Highly active antimycobacterial derivatives of benzoxazine

Article abstract of DOI:10.1016/j.bmc.2010.10.017

New 3-(4-alkylphenyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-dithiones were synthesized. The compounds were tested for in vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium a

Full text of DOI:10.1016/j.bmc.2010.10.017

Bioorganic & Medicinal Chemistry 18 (2010) 8178–8187
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Highly active antimycobacterial derivatives of benzoxazine
Eva Petrlíková a, , Karel Waisser , Hana Divišová , Petra Husáková , Petra Vrabcová , Jirí Kuneš ,
⇑
a
a
a
b
a
ˇ
c
ˇ b
ˇ
ˇ
Karel Kolár , Jirina Stolaríková
a Department of Inorganic and Organic Chemistry, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
b Department of Chemistry, University of Hradec Králové, Rokitanského 62, 500 03 Hradec Králové, Czech Republic
c
ˇ
Regional Institute of Public Health, Partyzánské námestí 7, Ostrava, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
New 3-(4-alkylphenyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-(4-alkylphenyl)-2H-1,3-benzox-
azine-2,4(3H)-dithiones were synthesized. The compounds were tested for in vitro antimycobacterial
activity against Mycobacterium tuberculosis, Mycobacterium avium and two strains of Mycobacterium kans-
asii. The antimycobacterial activity increased with the replacement of the carbonyl group by the thiocar-
bonyl group in the starting 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-diones. The most active
derivatives were more active than isonicotinhydrazide (INH). Free-Wilson analysis was also carried out
and the activity contribution was examined.
Received 27 June 2010
Revised 28 September 2010
Accepted 6 October 2010
Available online 19 October 2010
Keywords:
Benzoxazine
Thioxo group
Ó 2010 Elsevier Ltd. All rights reserved.
Antimycobacterial activity
Tuberculosis
1. Introduction
system inhibitors.7,8 This type of mechanism of action is very
promising while such a type of the antibacterial effect is probably
Tuberculosis is an infectious bacterial disease that can be trans-
mitted from person to person because of the droplets from the
throat and lungs of people suffering from the active respiratory dis-
ease. Tuberculosis was always a serious problem in developing
countries and the multidrug resistant strains of Mycobacterium
tuberculosis are transferred by the migration of a population to Eur-
ope and North America. More than two billion people (one third of
the world’s population) are, according to WHO, infected with TB
bacilli, the microbes that cause tuberculosis.1 WHO also estimated
that 9.37 million incident cases of TB and 11.09 million prevalent
cases of TB occurred in the year 2008. There were 1.32 million
deaths from TB in the same year.2 Hence, there is a great need to
develop new drugs and found new strategies to control this out-
break of tuberculosis.3
Many antituberculosis agents containing a thioxo group were
important in the history of antituberculosis drugs. For example,
p-acetamidobenzaldehyde thiosemicarbazone (Thiacetazone),
4,40-bis(isopentoxy)thiocarbanilide (Isoxyl), or 2-ethylpyridine-4-
carbotioamide (Ethionamide) are known to inhibit the mycolic
acids biosynthesis.4–6
different from the effect of other antibacterial drugs.6,9,10
2. Chemistry
The starting 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-
diones were prepared by the reaction of N-(4-alkylphenyl)salicyla-
mides with methyl chloroformate in dry pyridine and purified by
crystallization from ethanol. Several methods have been reported
for the preparation of these heterocycles.11–14 3-(4-Alkylphenyl)-
4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-(4-alkylphenyl)-
2H-1,3-benzoxazine-2,4(3H)-dithiones were synthesized by the
treatment of 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-diones
with phosphorus pentasulfide. The published methods include thi-
onation using P2S5 and Lawesson’s reagent.15,16 The structural
assignment is based on 1H NMR, 13C NMR, and IR spectra. The pur-
ity of the compounds was confirmed by means of elemental anal-
ysis. An overview of the synthesis is in Scheme 1. The structures of
the prepared compounds are summarized in Tables 1–3.
3. Antimycobacterial activity
This study is oriented on the derivatives of benzoxazinediones
in which one or both oxo groups were replaced by the thioxo
group. Since the compounds are cyclic derivatives of salicylanilides
it can be supposed that they can serve as bacterial two-component
The in vitro antimycobacterial activity of the compounds was
investigated against M. tuberculosis CNCTC My 331/88 (identical
with H37RV and ATCC 27294), Mycobacterium kansasii CNCTC My
235/80 (identical with ATCC 12478, resistant to INH), Mycobacte-
rium avium CNCTC My 330/88 (identical with ATCC 25291, resis-
tant to INH), and M. kansasii 6509/96. All the strains were
⇑
Corresponding author. Tel.: +420 495067335; fax: +420 465067166.
E-mail address: Eva.Petrlikova@faf.cuni.cz (E. Petrlíková).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.017

E. Petrlíková et al. / Bioorg. Med. Chem. 18 (2010) 8178–8187
8179
R2
O
COOH
OH
NH2
PCl3
N
H
+
OH
R1
R1
O
O
S
O
R2
N
2
O
O
O
R1
P2S5
Cl
O
N
R2
R1
O
S
1
R2
N
3
R1
S
R2
Scheme 1. Synthesis of 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-diones (1), 3-(4-alkylphenyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones (2), and 3-(4-alkylphenyl)-
2H-1,3-benzoxazine-2,4(3H)-dithiones (3).
Table 1
In vitro antimycobacterial activity of 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-diones.
Compound
MIC (lmol/L)
Incubation time 14 d/21 d
R1
R2
M. tuberculosis
M. avium
M. kansasii
M. kansasii
My 331/88
My 330/88
My 235/80
6509/96
d
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
1s
6-Br
6-Br
6-Cl
7-Cl
6-Br
6-Cl
7-Cl
6-F
7-CH3
6-Br
6-Cl
7-Cl
6-F
Methyl
Ethyl
Ethyl
Ethyl
16/16a
8/8
31/31a
32/32
16/32c
8/16
—62/62a
8/16
16/16c
16/16
8/8
—
16/16
16/16c
16/16
4/8
8/16c
8/8
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
sec-Butyl
sec-Butyl
sec-Butyl
sec-Butyl
sec-Butyl
Butyl
2/4
8/16
4/8c
4/4
32/32c
8/16
8/16c
8/16
8/8
8/16c
8/8
8/8
n/n
8/16
n/n
16/32
32/32c
8/8
8/16
32/n
8/16
16/16c
8/8
n/n
4/8
8/8
4/8c
4/4
16/16c
8/8
8/8
n/n
8/8
n/n
8/8
4/8
7-CH3
6-Cl
6-Cl
7-Cl
6-F
n/n
32/n
8/16b
8/8b
8/8b
8/16
n/n
16/16b
8/16b
4/4b
16/16
n/n
16/16b
16/32b
4/8b
8/8b
tert-Butyl
tert-Butyl
tert-Butyl
tert-Butyl
8/16b
8/16b
16/16
n/n
16/16
n/n
7-CH3
INH
0.5/0.5
>250/>250
>250/>250
8/8
a
b
c
Waisser, Hladu˚ vková (19).
Waisser, Matyk (17).
Kubicová (18).
d
Not tested, n: the MIC value could not be determined due to the limited solubility of the compound in the test medium.
obtained from the Czech National Collection of Type Cultures
(CNCTC), National Institute of Public Health, Prague. The only
exception is M. kansasii 6509/96 that was clinically isolated and,
that is, in contrast to the standard strain not resistant to isoniazid
(INH). The dilution micromethod was used for the determination of
minimum inhibitory concentrations (MIC) and the MIC values for
the standard INH were included for the sake of comparison. The
overview of the biological activity together with the activity of
INH is shown in Tables 1–3. The MIC values of compounds 1a,
1c, 1f, 1k, 1o, 1p, and 1q were taken from our previous papers17–
to complete the evaluation and to compare with the biological
activity.
The antimycobacterial activity of the starting 3-benzyl-2H-1,3-
benzoxazine-2,4(3H)-diones is in the range of 4–16 lmol/L. The
ment has a small effect on the activity. Figure 1 shows such an in-
crease in the activity in the case of M. tuberculosis. The MIC values
of the sulfur compounds are generally within the range of
0.25–125
lmol/L but the majority of MICs is in the range of
0.25–8 mol/L. All the sulfur derivatives are more active than INH.
l
4. Free-Wilson analysis
The Free-Wilson method20 with the Fujita–Ban modification21
was used to investigate the activity contribution in the case of sul-
fur derivatives (Tables 4 and 5). All the calculations were carried
out using the MULTIREG program for Microsoft Excel. Since the MIC
values after 14 d and 21 d incubation correlated with each other,
only the MICs after 14 d evaluation were taken for the calculations.
