Synthesis of benzofuro[6,7-d]thiazoles, benzofuro[7,6-d]thiazoles and 6-arylaminobenzo[d]thiazole-4,7-diones as antifungal agent

Article abstract of DOI:10.1248/cpb.c14-00146

Benzofuro[6,7-d]thiazoles, benzofuro[7,6-d]thiazoles and 6-arylaminobenzo[d]thiazole-4,7-diones were synthesized and tested for in vitro antifungal activity against Candida, Aspergillus species and Cryptococcus neoformans. Among them tested, many of synth

Full text of DOI:10.1248/cpb.c14-00146

668
Regular Article
Chem. Pharm. Bull. 62(7) 668–674 (2014)
Vol. 62, No. 7
Synthesis of Benzofuro[6,7-d]thiazoles, Benzofuro[7,6-d]thiazoles and
6-Arylaminobenzo[d]thiazole-4,7-diones as Antifungal Agent
Chung-Kyu Ryu,* Ji-Hee Nho, Guohua Jin, Sun Young Oh, and Soo Jung Choi
College of Pharmacy & Graduate School of Pharmaceutical Sciences, Ewha Womans University; 52 Ewhayeodae-gil,
Seodaemun-ku, Seoul 120–750, Republic of Korea.
Received February 12, 2014; accepted April 25, 2014; advance publication released online May 1, 2014
Benzofuro[6,7-d]thiazoles, benzofuro[7,6-d]thiazoles and 6-arylaminobenzo[d]thiazole-4,7-diones were
synthesized and tested for in vitro antifungal activity against Candida, Aspergillus species and Cryptococ-
cus neoformans. Among them tested, many of synthesized compounds showed potent antifungal activity.
The compounds 4d, 6e and 6h completely inhibited the growth of all Candida and Aspergillus species tested
at the MIC level of 6.3µg/mL. The results suggest that benzofuro[6,7-d]thiazoles and 6-arylaminobenzo[d]-
thiazole-4,7-diones would be promising antifungal agents.
Key words benzofuro[6,7-d]thiazole; benzofuro[7,6-d]thiazole; benzo[d]thiazole-4,7-dione; antifungal; anti-
microbial; fungi
The recent increase of fungal infections, especially among
Structure–activity relationship studies from heterocyclic
patients undergoing anticancer chemotherapy and AIDS pa- quinonoid compounds indicated that the number and position
tients, has generated a renewed interest in antifungal drugs, of nitrogen (N) or sulfur (S) atoms substituted in the hetero-
including development of new antifungal agents in the de- cyclic ring were considerably important factors to affect the
velopment of resistance to drugs.1,2) In a program aimed at biological activities.10,11) Generally, increasing the number of
identifying novel antifungal agents, we focused on develop- substituent nitrogen or sulfur atoms in the ring enhances the
ing benzofuro[6,7-d]thiazoles, benzofuro[7,6-d]thiazoles and activities. We speculated that incorporation of thiazole into
6-arylaminobenzo[d]thiazole-4,7-diones with new mode of the skeleton in compounds 2 or 3 would change the physico-
antifungal action.
N-Myristoyltransferase has been showed to be essential for 7-amino-4-hydroxybenzofuro[6,7-d]thiazole-6-carboxylates
the viability of fungi, including medically important patho- and alkyl 7-amino-4-hydroxy-2-methylbenzofuro[7,6-d]-
chemical properties, and lead to a new pharmacophore alkyl
4
genic fungi, Candida albicans3) and Cryptococcus neofor- thiazole-6-carboxylates 5 with a different biological profile
mans.4,5) It could be a target for the development of antifungal from scaffolds 2 or 3. The compounds 4 and 5 which would
agents with a novel mode of action. A benzofuran derivative be bioisosteres of compound 3 could metabolize to benzo[d]-
1, a novel myristoyltransferase inhibitor, has been reported thiazole-4,7-dione derivatives with a quinonoid structure in
as antifungal agent6,7) as well as antibacterial agent8,9) (Fig. fungi. Quinonoid benzo[d]thiazole-4,7-diones display potent
1). Benzofuran-5-ol
3 scaffolds10) have demonstrated potent antifungal activity antibacterial activity.10) The thiazole moiety of quinonoid
against pathogenic fungi. compounds could improve upon the biological activities. We
2 and 5-hydroxyfuro[2,3-f]quinoline biological properties including antifungal, antimalarial and
Fig. 1. Benzofuran, 4-Hydroxybenzofuro[6,7-d]thiazoles, 4-Hydroxybenzofuro[7,6-d]thiazoles and 6-Arylaminobenzo[d]thiazole-4,7-diones
The authors declare no conflict of interest.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: ckryu@ewha.ac.kr

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Reagents and conditions: (a) c-HCl/NaClO4/rt/0.5h, (b) alkyl cyanoacetate/NH4OH/CeCl3/EtOH/rt/30min, (c) arylthiol (1eq)/EtOH/hydrazine/reflux/5h, (d) arylamine
(1 eq)/CeCl3/EtOH/rt/20min, (e) hydrazine hydrate/EtOH/rt/2h.
