Synthesis and antifungal activity of 6-arylthio-/6-arylamino-4,7-dioxobenzothiazoles

Article abstract of DOI:10.1016/S0968-0896(03)00390-0

6-Arylthio-/6-arylamino-4,7-dioxobenzothiazoles were synthesized and tested for in vitro antifungal activity against Candida species and Aspergillus niger. 6-Arylamino-4,7-dioxobenzothiazoles 5 and 6 showed, in general, more potent antifungal activity than 6-arylthio-4,7-dioxobenzothiazoles 3 and 4. The 6-arylamino-substituted compounds 5 and 6 exhibited the greatest activity. In contrast, 6-arylthio-, 2-/5-methyl- or 5-methoxy-moieties of compounds 3-4 did not improve their antifungal activity significantly. The results of this study suggest that 6-arylamino-4,7-dioxobenzothiazoles would be potent antifungal agents.

Full text of DOI:10.1016/S0968-0896(03)00390-0

Bioorganic & Medicinal Chemistry 11 (2003) 4003–4008
Synthesis and Antifungal Activity
of 6-Arylthio-/6-Arylamino-4,7-dioxobenzothiazoles
Chung-Kyu Ryu,* Ko Un Choi, Ju-Yeon Shim,
Hea-Jung You, Ik Hwa Choi and Mi Jin Chae
College of Pharmacy, Ewha Womans University, Seodaemun-ku, Seoul 120-750, South Korea
Received 31 March 2003; accepted 4 June 2003
Abstract—6-Arylthio-/6-arylamino-4,7-dioxobenzothiazoles were synthesized and tested for in vitro antifungal activity against
Candida species and Aspergillus niger. 6-Arylamino-4,7-dioxobenzothiazoles 5 and 6 showed, in general, more potent antifungal
activity than 6-arylthio-4,7-dioxobenzothiazoles 3 and 4. The 6-arylamino-substituted compounds 5 and 6 exhibited the greatest
activity. In contrast, 6-arylthio-, 2-/5-methyl- or 5-methoxy-moieties of compounds 3–4 did not improve their antifungal activity
significantly. The results of this study suggest that 6-arylamino-4,7-dioxobenzothiazoles would be potent antifungal agents.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
substituents were designed and synthesized to elucidate
their contribution to the antifungal activity (Schemes 1
and 2).
Heterocyclic quinone compounds represent an important
class of biologically active molecules.1 The quinones
such as 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole
(UHDBT, 1) blockade a mitochondrial electron trans-
port in Saccaromyces cerevisiae.2 The UHDBT (1) has
been reported as inhibitors of mitochondrial cyto-
chrome complex in yeast,3,4 malaria,5 bacteria6 and
mammalians.7 In our previous reports,8 5-arylamino-
4,7-dioxobenzothiazoles 2, which could be analogues of
UHDBT, have demonstrated potent antifungal activity
against pathogenic fungi (Fig. 1). The arylthio/amino-
substituents of quinones improve sometimes the activ-
ities.8,9 On the line of this study, we further extended to
synthesize, 6-arylthio-4,7-dioxobenzothiazoles 3–4 and
6-arylamino-4,7-dioxobenzothiazoles 5–6, which would
be bioisosteres of quinones 2, and evaluated their anti-
fungal activity.
The in vitro antifungal activity of the 4,7-dioxo-
benzothiazoles 3–6 against pathogenic fungi was deter-
mined by the 2-fold broth dilution method. Additional
data for properties and antifungal activity of 4,7-dioxo-
benzothiazoles are provided.
Results and Discussion
Chemistry
A method for the synthesis of 6-arylthio-4,7-dioxo-
benzothiazoles 3a–3e, and 4a–4e (Table 1) is shown in
Scheme 1. Nitration of 2,5-dimethylbenzothiazole (7) by
HNO3/H2SO4 afforded 4-nitro-2,5-dimethylbenzothi-
azole (8) in about 92% yield. 4-Amino-2,5-dimethyl-
benzothiazole (9) was prepared by reduction of the
compound 8 with SnCl2/HCl variation in about 70%
yield. 2,5-Dimethyl-4,7-dioxobenzothiazole (10) was
synthesized by oxidizing the compound 9 with Fremy’s
salt (potassium nitrosodisulfonate) in 59% yield. The
6-arylthio-2,5-dimethyl-4,7-dioxobenzothiazoles 3a–3e
were prepared from the compound 10. The 4,7-dioxo-
benzothiazoles 3a–3e were synthesized by regioselective
nucleophilic substitution of the compound 10 with
appropriate arylthiols.
