Synthesis of Benzofuro[6,7-d]thiazoles, Benzofuro[7,6-d]thiazoles and 6-Arylaminobenzo[d]thiazole-4,7-diones as Antifungal Agent

Abstract

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 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 6.3 µg/mL. The results suggest that benzofuro[6,7-d]thiazoles and 6-arylaminobenzo[d]thiazole-4,7-diones would be promising antifungal agents.

The recent increase of fungal infections, especially among patients undergoing anticancer chemotherapy and AIDS patients, has generated a renewed interest in antifungal drugs, including development of new antifungal agents in the development of resistance to drugs.^1,2) In a program aimed at identifying novel antifungal agents, we focused on developing benzofuro[6,7-d]thiazoles, benzofuro[7,6-d]thiazoles and 6-arylaminobenzo[d]thiazole-4,7-diones with new mode of antifungal action.

N-Myristoyltransferase has been showed to be essential for the viability of fungi, including medically important pathogenic fungi, Candida albicans³⁾ and Cryptococcus neoformans.^4,5) It could be a target for the development of antifungal agents with a novel mode of action. A benzofuran derivative 1, a novel myristoyltransferase inhibitor, has been reported as antifungal agent^6,7) as well as antibacterial agent^8,9) (Fig. 1). Benzofuran-5-ol 2 and 5-hydroxyfuro[2,3-f]quinoline 3 scaffolds¹⁰⁾ have demonstrated potent antifungal activity against pathogenic fungi.

Fig. 1. Benzofuran, 4-Hydroxybenzofuro[6,7-d]thiazoles, 4-Hydroxybenzofuro[7,6-d]thiazoles and 6-Arylaminobenzo[d]thiazole-4,7-diones

Structure–activity relationship studies from heterocyclic quinonoid compounds indicated that the number and position of nitrogen (N) or sulfur (S) atoms substituted in the heterocyclic ring were considerably important factors to affect the biological activities.^10,11) Generally, increasing the number of substituent nitrogen or sulfur atoms in the ring enhances the activities. We speculated that incorporation of thiazole into the skeleton in compounds 2 or 3 would change the physicochemical properties, and lead to a new pharmacophore alkyl 7-amino-4-hydroxybenzofuro[6,7-d]thiazole-6-carboxylates 4 and alkyl 7-amino-4-hydroxy-2-methylbenzofuro[7,6-d]thiazole-6-carboxylates 5 with a different biological profile from scaffolds 2 or 3. The compounds 4 and 5 which would be bioisosteres of compound 3 could metabolize to benzo[d]thiazole-4,7-dione derivatives with a quinonoid structure in fungi. Quinonoid benzo[d]thiazole-4,7-diones display potent biological properties including antifungal, antimalarial and antibacterial activity.¹⁰⁾ The thiazole moiety of quinonoid compounds could improve upon the biological activities. We assumed that alkyl 4-hydroxybenzofuro[6,7-d]thiazoles 4 and 4-hydroxy-2-methylbenzofuro[7,6-d]thiazole 5 could have similar biological activities with those of benzo[d]thiazole-4,7-diones.

There have not been any reports on alkyl 7-amino-4-hydroxybenzofuro[6,7-d]thiazole 4 and 7-amino-4-hydroxybenzofuro[7,6-d]thiazole scaffolds 5 to the best of our knowledge. The presence of thio, alkyl, amino group or halogen atoms on quinonoid compounds such as benzo[d]thiazole-4,7-diones,¹²⁾ 1,4-naphthoquinones¹³⁾ and 5-hydroxyfuro[2,3-f]quinoline 3 significantly affects their antifungal activity. Thus, a variety of alkyl 7-amino-4-hydroxybenzofuro[6,7-d]thiazoles 4a–e, alkyl 7-amino-4-hydroxy-2-methylbenzofuro[7,6-d]thiazole-6-carboxylates 5a–b and 5-chloro-6-arylaminobenzo[d]thiazole-4,7-diones 6a–j with different substituents could exhibit the biological activities through different actions and sometimes improve upon the activities (Chart 1, Table 1).

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-arylaminobenzo[d]thiazole-4,7-diones

Reagents and conditions: (a) c-HCl/NaClO₄/rt/0.5 h, (b) alkyl cyanoacetate/NH₄OH/CeCl₃/EtOH/rt/30 min, (c) arylthiol (1 eq)/EtOH/hydrazine/reflux/5 h, (d) arylamine (1 eq)/CeCl₃/EtOH/rt/20 min, (e) hydrazine hydrate/EtOH/rt/2 h.