Chart 1. Synthesis of Alkyl 7-Amino-4-hydroxybenzofuro[6,7-d]thiazoles, 7-Amino-4-hydroxybenzofuro[7,6-d]thiazoles and 5-Chloro-6-arylamino-
benzo[d]thiazole-4,7-diones
assumed that alkyl 4-hydroxybenzofuro[6,7-d]thiazoles 4 and through different actions and sometimes improve upon the
4-hydroxy-2-methylbenzofuro[7,6-d]thiazole
5 could have activities (Chart 1, Table 1).
similar biological activities with those of benzo[d]thiazole-4,7-
Based on this speculation, compounds 4a–e, 5a,b and 6a–j
diones.
with various substituents were designed and synthesized to
There have not been any reports on alkyl 7-amino-4- elucidate their contribution to the antifungal activity.
hydroxybenzofuro[6,7-d]thiazole 4 and 7-amino-4-hydroxy-
The in vitro antifungal activity of new compounds 4a–e,
benzofuro[7,6-d]thiazole scaffolds 5 to the best of our 5a,b and 6a–j against pathogenic fungi was determined by
knowledge. The presence of thio, alkyl, amino group or the twofold broth dilution method. Additional data for antifun-
halogen atoms on quinonoid compounds such as benzo[d]- gal activity are provided.
thiazole-4,7-diones,12) 1,4-naphthoquinones13) and 5-hydroxy-
furo[2,3-f]quinoline 3 significantly affects their antifungal
activity. Thus, variety of alkyl 7-amino-4-hydroxy-
Results and Discussion
Chemistry Methods for the synthesis of compounds 4a–e,
a
benzofuro[6,7-d]thiazoles 4a–e, alkyl 7-amino-4-hydroxy-2- 5a,b and 6a–j are shown in Chart 1 and Table 1. 6-Methoxy-
methylbenzofuro[7,6-d]thiazole-6-carboxylates 5a–b and 7-aminobenzothiazole (7) was prepared from commer-
5-chloro-6-arylaminobenzo[d]thiazole-4,7-diones 6a–j with cially available 6-methoxyobenzothiazole.14) 5,6-Dichloro-2-
different substituents could exhibit the biological activities methylbenzo[d]thiazole-4,7-dione (8) was synthesized by

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Vol. 62, No. 7
Table 1. Structures and Antifungal Activity for 7-Amino-4-hydroxybenzofuro[6,7-d]thiazoles, 7-Amino-4-hydroxybenzofuro[7,6-d]thiazoles and 5-Chlo-
ro-6-arylaminobenzo[d]thiazole-4,7-diones
MICa) (µg/mL)
Compound
R1
R2
b)
C. albicans
C. tropicalis
C. krusei
C. neoformans
A. niger
A. flavus
4a
CH3CH2
CH3
F
12.5
12.5
6.3
12.5
6.3
25.0
50.0
1.6
12.5
12.5
12.5
3.2
12.5
12.5
6.3
50.0
12.5
25.0
3.2
4b
CH3CH2
4c
CH3CH2
CH3O
CH3
CH3
H
12.5
6.3
4d
CH3CH2
3.2
6.3
6.3
4e
CH3
CH3CH2
CH3
H
25.0
100.0
100.0
12.5
12.5
12.5
3.2
12.5
50.0
25.0
6.3
12.5
25.0
25.0
6.3
6.3
50.0
100.0
100.0
25.0
25.0
25.0
6.3
12.5
50.0
50.0
12.5
12.5
1.6
5a
25.0
25.0
12.5
12.5
6.3
5b
H
6a
CH3
H
6b
H
6.3
6.3
6c
H
F
25.0
12.5
6.3
3.2
6d
H
Br
12.5
6.3
6.3
12.5
1.6
6e
H
CH3O
CN
H
1.6
6.3
1.6
6f
H
3.2
25.0
6.3
12.5
100.0
6.3
12.5
12.5
6.3
3.2
6.3
6g
F
12.5
3.2
6.3
25.0
6.3
6h
CH3
H
CH3
I
3.2
1.6
6i
3.2
25.0
12.5
50.0
6.3
12.5
50.0
100.0
25.0
6.3
12.5
1.6
3.2
6.3
6j
7
H
Cl
50.0
100.0
25.0
3.2
25.0
>100.0
25.0
6.3
50.0
>100.0
25.0
6.3
—
—
>100.0
6.3
Fluconazole
5-FCc)
—
—
—
—
3.2
12.5
a) The MIC value was defined as the lowest concentration of the antifungal agent. MIC values were read after 1d for Candida species and Cryptococcus neoformans, and 2d
for Aspergillus species in 37°C. The inoculum sizes contained approximately 1×105 cells/mL. Culture media tested were the modified Sabouraud dextrose broth (Difco Lab.).
The final concentration of antifungal agents was between 0.2 and 50.0µg/mL. b) Fungi tested: Candida albicans BERKOUT KCCM 50235, C. tropicalis BERKOUT KCCM 50662,
C. krusei BERKOUT KCCM 11655, Cryptococcus neoformans KCCM 50564, Aspergillus niger KCTC 1231 and Aspergillus flavus KCCM 11899. c) 5-FC: 5-fluorocytosine.
chlorooxidizing compound 7 with HCl/NaClO4 resulting in
In a similar manner, alkyl 7-amino-4-hydroxy-2-methyl-
70% yields. Consequently, alkyl 2-(5-chloro-2-methyl-4,7- benzofuro[7,6-d]thiazole-6-carboxylates 5a,b were syn-
dioxo-4,7-dihydrobenzo[d]thiazol-6-yl)-2-cyanoacetates 9a,b thesized from 5-methoxy-2-methylbenzo[d]thiazole-4,7-dione
were synthesized by nucleophilic substitution of com- (12). Alkyl 2-cyano-2-(2-methyl-4,7-dioxo-4,7-dihydrobenzo-
pound 8 with 1eq of alkyl cyanoacetate in the presence of [d]thiazol-5-yl)acetates 13a,b were synthesized by nu-
NH4OH and CeCl3. Alkyl 7-amino-5-arylthio-4-hydroxy-2- cleophilic substitution of compound 12 with 1eq of alkyl
methylbenzofuro[6,7-d]thiazole-6-carboxylates 4a–c were cyanoacetate in the presence of NH4OH. Alkyl 2-cyano-2-
synthesized by nucleophilic substitution and cyclization of the (2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]thiazol-5-yl)acetates
compound 9a or 9b with appropriate arylthiols in EtOH. To a 5a,b were synthesized by cyclization of the compounds 13a,b
solution of the compound 9a or 9b in EtOH, 1eq of arylthiol with hydrazine hydrate in EtOH. 6-Arylamino-5-chloro-2-
was added. The mixture was refluxed for 5h and concentrated methylbenzo[d]thiazole-4,7-diones 6a–j were prepared from
in vacuo. Purification of residual crude product by column the compound 8. The compounds 6a–j were formed by regi-
chromatography yielded compounds 4a–c. Most of these reac- oselective nucleophilic substitution of the compound 8 with
tions went as expected and had overall low yields.