The MICs of sulfur derivatives of 6-bromo-3-(4-propylphenyl)-1,
3-benzoxazine-2,4(3H)-dione, 6-bromo-3-(4-butylphenyl)-1,
19
replacement of one oxo group with a thioxo group strongly
increases the antimycobacterial activity. The additional replace-

8180
E. Petrlíková et al. / Bioorg. Med. Chem. 18 (2010) 8178–8187
Table 2
In vitro antimycobacterial activity of 3-(4-alkylphenyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones
Compound
MIC (lmol/L)
Incubation time 14 d/21 d
R1
R2
M. tuberculosis
M. avium
M. kansasii
M. kansasii
My 331/88
My 330/88
My 235/80
6509/96
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
6-Br
6-Br
6-Cl
7-Cl
6-Br
6-Cl
7-Cl
6-F
7-CH3
6-Br
6-Cl
7-Cl
6-F
Methyl
Ethyl
Ethyl
Ethyl
0.5/1
32/32
32/32
16/16
8/8
4/8
32/125
8/8
0.5/1
0.5/1
16/32
32/32
8/8
2/4
8/16
8/8
2/4
4/8
4/4
8/8
4/8
1/2
4/8
4/4
1/2
2/4
4/8
1/2
2/4
0.5/1
4/4
4/8
2/4
8/8
1/1
0.5/0.5
0.5/0.5
0.5/0.5
0.25/0.5
0.5/1
8/8
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
sec-Butyl
sec-Butyl
sec-Butyl
sec-Butyl
sec-Butyl
Butyl
32/62.5
8/16
8/8
2/4
2/4
4/8
8/8
4/8
4/4
1/2
8/16
8/8
4/8
4/8
0.5/1
0.25/0.5
0.25/0.5
0.25/0.5
0.5/1
1/2
0.25/0.5
0.25/0.5
1/2
0.25/0.5
2/2
1/1
7-CH3
6-Cl
6-Cl
7-Cl
6-F
0.5/0.5
8/16
1/2
4/8
0.5/1
0.25/0.5
tert-Butyl
tert-Butyl
tert-Butyl
tert-Butyl
0.25/0.5
0.25/0.5
7-CH3
2/2
INH
1/1
>250/>250
>250/>250
8/8
Table 3
In vitro antimycobacterial activity of 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-dithiones
Compound
MIC (lmol/L)
Incubation time 14 d/21 d
R1
R2
M. tuberculosis
M. avium
M. kansasii
M. kansasii
My 331/88
My 330/88
My 235/80
6509/96
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
6-Br
6-Br
6-Cl
7-Cl
6-Br
6-Cl
7-Cl
6-F
7-CH3
6-Br
6-Cl
7-Cl
6-F
Methyl
Ethyl
Ethyl
Ethyl
0.5/1
1/2
0.5/0.5
0.5/0.5
0.5/1
0.5/1
0.5/1
0.25/0.5
0.5/1
1/2
15/30
62.5/62.5
8/16
4/8
8/16
4/4
8/16
8/8
8/16
32/62.5
8/16
4/8
4/8
2/4
4/8
8/16
4/8
2/4
2/2
8/8
8/8
2/4
8/8
2/2
4/4
4/8
4/6
8/16
4/8
1/2
4/4
2/4
1/2
2/4
4/8
1/2
2/4
0.5/1
8/8
8/8
2/4
4/8
1/2
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
sec-Butyl
sec-Butyl
sec-Butyl
sec-Butyl
sec-Butyl
Butyl
32/125
8/8
0.5/1
0.5/0.5
32/32
16/32
4/8
0.5/1
0.25/0.5
8/16
1/2
2/4
0.5/1
0.5/1
1/2
1/1
0.25/0.25
0.25/0.5
2/4
0.25/0.5
0.5/0.5
0.5/0.5
0.25/0.5
7-CH3
6-Cl
6-Cl
7-Cl
6-F
tert-Butyl
tert-Butyl
tert-Butyl
tert-Butyl
7-CH3
INH
1/1
>250/>250
>250/>250
8/8
3-benzoxazine-2,4(3H)-dione, and 7-chloro-3-(4-butylphenyl)-1,3-
benzoxazine-2,4(3H)-dione were taken from the literature22 and
used for the calculation. Chlorine as the R1 substituent at the posi-
tion 7 on the benzoxazine ring and propyl as the R2 substituent
were used as the standards. The results show that the biological
activity increases with the fluorine or methyl moiety as the R1 sub-
stituents. The increase in activity is also connected with the
branched R2 substituents.
(3a–3s) possess a better biological activity than INH, the standard
used for the sake of comparison. An advantage is the presumption
that these compounds can be of different mechanisms of action
from the antituberculosis drugs already in use. (4-sec-Butyl-
phenyl)-7-methyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-one (2n)
seems to be the most prospective compound as it shows the best
activity against all mycobacterial strains under tests. This com-
pound has been selected for future investigation.
5. Conclusion
6. Experimental protocols
The replacement of the carbonyl group by the thiocarbonyl
group in 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-diones in-
creases the antimycobacterial activity. The derivatives of 3-(4-
6.1. Chemistry, general information
The melting points were determined on a Kofler apparatus. The
samples for the analyzes and antimycobacterial tests were dried
over P2O5 at 61 °C and 66 Pa for 24 h. Elemental analyzes (C, H,
alkylphenyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones
(2a–2s)
and 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-dithiones

E. Petrlíková et al. / Bioorg. Med. Chem. 18 (2010) 8178–8187
8181
Figure 1. Increase in the biological activity after the replacement of the oxo group with the thioxo group.
Table 4
Activity contribution of the free-Wilson analyzes of 3-(4-alkylphenyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and statistical significances of correlation
Parameter
D
log MIC (
lmol/L) 14 d
M. tuberculosis
M. avium
M. kansasii
M. kansasii
My 331/88
My 330/88
My 235/80
6509/96
R1
7-Cl
0
0
0
0
6-Cl
6-Br
6-F
ꢀ0.482 ( 0.197)
ꢀ0.295 ( 0.212)
ꢀ0.563 ( 0.237)
ꢀ0.563 ( 0.237)
ꢀ0.301 ( 0.440)
ꢀ0.337 ( 0.380)
ꢀ0.396 ( 0.371)
ꢀ0.335 ( 0.371)
ꢀ0.269 ( 0.394)
0
0.180 ( 0.208)
0.176 ( 0.224)
ꢀ0.949 ( 0.251)
ꢀ1.150 ( 0.251)
ꢀ0.028 ( 0.466)
ꢀ0.244 ( 0.403)
ꢀ0.499 ( 0.393)
ꢀ0.318 ( 0.393)
ꢀ0.925 ( 0.417)
0
0.060 ( 0.144)
0.202 ( 0.155)
ꢀ0.292 ( 0.173)
ꢀ0.594 ( 0.173)
ꢀ0.602 ( 0.322)
0.115 ( 0.278)
0.206 ( 0.272)
ꢀ0.034 ( 0.272)
0.108 ( 0.288)
0
ꢀ0.060 ( 0.161)
0.133 ( 0.173)
0.218 ( 0.194)
ꢀ0.485 ( 0.194)
ꢀ0.602 ( 0.359)
ꢀ0.092 ( 0.311)
ꢀ0.370 ( 0.304)
ꢀ0.551 ( 0.304)
ꢀ0.237 ( 0.322)
0
7-CH3
Methyl
Ethyl
i-Propyl
sec-Butyl
tert-Butyl
Propyl
Butyl
R2
0.767 ( 0.380)
ꢀ0.444 ( 0.403)
0.315 ( 0.278)
0.109 ( 0.311)
l0
r
s
F
n
0.295 ( 0.376)
1.329 ( 0.398)
0.701 ( 0.275)
0.770 ( 0.308)
0.90
0.311
4.94
22
0.94
0.329
8.92
22
0.89
0.227
4.06
22
0.88
0.254
3.58
22
Table 5
Activity contribution of the Free-Wilson analyzes of 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-dithiones and statistical significances of correlation
Parameter
D
log MIC (
lmol/L) 14 d
M. tuberculosis
M. avium
M. kansasii
M. kansasii
My 331/88
My 330/88
My 235/80
6509/96
R1
7-Cl
0
0
0
0
6-Cl
6-Br
6-F
ꢀ0.180 ( 0.168)
ꢀ0.234 ( 0.181)
ꢀ0.280 ( 0.202)
ꢀ0.280 ( 0.202)
ꢀ0.301 ( 0.375)
ꢀ0.016 ( 0.324)
ꢀ0.026 ( 0.317)
0.095 ( 0.317)
ꢀ0.323 ( 0.336)
0
0.301 ( 0.200)
0.830 ( 0.216)
ꢀ0.888 ( 0.241)
ꢀ0.988 ( 0.241)
ꢀ0.329 ( 0.447)
0.048 ( 0.387)
0.136 ( 0.378)
ꢀ0.045 ( 0.378)
ꢀ0.357 ( 0.401)
0
0.241 ( 0.135)
0.299 ( 0.146)
0.047 ( 0.163)
ꢀ0.254 ( 0.163)
ꢀ0.301 ( 0.303)
0.119 ( 0.262)
ꢀ0.009 ( 0.256)
ꢀ0.129 ( 0.256)
ꢀ0.011 ( 0.271)
0
0.120 ( 0.150)
0.200 ( 0.162)
0.096 ( 0.181)
ꢀ0.406 ( 0.181)
ꢀ0.301 ( 0.336)
ꢀ0.108 ( 0.291)
ꢀ0.464 ( 0.284)
ꢀ0.525 ( 0.284)
ꢀ0.204 ( 0.302)
0
7-CH3
Methyl
Ethyl
i-Propyl
sec-Butyl
tert-Butyl
Propyl
Butyl
R2
1.120 ( 0.324)
ꢀ0.149 ( 0.387)
0.320 ( 0.262)
0.194 ( 0.291)
l0
r
s
F
n
ꢀ0.234 ( 0.321)
0.676 ( 0.383)
0.604 ( 0.259)
0.703 ( 0.288)
0.93
0.265
6.57
22
0.95
0.316
10.85
22
0.86
0.214
3.12
22
0.90
0.238
4.56
22

8182
E. Petrlíková et al. / Bioorg. Med. Chem. 18 (2010) 8178–8187
N) were performed on a CHNS-O CE elemental analyzer (Fisions EA
1110, Milan) and were within 0.4% of the theoretical values. The
IR spectra were measured in KBr pellets on a Nicolet Impact 400
apparatus; the wavenumbers are given in cmꢀ1. TLC was per-
formed on silica gel plates precoated with the fluorescent indicator
Silufol UV 254 + 366 (Kavalier Votice, Czech Republic), hexane/ace-
tone (3:1) was used as the mobile phase. Crystallization of prod-
ucts was carried out from ethanol. The 1H NMR and 13C NMR
spectra of new compounds were recorded in DMSO-d6 solutions
at ambient temperature on a Varian Mercury-Vx BB 300 spectrom-
eter 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).
J = 6.9 Hz, CH3); 13C NMR (75 MHz, DMSO) d 160.3, 153.3, 149.2,
147.6, 140.5, 132.8, 129.3, 128.5, 127.1, 125.8, 116.8, 114.2, 33.4,
24.0. Anal. Calcd for C17H14ClNO3 (315.76): C, 64.67; H, 4.47; N,
4.44. Found: C, 64.62; H, 4.43; N, 4.20.
6.2.5. 6-Fluoro-3-(4-isopropylphenyl)-1,3-benzoxazine-2,4(3H)-
dione (1h)
White solid, yield 60%, mp 193–194 °C, IR (m ;
CO) 1770, 1700 cmꢀ1
1HNMR(300 MHz, DMSO)d7.79–7.69(2H, m, H5,H7), 7.62–7.54(1H,
m,H8), 7.41–7.34(2H, m,AA0, BB0, H20, H60),7.34–7.28(2H, m,AA0, BB0,
H30, H50), 3.04–2.87 (1H, m, CH), 1.25 (6H, d, J = 6.9 Hz, CH3). Anal.
Calcd for C17H14FNO3 (299.3): C, 68.22; H, 4.71; N, 4.68. Found: C,
68.03; H, 4.71; N, 4.55.