A variety of quinones with different substituents could
exhibit the activities with different action and sometimes
improve the activities. The presence of thio, amino, halo
and alkyl substituents of quinones improved sometimes
their antifungal activity.8,9 Based on these considera-
tions, the 4,7-dioxobenzothiazoles 3–6 with various
*Corresponding author. Tel.: +82-2-3277-3027; fax: +82-2-3277-
3051; e-mail: ckryu@mm.ewha.ac.kr
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00390-0

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C.-K. Ryu et al. / Bioorg. Med. Chem. 11 (2003) 4003–4008
Table 1. Structures and in vitro antifungal activity for 6-arylthio-4,7-
dioxobenzothiazoles 3 and 4
Compd
R1 R2 R3
MICa (mg/mL)
C. albicans b C. tropicalis C. krusei A. niger
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
10
H
H
H
F
Cl
H
H
H
H
H
H
H
H
H
H
CH3
Cl
F
F
H
50
100
12.5
50
25
>100
>100
>100
>100
>100
>100
12.5
0.8
3.2
0.8
>100
>100
50
25
100
12.5
50
100
100
>100
>100
>100
>100
>100
100
0.8
>100
>100
50
>100
>100
>100
>100
H OCH3
12.5
12.5
25
12.5
12.5
25
H
H
F
CH3
H
H
Ketoconazole
6.3
6.3
12.5
12.5
aThe MIC value was defined as the lowest concentration of the anti-
fungal agent at which there showed optically clear. MIC values were
read after 1 day for Candida species and 2days for A. niger in 37 ꢀC.
The inoculum sizes contained approximately 1ꢁ105 CFU/mL. Culture
media tested were the modified Sabouraud dextrose broth (Difco
Lab.). The final concentration of antifungal agents was between 0.4
and 100 mg/mL.
Figure 1. Antifungal 4,7-dioxobenzothiazole derivatives.
bFungi tested: C. albicans ATCC 10231, C. tropicalis ATCC 28775, C.
krusei ATCC 749 and Aspergillus niger KCTC 1231.
5-Methoxy-2-methyl-4,7-dioxobenzothiazole (11) was
prepared according to previously reported method.8
6-Arylthio-5-methoy-2-methyl-4,7-dioxobenzothiazoles
4a–4e were formed by regioselective nucleophilic sub-
stitution of the compound 11 with the appropriate
arylthiols. Most of these substitutions went as expected
and had overall high yields.
A convenient method for the synthesis of 6-arylamino-
4,7-dioxobenzothiazoles 5a–5l and 6a–6e (Table 2) is
shown in Scheme 2.
6-Arylamino-2,5-dimethyl-4,7-dioxobenzothiazoles 5a–
5l were synthesized by nucleophilic substitutions of the
2,5-dimethyl-4,7-dioxobenzothiazole (10) with the
appropriate arylamines.
Scheme 1. Synthesis of 6-arylthio-4,7-dioxobenzothiazoles 3 and 4.
Reagents and conditions: (a) HNO3/H2SO4/rt; (b) SnCl2/HCl/60 ꢀC;
(c) Fremy’s salt (2equiv) in 0.3 M KH 2PO4/acetone/rt; (d) arylthiol (1
equiv)/EtOH/reflux/4–10 h.
6-Methoxy-4,7-dioxobenzothiazole (12) was prepared
according to previously reported method.5 6-Aryl-
amino-4,7-dioxobenzothiazoles 6a–6e were synthesized
by regioselective nucleophilic substitution of the com-
pound 12 with the appropriate arylamines.
Biological activities
The synthesized 4,7-dioxobenzothiazoles 3–6 were tes-
ted in vitro for their growth inhibitory activity against
pathogenic fungi by the standard method.10 The MIC
(minimum inhibitory concentration) values were deter-
mined by comparison with ketoconazole as a standard
agent. As indicated in Tables 1 and 2, the most active
potential among the 4,7-dioxobenzothiazole series 3–6
was found for 6-arylamino-4,7-dioxobenzothiazoles 5
and 6, which showed generally good activity against all
tested Candida species and A. niger. The 6-arylthio-4,7-
dioxobenzothiazoles 3a–3e showed also antifungal
Scheme 2. Synthesis of 6-arylamino-4,7-dioxobenzothiazoles 5 and 6.
Reagents and conditions: (a) arylamine (1 equiv)/EtOH/reflux/4–6 h.

C.-K. Ryu et al. / Bioorg. Med. Chem. 11 (2003) 4003–4008
4005
Table 2. Structures and in vitro antifungal activity for 6-arylamino-
4,7-dioxobenzothiazoles 5 and 6
In addition, the 4,7-dioxobenzothiazole 10 without a
6-arylamino group exhibited poor antifungal activity.
Thus, 6-arylamino moiety of 4,7-dioxobenzothiazoles 5
and 6 improves the antifungal activity significantly. The
2,5-dimethyl moieties of compounds 5 did not appear to
contribute partially toward biological potency. The
structure–activity relationship may not exist between
properties of substituents (R: F, Cl, Br, ...) for the
6-arylamino moieties of the 4,7-dioxobenzothiazoles
3–6.
Compd
R1 R2 R3
MICa (mg/mL)
C. albicansb C. tropicalis C. krusei A. niger
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
6a
6b
6c
H H OCH3
H H OC2H5
H H CH3
25
12.5
6.3
12.5
6.3
12.5
3.21.6
50
12.5
6.3
50
3.2
3.2
1.6
3.2
1.6
1.6
25
25
6.3
12.5
12.5
25
3.2
3.2
6.3
3.2
12.5
12.5
H H
F H
F H
F
H
F
H H Cl
H Cl Cl
6.3
6.3
>100 >100
>100
1.6
3.2
.5 3.2126.3
3.1
1.6
H H
H H Br
H Br
H H C4H9
I
3.2
Conclusion
H
>100 >100
>100 >100
The 6-arylthio-4,7-dioxobenzothiazoles 3 and 4 were
synthesized by regioselective nucleophilic substitution of
the 2,5-dimethyl-4,7-dioxobenzothiazole (10) and
5-methoxy-2-methyl-4,7-dioxobenzothiazole (11) with
the appropriate arylthiols. In similar manner, the
6-arylamino-4,7-dioxobenzothiazoles 5 and 6 were pre-
pared by the regioselective substitution of compound 10
and 6-methoxy-4,7-dioxobenzothiazole (12) with the
appropriate arylamines. Most of these substitutions
went as expected and had overall high yields.