the appropriate arylamines in the presence of CeCl3. The sub-
2,5-Dimethylbenzo[d]thiazole-4,7-dione (10) was prepared stitutions gave exclusively or mainly 6-arylamino-substituted
according to previously reported method.12) Consequently, compounds 6a–j along with traces of 7-arylamino-substituted
alkyl 2-cyano-2-(2,5-dimethyl-4,7-dioxo-4,7-dihydrobenzo[d]- compounds as regioisomer. Compounds 6a–j were purified by
thiazol-6-yl)acetates 11a,b were synthesized by nucleophilic column chromatography. Most of these substitutions went as
substitution of compound 10 with 1eq of alkyl cyanoacetate expected and had overall high yields.
in the presence of NH4OH. Alkyl 7-amino-4-hydroxy-2,5-
Antifungal Evaluation The synthesized alkyl 7-amino-4-
dimethylbenzofuro[6,7-d]thiazole-6-carboxylates 4d,e were hydroxybenzofuro[6,7-d]thiazoles 4a–e, 7-amino-4-hydro-
synthesized by cyclization of compounds 11a,b with hydra- xybenzofuro[7,6-d]thiazoles 5a,b, 5-chloro-6-arylamino-
zine hydrate in EtOH. To a solution of the compounds 11a,b benzo[d]thiazole-4,7-diones 6a–j and 7 were tested in vitro for
in EtOH, 1eq of hydrazine was added.
their growth inhibitory activity against pathogenic fungi using

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671
the standard method.15) The minimum inhibitory concentration dimethylbenzofuro[6,7-d]thiazole-6-carboxylates 4d–e were
(MIC) values were determined by comparison with flucon- synthesized by cyclization of the compounds 11a,b with
azole and 5-fluorocytosine as standard agents.
hydrazine in EtOH. 6-Arylamino-5-chloro-2-methylbenzo[d]-
As indicated in Table 1, many of compounds 4a–e, 5a,b and thiazole-4,7-diones 6a–j were prepared by regioselective
6a–j showed potent antifungal activity against tested fungi. nucleophilic substitution of the compound 8 with the ap-
Actually, the activity of compounds 4d, 6e and 6h was supe- propriate arylamines in the presence of CeCl3. Most of these
rior or comparable to that of fluconazole against fungi. The substitutions went as expected and had overall high yields.
compounds 4d, 6e and 6h completely inhibited the growth We have identified a lead compound that has antifungal ac-
of all against Candida and Aspergillus species tested at the tivity by screening of our compounds 4a‒e, 5a,b and 6a–j.
MIC level of 6.3µg/mL. The activity of many compounds Among them tested, most of 5-chloro-6-arylaminobenzo[d]-
among them tested was comparable to that of 5-fluorocytosine thiazole-4,7-diones showed potent antifungal activity. The
against some strain of fungi. Many of compounds 4a–e, 5a,b results suggest that benzofuro[6,7-d]thiazole and 5-chloro-6-
and 6a–j also were comparable to those of fluconazole against arylaminobenzo[d]thiazole-4,7-dione scaffolds would be
Candida species, Cryptococcus neoformans, and Aspergillus promising leads for the development of antifungal agents.
species. Actually, the activity of compounds 6c and 6f was
superior to that of 5-fluorocytosine against C. neoformans
and A. flavus. The alkyl 7-amino-4-hydroxybenzofuro[6,7-d]-
Experimental
All melting points were measured with Büchi melting point
thiazoles 4a–e, showed also antifungal activity. In contrast, B-545 and were uncorrected. 1H-NMR spectra, 13C-NMR
7-amino-4-hydroxybenzofuro[7,6-d]thiazoles 5a,b did not and 19F-NMR spectra were recorded on Varian Unity INOVA
show significant antifungal activity, although some com- 400MHz FT-NMR spectrometer with tetramethylsilane
pounds of them exhibited good activity against C. krusei and (TMS) or CFCl3. High resolution (HR)-MS spectra were
C. neoformans.
recorded with a Agilent 6220 Accurate-Mass time-of-flight
In terms of structure–activity relationship, the 5-chloro-6- (TOF)/LC-MS equipped with an electrospray ionisation ion
arylaminobenzo[d]thiazole-4,7-diones 6a–j showed, in gen- source used. Mass spectra were taken with Jeol JMS AX505
eral,
a more potent antifungal activity than the other WA. The IR spectra were taken from PerkinElmer, Inc. 1420r
alkyl 7-amino-4-hydroxybenzofuro[6,7-d]thiazoles 4a–e or IR spectrometer with KBr pellets or Nujol.
