P. Demers, S.A. Fraga-Spano, D.J. Hlasta, S.G. Johnson, R.
M. Konojia, R.K. Russell, Z. Sui, M.A. Weidner-Wells, H. Werblood, B.D. Folen, R.
M. Golschmidt, M.J. Loeloff, G.C. Webb, J.F. Barred, J. Med. Chem. 41 (1998)
2939e2945.
5.2.6. Method of evaluation
We used different methods to measure cell proliferation and
cytotoxicity. For estimating the influence of chemical compounds
on cell proliferation of K-562, the metabolic capacity of viable cells
present in multiwell plates were measured using the CellTiter-
BlueÒ assay (PROMEGA), employing the indicator dye resazurin.
Viable cells of untreated control retain the ability to reduce resa-
zurin into resorufin, whereas nonviable cells rapidly lose their
metabolic capacity and do not reduce the indicator dye.
[7] S. Houston, A. Franning, Drugs 48 (1994) 689e708.
[8] G. Winder, P.B. Collins, J. Wheland, J. Gen. Microbiol. 66 (1971) 379e380.
[9] E. Wang, R. Langley, G. Gulten, L.G. Dover, G.S. Besra, W.R. Jacobs Jr., J.
C. Sacchettini, J. Exp. Med. 204 (2007) 71e78.
ꢀ
ꢀ
ꢀ
ꢀ
[10] K. Waisser, M. Perina, P. Holý, M. Pour, O. Bures, J. Kunes, V. Klimesová,
V. Buchta, P. Kubanová, J. Kaustová, Arch. Pharm. 336 (2003) 322e335.
ꢀ
ꢀ
ꢀ
[11] K. Waisser, M. Perina, J. Kunes, V. Klimesová, J. Kaustová, Farmaco 58 (2003)
1137e1149.
ꢀ
ꢀ
[12] K. Waisser, M. Perina, V. Klimesová, J. Kaustová, Collect. Czech, Chem.
Commun. 68 (2003) 1275e1294.