>100
H H
H F
H H
F
H
H
12.5
12.5
12.5
12.5
12.5
6.3
6.3
3.2
3.2
25
3.2
6.3
12.5
3.2
1.6
3.2
12.5
3.2
12.5
12.5
12.5
12.5
12.5
6.3
6d
6e
H H Cl
H H CF3
Ketoconazole
aThe MIC value was defined as the lowest concentration of the anti-
fungal agent at which there showed optically clear. MIC values were
ꢀ
read after 1 day for Candida species and 2days for A. niger in 37
.
The inoculum sizes contained approximately 1ꢁ105 CFU/mL. Culture
media tested were the modified Sabouraud dextrose broth (Difco
Lab.). The final concentration of antifungal agents was between 0.4
and 100 mg/mL.
6-Arylamino-4,7-dioxobenzothiazoles 5 and 6 showed
generally more potent antifungal activity than 6-arylthio-
4,7-dioxobenzothiazoles 3 and 4. The 6-arylamino-moi-
eties of compounds 5 and 6 improved their antifungal
activity significantly. The results of this study suggest
that 6-arylamino-4,7-dioxobenzothiazoles would be
potent antifungal agents. Moreover, the results should
encourage the synthesis of 4,7-dioxobenzothiazoles
analogues for improving antifungal properties.
bFungi tested: Candida albicans ATCC 10231, C. tropicalis ATCC
28775, C. krusei ATCC 749 and Aspergillus niger KCTC 1231.
activity. In contrast, compounds 4a–4e did not show
significant antifungal activity, although many com-
pounds of them exhibited good activity against Can-
dida tropicalis. Most of the 6-arylamino-4,7-
dioxobenzothiazoles 5a–5l and 6a–6e showed potent
antifungal activity against all tested fungal species,
and the activity against C. tropicalis was prominent.
Most of compounds 5 and 6 had more potent anti-
fungal activity against C. tropicalis than ketocona-
zole. Actually, the activities of compounds 5c, 5e, 5g and
5j were superior or comparable to those of ketoconazole
against all tested fungi. The 4,7-dioxobenzothiazoles 5c
and 5g completely inhibited the growth of all fungal spe-
cies tested at the MIC level of 6.3 mg/mL.
Experimental
All melting points were measured in open capillary
tubes with Buchi melting point B-545 and were uncor-
rected. The TLC was performed on precoated silica gel
(60G 254, Merck) using CHCl3 for solvent. The com-
pounds were detected under UV light (254 nm) or by
heating at 110 ꢀC after spraying 30% H2SO4–vanillin
solution. Column chromatography was performed on
silica gel G60 (70–230 mesh, ASTM, Merck). The purity
of 4,7-dioxobenzothiazoles 3–6 was also verified by GC
(Hewlett Packard 5890A, HP-5 capillary column at
260 ꢀC, N2 gas, 17 mL/min as carrier gas, FID). The IR
spectra were taken from Perkin Elmer 1420r IR spec-
trometer with KBr pellets. 1H NMR spectra were
recorded on Brucker DPX 250 MHz spectrometer using
CDCl3 or DMSO-d6 as solvent, and chemical shifts are
given in ppm with TMS as a standard. High-resolution
mass spectra (HRMS EI) were taken from Jeol JMS
AX505 WA. 2,5-Dimethylbenzothiazole (7) was
obtained from TCI Co. CDCl3, DMSO-d6 and other
reagents were purchased from Aldrich Chemical Co.
The cytotoxic potential of compounds 3–6 was also
determined in human cancer cells according to the NCI
protocols.11 All of the tested compounds did not show
significant cytotoxic activity and showed the selectivity,
in that they possess potent antifungal activities without
the cytotoxicity in mammalian cells.12
In terms of structure–activity relationship, the 6-aryl-
amino-4,7-dioxobenzothiazole series 5a–5l and 6a–6e
showed, in general, more potent antifungal activity than
6-arylthio-4,7-dioxobenzothiazole series 3a–3e and
4a–4e. The 6-arylamino-substituted compounds 5 and 6
exhibited the greatest activity, indicating a correlation
that may offer insight into the mode of action of these
compounds. In contrast, 6-arylthio-, 2-/5-methyl- or
5-methoxy-moieties of compounds 3–4 did not improve
their antifungal activity in comparison to 6-arylamino-
4,7-dioxobenzothiazoles 5 significantly.
2,5 - Dimethyl - 4 - nitrobenzothiazole (8). 2,5 - Dimethyl -
benzothiazole (7, 5 g, 30.7 mmol) in 18 mL of C–H2SO4
and 18 mL of C–HNO3 was stirred at rt for 2h. The
precipitate was filtered and crystallized from CHCl3.