7-amino4-hydroxybenzofuro[7,6-d]thiazoles 5a,b. The activ-
6-Methoxy-7-aminobenzothiazole(7)waspreparedfromcom-
ity of alkyl 7-amino-4-hydroxybenzofuro[6,7-d]thiazoles 4a–e mercially available 6-methoxybenzothiazole. 5,6-Dichloro-2-
was better than that of 7-amino-4-hydroxybenzofuro[7,6-d]- methylbenzo[d]thiazole-4,7-dione (8) was synthesized by chlo-
thiazoles 5a,b. Thus, the benzo[d]thiazole-4,7-dione skeleton rooxidizing compound 7 with HCl/NaClO4. Dimethylbenzo[d]-
of compounds 6a–j appear to be important factor to affect thiazole-4,7-dione (10)14) and 5-methoxy-2-methylbenzo[d]-
their antifungal activity. The benzo[d]thiazole-4,7-dione skel- thiazole-4,7-dione (12)16) were prepared according to previ-
eton 6a–j exhibited good activity, indicating a correlation that ously reported method.
may offer insight into the mode of action of these compounds.
The products were separated by silica gel column chroma-
In addition, 6-methoxy-7-aminobenzothiazole (7) exhibited tography. Purity of products was determined both by to TLC
no or poor, if any, antifungal activity. In contrast, compounds and HPLC. The results showed that a single compound was
4a–e, 5a,b and 6a–j showed more potent antifungal activ- contained in each product. TLC was performed on precoated
ity than 6-methoxy-7-aminobenzothiazole (7). The quinonoid silica gel (60G 254, Merck) using n-hexane/EtOAc for solvent.
4-hydroxybenzofuro[6,7-d]thiazole, 4-hydroxybenzofuro[7,6- The compounds were detected under UV light (254nm).
d]thiazole or benzo[d]thiazole-4,7-dione moiety in compounds
4, 5 and 6 should be essential for the activity, for example, as droxy-2,5-dimethylbenzofuro[6,7-d]thiazole-6-carboxylates
nonquinonoid compound 7 lost the activity, indicating a cor-
To a solution of compound 8 (2.66mmol) and ethylcyano-
General Procedure for Synthesis of Alkyl 7-Amino-4-hy-
4
relation that may offer insight into the mode of action of these acetate or methylcyanoacetate (2.66mmol) in 100mL of EtOH,
1, R2, R3: H, X, Me, etc.) for 2mL of NH4OH solution was added dropwise. The mixture
compounds. The substituents (R
the compounds 4, 5 and 6 may not contribute partially toward was stirred at room temperature for 10min, 5mL of d-HCl
biological potency. Thus, the substituents appear to be not an was then added. The mixture was then extracted several times
important factor to affect their antifungal activity.
with CH2Cl2, and the organic layer was washed with water,
dried with anhydrous MgSO4, and concentrated in vacuo. The
product 9a or 9b was seperated by silica gel column chroma-
Conclusion
Alkyl 2-(5-chloro-2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]- tography (eluted from n-hexane:EtOAc=1:1 to 1:2).
thiazol-6-yl)-2-cyanoacetates 9a,b were synthesized by Ethyl 2-(5-Chloro-2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]-
nucleophilic substitution of 5,6-dichloro-2-methylbenzo[d]- thiazol-6-yl)-2-cyanoacetate (9a): Brown oil (87%). IR (Nujol)
thiazole-4,7-dione (8) with 1eq of alkyl cyanoacetate in the cm−1: 2262 (CN), 1697 (s, C=O), 1377, 1227 (s). 1H-NMR
presence of NH4OH. Alkyl 7-amino-5-arylthio-4-hydroxy-2- (CDCl3) δ: 4.38 (s, 1H, CH), 4.21 (q, 2H, J=7.2Hz, CH2), 2.81
methylbenzofuro[6,7-d]thiazole-6-carboxylates 4a–c were syn- (s, 3H, CH3), 1.31 (t, 3H, J=7.2Hz, CH3). 13C-NMR (DMSO-
thesized by nucleophilic substitution and cyclization of com- d6) δ: 187.4, 176.2, 171.9, 168.5, 148.9, 148.1, 143.8, 142.7, 62.9,
pound 9a or 9b with 1eq of appropriate arylthiols in EtOH. 23.1, 18.5, 14.4. HR-MS m/z: 325.0009 [(M+H)+] (Calcd for
Alkyl 2-cyano-2-(2,5-dimethyl-4,7-dioxo-4,7-dihydrobenzo[d]- C13H10ClN2O4S: 325.0005).
thiazol-6-yl)acetates 11a,b were synthesized by nucleophilic
Methyl 2-(5-Chloro-2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]-
substitution of 2,5-dimethylbenzo[d]thiazole-4,7-dione (10) thiazol-6-yl)-2-cyanoacetate (9b): Brown oil (73%). IR (Nujol)
with 1eq of alkyl cyanoacetate. Alkyl 7-amino-4-hydroxy-2,5- cm−1: 2257 (CN), 1692 (s, C=O), 1390, 1224 (s). 1H-NMR

672
Vol. 62, No. 7
(CDCl3) δ: 4.30 (s, 1H, CH), 4.10 (s, 3H, CH3), 2.80 (s, 3H, 148.3, 143.9, 142.2, 63.5, 23.3, 19.1, 14.7, 11.7. HR-MS m/z:
CH3). 13C-NMR (DMSO-d6) δ: 188.1, 176.4, 172.3, 168.4, 148.7, 305.0598 [(M+H)+] (Calcd for C14H13N2O4S: 305.0596).
