The oriented development of antituberculotics (Part II): Halogenated 3-(4-alkylphenyl)-1,3-benzoxazine-2,4-(3H)-diones

Article abstract of DOI:10.1002/ardp.200600002

Based on our previous studies, 21 new halogenated 3-(4-alkylphenyl)-1,3- benzoxazine-2,4-(3H)-diones were synthesized by the reaction of salicylanilides and methyl-chloroformate. All compounds were screened in vitro against three different strains of mycobacterium, and Free-Wilson method was used to establish structure-activity relationships. 6-Bromo-3-(4-butylphenyl)-1,3-benzoxazine-2, 4-(3H)-dione 3b proved to be the most active compound of the series.

Full text of DOI:10.1002/ardp.200600002

264
Arch. Pharm. Chem. Life Sci. 2007, 340, 264–267
Full Paper
The Oriented Development of Antituberculotics (Part II):
Halogenated 3-(4-Alkylphenyl)-1,3-benzoxazine-2,4-(3H)-
diones*
Karel Waisser1, Josef Matyk1, Hana DiviÐovꢀ1, Petra Husꢀkovꢀ1, Jirꢁ KuneÐ1, Vera KlimeÐovꢀ1,
Karel Palꢀt1, and Jarmila Kaustovꢀ2
1 Department of Inorganic and Organic Chemistry, Charles University in Prague, Faculty of Pharmacy in
Hradec Krꢀlovꢁ, Heyrovskꢁho, Czech Republic
2 Regional Institute of Public Health, Department for Diagnostic of Mycobacteria, Ostrava, Czech Republic
Based on our previous studies, 21 new halogenated 3-(4-alkylphenyl)-1,3-benzoxazine-2,4-(3H)-dio-
nes were synthesized by the reaction of salicylanilides and methyl-chloroformate. All com-
pounds were screened in vitro against three different strains of mycobacterium, and Free-Wilson
method was used to establish structure-activity relationships. 6-Bromo-3-(4-butylphenyl)-1,3-ben-
zoxazine-2,4-(3H)-dione 3b proved to be the most active compound of the series.
Keywords: Antimycobacterial activity / Antituberculotic activity / Benzoxazine / Tuberculostatics /
Received: October 18, 2006; accepted: January 19, 2007
DOI 10.1002/ardp.200600002
for several years now, we are investigating the antimyco-
bacterial arylbenzoxazine-2,4(3H)-diones [12].
Introduction
The goal of this paper is the synthesis and antimyco-
bacterial evaluation of 3-(4-alkyphenyl)-1,3-benzoxazine-
2,4(3H)-diones, substituted in the ring B by chlorine or
bromine, and to complete with this study our previous
work [1]. 3-Phenyl-1,3-benzoxazine-2,4(3H)-diones are the
cyclic carbamic derivatives of salicylanilides and the
mechanism of the antimycobacterial activity of 3-phenyl-
1,3-benzoxazine-2,4(3H)-diones is similar to that of salicy-
lanilides. [13].
The return of tuberculosis to Europe and North America
is one truly unpleasant development of the period dating
from 1985. New mycobacterial diseases, which recently
have been considered to be transferable to humans,
occurred during the last years (mycobacterioses pro-
duced by potentially pathogenic strains). The develop-
ment of new antituberculotic agents is the principal goal
of our group. Lately, we have studied a number of structu-
rally different compounds, such as the derivatives of sali-
cylamides [1], pyridine [2, 3], alkoxyphenylcarbamic
acids [4], tetrazoles [5, 6], dihydroindolethiones [7], and
other heterocycles [8, 9]. Our research was strongly
oriented to collaborate with German institutes like Tech-
nical University Dresden, Friedrich Schiller University
Jena, Hans Knꢀll Institute Jena, and Maxmilian Univer-
sity in Munich. Approximately ten years ago, we studied
antimycobacterial derivatives with the new pharmaco-
phore together with Hans-Dietrich Stachel [10, 11] and
Results and discussion
Chemistry
The synthesis of the title compounds is illustrated in
Scheme 1. The synthesis of the starting material, haloge-
nated N-(4-alkylphenyl)salicylamides, were described in
the previous paper [1]. Various N-(4-alkylphenyl)salicyl-
amides, being halogen substituted in ring A, were
reacted with methyl-chloroformate in dry pyridine. The
halogenated 3-(4-alkylphenyl)-1,3-benzoxazine-2,4(3H)-
Correspondence: Karel Waisser, Charles University in Prague, Faculty
of Pharmacy in Hradec Krꢀlovꢁ, Heyrovskꢁho 1203, CZ 500 05 Hradec
Krꢀlovꢁ, Czech Republic
E-mail: waisser@faf.cuni.cz
Fax: +420 495 514-330
* Dedicated to Prof. Hans-Dietrich Stachel (Munich) on the occasion of
his 80th birthday.