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C.-K. Ryu et al. / Bioorg. Med. Chem. 11 (2003) 4003–4008
2,5-Dimethyl-4-nitrobenzothiazole (8) was obtained (5.9
g, 92%): pale yellow powder; mp: 111–112 ꢀC; IR
6-[S-(4-Florophenyl)thio]-2,5-dimethyl-4,7-dioxobenzo-
thiazole (3c). Dark pink powder (66%); mp:
101–104 ꢀC; IR (KBr): n 3061 (w), 1659 (s, C¼O),
1
(KBr): n 3094 (s), 2990, 1550 (s, NO2), 1310 cmꢂ1; H
1
NMR (DMSO-d6): d 8.4 (s, 1H), 7.6 (s, 1H), 2.8 (s, 3H,
CH3), 2.4 (s, 3H, CH3); HRMS Anal. calcd for
C9H8N2O2S 208.0306, Found: 208.0306.
1428–1516, 1367 cmꢂ1; H NMR (DMSO-d6): d 7.4 (d,
2H), 7.2 (d, 2H), 2.8 (s, 3H, CH3), 2.3 (s, 3H, CH3);
HRMS Anal. calcd for C15H10FNO2S2 319.0137,
Found: 319.0138.
4-Amino-2,5-dimethylbenzothiazole (9). To 12g (64
mmol) of SnCl2 in 25 mL of C–HCl was added 2 g (9.6
mmol) of compound 8. The mixture was stirred at 60 ꢀC
for 2h and was extracted twice with CH 2Cl2. The
extract was evaporated and crystallized from CH2Cl2.
4-Amino-2,5-dimethylbenzothiazole (9) was obtained
6-[S-(2,4-Difluorophenyl)thio]-2,5-dimethyl-4,7-dioxoben-
zothiazole (3d). Red powder (45%); mp: 122–126 ꢀC; IR
(KBr): n 3081 (m), 1658 (s, C¼O), 1417-1516, 1365 cmꢂ1
;
1H NMR (DMSO-d6): d 7.6 (m, 1H), 7.4 (td, 1H), 7.1 (td,
1H), 2.8 (s, 3H, CH3), 2.3 (s, 3H, CH3); HRMS Anal.
calcd for C15H9F2NO2S2 337.0043, Found: 337.0043.
ꢀ
(1.2g, 70%): pale yellow plate; mp: 91–92 C; IR (KBr):
n 3451, 3329 (s, NH2), 3100 (s), 2915, 1473, 1326
1
cmꢂ1; H NMR (DMSO-d6): d 7.4 (s, 1H), 7.2(s, 1H),
6-[S-(2-Chlorophenyl)thio]-2,5-dimethyl-4,7-dioxobenzo-
thiazole (3e). Blight pink powder (61%); mp:
196–199 ꢀC; IR (KBr): n 3061 (m), 1663 (s, C¼O),
5.2(s, 2H, NH 2), 2.8 (s, 3H, CH3), 2.4 (s, 3H, CH3);
HRMS Anal. calcd for C9H10N2S 178.0565, Found:
178.0565.
1
1451–1516, 1252 cmꢂ1; H NMR (DMSO-d6): d 7.5 (m,
1H), 7.1 (t, 2H), 6.4 (m, 1H), 2.8 (s, 3H, CH3), 2.2 (s,
3H, CH3); HRMS Anal. calcd for C15H10ClNO2S
303.0121, Found: 303.0122.
2,5-Dimethyl-4,7-dioxobenzothiazole (10). To a solution
of compound 9 (0.5 g, 2.8 mmol) in 100 mL of
KH2PO4 buffer (0.3 M, 200 mL) was added a solution
of potassium nitrosodisulfonate (1.5 g, 5.60 mmol) in
the KH2PO4 buffer. The mixture was stirred at rt for
5 h and was extracted twice with CHCl3. The extract
was evaporated and purified by column chromato-
graphy with CHCl3. 2,5-Dimethyl-4,7-dioxobenzothi-
azole (10) was obtained (0.32g, 59%): yellow powder;
mp: 138–139 ꢀC. IR (KBr): n 3045 (w), 2922, 1670 (s,
General procedure for synthesis of 6-arylthio-5-methoxy-
2-methyl-4,7-dioxobenzothiazoles 4a–4e
A solution of compound 118 (0.209 g, 0.1 mmol) in 20
mL of 95% EtOH was added to a solution of the aryl-
thiol (1.1 mmol) in 10 mL of 95% EtOH and then
refluxed for 4–10 h. After the reaction mixture was kept
overnight, the precipitate was collected by the filtration.
The crude product was purified through silica gel col-
umn chromatography with CHCl3 or crystallized from
95% EtOH. Crystallization from aq EtOH afforded 6-
arylthio-5-methoxy-2-methyl-4,7-dioxobenzothiazoles
4a–4e (Table 1).
1
C¼O), 1475–1503 cmꢂ1; H NMR (CDCl3): d 6.8 (s,
1H, H6), 2.8 (s, 3H, CH3), 1.5 (s, 3H, CH3); HRMS
Anal. calcd for C9H7O2NS 193.0198, Found:
193.0196.