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-Chloro-6-arylaminobenzo[d]thiazole-4,7-diones

Compound	R1	R2	MICa) (µg/mL)
			C. albicans b)	C. tropicalis	C. krusei	C. neoformans	A. niger	A. flavus
4a	CH3CH2	CH3	12.5	12.5	25.0	12.5	12.5	50.0
4b	CH3CH2	F	12.5	6.3	50.0	12.5	12.5	12.5
4c	CH3CH2	CH3O	6.3	12.5	1.6	12.5	6.3	25.0
4d	CH3CH2	CH3	3.2	6.3	6.3	3.2	6.3	3.2
4e	CH3	CH3	25.0	12.5	12.5	6.3	50.0	12.5
5a	CH3CH2	H	100.0	50.0	25.0	25.0	100.0	50.0
5b	CH3	H	100.0	25.0	25.0	25.0	100.0	50.0
6a	H	CH3	12.5	6.3	6.3	12.5	25.0	12.5
6b	H	H	12.5	6.3	6.3	12.5	25.0	12.5
6c	H	F	12.5	25.0	3.2	6.3	25.0	1.6
6d	H	Br	3.2	12.5	12.5	6.3	6.3	12.5
6e	H	CH3O	1.6	6.3	6.3	6.3	1.6	1.6
6f	H	CN	3.2	25.0	12.5	12.5	3.2	6.3
6g	F	H	12.5	6.3	100.0	12.5	6.3	25.0
6h	CH3	CH3	3.2	3.2	6.3	6.3	1.6	6.3
6i	H	I	3.2	25.0	12.5	12.5	3.2	6.3
6j	H	Cl	50.0	12.5	50.0	1.6	25.0	50.0
7	—	—	100.0	50.0	100.0	>100.0	>100.0	>100.0
Fluconazole	—	—	25.0	6.3	25.0	6.3	25.0	25.0
5-FCc)	—	—	3.2	3.2	6.3	12.5	6.3	6.3

a) The MIC value was defined as the lowest concentration of the antifungal agent. MIC values were read after 1 d for Candida species and Cryptococcus neoformans, and 2 d 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 50.0 µg/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.

Based on this speculation, compounds 4a–e, 5a, b and 6a–j with various substituents were designed and synthesized to elucidate their contribution to the antifungal activity.

The in vitro antifungal activity of new compounds 4a–e, 5a, b and 6a–j against pathogenic fungi was determined by the twofold broth dilution method. Additional data for antifungal activity are provided.

Results and Discussion

Chemistry

Methods for the synthesis of compounds 4a–e, 5a, b and 6a–j are shown in Chart 1 and Table 1. 6-Methoxy-7-aminobenzothiazole (7) was prepared from commercially available 6-methoxyobenzothiazole.¹⁴⁾ 5,6-Dichloro-2-methylbenzo[d]thiazole-4,7-dione (8) was synthesized by chlorooxidizing compound 7 with HCl/NaClO₄ resulting in 70% yields. Consequently, alkyl 2-(5-chloro-2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]thiazol-6-yl)-2-cyanoacetates 9a, b were synthesized by nucleophilic substitution of compound 8 with 1 eq of alkyl cyanoacetate in the presence of NH₄OH and CeCl₃. Alkyl 7-amino-5-arylthio-4-hydroxy-2-methylbenzofuro[6,7-d]thiazole-6-carboxylates 4a–c were synthesized by nucleophilic substitution and cyclization of the compound 9a or 9b with appropriate arylthiols in EtOH. To a solution of the compound 9a or 9b in EtOH, 1 eq of arylthiol was added. The mixture was refluxed for 5 h and concentrated in vacuo. Purification of residual crude product by column chromatography yielded compounds 4a–c. Most of these reactions went as expected and had overall low yields.

2,5-Dimethylbenzo[d]thiazole-4,7-dione (10) was prepared according to previously reported method.¹²⁾ Consequently, alkyl 2-cyano-2-(2,5-dimethyl-4,7-dioxo-4,7-dihydrobenzo[d]thiazol-6-yl)acetates 11a, b were synthesized by nucleophilic substitution of compound 10 with 1 eq of alkyl cyanoacetate in the presence of NH₄OH. Alkyl 7-amino-4-hydroxy-2,5-dimethylbenzofuro[6,7-d]thiazole-6-carboxylates 4d, e were synthesized by cyclization of compounds 11a, b with hydrazine hydrate in EtOH. To a solution of the compounds 11a, b in EtOH, 1 eq of hydrazine was added.

In a similar manner, alkyl 7-amino-4-hydroxy-2-methylbenzofuro[7,6-d]thiazole-6-carboxylates 5a, b were synthesized from 5-methoxy-2-methylbenzo[d]thiazole-4,7-dione (12). Alkyl 2-cyano-2-(2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]thiazol-5-yl)acetates 13a, b were synthesized by nucleophilic substitution of compound 12 with 1 eq of alkyl cyanoacetate in the presence of NH₄OH. Alkyl 2-cyano-2-(2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]thiazol-5-yl)acetates 5a, b were synthesized by cyclization of the compounds 13a, b with hydrazine hydrate in EtOH. 6-Arylamino-5-chloro-2-methylbenzo[d]thiazole-4,7-diones 6a–j were prepared from the compound 8. The compounds 6a–j were formed by regioselective nucleophilic substitution of the compound 8 with the appropriate arylamines in the presence of CeCl₃. The substitutions gave exclusively or mainly 6-arylamino-substituted compounds 6a–j along with traces of 7-arylamino-substituted compounds as regioisomer. Compounds 6a–j were purified by column chromatography. Most of these substitutions went as expected and had overall high yields.

Antifungal Evaluation

The synthesized alkyl 7-amino-4-hydroxybenzofuro[6,7-d]thiazoles 4a–e, 7-amino-4-hydroxybenzofuro[7,6-d]thiazoles 5a, b, 5-chloro-6-arylaminobenzo[d]thiazole-4,7-diones 6a–j and 7 were tested in vitro for their growth inhibitory activity against pathogenic fungi using the standard method.¹⁵⁾ The minimum inhibitory concentration (MIC) values were determined by comparison with fluconazole and 5-fluorocytosine as standard agents.