6.2.6. 3-(4-Isopropylphenyl)-7-methyl-1,3-benzoxazine-
2,4(3H)-dione (1i)
6.2. Chemistry, synthetic procedures to preparation of 3-(4-
alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-diones (1b, 1d, 1e, 1g,
1h–1j, 1l–1n, 1r, 1s)
White solid, yield 67%, mp 212–213 °C, IR (m ;
CO) 1764, 1689 cmꢀ1
1HNMR(300 MHz, DMSO)d7.86(1H, d,J = 8.0 Hz,H5),7.41–7.24(6H,
m, H6, H8, H20, H30, H50, H60), 3.05–2.87 (1H, m, CH), 2.46 (3H, s, CH3),
1.25 (6H, d, J = 6.9 Hz, CH3); 13C NMR (75 MHz, DMSO) d 160.8, 152.7,
149.0, 148.0, 147.8, 133.0, 128.6, 127.4, 127.0, 126.5, 116.4, 112.4,
33.4, 24.0, 21.5. Anal. Calcd for C18H17NO3 (295.34): C, 73.20; H,
5.80; N, 4.74. Found: C, 72.81; H, 5.80; N, 4.57.
Methyl chloroformate (5.2 g, 48 mmol) was added dropwise to
a stirred solution of the corresponding salicylanilide (40 mmol)
in dry pyridine (20 mL) under ice cooling. The mixture was heated
on a steam bath for 1 h and then poured into 5% hydrochloric acid
(140 mL). After 24 h the product was filtered off, suspended in 5%
natrium carbonate solution and the solid was filtered off. The crude
product was purified by crystallization from ethanol.
6.2.7. 6-Bromo-3-(4-sec-butylphenyl)-1,3-benzoxazine-2,4(3H)-
dione (1j)
White solid, yield 64%, mp 208–209 °C, IR (m ;
CO) 1770, 1703 cmꢀ1
1H NMR (300 MHz, DMSO) d 8.07–7.98 (2H, m, H5, H7), 7.49 (1H, d,
J = 8.5 Hz, H8), 7.37–7.26 (4H, m, H20, H30, H50, H60), 2.75–2.59 (1H,
m, CH), 1.66–1.52 (2H, m, CH2), 1.22 (3H, d, J = 7.1 Hz, CH3), 0.81
(3H, t, J = 7.1 Hz, CH3); 13C NMR (75 MHz, DMSO) d 159.9, 151.9,
148.0, 147.5, 138.9, 132.8, 129.4, 128.4, 127.7, 119.2, 117.1, 117.0,
40.8, 30.7, 21.8, 12.3. Anal. Calcd for C18H16BrNO3 (374.24): C,
57.77; H, 4.31; N, 3.74. Found: C, 57.54; H, 4.27; N, 3.50.
6.2.1. 6-Bromo-3-(4-ethylphenyl)-1,3-benzoxazine-2,4(3H)-
dione (1b)
White solid, yield 59%, mp 219–222 °C, IR (m ;
CO) 1772, 1703 cmꢀ1
1H NMR (300 MHz, DMSO) d 8.07–7.97 (2H, m, H5, H7), 7.49 (1H, d,
J = 8.5 Hz, H8), 7.37–7.27 (4H, m, H20, H30, H50, H60), 2.67 (2H, q,
J = 7.6 Hz, CH2), 1.22 (3H, t, J = 7.6 Hz, CH3);13C NMR (75 MHz, DMSO)
d 159.9, 151.9, 147.5, 144.7, 138.9, 132.8, 129.5, 128.6, 128.4, 119.2,
117.1, 117.0, 28.1, 15.7. Anal. Calcd for C16H12BrNO3 (346.18): C,
55.51; H, 3.49; N, 4.05. Found: C, 55.67; H, 3.55; N, 3.80.
6.2.8. 3-(4-sec-Butylphenyl)-7-chloro-1,3-benzoxazine-2,4(3H)-
dione (1l)
White solid, yield 70%, mp 136–137 °C, IR (m ;
CO) 1776, 1696 cmꢀ1
6.2.2. 7-Chloro-3-(4-ethylphenyl)-1,3-benzoxazine-2,4(3H)-
dione (1d)
1H NMR (300 MHz, DMSO) d 7.98 (1H, d, J = 8.2 Hz, H5), 7.75 (1H, d,
J = 1.9 Hz, H8), 7.52 (1H, dd, J = 8.2 Hz, J = 1.9 Hz, H6), 7.37–7.24
(4H, m, H20, H30, H50, H60), 2.74–2.59 (1H, m, CH), 1.66–1.51 (2H, m,
CH2), 1.22 (3H, d, J = 7.1 Hz, CH3), 0.81 (3H, t, J = 7.1 Hz, CH3); 13C
NMR (75 MHz, DMSO) d 160.3, 153.3, 148.0, 147.6, 140.5, 132.8,
129.2, 128.4, 127.7, 125.8, 116.8, 114.2, 40.8, 30.7, 21.8, 12.3. Anal.
Calcd for C18H16ClNO3 (329.79): C, 65.56; H, 4.89; N, 4.25. Found: C,
65.27; H, 4.90; N, 4.07.
White solid, yield 58%, mp 185–189 °C, IR (
m
CO) 1776, 1708 cm
ꢀ1
;
1H NMR (300 MHz, DMSO) d 7.98 (1H, d, J = 8.4 Hz, H5), 7.75
(1H, d, J = 1.8 Hz, H8), 7.51 (1H, dd, J = 8.4 Hz, J = 1.8 Hz, H6),
7.37–7.27 (4H, m, H20, H30, H50, H60), 2.67 (2H, q, J = 7.6 Hz, CH2),
1.22 (3H, t, J = 7.6 Hz, CH3); 13C NMR (75 MHz, DMSO) d 160.3,
153.3, 147.6, 144.6, 140.5, 132.8, 129.2, 128.6, 128.5, 125.8,
116.8, 114.2, 28.1, 15.7. Anal. Calcd for C16H12ClNO3 (301.73): C,
63.69; H, 4.01; N, 4.46. Found: C, 63.59; H, 3.95; N, 4.36.
6.2.9. 3-(4-sec-Butylphenyl)-6-fluoro-1,3-benzoxazine-2,4(3H)-
dione (1m)
White solid, yield 63%, mp 114–116 °C, IR (m ;
CO) 1767, 1734 cmꢀ1
6.2.3. 6-Bromo-3-(4-isopropylphenyl)-1,3-benzoxazine-2,4(3H)-
1HNMR(300 MHz, DMSO)d7.82–7.68(2H, m, H5,H7), 7.64–7.54(1H,
m, H8), 7.37–7.27 (4H, m, H20, H30, H50, H60), 2.74–2.58 (1H, m, CH),
1.67–1.50 (2H, m, CH2), 1.22 (3H, d, J = 7.2 Hz, CH3), 0.81 (3H, d,
J = 7.2 Hz, CH3). Anal. Calcd for C18H16FNO3 (313.3): C, 69.00; H,
5.15; N, 4.47. Found: C, 68.93; H, 5.25; N, 4.37.
dione (1e)
White solid, yield 72%, mp 174–177 °C, IR (m ;
CO) 1763, 1703 cmꢀ1
1H NMR (300 MHz, DMSO) d 8.08–7.97 (2H, m, H5, H7), 7.49 (1H, d,
J = 8.5 Hz, H8), 7.41–7.34 (2H, m, AA0, BB0, H20, H60), 7.34–7.26 (2H,
m, AA0, BB0, H30, H50), 3.05–2.85 (1H, CH), 1.24 (6H, d, J = 6.9 Hz,
CH3); 13C NMR (75 MHz, DMSO) d 159.9, 151.9, 149.2, 147.5, 138.9,
132.8, 129.5, 128.4, 127.1, 119.2, 117.1, 117.0, 33.4, 24.0. Anal. Calcd
for C17H14BrNO3 (360.2): C, 56.69; H, 3.92; N, 3.89. Found: C, 56.47;
H, 3.97; N, 3.80.
6.2.10. 3-(4-sec-Butylphenyl)-7-methyl-1,3-benzoxazine-
2,4(3H)-dione (1n)
White solid, yield 71%, mp137–140 °C, IR (m ;
CO) 1761, 1694 cmꢀ1
1HNMR(300 MHz, DMSO)d7.86(1H, d,J = 8.0 Hz,H5),7.36–7.24(6H,
m, H6, H8, H20, H30, H50, H60), 2.74–2.58 (1H, m, CH), 2.46 (3H, s, CH3),
1.66–1.49 (2H, m, CH2), 1.22 (3H, d, J = 7.1 Hz, CH3), 1.81 (3H, d,
J = 7.1 Hz, CH3); 13C NMR (75 MHz, DMSO) d 160.8, 152.8, 148.0,
147.8, 133.1, 128.6, 127.4, 127.2, 126.5, 116.5, 112.4, 40.8, 30.8,
22.0, 12.3. Anal. Calcd for C19H19NO3 (309.37): C, 73.77; H, 6.19; N,
4.53. Found: C, 73.96; H, 6.28; N, 4.45.
6.2.4. 7-Chloro-3-(4-isopropylphenyl)-1,3-benzoxazine-2,4(3H)-
dione (1g)
White solid, yield 61%, mp 180–183 °C, IR (m ;
CO) 1770, 1704 cmꢀ1
1H NMR (300 MHz, DMSO) d 7.98 (1H, d, J = 8.5 Hz, H5), 7.74 (1H, d,
J = 1.8 Hz, H8), 7.51 (1H, dd, J = 8.5 Hz, J = 1.8 Hz, H6), 7.41–7.27
(4H, m, H20, H30, H50, H60), 3.04–2.88 (1H, m, CH), 1.25 (6H, d,

E. Petrlíková et al. / Bioorg. Med. Chem. 18 (2010) 8178–8187
8183
6.2.11. 3-(4-tert-Butylphenyl)-6-fluoro-1,3-benzoxazine-
2,4(3H)-dione (1r)
128.9, 128.0, 122.2, 119.2, 28.1, 15.6. Anal. Calcd for C16H12ClNO2S
(317.0): C, 60.47; H, 3.81; N, 4.41; S, 10.99. Found: C, 60.39; H,
3.84; N, 4.31; S, 11.28.