147.9, 143.5, 142.3, 61.7, 22.9, 18.9. HR-MS m/z: 310.9891 [(M+
Methyl
2-Cyano-2-(4,7-dihydro-2,5-dimethyl-4,7-dioxo-
H)+] (Calcd for C12H8ClN2O4S: 310.9893).
benzo[d]thiazol-6-yl)acetate (11b): Brown oil (80%). IR
To the solution of compound 9a or 9b (0.45mmol) in 100mL (Nujol) cm−1: 2259 (CN), 1698 (s, C=O), 1373, 1220 (s).
of 95% EtOH, appropriate arylthiol (0.45mmol) was added and 1H-NMR (CDCl3) δ: 5.39 (s, 1H, CH), 3.83 (s, 3H, OCH3),
the mixture was stirred at room temperature or refluxed for 2.85 (s, 3H, CH3), 2.19 (s, 3H, CH3). 13C-NMR (DMSO-d6)
4–5h. To the reaction mixture, 1eq of hydrazine was added. δ: 189.1, 176.7, 172.0, 167.1, 148.9, 147.8, 143.4, 142.2, 62.1,
The mixture was refluxed further for 5h and concentrated in 23.5, 19.3, 11.5. HR-MS m/z: 291.0437 [(M+H)+] (Calcd for
vacuo. The products 4a–c were purified by silica gel column C13H11N2O4S: 291.0439).
chromatography (eluted from n-hexane:EtOAc=2:1 to 1:2),
and crystalized from 95% EtOH.
One equivalent of hydrazine (0.45mmol) was added to the
solution of compound 11a or 11b (0.45mmol) in 100mL 95%
Ethyl
7-Amino-4-hydroxy-2-methyl-5-(p-tolylthio)benzo- EtOH. The mixture was stirred further at rt for 24h and con-
furo[6,7-d]thiazole-6-carboxylate (4a): Brown powder (54%). centrated in vacuo. The products 4d–e were purified by silica
mp 219–221°C. IR (KBr) cm−1: 3515–3270 (s, OH), 3315 (m, gel column chromatography (eluted from n-hexane:EtOAc=
1
NH2), 1692 (s, C=O), 1620 (w), 1228 (s). H-NMR (DMSO-d6) 2:1 to 1:2), and crystalized from 95% EtOH.
δ: 7.28 (d, 2H, J=8.8Hz), 6.86 (d, 2H, J=8.8Hz), 6.42 (s, 2H)
Ethyl
7-Amino-4-hydroxy-2,5-dimethylbenzofuro[6,7-d]-
5.10 (s, 1H), 4.08 (q, 2H, J=7.2Hz), 2.72 (s, 3H), 2.58 (s, 3H), thiazole-6-carboxylate (4d): Brown powder (73%). mp
1.10 (t, 3H, J=7.2Hz). 13C-NMR (DMSO-d6) δ: 166.1, 158.9, 212–213°C. IR (KBr) cm−1: 3452–3231 (s, OH), 3278 (m,
1
151.6, 150.2, 145.6, 139.9, 132.8, 131.4, 130.2, 129.5, 127.2, NH2), 1699 (s, C=O), 1215 (s). H-NMR (DMSO-d6) δ: 9.35 (s,
122.5, 115.3, 110.1, 61.3, 21.1, 14.7, 13.3. HR-MS m/z: 415.0783 1H, OH), 7.71 (s, 2H, NH2), 4.25 (q, 2H, J=7.2Hz, CH2), 2.78
[(M+H)+] (Calcd for C20H19N2O4S2: 415.0786).
(s, 3H, CH3), 2.54 (s, 3H, CH3), 1.31 (t, 3H, J=7.2Hz, CH3).
Ethyl 7-Amino-5-((4-fluorophenyl)thio)-4-hydroxy-2-methyl- 13C-NMR (DMSO-d6) δ: 166.5, 158.4, 152.6, 150.4, 146.3,
benzofuro[6,7-d]thiazole-6-carboxylate (4b): Brown powder 131.4, 127.7, 122.1, 116.3, 111.1, 60.2, 18.1, 14.3, 13.7. HR-MS
(45%). mp 204–209°C. IR (KBr) cm−1: 3496–3242 (s, OH), m/z: 307.0755 [(M+H)+] (Calcd for C14H15N2O4S: 307.0753).
1
3285 (m, NH2), 1695 (s, C=O), 1625 (w), 1220 (s). H-NMR
Methyl 7-Amino-4-hydroxy-2,5-dimethylbenzofuro[6,7-d]-
(DMSO-d6) δ: 7.32–6.78 (m, 4H) 6.42 (s, 2H), 4.11 (q, 2H, thiazole-6-carboxylate (4e): Brown powder (68%). mp
J=7.2Hz), 2.71 (s, 3H), 2.55 (s, 3H), 1.11 (t, 3H, J=7.2Hz). 205–206°C. IR (KBr) cm−1: 3427–3250 (s, OH), 3305 (m,
13C-NMR (DMSO-d6) δ: 166.4, 161.4, 158.5, 151.7, 150.7, NH2), 1708 (s, C=O), 1221 (s). H-NMR (400MHz) δ: 9.35 (s,
1
145.3, 139.4, 132.3, 131.7, 129.2, 127.4, 122.4, 115.3, 110.9, 1H, OH), 7.75 (s, 2H, NH2), 3.75 (s, 3H, OCH3), 2.78 (s, 3H,
60.3, 14.9, 13.2. 19F-NMR (376.3MHz, CDCl3) δ: −116.3. CH3), 2.54 (s, 3H, CH3). 13C-NMR (DMSO-d6) δ: 166.9, 157.6,
HR-MS m/z: 419.0537 [(M+H)+] (Calcd for C19H16FN2O4S2: 152.9, 151.4, 147.1, 131.9, 127.1, 123.5, 115.7, 112.5, 61.0, 19.2,
419.0535).
Ethyl 7-Amino-4-hydroxy-5-((4-methoxyphenyl)thio)-2-meth- 293.0596).