As indicated in Table 1, many of compounds 4a–e, 5a, b and 6a–j showed potent antifungal activity against tested fungi. Actually, the activity of compounds 4d, 6e and 6h was superior or comparable to that of fluconazole against fungi. The compounds 4d, 6e and 6h completely inhibited the growth of all against Candida and Aspergillus species tested at the MIC level of 6.3 µg/mL. The activity of many compounds among them tested was comparable to that of 5-fluorocytosine against some strain of fungi. Many of compounds 4a–e, 5a, b and 6a–j also were comparable to those of fluconazole against Candida species, Cryptococcus neoformans, and Aspergillus 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]thiazoles 4a–e, showed also antifungal activity. In contrast, 7-amino-4-hydroxybenzofuro[7,6-d]thiazoles 5a, b did not show significant antifungal activity, although some compounds of them exhibited good activity against C. krusei and C. neoformans.

In terms of structure–activity relationship, the 5-chloro-6-arylaminobenzo[d]thiazole-4,7-diones 6a–j showed, in general, a more potent antifungal activity than the other alkyl 7-amino-4-hydroxybenzofuro[6,7-d]thiazoles 4a–e or 7-amino4-hydroxybenzofuro[7,6-d]thiazoles 5a, b. The activity of alkyl 7-amino-4-hydroxybenzofuro[6,7-d]thiazoles 4a–e was better than that of 7-amino-4-hydroxybenzofuro[7,6-d]thiazoles 5a, b. Thus, the benzo[d]thiazole-4,7-dione skeleton of compounds 6a–j appear to be important factor to affect their antifungal activity. The benzo[d]thiazole-4,7-dione skeleton 6a–j exhibited good activity, indicating a correlation that may offer insight into the mode of action of these compounds.

In addition, 6-methoxy-7-aminobenzothiazole (7) exhibited no or poor, if any, antifungal activity. In contrast, compounds 4a–e, 5a, b and 6a–j showed more potent antifungal activity than 6-methoxy-7-aminobenzothiazole (7). The quinonoid 4-hydroxybenzofuro[6,7-d]thiazole, 4-hydroxybenzofuro[7,6-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 nonquinonoid compound 7 lost the activity, indicating a correlation that may offer insight into the mode of action of these compounds. The substituents (R¹, R², R³: H, X, Me, etc.) for the compounds 4, 5 and 6 may not contribute partially toward biological potency. Thus, the substituents appear to be not an important factor to affect their antifungal activity.

Conclusion

Alkyl 2-(5-chloro-2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]thiazol-6-yl)-2-cyanoacetates 9a, b were synthesized by nucleophilic substitution of 5,6-dichloro-2-methylbenzo[d]thiazole-4,7-dione (8) with 1 eq of alkyl cyanoacetate in the presence of NH₄OH. Alkyl 7-amino-5-arylthio-4-hydroxy-2-methylbenzofuro[6,7-d]thiazole-6-carboxylates 4a–c were synthesized by nucleophilic substitution and cyclization of compound 9a or 9b with 1 eq of appropriate arylthiols in EtOH. Alkyl 2-cyano-2-(2,5-dimethyl-4,7-dioxo-4,7-dihydrobenzo[d]thiazol-6-yl)acetates 11a, b were synthesized by nucleophilic substitution of 2,5-dimethylbenzo[d]thiazole-4,7-dione (10) with 1 eq of alkyl cyanoacetate. Alkyl 7-amino-4-hydroxy-2,5-dimethylbenzofuro[6,7-d]thiazole-6-carboxylates 4d–e were synthesized by cyclization of the compounds 11a, b with hydrazine in EtOH. 6-Arylamino-5-chloro-2-methylbenzo[d]thiazole-4,7-diones 6a–j were prepared by regioselective nucleophilic substitution of the compound 8 with the appropriate arylamines in the presence of CeCl₃. Most of these substitutions went as expected and had overall high yields. We have identified a lead compound that has antifungal activity by screening of our compounds 4a‒e, 5a, b and 6a–j. Among them tested, most of 5-chloro-6-arylaminobenzo[d]thiazole-4,7-diones showed potent antifungal activity. The results suggest that benzofuro[6,7-d]thiazole and 5-chloro-6-arylaminobenzo[d]thiazole-4,7-dione scaffolds would be promising leads for the development of antifungal agents.

Experimental

All melting points were measured with Büchi melting point B-545 and were uncorrected. ¹H-NMR spectra, ¹³C-NMR and ¹⁹F-NMR spectra were recorded on Varian Unity INOVA 400 MHz FT-NMR spectrometer with tetramethylsilane (TMS) or CFCl₃. High resolution (HR)-MS spectra were recorded with a Agilent 6220 Accurate-Mass time-of-flight (TOF)/LC-MS equipped with an electrospray ionisation ion source used. Mass spectra were taken with Jeol JMS AX505 WA. The IR spectra were taken from PerkinElmer, Inc. 1420r IR spectrometer with KBr pellets or Nujol.