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Arch. Pharm. Chem. Life Sci. 2007, 340, 264–267
Benzoxazines as Antituberculotics
265
Table 1. Yield, melting point, and carbonyl frequency of 3-(4-alkylphenyl)-1,3-benzoxazine-2,4(3H)-diones.
Compound
Yield
(%)
M. p.
(8C)
mC=O
(cm– 1
Compound Yield
(%)
M. p.
(8C)
mC=O
(cm– 1
)
)
1a
1b
1c
1d
1e
1g
2a
2b
2c
2d
2e
60
58
61
52
52
50
63
66
66
56
51
188–190
189–190
214–215
181–182
162–165
181–182
158–160
161–162
214–215
136–137
146–148
1773, 1703
1771, 1705
1770, 1709
1773, 1701
1776, 1699
1773, 1702
1777, 1696
1776, 1708
1767, 1716
1777, 1708
1776, 1708
2f
56
67
58
63
64
58
62
47
58
48
134–135
141–142
193–194
194–195
179–180
168–169
158–160
168–169
197–198
196–199
1777, 1708
1777, 1708
1774, 1700
1770, 1702
1777, 1709
1774, 1701
1774, 1701
1774, 1701
1780, 1701
1781, 1703
2g
3a
3b
3d
3e
3f
4b
4d
4e
Table 2. Minimum antimycobacterial inhibitory activity.
Compound
R2
MIC (lmol/L) Incubation time 14 d/21 d
R1
M. tuberculo- M. kansasii M. avium M. kansa-
sis
My 235/ 80My 330/ sii
88 6 509/ 96
My 331/ 88
1a
1b
1c
1d
1e
1g
2a
2b
2c
2d
2e
2f
2g
3a
3b
3d
3e
3f
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
7-Cl
7-Cl
7-Cl
7-Cl
7-Cl
7-Cl
propyl
butyl
tert-butyl
pentyl
hexyl
octyl
propyl
butyl
tert-butyl
pentyl
hexyl
heptyl
octyl
propyl
butyl
pentyl
hexyl
heptyl
butyl
16/16
8/8
8/16
8/4
8/8
8/8
8/8
8/8
4/4
8/8
8/8
8/8
16/16
8/16
8/16
8/8
8/16
8/16
4/4
8/16
8/16
8/8
8/16
8/8
8/16
8/16
8/16
8/8
8/8
8/8
8/8
16/32
8/16
8/8
8/16
8/16
8/16
4/8
8/16
8/16
8/16
8/8
8/8
16/16
16/16
4/8
4/8
4/8
16/32
8/16
16/16
8/8
8/8
4/8
Scheme 1. Synthesis of 3-(4-alkylphenyl)-1,3-benzoxazine-
2,4(3H)-diones.
4/8
8/8
8/8
16/32
8/16
16/16
4/4
4/4
4/8
7-Cl
8/16
8/16
8/8
8/16
8/16
8/16
6-Br
6-Br
6-Br
6-Br
6-Br
6,8-Br2
6,8-Br2
6,8-Br2
diones were purified by crystallization from ethanol.
Yield, melting point, and carbonyl frequency of the pro-
ducts are summarized in Table 1. Data from elemental
analysis and NMR spectra are described in the Support-
ing Information.
4/8
4/4
4/8
4b
4d
4e
INH
32/32
16/32
62.5/62.5
1/2
62.5/62.5 62.5/62.5 62.5/62.5
16/16
32/32
pentyl
hexyl
62.5
62.5
32/32
62.5/62.5
8/8
250/250 250/250
Biology
In vitro antimycobacterial activity of the compounds was
evaluated against Mycobacterium tuberculosis CNCTC My
331/88, Mycobacterium kansasii CNCTC My 235/80, Myco-
bacterium avium CNCTC My 330/88 and Mycobacterium
kansasii 6 509/96 using the micromethod for the determi-
nation of minimum inhibitory concentration (MIC). All
strains were obtained from the Czech National Collec-
tion of Type Cultures (CNCTC), National Institute of Pub-
lic Health, Prague with the exception of M. kansasii 6 509/
96. The minimum inhibitory concentrations are illu-
strated in Table 2. For the sake of comparison, we also
include the MIC values of the standard isoniazide (INH).