14.1. HR-MS m/z: 293.0597 [(M+H)+] (Calcd for C13H13N2O4S:
ylbenzofuro[6,7-d]thiazole-6-carboxylate (4c): Brown powder
General Procedure for Synthesis of Alkyl 7-Amino-
(67%). mp 221–225°C. IR (KBr) cm−1: 3490–3234 (s, OH), 3247 4-hydroxy-2-methylbenzofuro[7,6-d]thiazole-6-carboxyl-
1
(m, NH2), 1690 (s, C=O), 1616 (w), 1228 (s). H-NMR (DMSO- ates 5 To a solution of compound 12 (2.5mmol) and eth-
d6) δ: 7.48 (d, 2H, J=8.8Hz), 7.29 (d, 2H, J=8.8Hz), 6.71 (s, ylcyanoacetate or methylcyanoacetate (2.5mmol) in 100mL
2H), 5.10 (s, 1H), 4.08 (q, 2H, J=7.2Hz), 2.72 (s, 3H), 2.58 of EtOH, 2mL of NH4OH solution was added dropwise. The
(s, 3H), 1.10 (t, 3H, J=7.2Hz). 13C-NMR (DMSO-d6) δ: 166.4, mixture was stirred at rt for 20min, 5mL d-HCl was then
159.4, 158.5, 151.7, 150.7, 145.3, 139.4, 132.3, 131.7, 129.2 127.4, added. The mixture was then extracted several times with
122.4, 115.3, 110.9, 60.3, 55,8, 14.9, 13.2. HR-MS m/z: 431.0733 CH2Cl2. The combined organic layer was washed with water,
[(M+H)+] (Calcd for C20H19N2O5S2: 431.0735).
dried with anhydrous MgSO4, and concentrated. The product
General Procedure for Synthesis of Alkyl 7-Amino- 13a or 13b was seperated by silica gel column chromatogra-
4-hydroxy-2,5-dimethylbenzofuro[6,7-d]thiazole-6-carbox- phy (eluted from n-hexane:EtOAc=2:1 to 1:1).
ylate 4d and 4e To a solution of compound 10 (2.66mmol)
Ethyl 2-Cyano-2-(2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]-
and ethylcyanoacetate or methylcyanoacetate (2.66mmol) thiazol-5-yl)acetate (13a): Yellow oil (86%). IR (Nujol) cm−1:
1
in 100mL of EtOH, 2mL of NH4OH solution was added 2237 (CN), 1706 (s, C=O), 1372, 1214 (s). H-NMR (DMSO-
dropwise. The mixture was stirred at room temperature for d6) δ: 6.90 (s, 1H), 4.22 (q, 2H, J=7.3Hz, CH2), 4.10 (s, H),
20min, 5mL of d-HCl was then added. The mixture was then 2.79 (s, 3H, CH3), 1.30 (t, 3H, J=7.3Hz, CH3). 13C-NMR
extracted several times with CH2Cl2, and the organic layer (DMSO-d6) δ: 188.8, 177.8, 171.5 168.1, 148.1, 147.8, 143.5,
was washed with water, dried with anhydrous MgSO4, and 142.5, 63.7, 23.1, 18.7, 14.4. HR-MS m/z: 291.3027 [(M+H)+]
concentrated in vacuo. The product 11a or 11b was seper- (Calcd for C13H11N2O4S: 291.3024).
ated by silica gel column chromatography (eluted from n-
hexane:EtOAc=1:1 to 1:3).
Methyl 2-Cyano-2-(2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]-
thiazol-5-yl)acetate (13b): Yellow oil (72%). IR (Nujol) cm−1:
1
Ethyl 2-Cyano-2-(4,7-dihydro-2,5-dimethyl-4,7-dioxobenzo- 2260 (CN), 1705 (s, C=O), 1381, 1220 (s). H-NMR (DMSO-
[d]thiazol-6-yl)acetate (11a): Brown oil (85%). IR (Nujol) d6) δ: 6.84 (s, 1H), 4.30 (s, 3H), 4.15 (s, H), 2.81 (s, 3H, CH3).
cm−1: 2246 (CN), 1703 (s, C=O), 1385, 1214 (s). 1H-NMR 13C-NMR (DMSO-d6) δ: 188.3, 177.4, 172.6, 167.8, 148.5,
(CDCl3) δ: 5.38 (s, 1H, CH), 4.33 (q, 2H, J=7.2Hz, CH2), 2.32 148.1, 143.2, 143.1, 62.9, 23.4, 18.9. HR-MS m/z: 277.2755 [(M+
(s, 3H, CH3), 2.19 (s, 3H, CH3), 1.34 (t, 3H, J=7.2Hz, CH3). H)+] (Calcd for C12H9N2O4S: 277.2759).
13C-NMR (DMSO-d6) δ: 189.4, 177.2, 171.2, 167.4, 148.3,
One equivalent of hydrazine (0.45mmol) was added to the

July 2014
673
solution of compound 13a or 13b (0.45mmol) in 100mL of 2.60 (s, 3H). 13C-NMR (CD3OD) δ: 185.4, 175.1, 168.3, 153.1,
95% EtOH. The mixture was refluxed further for 5h and con- 149.4, 145.8, 133.6, 131.8, 117.2, 115.3, 113.8, 20.9. HR-MS m/z:
centrated in vacuo. The products 4d,e were purified by silica 382.9257 [(M+H)+] (Calcd for C14H9BrClN2O2S: 382.9256).
gel column chromatography (eluted from n-hexane:EtOAc=
5-Chloro-6-((4-methoxyphenyl)amino)-2-methylbenzo-
2:1 to 1:1) and crystalized from 95% EtOH.