6-Methoxy-7-aminobenzothiazole (7) was prepared from commercially available 6-methoxybenzothiazole. 5,6-Dichloro-2-methylbenzo[d]thiazole-4,7-dione (8) was synthesized by chlorooxidizing compound 7 with HCl/NaClO₄. Dimethylbenzo[d]thiazole-4,7-dione (10)¹⁴⁾ and 5-methoxy-2-methylbenzo[d]thiazole-4,7-dione (12)¹⁶⁾ were prepared according to previously reported method.

The products were separated by silica gel column chromatography. Purity of products was determined both by to TLC and HPLC. The results showed that a single compound was contained in each product. TLC was performed on precoated silica gel (60G 254, Merck) using n-hexane/EtOAc for solvent. The compounds were detected under UV light (254 nm).

General Procedure for Synthesis of Alkyl 7-Amino-4-hydroxy-2,5-dimethylbenzofuro[6,7-d]thiazole-6-carboxylates 4

To a solution of compound 8 (2.66 mmol) and ethylcyanoacetate or methylcyanoacetate (2.66 mmol) in 100 mL of EtOH, 2 mL of NH₄OH solution was added dropwise. The mixture was stirred at room temperature for 10 min, 5 mL of d-HCl was then added. The mixture was then extracted several times 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 chromatography (eluted from n-hexane : EtOAc=1 : 1 to 1 : 2).

Ethyl 2-(5-Chloro-2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]thiazol-6-yl)-2-cyanoacetate (9a): Brown oil (87%). IR (Nujol) cm⁻¹: 2262 (CN), 1697 (s, C=O), 1377, 1227 (s). ¹H-NMR (CDCl₃) δ: 4.38 (s, 1H, CH), 4.21 (q, 2H, J=7.2 Hz, CH₂), 2.81 (s, 3H, CH₃), 1.31 (t, 3H, J=7.2 Hz, CH₃). ¹³C-NMR (DMSO-d₆) δ: 187.4, 176.2, 171.9, 168.5, 148.9, 148.1, 143.8, 142.7, 62.9, 23.1, 18.5, 14.4. HR-MS m/z: 325.0009 [(M+H)⁺] (Calcd for C₁₃H₁₀ClN₂O₄S: 325.0005).

Methyl 2-(5-Chloro-2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]thiazol-6-yl)-2-cyanoacetate (9b): Brown oil (73%). IR (Nujol) cm⁻¹: 2257 (CN), 1692 (s, C=O), 1390, 1224 (s). ¹H-NMR (CDCl₃) δ: 4.30 (s, 1H, CH), 4.10 (s, 3H, CH₃), 2.80 (s, 3H, CH₃). ¹³C-NMR (DMSO-d₆) δ: 188.1, 176.4, 172.3, 168.4, 148.7, 147.9, 143.5, 142.3, 61.7, 22.9, 18.9. HR-MS m/z: 310.9891 [(M+H)⁺] (Calcd for C₁₂H₈ClN₂O₄S: 310.9893).

To the solution of compound 9a or 9b (0.45 mmol) in 100 mL of 95% EtOH, appropriate arylthiol (0.45 mmol) was added and the mixture was stirred at room temperature or refluxed for 4–5 h. To the reaction mixture, 1 eq of hydrazine was added. The mixture was refluxed further for 5 h and concentrated in vacuo. The products 4a–c were purified by silica gel column chromatography (eluted from n-hexane : EtOAc=2 : 1 to 1 : 2), and crystalized from 95% EtOH.

Ethyl 7-Amino-4-hydroxy-2-methyl-5-(p-tolylthio)benzofuro[6,7-d]thiazole-6-carboxylate (4a): Brown powder (54%). mp 219–221°C. IR (KBr) cm⁻¹: 3515–3270 (s, OH), 3315 (m, NH₂), 1692 (s, C=O), 1620 (w), 1228 (s). ¹H-NMR (DMSO-d₆) δ: 7.28 (d, 2H, J=8.8 Hz), 6.86 (d, 2H, J=8.8 Hz), 6.42 (s, 2H) 5.10 (s, 1H), 4.08 (q, 2H, J=7.2 Hz), 2.72 (s, 3H), 2.58 (s, 3H), 1.10 (t, 3H, J=7.2 Hz). ¹³C-NMR (DMSO-d₆) δ: 166.1, 158.9, 151.6, 150.2, 145.6, 139.9, 132.8, 131.4, 130.2, 129.5, 127.2, 122.5, 115.3, 110.1, 61.3, 21.1, 14.7, 13.3. HR-MS m/z: 415.0783 [(M+H)⁺] (Calcd for C₂₀H₁₉N₂O₄S₂: 415.0786).

Ethyl 7-Amino-5-((4-fluorophenyl)thio)-4-hydroxy-2-methylbenzofuro[6,7-d]thiazole-6-carboxylate (4b): Brown powder (45%). mp 204–209°C. IR (KBr) cm⁻¹: 3496–3242 (s, OH), 3285 (m, NH₂), 1695 (s, C=O), 1625 (w), 1220 (s). ¹H-NMR (DMSO-d₆) δ: 7.32–6.78 (m, 4H) 6.42 (s, 2H), 4.11 (q, 2H, J=7.2 Hz), 2.71 (s, 3H), 2.55 (s, 3H), 1.11 (t, 3H, J=7.2 Hz). ¹³C-NMR (DMSO-d₆) δ: 166.4, 161.4, 158.5, 151.7, 150.7, 145.3, 139.4, 132.3, 131.7, 129.2, 127.4, 122.4, 115.3, 110.9, 60.3, 14.9, 13.2. ¹⁹F-NMR (376.3 MHz, CDCl₃) δ: −116.3. HR-MS m/z: 419.0537 [(M+H)⁺] (Calcd for C₁₉H₁₆FN₂O₄S₂: 419.0535).