White solid, yield 58%, mp 145–147 °C, IR (m ;
CO) 1770, 1733 cmꢀ1
1H NMR (300 MHz, DMSO) d7.80–7.68 (1H, m, H5), 7.64–7.53 (1H, m,
H7), 7.55–7.48 (2H, m, AA0, BB0, H20, H60), 7.42–7.35 (1H, m, H8), 7.35–
7.28 (2H, m, AA0, BB0, H30, H50), 1.32 (9H, s, CH3). Anal. Calcd for
6.3.4. 7-Chloro-3-(4-ethylphenyl)-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2d)
Yellow solid, yield 39%, mp 188–190 °C, IR (m
CO) 1761 cmꢀ1; 1H
C18H16FNO3 (313.3): C, 69.00; H, 5.15; N, 4.47. Found: C, 68.90; H,
5.21; N, 4.39.
NMR (300 MHz, DMSO) d 8.28 (1H, d, J = 8.8 Hz, H5), 7.74 (1H, d,
J = 2.2 Hz, H8), 7.50 (1H, dd, J = 8.8 Hz, J = 2.2 Hz, H6), 7.38–7.30
(2H, m, AA0, BB0, H20, H60), 7.29–7.23 (2H, m, AA0, BB0, H30, H50),
2.67 (2H, q, J = 7.6 Hz, CH2), 1.53 (3H, t, J = 7.6 Hz, CH3); 13C NMR
(75 MHz, DMSO) d 191.9, 150.2, 144.5, 144.5, 140.7, 137.6, 133.0,
128.9, 128.1, 126.2, 120.1, 116.8, 28.1, 15.6. Anal. Calcd for
6.2.12. 3-(4-tert-Butylphenyl)-7-methyl-1,3-benzoxazine-
2,4(3H)-dione (1s)
White solid, yield 70%, mp 170–172 °C, IR (m ;
CO) 1753, 1707 cmꢀ1
1HNMR(300 MHz, DMSO)d7.86(1H, d,J = 8.0 Hz, H5),7.55–7.47(2H,
m, H20, H60), 7.35–7.24 (4H, m, H6, H8, H30, H50), 2.46(3H, s, CH3), 1.32
(9H, s, CH3); 13C NMR (75 MHz, DMSO) d 160.9, 152.8, 151.2, 148.1,
147.8, 132.8, 128.3, 127.5, 126.5, 126.0, 116.5, 112.4, 34.7, 31.3,
21.6. Anal. Calcd for C19H19NO3 (309.37): C, 73.77; H, 6.19; N, 4.53.
Found: C, 73.49; H, 6.17; N, 4.36.
C16H12ClNO2S (317.0): C, 60.47; H, 3.81; N, 4.41; S, 10.99. Found:
C, 60.33; H, 3.79; N, 4.22; S, 10.22.
6.3.5. 6-Bromo-3-(4-isopropylphenyl)-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2e)
Yellow solid, yield 26%, mp 199–201 °C, IR (m
CO) 1759 cmꢀ1; 1H
NMR (300 MHz, DMSO) d 8.35 (1H, d, J = 2.5 Hz, H5), 8.01 (1H, dd,
J = 8.8 Hz, J = 2.5 Hz, H7), 7.47 (1H, d, J = 8.8 Hz, H8), 7.42–7.34 (2H,
m, AA0, BB0, H20, H60), 7.32–7.24 (2H, m, AA0, BB0, H30, H50), 3.04–
2.87 (1H, m, CH), 1.24 (6H, d, J = 6.9 Hz, CH3); 13C NMR (75 MHz,
DMSO) d 191.3, 149.1, 149.0, 144.5, 138.6, 137.7, 133.0, 128.0,
127.4, 122.5, 119.4, 117.4, 33.4, 24.0. Anal. Calcd for C17H14BrNO2S
(376.27): C, 54.27; H, 3.75; N, 3.72; S, 8.52. Found: C, 54.04; H,
3.62; N, 3.62; S, 8.51.
6.3. Chemistry, synthetic procedures for the preparation of 3-(4-
alkylphenyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones (2a–2s)
and 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-dithiones
(3a–3s)
3-(4-Alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-diones
(3.8 mmol) were melted with P4S10 (7.6 mmol) for 20 min (175–
200 °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.,
40 mL). Column chromatography on silica gel yielded 3-(4-alkyl-
phenyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-(4-alkyl-
phenyl)-2H-1,3-benzoxazine-2,4(3H)-dithiones. The products
were crystallized from ethanol.
6.3.6. 6-Chloro-3-(4-isopropyl)-4-thioxo-2H-1,3-benzoxazine-
2(3H)-one (2f)
Yellow solid, yield 14%, mp 163–165 °C, IR (m
CO) 1751 cmꢀ1; 1H
NMR (300 MHz, DMSO) d 8.22 (1H, d, J = 2.6 Hz, H5), 7.91 (1H, dd,
J = 8.8 Hz, J = 2.6 Hz, H7), 7.54 (1H, d, J = 8.8 Hz, H8), 7.41–7.35 (2H,
m, AA0, BB0, H20, H60), 7.31–7.25 (2H, m, AA0, BB0, H30, H50), 3.04–
2.86 (1H, CH), 1.24 (6H, d, J = 6.9 Hz, CH3); 13C NMR (75 MHz,
DMSO) d 191.4, 149.0, 148.7, 144.6, 137.7, 135.8, 130.1, 129.7,
128.0, 127.4, 122.2, 119.2, 33.4, 24.0. Anal. Calcd for C17H14ClNO2S
(331.8): C, 61.53; H, 4.25; N, 4.22; S, 9.66. Found: C, 61.18; H, 4.27;
N, 3.77; S, 10.05.
6.3.1. 6-Bromo-3-(4-methylphenyl)-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2a)
Yellow solid, yield 36%, mp 259–261 °C, IR (m
CO) 1759 cmꢀ1; 1H
NMR (300 MHz, CDCl3) d 8.54 (d, 1H, J = 2.45 Hz, H5), 7.80 (dd, 1H,
J = 8.52 Hz, J = 2.45 Hz, H7), 7.40–7.34 (m, AA0, BB0, 2H, H20, H60),
7.20 (d, 1H, J = 8.52 Hz, H8), 7.16–7.11 (m, AA0, BB0, 2H, H30, H50),
2.45 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) d 190.0, 148.2, 144.6,
139.5, 138.4, 136.2, 134.3, 130.6, 127.3, 122.0, 118.8, 118.4, 21.5.
Anal. Calcd for C15H10BrNO2S (348.2): C, 51.74; H, 2.89; N, 4.02;
S, 9.21. Found: C, 51.22; H, 2.62; N, 3.98; S, 9.11.
6.3.7. 7-Chloro-3-(4-isopropylphenyl)-4-thioxo-2H-1,3-benz-
oxazine-2(3H)-one (2g)
Yellow solid, yield 31%, mp 198–199 °C, IR (m
CO) 1761 cmꢀ1; 1H
NMR (300 MHz, DMSO) d 8.28 (1H, d, J = 8.8 Hz, H5), 7.73 (1H, d,
J = 2.1 Hz, H8), 7.50 (1H, dd, J = 8.8 Hz, J = 2.1 Hz, H6), 7.46–7.34
(2H, m, AA0, BB0, H20, H60), 7.30–7.24 (2H, m, AA0, BB0, H30, H50),
3.03–2.87 (1H, m, CH), 1.24 (6H, t, J = 6.5 Hz, CH3); 13C NMR
(75 MHz, DMSO) d 192.3, 150.7, 149.5, 145.0, 141.2, 138.1, 133.5,
128.6, 127.9, 126.6, 120.6, 117.3, 33.9, 24.5. Anal. Calcd for
6.3.2. 6-Bromo-3-(4-ethylphenyl)-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2b)
Yellow solid, yield 42%, mp 227–229 °C, IR (m
CO) 1759 cmꢀ1; 1H
NMR (300 MHz, DMSO) d 8.35 (1H, d, J = 2.3 Hz, H5), 8.02 (1H, dd,
J = 8.8 Hz, J = 2.3 Hz, H7), 7.47 (1H, d, J = 8.8 Hz, H8), 7.38–7.30 (2H,
m, AA0, BB0, H20, H60), 7.30–7.23 (2H, m, AA0, BB0, H30, H50), 2.66 (2H,
q, J = 7.6 Hz, CH2), 1.22 (3H, t, J = 7.6 Hz, CH3); 13C NMR (75 MHz,
DMSO) d 191.4, 149.1, 144.5, 138.6, 137.6, 133.0, 128.9, 128.0,
122.5, 119.4, 117.4, 28.1, 15.6. Anal. Calcd for C16H12BrNO2S
(361.0): C, 52.05; H, 3.34; N, 3.87; S, 8.85. Found: C, 52.80; H,
3.33; N, 3.56; 8.73.
C17H14ClNO2S (331.8): C, 61.54; H, 4.25; N, 4.22; S, 9.66. Found:
C, 61.55; H, 4.07; N, 3.99; S, 9.91.
6.3.8. 6-Fluoro-3-(4-isopropylphenyl)-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2h)
Yellow solid, yield 58%, mp 197–200 °C, IR (m
CO) 1749 cmꢀ1; 1H
NMR (300 MHz, DMSO) d 7.97 (1H, dd, J = 9.1 Hz, J = 2.9 Hz, H5),
7.76 (1H, dt, J = 9.1 Hz, J = 2.9 Hz, H7), 7.57 (1H, dd, J = 9.1 Hz,
J = 4.7 Hz, H8), 7.41–7.34 (2H, m, AA0, BB0, H20, H60), 7.32–7.25
(2H, m, AA0, BB0, H30, H50), 3.06–2.87 (1H, m, CH), 1.24 (6H, d,
J = 7.0 Hz, CH3); 13C NMR (75 MHz, DMSO) d 191.7 (d, J = 2.9 Hz),
158.8 (d, J = 242.5 Hz), 149.0, 146.3 (d, J = 1.7 Hz), 144.7, 137.8,
128.0, 127.4, 123.8 (d, J = 25.4 Hz), 122.0 (d, J = ꢀ9.2 Hz), 119.3
(d, J = 8.0 Hz), 116.2 (d, J = 26.5 Hz), 33.4, 24.0. Anal. Calcd for
6.3.3. 6-Chloro-3-(4-ethylphenyl)-4-thioxo-2H-1,3-benzo-
xazine-2(3H)-one (2c)
Yellow solid, yield 33%, mp 199–202 °C, IR (m
CO) 1761 cmꢀ1; 1H
NMR (300 MHz, DMSO) d 8.22 (1H, d, J = 2.6 Hz, H5), 7.91 (1H, dd,
J = 8.8 Hz, J = 2.6 Hz, H7), 7.55 (1H, d, J = 8.8 Hz, H8), 7.38–7.31 (2H,
m, AA0, BB0, H20, H60), 7.31–7.24 (2H, m, AA0, BB0, H30, H50), 2.67 (2H,
q, J = 7.6 Hz, CH2), 1.22 (3H, t, J = 7.6 Hz, CH3); 13C NMR (75 MHz,
DMSO) d 191.5, 148.7, 144.6, 144.5, 137.7, 135.8, 130.0, 129.7,
C17H14FNO2S (315.4): C, 64.75; H, 4.47; N, 4.44; S, 10.17. Found:
C, 64.74; H, 4.66; N, 4.27; S, 9.78.