General procedure for synthesis of 6-arylthio-2,5-di-
methyl-4,7-dioxobenzothiazoles 3a–3e
6-[S-(4-Methoxyphenyl)thio]-5-methoxy-2-methyl-4,7-di-
oxobenzothiazole (4a). Dark purple powder (36%); mp:
94–96 ꢀC; IR (KBr): n 3231 (s), 1680 (s, C¼O), 1491–1590,
A solution of compound 10 (0.193 g, 1 mmol) in 20 mL
of 95% EtOH was added to a solution of the arylthiol
(1.1 mmol) in 10 mL of 95% EtOH and then refluxed for
4ꢃ10 h. After the reaction mixture was kept overnight,
the precipitate was collected by the filtration. The crude
product was purified by silica gel column chromato-
graphy with CHCl3 or crystallized from 95% EtOH.
Crystallization from aq EtOH afforded 5-arylthio-2,5-
dimethyl-4,7-dioxobenzothiazoles 3a–3e (Table 1).
1
1330 cmꢂ1; H NMR (DMSO-d6): d 7.2(d, J=8.4 Hz,
2H), 6.8 (d, J=8.4 Hz, 2H), 3.9 (s, 3H, OCH3), 3.7 (s, 3H,
OCH3), 2.8 (s, 3H, OCH3); HRMS Anal. calcd for
C16H13NO4S2 347.0286, Found: 347.0286.
6-[S-(4-Methylphenyl)thio]-5-methoxy-2-methyl-4,7-diox-
obenzothiazole (4b). Dark purple powder (50%); mp:
102–103 ꢀC; IR (KBr): n 3014 (s), 1685 (s, C¼O),
1
6-[S-(4-Methylphenyl)thio]-2,5-dimethyl-4,7-dioxobenzo-
thiazole (3a). Dark orange powder (92%); mp:
88–90 ꢀC; IR (KBr): n 3088 (m), 1667 (s, C¼O),
1475–1555, 1314 cmꢂ1; H NMR (DMSO-d6): d 7.4 (d,
J=8 Hz, 2H), 7.2 (d, J=8 Hz, 2H), 4.0 (s, 3H, OCH3),
2.8 (s, 3H, CH3), 2.3 (s, 3H, CH3); HRMS Anal. calcd
for C16H13NO3S2 331.0337, Found: 331.0336.
1
1438-1514, 1364 cmꢂ1; H NMR (DMSO-d6): d 7.3 (d,
J=8 Hz, 2H), 7.1 (d, J=8 Hz, 2H), 2.8 (s, 3H, CH3),
2.2 (s, 3H, CH3); HRMS Anal. calcd for C16H13NO2S2
315.0388, Found: 315.0381.
6-[S-(Phenyl)thio]-5-methoxy-2-methyl-4,7-dioxobenzo-
thiazole (4c). Violet powder (41%); mp: 158–159 ꢀC; IR
(KBr): n 3233 (s), 1663 (s, C¼O), 1459-1540, 1308 cmꢂ1
;
6-[S-(4-Chlorophenyl)thio]-2,5-dimethyl-4,7-dioxobenzo-
thiazole (3b). Shining dark red powder (80%); mp:
153–154 ꢀC; IR (KBr): n 3083 (m), 1658 (s, C¼O),
1H NMR (DMSO-d6): d 7.0 (m, 1H), 7.3 (m, 3H), 4.0 (s,
3H, OCH3), 2.8 (s, 3H, CH3); HRMS Anal. calcd for
C15H11NO3S2 317.0181, Found: 317.0182.
1
1427–1516, 1364 cmꢂ1; H NMR (DMSO-d6): d 7.4 (d,
J=8.8 Hz, 2H), 7.3 (d, J=8.8 Hz, 2H), 2.8 (s, 3H,
CH3), 2.3 (s, 3H, CH3); HRMS Anal. calcd for
C15H10O2N2ClS2 334.9841, Found: 334.9844.
6-[S-(3-Fluorophenyl)thio]-5-methoxy-2-methyl-4,7-diox-
obenzothiazole (4d). Brown powder (40%); mp:
163–164 ꢀC; IR (KBr): n 3249 (s), 1674 (s, C¼O),

C.-K. Ryu et al. / Bioorg. Med. Chem. 11 (2003) 4003–4008
4007
1
1
1495–1545, 1303 cmꢂ1; H NMR (DMSO-d6): d 7.2(t,
1459–1515 cmꢂ1; H NMR (DMSO-d6): d 9.1 (s, 1H,
NH), 7.1–7.2(m, 4H), .28 (s, 3H, CH 3), 1.6 (s, 3H,
CH3); HRMS Anal. calcd for C15H11FN2O2S 302.0525,
Found: 302.0526.
1H), 7.0 (d, 1H), 6.9 (s, 1H), 6.8 (d, 1H), 3.8 (s, 3H,
OCH3), 2.8 (s, 3H, CH3); HRMS Anal. calcd for
C15H10FNO3S2 335.0086, Found: 335.0086.
6-[S-(2-Naphthyl)thio]-5-methoxy-2-methyl-4,7-dioxoben-
zothiazole (4e). Dark red needle (47%); mp: 114–116 ꢀC;
IR (KBr): n 3056 (s), 1692(s, C ¼O), 1491–1567, 1301
6-[N-(2,4-Difluorophenyl)amino]-2,5-dimethyl-4,7-dioxo-
benzothiazole (5f). Dark violet powder (72%); mp:
161–162 ꢀC; IR (KBr): n 3329 (s, NH), 3063 (w), 1670 (s,
C¼O), 1454–1515 cmꢂ1; 1H NMR (DMSO-d6): d 9.0 (s,
1H, NH), 7.0–7.2 (m, 2H), 6.9 (d, 1H), 2.8 (s, 3H, CH3),
1.6 (s, 3H, CH3); HRMS Anal. calcd for
C15H10F2N2O2S 320.0431, Found: 320.0430.