Ethyl 7-Amino-4-hydroxy-5-((4-methoxyphenyl)thio)-2-methylbenzofuro[6,7-d]thiazole-6-carboxylate (4c): Brown powder (67%). mp 221–225°C. IR (KBr) cm⁻¹: 3490–3234 (s, OH), 3247 (m, NH₂), 1690 (s, C=O), 1616 (w), 1228 (s). ¹H-NMR (DMSO-d₆) δ: 7.48 (d, 2H, J=8.8 Hz), 7.29 (d, 2H, J=8.8 Hz), 6.71 (s, 2H), 5.10 (s, 1H), 4.08 (q, 2H, J=7.2 Hz), 2.72 (s, 3H), 2.58 (s, 3H), 1.10 (t, 3H, J=7.2 Hz). ¹³C-NMR (DMSO-d₆) δ: 166.4, 159.4, 158.5, 151.7, 150.7, 145.3, 139.4, 132.3, 131.7, 129.2 127.4, 122.4, 115.3, 110.9, 60.3, 55,8, 14.9, 13.2. HR-MS m/z: 431.0733 [(M+H)⁺] (Calcd for C₂₀H₁₉N₂O₅S₂: 431.0735).

General Procedure for Synthesis of Alkyl 7-Amino-4-hydroxy-2,5-dimethylbenzofuro[6,7-d]thiazole-6-carboxylate 4d and 4e

To a solution of compound 10 (2.66 mmol) and ethylcyanoacetate or methylcyanoacetate (2.66 mmol) in 100 mL of EtOH, 2 mL of NH₄OH solution was added dropwise. The mixture was stirred at room temperature for 20 min, 5 mL of d-HCl was then added. The mixture was then extracted several times with CH₂Cl₂, and the organic layer was washed with water, dried with anhydrous MgSO₄, and concentrated in vacuo. The product 11a or 11b was seperated by silica gel column chromatography (eluted from n-hexane : EtOAc=1 : 1 to 1 : 3).

Ethyl 2-Cyano-2-(4,7-dihydro-2,5-dimethyl-4,7-dioxobenzo[d]thiazol-6-yl)acetate (11a): Brown oil (85%). IR (Nujol) cm⁻¹: 2246 (CN), 1703 (s, C=O), 1385, 1214 (s). ¹H-NMR (CDCl₃) δ: 5.38 (s, 1H, CH), 4.33 (q, 2H, J=7.2 Hz, CH₂), 2.32 (s, 3H, CH₃), 2.19 (s, 3H, CH₃), 1.34 (t, 3H, J=7.2 Hz, CH₃). ¹³C-NMR (DMSO-d₆) δ: 189.4, 177.2, 171.2, 167.4, 148.3, 148.3, 143.9, 142.2, 63.5, 23.3, 19.1, 14.7, 11.7. HR-MS m/z: 305.0598 [(M+H)⁺] (Calcd for C₁₄H₁₃N₂O₄S: 305.0596).

Methyl 2-Cyano-2-(4,7-dihydro-2,5-dimethyl-4,7-dioxobenzo[d]thiazol-6-yl)acetate (11b): Brown oil (80%). IR (Nujol) cm⁻¹: 2259 (CN), 1698 (s, C=O), 1373, 1220 (s). ¹H-NMR (CDCl₃) δ: 5.39 (s, 1H, CH), 3.83 (s, 3H, OCH₃), 2.85 (s, 3H, CH₃), 2.19 (s, 3H, CH₃). ¹³C-NMR (DMSO-d₆) δ: 189.1, 176.7, 172.0, 167.1, 148.9, 147.8, 143.4, 142.2, 62.1, 23.5, 19.3, 11.5. HR-MS m/z: 291.0437 [(M+H)⁺] (Calcd for C₁₃H₁₁N₂O₄S: 291.0439).

One equivalent of hydrazine (0.45 mmol) was added to the solution of compound 11a or 11b (0.45 mmol) in 100 mL 95% EtOH. The mixture was stirred further at rt for 24 h and concentrated in vacuo. The products 4d–e were purified by silica gel column chromatography (eluted from n-hexane : EtOAc=2 : 1 to 1 : 2), and crystalized from 95% EtOH.

Ethyl 7-Amino-4-hydroxy-2,5-dimethylbenzofuro[6,7-d]thiazole-6-carboxylate (4d): Brown powder (73%). mp 212–213°C. IR (KBr) cm⁻¹: 3452–3231 (s, OH), 3278 (m, NH₂), 1699 (s, C=O), 1215 (s). ¹H-NMR (DMSO-d₆) δ: 9.35 (s, 1H, OH), 7.71 (s, 2H, NH₂), 4.25 (q, 2H, J=7.2 Hz, CH₂), 2.78 (s, 3H, CH₃), 2.54 (s, 3H, CH₃), 1.31 (t, 3H, J=7.2 Hz, CH₃). ¹³C-NMR (DMSO-d₆) δ: 166.5, 158.4, 152.6, 150.4, 146.3, 131.4, 127.7, 122.1, 116.3, 111.1, 60.2, 18.1, 14.3, 13.7. HR-MS m/z: 307.0755 [(M+H)⁺] (Calcd for C₁₄H₁₅N₂O₄S: 307.0753).