thiazole-4,7-dione (6e): Black powder (75%). IR (KBr) cm−1:
1
Ethyl
7-Amino-4-hydroxy-2-methylbenzofuro[7,6-d]thia- 3245 (w, NH), 3030 (w), 1695 (s, C=O), 1490–1555. H-NMR
zole-6-carboxylate (5a): Pale brown powder (35%). mp 300– (CDCl3) δ: 7.20 (d, 2H, J=8.0Hz), 7.08 (d, 2H, J=8.0Hz), 3.88
304°C. IR (KBr) cm−1: 3432–3259 (s, OH), 3297 (m, NH2), (s, 3H), 2.55 (s, 3H). 13C-NMR (CD3OD) δ: 185.8, 177.2, 170.3,
1
1703 (s, C=O), 1228 (s). H-NMR (DMSO-d6) δ: 10.11 (s, 1H, 157.0, 151.7, 147.1, 146.9, 137.8, 117.2, 114.9, 112.3, 57.3, 21.7.
OH), 7.68 (s, 2H, NH2), 7.13 (s, 1H, quinone), 4.25 (q, 2H, HR-MS m/z: 335.0257 [(M+H)+] (Calcd for C15H12ClN2O3S:
J=7.20Hz, CH2), 2.78 (s, 3H, CH3), 1.34 (t, 3H, J=7.20Hz, 335.0259).
CH3). 13C-NMR (DMSO-d6) δ: 167.3, 158.8, 151.3, 150.7,
4-((5-Chloro-2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]-
147.3, 132.1, 129.5, 122.4, 114.2, 112.1, 61.2, 17.9, 13.9. HR-MS thiazol-6-yl)amino)benzonitrile (6f): Black powder (47%). IR
m/z: 293.0594 [(M+H)+] (Calcd for C13H13N2O4S: 293.0596).
(KBr) cm−1: 3240 (w, NH), 3036 (w), 2130 (CN), 1692 (s, C=
1
Methyl 7-Amino-4-hydroxy-2-methylbenzofuro[7,6-d]thia- O), 1496–1550. H-NMR (CDCl3) δ: 7.72 (d, 2H, J=8.0Hz),
zole-6-carboxylate (5b): Pale brown powder (30%). mp 245– 6.93 (d, 2H, J=8.0Hz), 3.66 (s, 1H), 264 (s, 3H). 13C-NMR
258°C. IR (KBr) cm−1: 3430‒3246 (s, OH), 3273 (m, NH2), (CDCl3) δ: 187.2, 176.2, 168.7, 152.7, 149.6, 146.7, 133.6, 130.2,
1695 (s, C=O), 1236 (s). 1H-NMR (DMSO-d6) δ: 10.12 (s, 121.9, 119.4. 116.1, 111.9, 19.2. HR-MS m/z: 330.0108 [(M+
1H, OH), 7.73 (s, 2H, NH2), 7.11 (s, 1H, quinone), 3.78 (s, 3H, H)+] (Calcd for C15H9ClN3O2S: 330.0104).
OCH3), 2.78 (s, 3H, CH3). 13C-NMR (DMSO-d6) δ: 166.4,
5-Chloro-6-((3-fluorophenyl)amino)-2-methylbenzo-
155.7, 153.2, 151.0, 149.2, 131.3, 128.7, 124.2, 113.7, 111.2, 60.8, thiazole-4,7-dione (6g): Black purple powder (54%). IR (KBr)
18.8. HR-MS m/z: 279.0437 [(M+H)+] (Calcd for C12H11N2O4S: cm−1: 3275 (NH), 3040 (w), 1695 (s, C=O), 1595–1470, 1235.
279.0439).
1H-NMR (CD3OD) δ: 7.47–7.41 (m, 1H), 7.01 (td, 1H, J=6.4,
General Procedure for Synthesis of 6-Arylamino- 2.0Hz), 6.67 (d, 1H, J=8.0Hz), 6.63 (d, 1H, J=8.0Hz), 2.61
5-chloro-2-methylbenzo[d]thiazole-4,7-diones 6 To a solu- (s, 3H). 13C-NMR (CD3OD) δ: 187.9, 175.8, 169.6, 157.2,
tion of compound 8 (100mg, 0.40mmol) in 100mL of EtOH 152.9, 148.2, 145.8, 135.2, 133.8, 115.1, 112.6, 111.2, 110.6,
in the presence of CeCl3 (0.02mmol), appropriate arylamine 20.2. 19F-NMR (376.3MHz, CDCl3) δ: −114.2. HR-MS m/z:
(0.4mmol) was added and the mixture was stirred at room 323.0058 [(M+H)+] (Calcd for C14H9ClFN2O2S: 323.0057).
temperature for 20–60min. The solvent was evaporated off
5-Chloro-6-((3,4-dimethylphenyl)amino)-2-methylbenzo[d]-
and the reaction was quenched with water and extracted with thiazole-4,7-dione (6h): Black powder (58%). IR (KBr) cm−1:
1
EtOAc. The organic layer was washed with water, dried over 3310 (s, NH), 3030 (w), 1665 (s, C=O), 1450–1560. H-NMR
MgSO4, and concentrated in vacuo. The residue was purified (CDCl3) δ: 7.22 (m, 1H), 6.75 (s, 1H), 6.71–6.70 (m, 1H), 4.01
by column chromatography (EtOAc:MeOH=15:1) to give (s, 1H), 2.58 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.30 (s, 3H, CH3).
compound 6.