8184
E. Petrlíková et al. / Bioorg. Med. Chem. 18 (2010) 8178–8187
6.3.9. 3-(4-Isopropylphenyl)-7-methyl-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2i)
6.3.14. 3-(4-sec-Butylphenyl)-7-methyl-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2n)
Yellow solid, yield 20%, mp 203–207 °C, IR (
m
CO) 1746 cmꢀ1; 1H
Yellow solid, yield 9%, mp 179–182 °C, IR (m ;
CO) 1749 cmꢀ1 1H
NMR (300 MHz, DMSO) d 8.17 (1H, d, J = 8.0 Hz, H5), 7.41–7.22 (6H,
m, H6, H8, H20, H30, H50, H60), 3.03–2.87 (1H, m, CH), 2.44 (3H, s,
CH3), 1.24 (6H, d, J = 7.1 Hz, CH3); 13C NMR (75 MHz, DMSO) d
192.6, 149.8, 148.8, 148.1, 145.0, 137.8, 131.4, 128.2, 127.3,
127.0, 118.9, 116.4, 33.4, 24.0, 21.5. Anal. Calcd for C18H17NO2S
(311.4): C, 69.43; H, 5.50; N, 4.50; S, 10.30. Found: C, 69.06; H,
5.41; N, 4.54; S, 10.50.
NMR (300 MHz, DMSO) d 8.18 (1H, d, J = 8.2 Hz, H5), 7.35–7.22
(6H, m, H6, H8, H20, H30, H50, H60), 2.72–2.59 (1H, m, CH), 2.44
(3H, s, CH3), 1.66–1.49 (2H, m, CH2), 1.23 (3H, d, J = 7.2 Hz, CH3),
0.81 (3H, t, J = 7.2 Hz, CH3); 13C NMR (75 MHz, DMSO) d 192.6,
149.8, 148.1, 147.6, 145.0, 137.8, 131.4, 128.2, 127.8, 127.0,
118.9, 116.4, 40.7, 30.7, 21.7, 21.4, 12.3. Anal. Calcd for C19H19NO2S
(325.43): C, 70.13; H, 5.88; N, 4.30; S, 9.85. Found: C, 69.79; H,
5.96; N, 4.22; S, 9.50.
6.3.10. 6-Bromo-3-(4-sec-butylphenyl)-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2j)
6.3.15. 3-(4-Butylphenyl)-6-chloro-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2o)
Yellow solid, yield 24%, mp 189–190 °C, IR (m
CO) 1756 cmꢀ1; 1H
Yellow solid, yield 29%, mp 149–152 °C, IR (m
CO) 1771 cmꢀ1; 1H
NMR (300 MHz, DMSO) d 8.35 (1H, d, J = 2.5 Hz, H5), 8.02 (1H, dd,
J = 8.8 Hz, J = 2.5 Hz, H7), 7.47 (1H, d, J = 8.8 Hz, H8), 7.37–7.30 (2H,
m, AA0, BB0, H20, H60), 7.30–7.24 (2H, m, AA0, BB0, H30, H50), 2.73–
2.59 (1H, m, CH), 1.65–1.51 (2H, m, CH2), 1.23 (3H, d, J = 7.2 Hz,
CH2), 0.80 (3H, t, J = 7.2 Hz, CH3); 13C NMR (75 MHz, DMSO) d
191.4, 149.1, 147.9, 144.5, 138.6, 137.7, 133.0, 128.0, 122.5,
119.4, 117.4, 40.8, 30.7, 21.7, 12.3. Anal. Calcd for C18H16BrNO2S
(390.30): C, 55.39; H, 4.13; N, 3.59; S, 8.22. Found: C, 55.08; H,
4.15; N, 3.44; S, 8.43.
NMR (300 MHz, DMSO) d 8.22 (1H, d, J = 2.6 Hz, H5), 7.91 (1H, dd,
J = 8.8 Hz, J = 2.6 Hz, H7), 7.55 (1H, d, J = 8.8 Hz, H8), 7.36–7.29 (2H,
m, AA0, BB0, H20, H60), 7.29–7.22 (2H, m, AA0, BB0, H30, H50), 2.63 (2H,
t, J = 7.5 Hz, CH2), 1.67–1.52 (2H, m, CH2), 1.42–1.26 (2H, m, CH2),
0.92 (3H, t, J = 7.5 Hz, CH3); 13C NMR (75 MHz, DMSO) d 191.5,
148.7, 144.5, 143.2, 137.6, 135.8, 13.0, 129.7, 129.4, 127.9, 122.2,
119.2, 34.7, 33.2, 22.0, 14.0. Anal. Calcd for
C18H16ClNO2S
(345.8): C, 62.51; H, 4.66; N, 4.05. Found: C, 62.38; H, 4.73; N, 3.91.
6.3.16. 3-(4-tert-Butylphenyl)-6-chloro-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2p)
6.3.11. 3-(4-sec-Butylphenyl)-6-chloro-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2k)
Yellow solid, yield 25%, mp 226–229 °C, IR (m
CO) 1753 cmꢀ1; 1H
Yellow solid, yield 32%, mp 154–155 °C, IR (m
CO) 1759 cmꢀ1; 1H
NMR (300 MHz, DMSO) d 8.22 (1H, d, J = 2.9 Hz, H5), 7.91 (1H, dd,
J = 8.8 Hz, J = 2.9 Hz, H7), 7.58–7.48 (3H, m, H8, H20, H60), 7.33–7.24
(2H, m, H30, H50), 1.32 (9H, s, CH3); 13C NMR (75 MHz, DMSO) d
191.4, 151.2, 148.7, 144.5, 137.4, 135.8, 130.0, 129.7, 127.7,
126.4, 122.1, 119.2, 34.7, 31.3. Anal. Calcd for C18H16ClNO2S
(345.8): C, 62.51; H, 4.66; N, 4.05; S, 9.27. Found: C, 62.81; H,
4.74; N, 3.82; S, 9.43.
NMR (300 MHz, DMSO) d 8.22 (1H, d, J = 2.5 Hz, H5), 7.91 (1H, dd,
J = 8.8 Hz, J = 2.5 Hz, H7), 7.54 (1H, d, J = 8.8 Hz, H8), 7.37–7.30 (2H,
m, AA0, BB0, H20, H60), 7.30–7.25 (2H, m, AA0, BB0, H30, H50), 2.74–
2.59 (1H, m, CH), 1.66–1.51 (2H, m, CH2), 1.23 (3H, d, J = 7.2 Hz,
CH2), 0.80 (3H, t, J = 7.2 Hz, CH3); 13C NMR (75 MHz, DMSO) d
191.5, 148.7, 147.9, 144.6, 137.7, 135.8, 130.0, 129.7, 128.0,
122.2, 119.2, 40.8, 30.8, 21.7, 12.3. Anal. Calcd for C18H16ClNO2S
(345.85): C, 62.51; H, 4.66; N, 4.05; S, 9.27. Found: C, 62.29; H,
4.64; N, 3.92; S, 9.47.
6.3.17. 3-(4-tert-Butylphenyl)-7-chloro-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2q)
Yellow solid, yield 33%, mp 147–148 °C, IR (m
CO) 1745 cmꢀ1; 1H
NMR (300 MHz, DMSO) d 8.28 (1H, d, J = 8.5 Hz, H5), 7.73 (1H, d,
J = 1.9 Hz, H8), 7.56–7.47 (3H, m, H6, H20, H60), 7.31–7.25 (2H, m,
H30, H50), 1.32 (9H, s, CH3); 13C NMR (75 MHz, DMSO) d 191.8,
151.2, 150.2, 144.5, 140.7, 137.4, 133.0, 128.4, 127.8, 126.3,
120.1, 116.8, 34.7, 31.3. Anal. Calcd for C18H16ClNO2S (345.8): C,
62.51; H, 4.66; N, 4.05; S, 9.27. Found: C, 62.30; H, 4.63; N, 4.10;
S, 9.61.
6.3.12. 3-(4-sec-Butylphenyl)-7-chloro-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2l)
Yellow solid, yield 18%, mp 114–116 °C, IR (m
CO) 1751 cmꢀ1; 1H
NMR (300 MHz, DMSO) d 8.28 (1H, d, J = 8.8 Hz, H5), 7.74 (1H, d,
J = 2.3 Hz, H8), 7.51 (1H, dd, J = 8.8 Hz, J = 2.3 Hz, H6), 7.36–7.30
(2H, m, AA0, BB0, H20, H60), 7.30–7.24 (2H, m, AA0, BB0, H30, H50),
2.74–2.58 (1H, m, CH), 1.69–1.49 (2H, m, CH2), 1.23 (3H, d,
J = 6.9 Hz, CH3), 0.80 (3H, d, J = 6.9 Hz, CH3); 13C NMR (75 MHz,
DMSO) d 191.8, 150.2, 147.8, 144.5, 140.7, 137.7, 133.0, 128.0,
127.9, 126.1, 120.1, 116.8, 40.8, 30.7, 21.7, 12.3. Anal. Calcd for
6.3.18. 3-(4-tert-Butylphenyl)-6-fluoro-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2r)
Yellow solid, yield 29%, mp 161–163 °C, IR (m
CO) 1754 cmꢀ1; 1H
C18H16ClNO2S (345.8): C, 62.51; H, 4.66; N, 4.05; S, 9.27. Found:
NMR (300 MHz, DMSO) d 7.98 (1H, dd, J = 9.4 Hz, J = 3.5 Hz, H5),
7.81–7.71 (1H, m, H7), 7.57 (1H, dd overlapped, J = 9.4 Hz,
J = 4.7 Hz, H8), 7.56–7.49 (2H, m, AA0, BB0, H20, H60), 7.33–7.25
(2H, m, AA0, BB0, H30, H50), 1.32 (9H, s, CH3); 13C NMR (75 MHz,
DMSO) d 191.7 (d, J = 2.9 Hz), 158.8 (d, J = 242.5 Hz), 151.2, 146.3,
144.7, 137.5, 127.7, 126.4, 123.7 (d, J = 24.8 Hz), 120.0 (d,
J = 8.7 Hz), 119.3 (d, J = 8.1 Hz), 116.2 (d, J = 26.5 Hz), 34.7, 31.3.