1
cmꢂ1; H NMR (DMSO-d6): d 7.8 (m, 4H, naphtha-
lene), 7.5 (m, 3H, naphthalene), 4.0 (s, 3H, OCH3), 2.8
(s, 3H, CH3); HRMS Anal. calcd for C19H13NO3S2
367.0337, Found: 367.0337.
6-[N-(4-Chlorophenyl)amino]-2,5-dimethyl-4,7-dioxoben-
zothiazole (5g). Black powder (86%); mp: 207–208 ꢀC;
IR (KBr): n 3348 (s, NH), 3056 (w), 1658 (s, C¼O),
General procedure for synthesis of 5-arylamino-2-
methyl-4,7-dioxobenzothiazoles 5a–5l
1
A solution of compound 10 (0.193 g, 1 mmol) in 20 mL
of 95% EtOH was added to a solution of the arylamine
(1.1 mmol) in 10 mL of 95% EtOH and then refluxed
for 4ꢃ6 h. After the reaction mixture was kept over-
night, the precipitate was collected by the filtration. The
crude product was purified by silica gel column chroma-
tography with CHCl3 or crystallized from 95% EtOH.
Crystallization from aq EtOH afforded 5-arylamino-2-
methyl-4,7-dioxobenzothiazoles 5a–5l (Table 2).
1491–1599, 1369 cmꢂ1; H NMR (DMSO-d6): d 9.1 (s,
1H, NH), 7.0 (m, J=8.8 Hz, 2H), 6.9 (d, J=8.8 Hz,
2H), 2.8 (s, 3H, CH3), 1.6 (s, 3H, CH3); HRMS Anal.
calcd for C15H11ClN2O2S 318.0230, Found: 318.0227.
6-[N-(3,4-Dichlorophenyl)amino]-2,5-dimethyl-4,7-dioxo-
benzothiazole (5h). Dark violet powder (90%); mp:
210–213 ꢀC; IR (KBr): n 3331 (s, NH), 3066 (w), 1660 (s,
1
C¼O), 1469–1568, 1384 cmꢂ1; H NMR (DMSO-d6): d
9.1 (s, 1H, NH), 7.5 (m, J=8.8 Hz, 2H), 7.2 (s, 1H), 6.9
(dd, J=8.8 Hz, 1H), 2.8 (s, 3H, CH3), 1.6 (s, 3H, CH3);
HRMS Anal. calcd for C15H10Cl2N2O2S 351.9840,
Found: 351.9841.
6-[N-(4-Methoxyphenyl)amino]-2,5-dimethyl-4,7-dioxo-
benzothiazole (5a). Dark violet powder (53%); mp:
195–196 ꢀC; IR (KBr): n 3243 (w, NH), 3016 (w), 1694
(s, C¼O), 1486–1554 cmꢂ1; 1H NMR (DMSO-d6): d 9.1
(s, 1H, NH), 7.0 (d, J=8.8 Hz, 2H), 6.9 (d, J=8.8 Hz,
2H), 3.7 (s, 3H, OCH3), 2.8 (s, 3H, CH3), 1.5 (s, 3H,
CH3); HRMS Anal. calcd for C16H14N2O3S 314.0725,
Found: 314.0725.
6-[N-(4-Iodophenyl)amino]-2,5-dimethyl-4,7-dioxobenzo-
thiazole (5i). Black powder (76%); mp: 202–204 ꢀC; IR
(KBr): n 3354 (s, NH), 3056 (w), 1658 (s, C¼O),
1
1497–1588, 1271 cmꢂ1; H NMR (DMSO-d6): d 9.1 (s,
1H, NH), 7.6 (d, J=8.4 Hz, 2H), 6.8 (d, 2H), 2.8 (s, 3H,
CH3), 1.6 (s, 3H, CH3); HRMS Anal. calcd for
C15H11IN2O2S 409.9586, Found: 409.9587.
6-[N-(4-Ethoxyphenyl)amino]-2,5-dimethyl-4,7-dioxoben-
zothiazole (5b). Black powder (52%); mp: 202–203 ꢀC;
IR (KBr): n 3253 (s, NH), 3073 (w), 1667 (s, C¼O),
1
1470–1554, 1324 cmꢂ1; H NMR (DMSO-d6): d 9.1 (s,
6-[N-(4-Bromophenyl)amino]-2,5-dimethyl-4,7-dioxoben-
zothiazole (5j). Shining dark black powder (80%); mp:
215–217 ꢀC; IR (KBr): n 3352(s, NH), 3088 (w), 1647 (s,
C¼O), 1493–1599, 1369 cmꢂ1; 1H NMR (DMSO-d6): d
9.1 (s, 1H, NH), 7.4 (d, J=8.4 Hz, 2H), 6.9 (d, J=8.4
Hz, 2H), 2.8 (s, 3H, CH3), 1.6 (s, 3H, CH3); HRMS
Anal. calcd for C15H11BrN2O2S 363.9705, Found:
363.9730.