Discussion
The study was incited by the previous synthesis of anti-
mycobacterial salicylanilides. For the synthesis of 3-(4-
alkylphenyl)-1,3-benzoxazine-2,4(3H)diones
salicylani-
lides are reacted with chloroformate. The 1,3-benzoxa-
zine-2,4(3H)diones are cyclic carbamates of salicylani-
lides. The structures of products were confirmed by ele-
mental analyses and by IR, 1H- and13C-NMR spectral meth-
ods. All halogenated 3-(4-alkylphenyl)-1,3-benzoxazine-
2,4(3H)diones showed characteristic absorption maxima
of two C=O groups at 1767–1777 cm1 and 1696–
1716 cm1 with the exception of compound 3d. Two
absorption maxima of the C=O groups in this region are a
characteristic feature for 1,3-benzoxazine-2,4(3H)diones.
The other confirmation of the structure was by NMR
Calculation
All calculations were carried out with the use of the Mul-
tireg H program (Klemera) for Microsoft Excel. The results
of Free–Wilson method are summarized in Table 3.
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266
K. Waisser et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 264–267
Table 3. Activity contribution of Free-Wilson analyzes of benzoxazinediones and statistical significants of correlations.
Parameter
D log MIC (lmol/L)
For incubation time 14 d/21 d
M. tuberculosis
My 331/88
M. kansasii
My 235/80
M. avium
My 330/88
M. kansasii
6509/96
R1: 6-Cl
7-Cl
6-Br
0.0209/-0.207
–0.0935/–0.0842
–0.2664/–0.2469
0.6204/0.6494
0.2701/0.1744
–0.0444/–0.1423
–0.1566/–0.0904
–0.1132/–0.1423
–0.0382/0.0078
0.1371/0.3227
–0.0066/0.0596
0.943/1.043
–0.1121a)
–0.0638a)
0.0023a)
0.5133a)
0.0152a)
0.0901a)
0.0165a)
–0.0597a)
0.0153a)
0.0025a)
–0.1335a)
0.9714a)
0.928a)
–0.0435/–0.0646
–0.1504/–0.0569
–0.2318/–0.2654
0.8247/0.7043
0.2991/0.1718
0.0575/–0.0365
–0.0458/0.1037
–0.0926/–0.0365
–0.0962/–0.115
0.0483/0.054
–0.0458/–0.0464
1.043/1.157
–0.1383/–0.062
–0.0696/–0.006
–0.1459/–0.235
0.7451/0.5321
0.0322/0.1585
0.0572/0.679
0.0182/–0.208
–0.0178/0.0002
0.0572/0.0002
–0.128/0.028
–0.1318/–0.207
0.1015/0.1724
0.971/0.901
6,8-Br2
R2: propyl
butyl
tert-butyl
pentyl
hexyl
heptyl
octyl
lo
r
s
F
0.881/0.878
0.2033/0.2087
4.24/4.12
0.915/0.860
0.211/0.238
6.25/3.48
0.1253a)
0.1015/0.1724
20.42/5.25
7.57a)
n
21/21
21/21
21/21
21/21
a)
Correlation is not statistically significant.
1
methods: The H- and 13C-NMR spectra of all compounds butylphenyl)-1,3-benzoxazine-2,4(3H)-dione 3b. Accord-
(see Table 1) are in the good agreement with the proposed ing to Free-Wilson calculations, we can assume that 6-
structures. In addition, elemental analyses of all com- bromo-3-(4-tert-butylphenyl)-1,3-benzoxazine-2,4(3H)-
pounds correspond to the calculated values.
dione will be more active than the compounds under
The values of antimycobacterial activity of the haloge- study.
nated
3-(4-alkylphenyl)-1,3-benzoxazine-2,4(3H)-diones
are shown in Table 2. For the sake of comparison, we also This work is a part of the research project No. MSM0021620822
included the MIC values of the standard isoniazide (INH). of the Ministry of Education of the Czech Republic.