Anal. Calcd for C18H16FNO2S (329.4): C, 65.63; H, 4.90; N, 4.25; S,
9.73. Found: C, 65.49; H, 4.96; N, 4.15; S, 9.39.
C, 62.12; H, 4.92; N, 3.82; S, 9.25.
6.3.13. 3-(4-sec-Butylphenyl)-6-fluoro-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2m)
Yellow solid, yield 42%, mp 141–142 °C, IR (m ;
CO) 1753 cmꢀ1
1H NMR (300 MHz, DMSO) d 7.97 (1H, dd, J = 9.4 Hz, J = 2.9 Hz,
H5), 7.80–7.72 (1H, m, H7), 7.57 (1H, dd, J = 9.4 Hz, J = 4.7 Hz,
H8), 7.37–7.31 (2H, m, AA0, BB0, H20, H60), 7.31–7.25 (2H, m, AA0,
BB0, H30, H50), 2.75–2.58 (1H, m, CH), 1.68–1.50 (2H, m, CH2),
1.23 (3H, d, J = 6.8 Hz, CH3), 0.80 (3H, d, J = 6.8 Hz, CH3); 13C
6.3.19. 3-(4-tert-Butylphenyl)-7-methyl-4-thioxo-2H-1,3-
benzoxazine-2(3H)-one (2s)
NMR (75 MHz, DMSO)
d
191.7 (d, J = 3.5 Hz), 158.8 (d,
Yellow solid, yield 31%, mp 156–159 °C, IR (m
CO) 1746 cmꢀ1; 1H
J = 242.5 Hz), 147.8, 146.3, 146.3, 144.7, 137.8, 128.0, 123.7 (d,
J = 25.3 Hz), 122.0 (d, J = 8.6 Hz), 119.3 (d, J = 8.0 Hz), 116.2 (d,
J = 26.5 Hz), 40.7, 30.7, 21.6, 12.3. Anal. Calcd for C18H16FNO2S
(329.4): C, 65.63; H, 4.90; N, 4.25; S, 9.73. Found: C, 65.42; H,
4.91; N, 4.07; S, 9.43.
NMR (300 MHz, DMSO) d 8.18 (1H, d, J = 8.0 Hz, H5), 7.55–7.47 (2H,
m, H20, H60), 7.32–7.23 (4H, m, H6, H8, H30, H50), 2.44 (3H, s, CH3),
1.33 (9H, s, CH3); 13C NMR (75 MHz, DMSO) d 192.5, 151.0, 149.7,
148.1, 145.0, 137.5, 131.4, 127.9, 127.0, 126.2, 118.9, 116.4, 34.6,

E. Petrlíková et al. / Bioorg. Med. Chem. 18 (2010) 8178–8187
8185
31.3, 21.4. Anal. Calcd for C19H19NO2S (325.43): C, 70.13; H, 5.88;
N, 4.30; S, 9.85. Found: C, 70.18; H, 5.98; N, 4.20; S, 9.50.
7.63 (1H, d, J = 8.8 Hz, H8), 7.41–7.34 (2H, m, AA0, BB0, H20, H60),
7.26–7.19 (2H, m, AA0, BB0, H30, H50), 3.04–2.85 (1H, CH), 1.24
(6H, d, J = 6.5 Hz, CH3); 13C NMR (75 MHz, DMSO) d 187.0, 177.8,
148.9, 148.7, 141.6, 136.1, 130.7, 130.0, 127.8, 127.7, 123.6,
118.9, 33.3, 24.0. Anal. Calcd for C17H14ClNOS2 (347.89): C, 58.69;
H, 4.06; N, 4.03; S, 18.43. Found: C, 58.27; H, 3.90; N, 4.07; S, 18.75.
6.3.20. 6-Bromo-3-(4-methylphenyl)-1,3-benzoxazine-2,4(3H)-
dithione (3a)
Red solid, yield 31%, mp 246–248 °C; 1H NMR (300 MHz, CDCl3)
d 8.45 (d, 1H, J = 2.40 Hz, H5), 7.81 (dd, 1H, J = 8.77 Hz, J = 2.40 Hz,
H7), 7.39–7.34 (m, AA0, BB0, 2H, H20, H60), 7.25 (d, 1H, J = 8.77 Hz,
H8), 7.12–7.06 (m, AA0, BB0, 2H, H30, H50), 2.46 (s, 3H, CH3); 13C
NMR (75 MHz, CDCl3) d 185.5, 177.0, 148.5, 140.5, 139.2, 138.6,
134.2, 130.7, 127.2, 123.3, 119.5, 118.0, 21.6. Anal. Calcd for
6.3.26. 7-Chloro-3-(4-isopropylphenyl)-1,3-benzoxazine-
2,4(3H)-dithione (3g)
Red solid, yield 24%, mp 200–202 °C; 1H NMR (300 MHz, DMSO)
d 8.19 (1H, d, J = 8.2 Hz, H5), 7.83 (1H, d, J = 2.3 Hz, H8), 7.53 (1H,
dd, J = 8.2 Hz, J = 2.3 Hz, H6), 7.41–7.33 (2H, m, AA0, BB0, H20, H60),
7.25–7.19 (2H, m, AA0, BB0, H30, H50), 3.04–2.86 (1H, m, CH), 1.24
(6H, d, J = 6.4 Hz, CH3); 13C NMR (75 MHz, DMSO) d 187.4, 177.9,
150.1, 148.8, 141.5, 141.2, 133.1, 127.9, 127.7, 127.1, 121.5,
C15H10BrNOS2 (364.3): C, 49.46; H, 2.77; N, 3.85; S, 17.60. Found:
C, 49.23; H, 2.96; N, 3.62; S, 17.32.
6.3.21. 6-Bromo-3-(4-ethylphenyl)-1,3-benzoxazine-2,4(3H)-
dithione (3b)
116.5, 33.3, 24.0. Anal. Calcd for
C17H14ClNOS2 (347.89):
Red solid, yield 17%, mp 195 °C; 1H NMR (300 MHz, DMSO) d
8.26 (1H, d, J = 2.3 Hz, H5), 8.05 (1H, dd, J = 8.8 Hz, J = 2.3 Hz, H7),
7.55 (1H, d, J = 8.8 Hz, H8), 7.37–7.29 (2H, m, AA0, BB0, H20, H60),
7.25–7.17 (2H, m, AA0, BB0, H30, H50), 2.66 (2H, q, J = 7.6 Hz, CH2),
1.22 (3H, t, J = 7.6 Hz, CH3); 13C NMR (75 MHz, DMSO) d 186.8,
177.8, 149.1, 144.3, 141.5, 138.9, 133.1, 129.2, 127.8, 123.9,
119.0, 118.5, 28.0, 15.5. Anal. Calcd for C16H12BrNOS2 (378.3): C,
50.80; H, 3.20; N, 3.70; S, 16.95. Found: C, 50.64; H, 3.25; N,
3.44; S, 16.78.
C, 57.69; H, 4.06; N, 4.03; S, 18.43. Found: C, 57.45; H, 3.89; N,
3.86; S, 18.56.
6.3.27. 6-Fluoro-3-(4-isopropylphenyl)-1,3-benzoxazine-
2,4(3H)-dithione (3h)
Red solid, yield 17%, mp 141–143 °C; 1H NMR (300 MHz, DMSO)
d 7.90 (1H, dd, J = 9.4 Hz, J = 2.9 Hz, H5), 7.84–7.76 (1H, m, H7),
7.67 (1H, dd, J = 9.4 Hz, J = 4.1 Hz, H8), 7.41–7.33 (2H, m, AA0, BB0,
H20, H60), 7.27–7.20 (2H, m, AA0, BB0, H30, H50), 3.04–2.85 (1H, m,
CH), 1.24 (6H, d, J = 7.0 Hz, CH3); 13C NMR (75 MHz, DMSO) d
187.2 (d, J = 3.5 Hz), 177.9, 159.4 (d, J = 244.1 Hz), 148.8, 146.5 (d,
J = 1.7 Hz), 141.6, 127.8, 127.7, 124.1 (d, J = 25.3 Hz), 123.6 (d,
J = ꢀ9.2 Hz), 119.2 (d, J = 8.7 Hz), 116.2 (d, J = 27.0 Hz), 33.3, 24.0.
Anal. Calcd for C17H14FNO2S (331.4): C, 61.61; H, 4.26; N, 4.23; S,
19.35. Found: C, 61.74; H, 4.37; N, 3.96; S, 19.43.
6.3.22. 6-Chloro-3-(4-ethylphenyl)-1,3-benzoxazine-2,4(3H)-
dithione (3c)
Red solid, yield 21%, mp 173–174 °C; 1H NMR (300 MHz, DMSO)
d 8.12 (1H, d, J = 2.3 Hz, H5), 7.93 (1H, dd, J = 8.8 Hz, J = 2.3 Hz, H7),
7.63 (1H, d, J = 8.8 Hz, H8), 7.38–7.29 (2H, m, AA0, BB0, H20, H60),
7.25–7.17 (2H, m, AA0, BB0, H30, H50), 2.66 (2H, q, J = 7.6 Hz, CH2),
1.22 (3H, t, J = 7.6 Hz, CH3); 13C NMR (75 MHz, DMSO) d 186.9,
177.8, 148.7, 144.3, 141.5, 136.2, 130.7, 130.0, 129.1, 127.8,
123.6, 118.9, 28.0, 15.4. Anal. Calcd for C16H12ClNOS2 (333.9): C,
57.56; H, 3.62; N, 4.20; S, 19.21. Found: C, 57.45; H, 3.53; N,
4.04; S, 18.82.
6.3.28. 3-(4-Isopropylphenyl)-7-methyl-1,3-benzoxazine-
2,4(3H)-dithione (3i)
Red solid, yield 29%, mp 195–197 °C; 1H NMR (300 MHz, DMSO)
d 8.10 (1H, d, J = 8.2 Hz, H5), 7.40–7.26 (4H, m, H6, H8, H20, H60),
7.25–7.18 (2H, m, H30, H50), 3.01–2.86 (1H, m, CH), 2.44 (3H, s,
CH3), 1.24 (6H, d, J = 6.9 Hz, CH3); 13C NMR (75 MHz, DMSO) d
188.0, 178.4, 149.8, 148.7, 148.6, 141.6, 131.4, 128.1, 128.0,
127.6, 120.4, 116.0, 33.3, 24.0, 21.5. Anal. Calcd for C18H17NOS2
(327.47): C, 66.02; H, 5.23; N, 4.28; S, 19.58. Found: C, 66.00; H,
5.34; N, 4.18; S, 19.19.