1H, NH), 7.0 (d, J=8.8 Hz, 2H), 6.9 (d, J=8.8 Hz, 2H),
4.0 (q, J=7.8 Hz, 2H, CH2), 2.8 (s, 3H, CH3), 1.5 (s,
3H, CH3), 1.4 (t, J=7.8 Hz, 3H, CH3); HRMS Anal.
calcd for C17H16N2O3S 328.0882, Found: 328.0881.
6-[N-(4-Methylphenyl)amino]-2,5-dimethyl-4,7-dioxoben-
zothiazole (5c). Dark violet flake (56%); mp: 190–192 ꢀC;
IR (KBr): n 3314 (s, NH), 3029 (w), 1660 (s, C¼O),
1
1454–1554 cmꢂ1; H NMR (DMSO-d6): d 9.1 (s, 1H,
6-[N-(3-Bromophenyl)amino]-2,5-dimethyl-4,7-dioxoben-
zothiazole (5k). Shining black powder (88%); mp:
194–198 ꢀC; IR (KBr): n 3313 (s, NH), 3077 (w), 1661 (s,
NH), 7.1 (d, J=8.4 Hz, 2H), 6.9 (d, J=8.4 Hz, 2H), 2.8 (s,
3H, CH3), 2.3 (s, 3H, CH3), 1.6 (s, 3H, CH3); HRMS
Anal. calcd for C16H14N2O2S 298.0776, Found: 298.0776.
1
C¼O), 1454–1567, 1310 cmꢂ1; H NMR (DMSO-d6): d
9.1 (s, 1H, NH), 7.2(m, 2H), 7.12(d, 1H), 7.0 (d, 1H),
2.8 (s, 3H, CH3), 1.6 (s, 3H, CH3); HRMS Anal. calcd
for C15H11BrN2O2S 363.9705, Found: 363.9730.
6-[N-(4-Fluorophenyl)amino]-2,5-dimethyl-4,7-dioxoben-
zothiazole (5d). Black powder (76%); mp: 171–172 ꢀC;
IR (KBr): n 3320 (s, NH), 3061 (w), 1653 (s, C¼O),
1
1455-1591 cmꢂ1; H NMR (DMSO-d6): d 9.1 (s, 1H,
6-[N-(4-Butylphenyl)amino]-2,5-dimethyl-4,7-dioxobenzo-
thiazole (5l). Black plate (51%); mp: 118–120 ꢀC; IR
(KBr): n 3323 (s, NH), 2927 (w), 1658 (s, C¼O),
NH), 7.1 (d, 2H), 6.9 (d, 2H), 2.8 (s, 3H, CH3), 1.5 (s,
3H, CH3); HRMS Anal. calcd for C15H11FN2O2S
302.0525, Found: 302.0526.
1
1454–1554, 1308 cmꢂ1; H NMR (DMSO-d6): d 9.1 (s,
1H, NH), 7.1 (d, 2H), 6.9 (d, 2H), 2.8 (s, 3H, CH3), 2.7
(t, 2H, CH2), 2.6 (s, 3H, CH3), 2.5 (quintet, 3H, CH3),
2.3 (sextet, 2H, CH2), 0.9 (t, 3H, CH3); HRMS Anal.
calcd for C19H20N2O2S 340.1246, Found: 340.1246.
6-[N-(2-Fluorophenyl)amino]-2,5-dimethyl-4,7-dioxoben-
zothiazole (5e). Black powder (54%); mp: 142–143 ꢀC;
IR (KBr): n 3357 (s, NH), 3072(w), 1660 (s, C ¼O),

4008
C.-K. Ryu et al. / Bioorg. Med. Chem. 11 (2003) 4003–4008
General procedure for synthesis of 6-arylamino-4,7-diox-
obenzothiazoles 6a–6e
activities were tested in modified Sabouraud dextrose
broth against the following fungal strains: C. albicans
ATCC 10231, C. krusei ATCC 749, C tropicalis ATCC
28775 and A. niger KCTC 1231. Ketoconazole as an
antifungal standard agent was used. The compounds
were tested in the 0.1–100 mg/mL range. That was added
to the modified Sabouraud dextrose broth (Difco Lab.)
for fungi over a final concentration range of 0.1–100 mg/
mL. The inoculum sizes contained approximately 1ꢁ105
CFU/mL. They were incubated at 37 ꢀC for appropriate
periods of time that sufficed to show clearly visible
growth on drug-free control broths. The MIC value was
defined, as the lowest concentration of the antifungal
agent at which there showed optically clear. MIC values
were read after 1 day for Candida species and 2days for
A. niger in 37 ꢀC.
A solution of compound 125 (0.195 g, 1 mmol) in 20 mL
of 95% EtOH was added to a solution of the arylamine
(1.1 mmol) in 10 mL of 95% EtOH and then refluxed
for 4–6 h. After the reaction mixture was kept over-
night, the precipitate was collected by the filtration. The
crude product was purified by silica gel column chro-
matography with CHCl3 or crystallized from 95%
EtOH. Crystallization from aq EtOH afforded 6-aryl-
amino-4,7-dioxobenzothiazoles 6a–6e (Table 2).
6 - [N - (4 - Fluorophenyl)amino] - 4,7 - dioxobenzothiazole
(6a). Black powder (80%); mp: 242–243 ꢀC; IR (KBr):
3270 (NH), 3050 (w), 1694 (s, C¼O), 1590–1470, 1230
1
cmꢂ1; H NMR (DMSO-d6): d 9.5 (s, 1H, H2), 9.3 (d,
1H, NH), 7.5–7.3 (m, 4H), 5.8 (s, 1H, H5); HRMS
Anal. calcd for C13H7FN2O2S 274.0212, Found:
274.0211.