Methyl 7-Amino-4-hydroxy-2,5-dimethylbenzofuro[6,7-d]thiazole-6-carboxylate (4e): Brown powder (68%). mp 205–206°C. IR (KBr) cm⁻¹: 3427–3250 (s, OH), 3305 (m, NH₂), 1708 (s, C=O), 1221 (s). ¹H-NMR (400 MHz) δ: 9.35 (s, 1H, OH), 7.75 (s, 2H, NH₂), 3.75 (s, 3H, OCH₃), 2.78 (s, 3H, CH₃), 2.54 (s, 3H, CH₃). ¹³C-NMR (DMSO-d₆) δ: 166.9, 157.6, 152.9, 151.4, 147.1, 131.9, 127.1, 123.5, 115.7, 112.5, 61.0, 19.2, 14.1. HR-MS m/z: 293.0597 [(M+H)⁺] (Calcd for C₁₃H₁₃N₂O₄S: 293.0596).

General Procedure for Synthesis of Alkyl 7-Amino-4-hydroxy-2-methylbenzofuro[7,6-d]thiazole-6-carboxylates 5

To a solution of compound 12 (2.5 mmol) and ethylcyanoacetate or methylcyanoacetate (2.5 mmol) in 100 mL of EtOH, 2 mL of NH₄OH solution was added dropwise. The mixture was stirred at rt for 20 min, 5 mL d-HCl was then added. The mixture was then extracted several times with CH₂Cl₂. The combined organic layer was washed with water, dried with anhydrous MgSO₄, and concentrated. The product 13a or 13b was seperated by silica gel column chromatography (eluted from n-hexane : EtOAc=2 : 1 to 1 : 1).

Ethyl 2-Cyano-2-(2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]thiazol-5-yl)acetate (13a): Yellow oil (86%). IR (Nujol) cm⁻¹: 2237 (CN), 1706 (s, C=O), 1372, 1214 (s). ¹H-NMR (DMSO-d₆) δ: 6.90 (s, 1H), 4.22 (q, 2H, J=7.3 Hz, CH₂), 4.10 (s, H), 2.79 (s, 3H, CH₃), 1.30 (t, 3H, J=7.3 Hz, CH₃). ¹³C-NMR (DMSO-d₆) δ: 188.8, 177.8, 171.5 168.1, 148.1, 147.8, 143.5, 142.5, 63.7, 23.1, 18.7, 14.4. HR-MS m/z: 291.3027 [(M+H)⁺] (Calcd for C₁₃H₁₁N₂O₄S: 291.3024).

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: 2260 (CN), 1705 (s, C=O), 1381, 1220 (s). ¹H-NMR (DMSO-d₆) δ: 6.84 (s, 1H), 4.30 (s, 3H), 4.15 (s, H), 2.81 (s, 3H, CH₃). ¹³C-NMR (DMSO-d₆) δ: 188.3, 177.4, 172.6, 167.8, 148.5, 148.1, 143.2, 143.1, 62.9, 23.4, 18.9. HR-MS m/z: 277.2755 [(M+H)⁺] (Calcd for C₁₂H₉N₂O₄S: 277.2759).

One equivalent of hydrazine (0.45 mmol) was added to the solution of compound 13a or 13b (0.45 mmol) in 100 mL of 95% EtOH. The mixture was refluxed further for 5 h and concentrated in vacuo. The products 4d, e were purified by silica gel column chromatography (eluted from n-hexane : EtOAc=2 : 1 to 1 : 1) and crystalized from 95% EtOH.

Ethyl 7-Amino-4-hydroxy-2-methylbenzofuro[7,6-d]thiazole-6-carboxylate (5a): Pale brown powder (35%). mp 300–304°C. IR (KBr) cm⁻¹: 3432–3259 (s, OH), 3297 (m, NH₂), 1703 (s, C=O), 1228 (s). ¹H-NMR (DMSO-d₆) δ: 10.11 (s, 1H, OH), 7.68 (s, 2H, NH₂), 7.13 (s, 1H, quinone), 4.25 (q, 2H, J=7.20 Hz, CH₂), 2.78 (s, 3H, CH₃), 1.34 (t, 3H, J=7.20 Hz, CH₃). ¹³C-NMR (DMSO-d₆) δ: 167.3, 158.8, 151.3, 150.7, 147.3, 132.1, 129.5, 122.4, 114.2, 112.1, 61.2, 17.9, 13.9. HR-MS m/z: 293.0594 [(M+H)⁺] (Calcd for C₁₃H₁₃N₂O₄S: 293.0596).

Methyl 7-Amino-4-hydroxy-2-methylbenzofuro[7,6-d]thiazole-6-carboxylate (5b): Pale brown powder (30%). mp 245–258°C. IR (KBr) cm⁻¹: 3430‒3246 (s, OH), 3273 (m, NH₂), 1695 (s, C=O), 1236 (s). ¹H-NMR (DMSO-d₆) δ: 10.12 (s, 1H, OH), 7.73 (s, 2H, NH₂), 7.11 (s, 1H, quinone), 3.78 (s, 3H, OCH₃), 2.78 (s, 3H, CH₃). ¹³C-NMR (DMSO-d₆) δ: 166.4, 155.7, 153.2, 151.0, 149.2, 131.3, 128.7, 124.2, 113.7, 111.2, 60.8, 18.8. HR-MS m/z: 279.0437 [(M+H)⁺] (Calcd for C₁₂H₁₁N₂O₄S: 279.0439).