6.3.23. 7-Chloro-3-(4-ethylphenyl)-1,3-benzoxazine-2,4(3H)-
dithione (3d)
Red solid, yield 50%, mp 205–208 °C; 1H NMR (300 MHz, DMSO)
d 8.20 (1H, d, J = 8.8 Hz, H5), 7.84 (1H, d, J = 1.8 Hz, H8), 7.53 (1H,
dd, J = 8.8 Hz, J = 1.8 Hz, H6), 7.37–7.28 (2H, m, AA0, BB0, H20, H60),
7.26–7.17 (2H, m, AA0, BB0, H30, H50), 2.66 (2H, q, J = 7.6 Hz, CH2),
1.22 (3H, t, J = 7.6 Hz, CH3); 13C NMR (75 MHz, DMSO) d 187.4,
177.9, 150.2, 144.3, 141.5, 141.2, 133.1, 129.1, 127.9, 127.1,
121.5, 116.5, 28.0, 15.4. Anal. Calcd for C16H12ClNOS2 (333.9): C,
57.56; H, 3.62; N, 4.20; 19.21. Found: C, 57.27; H, 3.53; N, 3.99;
S, 19.03.
6.3.29. 6-Bromo-3-(4-sec-butylphenyl)-1,3-benzoxazine-
2,4(3H)-dithione (3j)
Red solid, yield 38%, mp 161–162 °C; 1H NMR (300 MHz, DMSO)
d 8.26 (1H, d, J = 2.5 Hz, H5), 8.04 (1H, dd, J = 8.8 Hz, J = 2.5 Hz, H7),
7.55 (1H, d, J = 8.8 Hz, H8), 7.36–7.28 (2H, m, AA0, BB0, H20, H60),
7.26–7.18 (2H, m, AA0, BB0, H30, H50), 2.73–2.58 (1H, m, CH),
1.67–1.47 (2H, m, CH2), 1.23 (3H, d, J = 7.2 Hz, CH2), 0.78 (3H, t,
J = 7.2 Hz, CH3); 13C NMR (75 MHz, DMSO) d 186.8, 177.7, 149.1,
147.7, 141.6, 138.9, 133.0, 128.3, 127.7, 123.9, 119.0, 118.5, 40.7,
30.8, 21.6, 12.2. Anal. Calcd for C18H16BrNOS2 (406.37): C, 53.20;
H, 3.97; N, 3.45; S, 15.78. Found: C, 52.90; H, 3.95; N, 3.31; S, 15.80.
6.3.24. 6-Bromo-3-(4-isopropylphenyl)-1,3-benzoxazine-
2,4(3H)-dithione (3e)
Red solid, yield 32%, mp 181–182 °C; 1H NMR (300 MHz, DMSO)
d 8.26 (1H, d, J = 2.5 Hz, H5), 8.04 (1H, dd, J = 8.8 Hz, J = 2.5 Hz, H7),
7.55 (1H, d, J = 8.8 Hz, H8), 7.41–7.33 (2H, m, AA0, BB0, H20, H60),
7.26–7.18 (2H, m, AA0, BB0, H30, H50), 3.03–2.86 (1H, m, CH), 1.24
(6H, d, J = 6.9 Hz, CH3); 13C NMR (75 MHz, DMSO) d 186.8, 177.8,
149.1, 148.9, 141.5, 138.9, 133.0, 127.8, 127.7, 123.9, 119.0,
6.3.30. 3-(4-sec-Butylphenyl)-6-chloro-1,3-benzoxazine-
2,4(3H)-dithione (3k)
Red solid, yield 27%, mp 153–154 °C; 1H NMR (300 MHz, DMSO)
d 8.22 (1H, d, J = 2.5 Hz, H5), 7.91 (1H, dd, J = 8.8 Hz, J = 2.5 Hz, H7),
7.54 (1H, d, J = 8.8 Hz, H8), 7.37–7.30 (2H, m, AA0, BB0, H20, H60),
7.30–7.24 (2H, m, AA0, BB0, H30, H50), 2.73–2.57 (1H, m, CH),
1.66–1.50 (2H, m, CH2), 1.23 (3H, d, J = 7.2 Hz, CH2), 0.80 (3H, t,
J = 7.2 Hz, CH3); 13C NMR (75 MHz, DMSO) d 186.9, 177.8, 148.7,
147.7, 141.6, 136.2, 130.7, 130.0, 128.3, 127.8, 123.6, 118.9, 40.7,
30.8, 21.6, 12.2. Anal. Calcd for C18H16ClNOS2 (361.92): C, 59.74;
H, 4.46; N, 3.87; S, 17.72. Found: C, 59.48; H, 4.36; N, 3.80; S, 17.95.
118.5, 33.3, 24.0. Anal. Calcd for
C17H14BrNOS2 (392.34): C,
52.04; H, 3.60; N, 3.57; S, 16.35. Found: C, 51.78; H, 3.48; N,
3.47; S, 16.20.
6.3.25. 6-Chloro-3-(4-isopropylphenyl)-1,3-benzoxazine-
2,4(3H)-dithione (3f)
Red solid, yield 28%, mp 163–166 °C; 1H NMR (300 MHz, DMSO)
d 8.13 (1H, d, J = 2.6 Hz, H5), 7.94 (1H, dd, J = 8.8 Hz, J = 2.6 Hz, H7),

8186
E. Petrlíková et al. / Bioorg. Med. Chem. 18 (2010) 8178–8187
6.3.31. 3-(4-sec-Butylphenyl)-7-chloro-1,3-benzoxazine-
2,4(3H)-dithione (3l)
13C NMR (75 MHz, DMSO) d 187.4, 177.8, 151.1, 150.1, 141.2,
141.2, 133.1, 127.5, 127.1, 126.6, 121.5, 116.5, 34.7, 31.3. Anal.
Calcd for C18H16ClNOS2 (361.9): C, 59.74; H, 4.46; N, 3.87; S,
17.72. Found: C, 59.83; H, 4.38; N, 3.99; S, 18.15.
Red solid, yield 32%, mp 169–170 °C; 1H NMR (300 MHz, DMSO)
d 8.20 (1H, d, J = 8.8 Hz, H5), 7.83 (1H, d, J = 1.8 Hz, H8), 7.53 (1H,
dd, J = 8.8 Hz, J = 1.8 Hz, H6), 7.35–7.28 (2H, m, AA0, BB0, H20, H60),
7.25–7.18 (2H, m, AA0, BB0, H30, H50), 2.75–2.56 (1H, m, CH),
1.68–1.47 (2H, m, CH2), 1.22 (3H, d, J = 6.9 Hz, CH3), 0.78 (3H, d,
J = 6.9 Hz, CH3); 13C NMR (75 MHz, DMSO) d 187.4, 177.9, 150.2,
147.6, 141.6, 141.2, 133.1, 128.2, 127.8, 127.1, 121.5, 116.5, 40.7,
30.8, 21.6, 12.2. Anal. Calcd for C18H16ClNOS2 (361.9): C, 59.74;
H, 4.46; N, 3.87; S, 17.72. Found: C, 59.76; H, 4.60; N, 3.64; S, 17.36.
6.3.37. 3-(4-tert-Butylphenyl)-6-fluoro-1,3-benzoxazine-
2,4(3H)-dithione (3r)
Red solid, yield 40%, mp 168–169 °C; 1H NMR (300 MHz, DMSO)
d 7.90 (1H, dd, J = 8.9 Hz, J = 2.9 Hz, H5), 7.80 (1H, dt, J = 8.9 Hz,
J = 2.9 Hz, H7), 7.66 (1H, dd, J = 8.9 Hz, J = 4.7 Hz, H8), 7.56–7.48
(2H, m, AA0, BB0, H20, H60), 7.27–7.20 (2H, m, AA0, BB0, H30, H50),
1.32 (9H, s, CH3); 13C NMR (75 MHz, DMSO)
d 187.2 (d,
6.3.32. 3-(4-sec-Butylphenyl)-6-fluoro-1,3-benzoxazine-
2,4(3H)-dithione (3m)
J = 3.5 Hz), 177.9, 159.4 (d, J = 24.4 Hz), 151.1, 146.5, 141.4, 127.5,
126.6, 124.1 (d, J = 24.8 Hz), 123.5 (d, J = 9.2 Hz), 119.2 (d,
J = 8.7 Hz), 116.2 (d, J = 26.5 Hz), 34.7, 31.3. Anal. Calcd for
Red solid, yield 25%, mp 107–109 °C; 1H NMR (300 MHz, DMSO)
d 7.90 (1H, dd, J = 8.8 Hz, J = 2.9 Hz, H5), 7.85–7.75 (1H, m, H7),
7.66 (1H, dd, J = 8.8 Hz, J = 4.7 Hz, H8), 7.36–7.28 (2H, m, AA0, BB0,
H20, H60), 7.26–7.18 (2H, m, AA0, BB0, H30, H50), 2.73–2.57 (1H, m,
CH), 1.67–1.47 (2H, m, CH2), 1.23 (3H, d, J = 7.3 Hz, CH3), 0.78
(3H, d, J = 7.3 Hz, CH3); 13C NMR (75 MHz, DMSO) d 187.2 (d,
J = 3.5 Hz), 177.9, 159.4 (d, J = 244.1 Hz), 147.7, 146.5, 141.7,
128.2, 127.8, 124.1 (d, J = 24.8 Hz), 123.6 (d, J = 8.7 Hz), 119.2 (d,
J = 8.7 Hz), 116.2 (d, J = 26.5 Hz), 40.7, 30.8, 21.6, 12.2. Anal. Calcd
for C18H16FNOS2 (345.5): C, 62.58; H, 4.67; N, 4.05; S, 18.56. Found:
C, 62.48; H, 4.38; N, 4.18; S, 18.24.
C18H16FNOS2 (345.5): C, 62.58; H, 4.67; N, 4.05; S, 18.56. Found:
C, 62.50; H, 4.60; N, 3.88; S, 18.21.