Acknowledgements
This work was supported by Korea Research Founda-
tion Grant (KRF-2002-041- E00292).
6 - [N - (3- Fluorophenyl)amino] - 4,7 - dioxobenzothiazole
(6b). Violet powder (84%); mp: 236–238 ꢀC; IR (KBr):
3270 (NH), 3050 (w), 1694 (s, C¼O), 1590–1470, 1230
1
cmꢂ1; H NMR (DMSO-d6): d 9.6 (s, 1H, H2), 9.4 (s,
References and Notes
1H, NH), 7.5–7.3 (m, 4H), 6.0 (s, 1H, H5); HRMS
Anal. calcd for C13H7FN2O2S 274.0212, Found:
274.0212.
1. Middleton, R. W.; Parrick, J. In The Chemistry of The
Quinonoid Compounds; Patai, S., Rappoport, Z., Eds.; John
Wiley & Sons: London, 1988; Part 2, p 1019.
2. Roberts, H.; Choo, W. M.; Smith, S. C.; Mrzuki, S.; Lin-
nane, A. W.; Porter, T. H.; Folkers, K. Arch. Biochem. Bio-
phys. 1978, 191, 306.
3. Di Rigo, J.-P.; Bruel, C.; Graham, L. A.; Slonimski, P.;
Trumpower, B. L. J. Biol. Chem. 1996, 271, 15341.
4. Tsai, A. L.; Kauten, R.; Palmer, G. Biochim. Biophys. Acta
1985, 806, 418.
6-[N-(Phenyl)amino]-4,7-dioxobenzothiazole (6c). Black
powder (72%); mp: 219–221 ꢀC; IR (KBr): 3270 (NH),
1
3050 (w), 1693 (s, C¼O), 1553–1470 cmꢂ1; H NMR
(DMSO-d6): d 9.5 (s, 1H, H2), 9.4 (s, 1H, NH), 7.4–7.5
(m, 5H), 5.9 (s, 1H, H5); HRMS Anal. calcd for
C13H8N2O2S 256.0307, Found: 256.0308.
5. Friedman, M. D.; Stotter, P. L.; Porter, T. H.; Folkers, K.
J. Med. Chem. 1973, 16, 1314.
6. Musser, S. M.; Stowell, M. H. B.; Lee, H. K.; Rumbley,
J. N.; Chan, S. I. Biochemistry 1997, 36, 894.
7. Kim, H.; Xia, D.; Yu, C.-A.; Xia, J.-X.; Kachurin, A. M.;
Zhang, L.; Yu, L.; Deisenhofer, H. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 8026. Yang, F. D.; Yu, L.; Yu, C.-A. J. Biol.
Chem. 1989, 264, 891.
8. Ryu, C.-K.; Kang, H.-Y.; Yi, Y.-J.; Shin, K.-H.; Lee, B.-H.
Bioorg. Med. Chem. Lett. 2000, 10, 1589.
6 - [N - (4 - Chlorophenyl)amino] - 4,7 - dioxobenzothiazole
(6d). Dark violet powder (85%); mp: 216–217 ꢀC; IR
(KBr): 3277 (NH), 3000 (w), 1694 (s, C¼O), 1590–1470
1
cmꢂ1; H NMR (DMSO-d6): d 9.5 (d, 1H, H2), 9.4 (s,
1H, NH), 7.5–7.4 (m, 4H), 6.2(s, 1H, H5); HRMS
Anal. calcd for C13H7ClN2O2S 289.9917, Found:
289.9917.
6-[N-(4-Trifluoromethylphenyl)amino]-4,7-dioxobenzo-
thiazole (6e). Purple powder (87%); mp: 227–228 ꢀC;
IR (KBr): 3270 (NH), 3000 (w), 1695 (s, C¼O), 1590–
9. Ryu, C.-K.; Kim, H. J. Arch Pharm. Res. 1994, 17, 139.
Ryu, C.-K.; Sun, Y.-J.; Shim, J.-Y.; You, H.-J.; Choi, K. U.;
Lee, H. Arch. Pharm. Res. 2002, 25, 784.
10. Mcginnis, M. R.; Rindali, M. G. In: Antibiotics in
Laboratory Medicine, 4th ed.; Lorian, V., Ed.; Williams and
Wilkins: Baltimore, 1996; p 176.
11. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.;
McMahon, J.; Vistica, D.; Warren, T. W.; Bokesch, H.; Ken-
ney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107.
12. Unpublished data; we also tested cytotoxic activity of
compounds 3–6 against human tumor cell lines such as A 549,
HL-60 and HepG2according to the NCI protocols. 11 All of the
tested compounds did not show significant cytotoxic activity.
1
1470, 1270, 1230 cmꢂ1; H NMR (DMSO-d6): d 9.6 (s,
1H, H2), 9.4 (s, 1H, NH), 7.5–7.4 (m, 4H), 6.0 (s, 1H,
H5); HRMS Anal. calcd for C14H7F3N2O2S 324.0180,
Found: 324.0181.
Antifungal in vitro susceptibility testing
The MIC values of compounds 3–6 were determined by
the standard broth dilution method.10 The antifungal