The results revealed that the compounds exhibited in
vitro activity against all tested mycobacterial strains. The
values of MICs are generally within the range 4-
62.5 lmol/L, most often between 4-8 lmol/L. The com-
Experimental
pounds were less active than INH against M. tuberculosis
331/88, on the other hand, the compounds possessed a
better activity against M. kansasii 235/80 and M. avium
330/88 than INH. The activities of newly prepared com-
pounds against M. kansasii 6509/ 96 (clinical isolate) are
comparable with that of INH. The newly synthesized com-
pounds form the new promising group of antimycobac-
terials with the broad spectrum of activity. It is worth to
note, that the monohalogenated compounds are more
active than the dihalogenated ones. The antimycobacter-
ial activity increases if 1,3-benzoxazine-2,4(3H)diones are
substituted in position 6 with bromine. Bromo deriv-
atives were more active than chloro derivatives. Contrary
to the starting salicylanilides, the antimycobacterial
activity mostly increases when the phenyl ring is substi-
tuted by butyl. (In case of salicylanilides it was propyl).
The most active in the group of halogenated 3-(4-alkyl-
phenyl)-1,3-benzoxazine-2,4(3H)dione was 6-bromo-3-(4-
Melting points were determined on a Kofler block (C. Reichert,
Vienna, Austria) and are uncorrected. The IR spectra were meas-
ured in KBr pellets or in CHCl3 solutions on a Nicolet Impact 400
apparatus (Nicolet, Madison, WI, USA); the wave numbers are
given in cm1. The NMR spectra were recorded on a Varian Mer-
cury–Vx BB 300 spectrometer operating at 300 MHz for 1H and
75 MHz for 13C in d6-DMSO (Varian Inc., Palo Alto, CA, USA). Che-
mical shifts were recorded as d values in ppm and were indir-
ectly referenced to tetramethylsilane via the solvent signal (7.26
for 1H and 77.0 for 13C). The coupling constants J are given in Hz.
Elemental analyses were done on a CHNS-O CE elemental analy-
zer (FISONS EA1110, Milano, Italy). Analyses of the C, H, N, S con-
tent were within l0.4% of the theoretical values. TLC was per-
formed on silica gel plates precoated with a fluorescent indica-
tor, Silufol UV 254 + 366 (Kavalier, Votice, The Czech Republic),
in cyclohexane/acetone 3:1, to check the purity of the products.
General procedure for the preparation 3-(4-
alkylphenyl)-1,3-benzoxazine-2,4(3H)-diones
Methyl-chloroformate (5.2 g, 48 mmol) was added dropwise to a
stirred solution of the corresponding salicylanilide (40 mmol) in
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Arch. Pharm. Chem. Life Sci. 2007, 340, 264–267
Benzoxazines as Antituberculotics
267
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% potassium hydroxide solution, and the solid was filtered
off. The crude products were purified by the crystallization from
EtOH-water (yields 55–65%).
ˇ
ˇ
[4] K. Waisser, R. Dolezal, J. Cizmꢁrik, J. Kaustovꢁ, Folia
Microbiol. 2006, 51, 21–24.
[5] K. Waisser, J. Adamec, R. Doleˇzal, J. Kaustovꢁ, Folia Micro-
biol. 2005, 50, 195–197.
[6] J. Adamec, K. Waisser, J. KuneÐ, J. Kaustovꢁ, Arch. Pharm.
Chem. Life. Sci. 2005, 338, 385–389.
[7] K. Waisser, L. Heinisch, M. ꢃlosꢁrek, J. Janota, Folia Micro-
biol. 2006, 51, 25–26.
Antimycobacterial susceptibility testing
The methods of the experiments are described in our previous
paper [12]. The minimum inhibitory concentrations (MIC) were
determined after incubation at 378C for 14 and 21 days.
[8] J. Matyk, K. Waisser, K. Draˇzkovꢁ, J. KuneÐ, et al., Farmaco
2005, 60, 399–408.
[9] K. Waisser, L. Heinisch, M. ꢃlosꢁrek, J. Janota, Folia Micro-
biol. 2005, 50, 479–481.
ˇ
[10] H.-D. Stachel, Z. Odlerovꢁ, K. Waisser, Collect. Czech. Chem.
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