General Procedure for Synthesis of 6-Arylamino-5-chloro-2-methylbenzo[d]thiazole-4,7-diones 6

To a solution of compound 8 (100 mg, 0.40 mmol) in 100 mL of EtOH in the presence of CeCl₃ (0.02 mmol), appropriate arylamine (0.4 mmol) was added and the mixture was stirred at room temperature for 20–60 min. The solvent was evaporated off and the reaction was quenched with water and extracted with EtOAc. The organic layer was washed with water, dried over MgSO₄, and concentrated in vacuo. The residue was purified by column chromatography (EtOAc : MeOH=15 : 1) to give compound 6.

5-Chloro-2-methyl-6-(p-tolylamino)benzothiazole-4,7-dione (6a): Black powder (70%). IR (KBr) cm⁻¹: 3315 (s, NH), 3031 (w), 1662 (s, C=O), 1452–1558. ¹H-NMR (CD₃OD) δ: 7.34 (d, 2H, J=8.0 Hz), 7.02 (d, 2H, J=8.0 Hz), 2.55 (s, 3H), 2.44 (s, 3H). ¹³C-NMR (CD₃OD) δ: 189.2, 175.2, 168.5, 152.7, 149.2, 148.8, 133.7, 130.2, 116.4, 116.1, 111.9, 22.0, 19.4. HR-MS m/z: 319.0307 [(M+H)⁺] (Calcd for C₁₅H₁₂ClN₂O₂S: 319.0308).

5-Chloro-2-methyl-6-phenylaminobenzothiazole-4,7-dione (6b): Black powder (65%). IR (KBr) cm⁻¹: 3275 (NH), 3050 (w), 1690 (s, C=O), 1550–1470. ¹H-NMR (CDCl₃) δ: 7.55 (d, 1H, J=6.8 Hz), 7.48 (d, 1H, J=7.6 Hz), 7.35 (d, 1H, J=6.8 Hz), 7.33–7.31 (m, 1H), 6.76 (d, 1H, J=7.6 Hz), 2.59 (s, 3H). ¹³C-NMR (CDCl₃) δ: 187.2, 173.5, 169.7, 151.5, 149.8, 147.3, 135.2, 128.8, 114.2, 113.9, 110.3, 21.7. δ: HR-MS m/z: 305.0152 [(M+H)⁺] (Calcd for C₁₄H₁₀ClN₂O₂S: 305.0151).

5-Chloro-6-((4-fluorophenyl)amino)-2-methylbenzothiazole-4,7-dione (6c): Black purple powder (72%). IR (KBr) cm⁻¹: 3275 (NH), 3045 (w), 1695 (s, C=O), 1590–1475, 1230. ¹H-NMR (CD₃OD) δ: 7.25–7.20 (m, 2H), 7.02–7.01 (m, 2H) 2.57 (s, 3H). ¹³C-NMR (CD₃OD) δ: 187.2, 177.6, 169.1, 155.9, 152.1, 147.9, 145.0, 139.2, 116.8, 113.3, 111.8, 21.0. ¹⁹F-NMR (376.3 MHz, CDCl₃) δ: −116.9. HR-MS m/z: 323.0059 [(M+H)⁺] (Calcd for C₁₄H₉ClFN₂O₂S: 323.0057).

6-((4-Bromophenyl)amino)-5-chloro-2-methylbenzothiazole-4,7-dione (6d): Black powder (56%). IR (KBr) cm⁻¹: 3350 (s, NH), 3080 (w), 1650 (s, C=O), 1495–1560, 1370. ¹H-NMR (CD₃OD) δ: 7.64 (d, 2H, J=8.4 Hz), 6.96 (d, 2H, J=8.4 Hz), 2.60 (s, 3H). ¹³C-NMR (CD₃OD) δ: 185.4, 175.1, 168.3, 153.1, 149.4, 145.8, 133.6, 131.8, 117.2, 115.3, 113.8, 20.9. HR-MS m/z: 382.9257 [(M+H)⁺] (Calcd for C₁₄H₉BrClN₂O₂S: 382.9256).

5-Chloro-6-((4-methoxyphenyl)amino)-2-methylbenzothiazole-4,7-dione (6e): Black powder (75%). IR (KBr) cm⁻¹: 3245 (w, NH), 3030 (w), 1695 (s, C=O), 1490–1555. ¹H-NMR (CDCl₃) δ: 7.20 (d, 2H, J=8.0 Hz), 7.08 (d, 2H, J=8.0 Hz), 3.88 (s, 3H), 2.55 (s, 3H). ¹³C-NMR (CD₃OD) δ: 185.8, 177.2, 170.3, 157.0, 151.7, 147.1, 146.9, 137.8, 117.2, 114.9, 112.3, 57.3, 21.7. HR-MS m/z: 335.0257 [(M+H)⁺] (Calcd for C₁₅H₁₂ClN₂O₃S: 335.0259).