6.3.38. 3-(4-tert-Butylphenyl)-7-methyl-1,3-benzoxazine-
2,4(3H)-dithione (3s)
Red solid, yield 16%, mp 260–262 °C; 1H NMR (300 MHz, DMSO)
d 8.11 (1H, d, J = 8.3 Hz, H5), 7.55–7.47 (2H, m, AA0, BB0, H20, H60),
7.39 (1H, s, H8), 7.34–7.26 (1H, m, H6), 7.26–7.18 (2H, m, AA0,
BB0, H30, H50), 2.45 (3H, s, CH3), 1.32 (9H, s, CH3); 13C NMR
(75 MHz, DMSO) d 188.0, 178.3, 151.0, 149.8, 148.6, 141.3, 131.4,
128.1, 127.7, 126.5, 120.3, 116.0, 34.7, 31.4, 21.5. Anal. Calcd for
6.3.33. 3-(4-sec-Butylphenyl)-7-methyl-1,3-benzoxazine-
2,4(3H)-dithione (3n)
C19H19NOS2 (341.50): C, 66.83; H, 5.61; N, 4.10; S, 18.78. Found:
C, 67.13; H, 5.59; N, 4.26; S, 18.53.
Red solid, yield 40%, mp 169–170 °C; 1H NMR (300 MHz, DMSO)
d 8.11 (1H, d, J = 8.3 Hz, H5), 7.39–7.27 (4H, m, H6, H8, H20, H60),
7.25–7.17 (2H, m, H30, H50), 2.73–2.59 (1H, m, CH), 2.45 (3H, s,
CH3), 1.65–1.48 (2H, m, CH2), 1.23 (3H, d, J = 7.1 Hz, CH3), 0.79
(3H, t, J = 7.1 Hz, CH3); 13C NMR (75 MHz, DMSO) d 188.0, 178.3,
149.8, 148.6, 147.5, 141.6, 131.4, 128.1, 128.0, 120.3, 116.0, 40.7,
30.8, 21.6, 21.5, 12.2. Anal. Calcd for C19H19NOS2 (341.50): C,
66.83; H, 5.61; N, 4.10; S, 18.78. Found: C, 66.77; H, 5.52; N,
4.27; S, 18.49.
6.4. 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 12478), 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. kans-
asii 6509/96. The antimycobacterial activity of the compounds was
determined in the Šula semisynthetic medium (SEVAC, Prague).
Each strain was simultaneously inoculated into a Petri dish con-
taining the Löwenstein-Jensen 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
6.3.34. 3-(4-Butylphenyl)-6-chloro-1,3-benzoxazine-2,4(3H)-
dithione (3o)
Red solid, yield 26%, mp 125–127 °C; 1H NMR (300 MHz, DMSO)
d 8.13 (1H, d, J = 2.6 Hz, H5), 7.94 (1H, dd, J = 8.8 Hz, J = 2.6 Hz, H7),
7.63 (1H, d, J = 8.8 Hz, H8), 7.35–7.27 (2H, m, AA0, BB0, H20, H60),
7.24–7.16 (2H, m, AA0, BB0, H30, H50), 2.63 (2H, t, J = 7.5 Hz, CH2),
1.67–1.51 (2H, m, CH2), 1.41–1.24 (2H, m, CH2), 0.91 (3H, t,
J = 7.5 Hz, CH3); 13C NMR (75 MHz, DMSO) d 186.9, 177.8, 148.7,
143.0, 141.5, 136.2, 130.7, 130.0, 129.6, 127.8, 123.6, 118.9, 34.7,
33.1, 22.0, 14.0. Anal. Calcd for C18H16ClNOS2 (361.9): C, 59.74;
H, 4.46; N, 3.87. Found: C, 59.56; H, 4.44; N, 3.55.
1000, 500, 250, 125, 62.5, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 lmol/L.
The MICs were determined after incubation at 37 °C for 14 days
and 21 days. The MIC was the lowest concentration of the antimy-
cobacterially effective substance (on the above concentration
scale), at which the inhibition of the growth of mycobacteria oc-
curred. The evaluation was repeated three times and the values
of the MIC were the same.
6.3.35. 3-(4-tert-Butylphenyl)-6-chloro-1,3-benzoxazine-
2,4(3H)-dithione (3p)
Red solid, yield 22%, mp 219–222 °C; 1H NMR (300 MHz, DMSO)
d 8.13 (1H, d, J = 2.9 Hz, H5), 7.94 (1H, dd, J = 8.8 Hz, J = 2.9 Hz, H7),
7.63 (d, 1H, J = 8.8 Hz, H8), 7.56–7.48 (2H, m, AA0, BB0, H20, H60),
7.26–7.20 (2H, m, AA0, BB0, H30, H50), 1.32 (9H, s, CH3); 13C NMR
(75 MHz, DMSO) d 186.9, 177.8, 151.2, 148.7, 141.3, 136.1, 130.7,
130.0, 127.5, 126.6, 123.5, 118.9, 34.7, 31.3. Anal. Calcd for
Acknowledgments
This work is financially supported by project no. MSM
0021620822 of the Ministry of Education of the Czech Republic,
by grant no. SVV-2010-261-001 and Grant Agency of Charles Uni-
versity (GAUK 11809/2009/B-CH/FAF).
C18H16ClNOS2 (361.9): C, 59.74; H, 4.46; N, 3.87; S, 17.72. Found:
The authors have declared no conflict of interest.
C, 59.60; H, 4.45; N, 3.71; S, 17.48.
References and notes
6.3.36. 3-(4-tert-Butylphenyl)-7-chloro-1,3-benzoxazine-
2,4(3H)-dithione (3q)
1. World Health Organization: 10 facts about tuberculosis [cit. 2009-10-23].
Red solid, yield 29%, mp 255–256 °C; 1H NMR (300 MHz, DMSO)
d 8.20 (1H, d, J = 8.8 Hz, H5), 7.82 (1H, d, J = 1.9 Hz, H8), 7.56–7.48
(3H, m, H6, H20, H60), 7.27–7.19 (2H, m, H30, H50), 1.32 (9H, s, CH3);
Available
from:
http://www.who.int/features/factfiles/tuberculosis/en/
index.html.
2. WHO Fact Sheet No 104, 2010 [cit. 2010-06-02]. Available from: http://
www.who.int/mediacentre/factsheets/fs104/en/index.html.

E. Petrlíková et al. / Bioorg. Med. Chem. 18 (2010) 8178–8187
8187
3. World Health Organization: The global plan to stop TB 2006-2015 [cit. 2010-
09-23]. Available from: http://www.stoptb.org/assets/documents/global/plan/
GlobalPlanFinal.pdf.
4. Alahari, A.; Trivelli, X.; Guérardel, Y.; Dover, G. L.; Besra, G. S.; Sacchetini, J. C.;
Reynolds, R. C.; Coxon, G. D.; Kremer, L. Plos One 2007, 12.
11. Katritzky, A. R.; Singh, S. K.; Akhmedova, R.; Cai, Ch.; Bobrov, S. ARKIVOC 2007,
6.
12. Boontheung, P.; Perlmutter, P. Tetrahedron Lett. 1998, 39, 2629.
ˇ
13. Waisser, K.; Machácek, M.; Dostál, H.; Gregor, J.; Kubicová, L.; Klimešová, V.;
Kuneš, J.; Palát, K.; Hladu˚ vková, J.; Kaustová, J.; Moellmann, U. Collect. Czech.
5. Phetsuksiri, B.; Jackson, M.; Scherman, H.; McNeil, M.; Besra, G. S.; Baulard, A.
R.; Slayden, R. A.; DeBarber, A. E.; Barry, C. E.; Baird, M. S.; Crick, D. C.; Brennan,
P. J. J. Biol. Chem. 2003, 278, 53123.
6. Schroeder, E. K.; Souza de, O. N.; Santos, D. S.; Blanchard, J. S.; Basso, L. A. Curr.
Pharm. Biotechnol. 2002, 3, 197.
Chem. Commun. 1999, 64, 1902.
14. Resck, I. S.; dos Santos, M. L.; Romeiro, L. A. S. Heterocycles 2005, 65, 311.
15. Wagner, G.; Singer, D.; Weuffen, W. Pharmazie 1966, 21, 161.
16. Saeed, A.; Ashraf, Z. J. Heterocycl. Chem. 2008, 45, 679.
17. Waisser, K.; Matyk, J.; Divišová, H.; Husáková, P.; Kuneš, J.; Klimešová, V.; Palát,
7. Hlasta, D. J.; Demers, J. P.; Foleno, B. D.; Fraga-Spano, S. A.; Guan, J.; Hilliard, J. J.;
Macielang, M. J.; Ohemeng, K. A.; Sheppard, C. M.; Sui, Z.; Webb, G. C.; Weidner-
Wells, M. A.; Werblood, H.; Barrett, J. F. Bioorg. Med. Chem. Lett. 1998, 8, 1923.
8. Macielang, M. J.; Demers, J. P.; Fraga-Spano, S. A.; Hlasta, D. J.; Johnson, S. G.;
Konojia, R. M.; Russel, R. K.; Sui, Z.; Weidner-Wells, M. A.; Werblood, H.; Foleno,
B. D.; Golschmidt, R. M.; Loeloff, M. J.; Webb, G. C.; Barrett, J. F. J. Med. Chem.
1998, 41, 2939.
K.; Kaustová, J. Arch. Pharm. Chem. Life Sci. 2007, 340, 264.
ˇ
18. Kubicová, L.; Šustr, M.; Machácek, M.; Waisser, K.; Buchta, V.; Kubanová, P.;
Kaustová, J.; Králová, K. International Electronic Conferences on Synthetic
Organic Chemistry, 2004; p 304.
19. Waisser, K.; Hladu˚ vková, J.; Holy´, P.; Machácek, M.; Karajannis, P.; Kubicová, L.;
ˇ
Klimešová, V.; Kuneš, J.; Kaustová, J. Chem. Pap. 2001, 55, 323.
20. Free, S. M.; Wilson, J. W., Jr. J. Med. Chem. 1964, 7, 395.
21. Fujita, T.; Ban, T. J. Med. Chem. 1971, 14, 148.
9. Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479.
ˇ
10. van den Boogaard, J.; Kibiki, G. S.; Kisanga, E. R.; Boeree, M. J.; Aarnoutse, R. E.
Antimicrob. Agents Chemother. 2009, 53, 849.
22. Waisser, K.; Matyk, J.; Kuneš, J.; Dolezal, R.; Kaustová, J.; Dahse, H. M. Arch.
Pharm. Chem. Life Sci. 2008, 341, 800.