13C-NMR (CDCl3) δ: 189.8, 173.6, 163.8, 154.5, 148.7, 149.2,
5-Chloro-2-methyl-6-(p-tolylamino)benzothiazole-4,7-dione 134.2, 131.9, 119.2, 115.7, 112.5, 119.6, 118.5, 22.1. HR-MS m/z:
(6a): Black powder (70%). IR (KBr) cm−1: 3315 (s, NH), 3031 333.0469 [(M+H)+] (Calcd for C16H14ClN2O2S: 333.0465).
1
(w), 1662 (s, C=O), 1452–1558. H-NMR (CD3OD) δ: 7.34 (d,
5-Chloro-6-((4-iodophenyl)amino)-2-methylbenzo-
2H, J=8.0Hz), 7.02 (d, 2H, J=8.0Hz), 2.55 (s, 3H), 2.44 (s, thiazole-4,7-dione (6i): Black powder (64%). IR (KBr) cm−1:
3H). 13C-NMR (CD3OD) δ: 189.2, 175.2, 168.5, 152.7, 149.2, 3355 (s, NH), 3052 (w), 1650 (s, C=O), 1495–1588, 1270.
148.8, 133.7, 130.2, 116.4, 116.1, 111.9, 22.0, 19.4. HR-MS m/z: 1H-NMR (CDCl3) δ: 7.75 (d, 2H, J=8.0Hz), 6.60 (d, 2H,
319.0307 [(M+H)+] (Calcd for C15H12ClN2O2S: 319.0308).
J=8.0Hz), 3.67 (s, 1H), 2.62 (s, 3H). 13C-NMR (CDCl3) δ:
5-Chloro-2-methyl-6-phenylaminobenzothiazole-4,7-dione 187.2, 173.5, 169.7, 151.5, 149.8, 147.3, 135.2, 128.8, 114.2,
(6b): Black powder (65%). IR (KBr) cm−1: 3275 (NH), 3050 113.9, 83.2, 21.7. HR-MS m/z: 430.9120 [(M+H)+] (Calcd for
1
(w), 1690 (s, C=O), 1550–1470. H-NMR (CDCl3) δ: 7.55 (d, C14H9ClIN2O2S: 430.9118).
1H, J=6.8Hz), 7.48 (d, 1H, J=7.6Hz), 7.35 (d, 1H, J=6.8Hz),
5-Chloro-6-((4-chlorophenyl)amino)-2-methylbenzo-
7.33–7.31 (m, 1H), 6.76 (d, 1H, J=7.6Hz), 2.59 (s, 3H). thiazole-4,7-dione (6j): Black powder (56%). IR (KBr) cm−1:
13C-NMR (CDCl3) δ: 187.2, 173.5, 169.7, 151.5, 149.8, 147.3, 3275 (NH), 3010 (w), 1695 (s, C=O), 1594–1475. 1H-NMR
135.2, 128.8, 114.2, 113.9, 110.3, 21.7. δ: HR-MS m/z: 305.0152 (CD3OD) δ: 7.43 (d, 2H, J=8.8Hz), 6.82 (d, 2H, J=8.8Hz),
[(M+H)+] (Calcd for C14H10ClN2O2S: 305.0151).
3.64 (s, 1H), 2.62 (s, 3H). 13C-NMR (CD3OD) δ: 188.3,
5-Chloro-6-((4-fluorophenyl)amino)-2-methylbenzothia- 176.2, 168.4, 152.2, 146.4, 144.2, 136.3, 126.2, 113.7, 112.6,
zole-4,7-dione (6c): Black purple powder (72%). IR (KBr) 111.0, 21.2. HR-MS m/z: 338.9764 [(M+H)+] (Calcd for
cm−1: 3275 (NH), 3045 (w), 1695 (s, C=O), 1590–1475, 1230. C14H9Cl2N2O2S: 338.9762).
1H-NMR (CD3OD) δ: 7.25–7.20 (m, 2H), 7.02–7.01 (m, 2H)
Antifungal in Vitro Susceptibility Testing The MIC
2.57 (s, 3H). 13C-NMR (CD3OD) δ: 187.2, 177.6, 169.1, 155.9, values of compounds 4–7 were determined by the standard
152.1, 147.9, 145.0, 139.2, 116.8, 113.3, 111.8, 21.0. 19F-NMR broth dilution method.15) The antifungal activities were tested
(376.3 MHz, CDCl3) δ: −116.9. HR-MS m/z: 323.0059 [(M+ in modified Sabouraud dextrose broth against the following
H)+] (Calcd for C14H9ClFN2O2S: 323.0057).
fungal strains: Candida albicans ATCC 10231, C. glabrata
6-((4-Bromophenyl)amino)-5-chloro-2-methylbenzothia- ATCC 2001, C. krusei ATCC 749, C tropicalis ATCC 28775
zole-4,7-dione (6d): Black powder (56%). IR (KBr) cm−1: 3350 and Aspergillus niger KCTC 1231. Fluconazole and 5-fluoro-
1
(s, NH), 3080 (w), 1650 (s, C=O), 1495–1560, 1370. H-NMR cytosine as standard agents were used. The compounds were
(CD3OD) δ: 7.64 (d, 2H, J=8.4Hz), 6.96 (d, 2H, J=8.4Hz), tested in the 0.1–100µg/mL range. That was added to the

674
Vol. 62, No. 7
12482–12491 (1998).
modified Sabouraud dextrose broth (Difco Lab.) for fungi over
a final concentration range of 0.1 to 100µg/mL. The inoculum
sizes contained approximately 1×105 CFU/mL. They were in-
cubated 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 1d for Candida species and 2d for A.
niger in 37°C.
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Acknowledgment This study was supported by a Grant
of the Korea Healthcare Technology R&D Project, Ministry
for Health, Welfare and Family Affairs, Republic of Korea
(A08-0414-AA1723-08N1-00010A).
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