4-((5-Chloro-2-methyl-4,7-dioxo-4,7-dihydrobenzo[d]thiazol-6-yl)amino)benzonitrile (6f): Black powder (47%). IR (KBr) cm⁻¹: 3240 (w, NH), 3036 (w), 2130 (CN), 1692 (s, C=O), 1496–1550. ¹H-NMR (CDCl₃) δ: 7.72 (d, 2H, J=8.0 Hz), 6.93 (d, 2H, J=8.0 Hz), 3.66 (s, 1H), 264 (s, 3H). ¹³C-NMR (CDCl₃) δ: 187.2, 176.2, 168.7, 152.7, 149.6, 146.7, 133.6, 130.2, 121.9, 119.4. 116.1, 111.9, 19.2. HR-MS m/z: 330.0108 [(M+H)⁺] (Calcd for C₁₅H₉ClN₃O₂S: 330.0104).

5-Chloro-6-((3-fluorophenyl)amino)-2-methylbenzothiazole-4,7-dione (6g): Black purple powder (54%). IR (KBr) cm⁻¹: 3275 (NH), 3040 (w), 1695 (s, C=O), 1595–1470, 1235. ¹H-NMR (CD₃OD) δ: 7.47–7.41 (m, 1H), 7.01 (td, 1H, J=6.4, 2.0 Hz), 6.67 (d, 1H, J=8.0 Hz), 6.63 (d, 1H, J=8.0 Hz), 2.61 (s, 3H). ¹³C-NMR (CD₃OD) δ: 187.9, 175.8, 169.6, 157.2, 152.9, 148.2, 145.8, 135.2, 133.8, 115.1, 112.6, 111.2, 110.6, 20.2. ¹⁹F-NMR (376.3 MHz, CDCl₃) δ: −114.2. HR-MS m/z: 323.0058 [(M+H)⁺] (Calcd for C₁₄H₉ClFN₂O₂S: 323.0057).

5-Chloro-6-((3,4-dimethylphenyl)amino)-2-methylbenzo[d]thiazole-4,7-dione (6h): Black powder (58%). IR (KBr) cm⁻¹: 3310 (s, NH), 3030 (w), 1665 (s, C=O), 1450–1560. ¹H-NMR (CDCl₃) δ: 7.22 (m, 1H), 6.75 (s, 1H), 6.71–6.70 (m, 1H), 4.01 (s, 1H), 2.58 (s, 3H, CH₃), 2.35 (s, 3H, CH₃), 2.30 (s, 3H, CH₃). ¹³C-NMR (CDCl₃) δ: 189.8, 173.6, 163.8, 154.5, 148.7, 149.2, 134.2, 131.9, 119.2, 115.7, 112.5, 119.6, 118.5, 22.1. HR-MS m/z: 333.0469 [(M+H)⁺] (Calcd for C₁₆H₁₄ClN₂O₂S: 333.0465).

5-Chloro-6-((4-iodophenyl)amino)-2-methylbenzothiazole-4,7-dione (6i): Black powder (64%). IR (KBr) cm⁻¹: 3355 (s, NH), 3052 (w), 1650 (s, C=O), 1495–1588, 1270. ¹H-NMR (CDCl₃) δ: 7.75 (d, 2H, J=8.0 Hz), 6.60 (d, 2H, J=8.0 Hz), 3.67 (s, 1H), 2.62 (s, 3H). ¹³C-NMR (CDCl₃) δ: 187.2, 173.5, 169.7, 151.5, 149.8, 147.3, 135.2, 128.8, 114.2, 113.9, 83.2, 21.7. HR-MS m/z: 430.9120 [(M+H)⁺] (Calcd for C₁₄H₉ClIN₂O₂S: 430.9118).

5-Chloro-6-((4-chlorophenyl)amino)-2-methylbenzothiazole-4,7-dione (6j): Black powder (56%). IR (KBr) cm⁻¹: 3275 (NH), 3010 (w), 1695 (s, C=O), 1594–1475. ¹H-NMR (CD₃OD) δ: 7.43 (d, 2H, J=8.8 Hz), 6.82 (d, 2H, J=8.8 Hz), 3.64 (s, 1H), 2.62 (s, 3H). ¹³C-NMR (CD₃OD) δ: 188.3, 176.2, 168.4, 152.2, 146.4, 144.2, 136.3, 126.2, 113.7, 112.6, 111.0, 21.2. HR-MS m/z: 338.9764 [(M+H)⁺] (Calcd for C₁₄H₉C_l2N₂O₂S: 338.9762).

Antifungal in Vitro Susceptibility Testing

The MIC values of compounds 4–7 were determined by the standard broth dilution method.¹⁵⁾ The antifungal activities were tested in modified Sabouraud dextrose broth against the following fungal strains: Candida albicans ATCC 10231, C. glabrata ATCC 2001, C. krusei ATCC 749, C tropicalis ATCC 28775 and Aspergillus niger KCTC 1231. Fluconazole and 5-fluorocytosine as standard agents were used. The compounds were tested in the 0.1–100 µg/mL range. That was added to the modified Sabouraud dextrose broth (Difco Lab.) for fungi over a final concentration range of 0.1 to 100 µg/mL. The inoculum sizes contained approximately 1×10⁵ 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 d for Candida species and 2 d for A. niger in 37°C.

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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