Synthesis and antifungal activity of benzofuran-5-ols

Article abstract of DOI:10.1016/j.bmcl.2010.08.129

Benzofuran-5-ol derivatives were synthesized and tested for in vitro antifungal activity against Candida, Aspergillus species, and Cryptococcus neoformans. Among them tested, many benzofuran-5-ols showed good antifungal activity. The results suggest that benzofuran-5-ols would be promising antifungal agents.

Full text of DOI:10.1016/j.bmcl.2010.08.129

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6777–6780
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antifungal activity of benzofuran-5-ols
⇑
Chung-Kyu Ryu , Ae Li Song, Jung Yoon Lee, Jung An Hong, Joo Hee Yoon, Aram Kim
College of Pharmacy, Ewha Womans University, Seodaemun-ku, Seoul 120-750, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 May 2010
Revised 18 August 2010
Accepted 27 August 2010
Available online 28 September 2010
Benzofuran-5-ol derivatives were synthesized and tested for in vitro antifungal activity against Candida,
Aspergillus species, and Cryptococcus neoformans. Among them tested, many benzofuran-5-ols showed
good antifungal activity. The results suggest that benzofuran-5-ols would be promising antifungal agents.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Benzofuran-5-ol
Antimicrobial compds
Antifungal
Fungi
Substitution effects
Benzofuran scaffolds 1 display potent biological properties
including antifungal activity1,2 as well as antibacterial,3 angiogen-
esis inhibitory properties4 (Fig. 1). A benzofuran derivative, a novel
myristoyltransferase inhibitor, has been reported as antifungal
agent.1,2 N-Myristoyltransferase has been proven to be essential
for the viability of fungi, including medically important pathogenic
fungi, Candida albicans5 and Cryptococcus neoformans6,7 making it a
possible target for the development of antifungal agents with a no-
vel mode of action.
An interesting sub-group of benzofurans is benzofuran-5-ols 2
which could metabolize to benzoquinone derivatives with a quino-
noid structure in fungi (Fig. 1). Quinonoid compounds display
potent biological properties including antifungal, antimalarial,
and antibacterial activity.8 We assumed that benzofuran-5-ols 2
could have similar biological activities with those of the quinonoid
compounds. The antifungal activity of benzofuran-5-ols 2 against
pathogenic fungi has not been reported to the best of our knowl-
edge. A variety of benzofuran-5-ols or quinonoid compounds with
different substituents could exhibit the biological activities
through different actions and sometimes improve upon the activi-
ties. The presence of aryl, thio, amino group or halogen atoms on
quinonoid compounds was considerably important factor to affect
their antifungal activity.9 Based on this speculation, benzofuran-5-
ols 2a–n with various substituents were designed and synthesized
to elucidate their contribution to the antifungal activity (Schemes
1 and 2). The in vitro antifungal activity of compounds 2a–n
against pathogenic fungi was determined by the twofold broth
dilution method. Additional data for antifungal activity of cyclo-
hexa-2,5-diene-1,4-dione derivatives 4, 5d, and 6 are provided.
A method for the synthesis of benzofuran-5-ols 2a–f is shown in
Scheme 1 and Table 1. The 2,3-dichloro-5,6-dimethylcyclohexa-2,5-
diene-1,4-dione (3)10 was prepared by oxidizing commercially
available 2,3-dimethylbenzene-1,4-diol (6) with HNO3/HCl variation.
Compound 4 was synthesized by nucleophilic substitution of
the compound 3 with equivalent of methyl cyanoacetate in EtOH
in the presence of NH4OH according to the reported method11 with
minor modification. When the equivalent amount of compound 4
and hydrazine hydrate were mixed in EtOH and refluxed for 2 h,
benzofuran-5-ol 2a was formed. 2-Amino-4-arylthio-5-hydrox-
R1
NH2
O
O
R4
R3
R1
R2
R2
R3
R4
OH
R = Ph, alkyl, X, ..
R = alkyl, Cl, CN, ...
1
2a-n
R1
R1
R2
O
O
R5
R4
R2
R5
R4
R3
R3
OH
O
Benzofuran-5-ols 2
benzoquinones
⇑
Corresponding author. Tel.: +82 2 3277 3027; fax: +82 2 3277 3051.
E-mail address: ckryu@ewha.ac.kr (C.-K. Ryu).
Figure 1. Benzofuran derivatives.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.129

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C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6777–6780
R1
N
C
NH2
O
O
O
HS
R2
O
CH3
H3C
H3C
Cl
Cl
H3C
H3C
O
C
N
H3C
H3C
a
CH3
O
+
O
O
b
Cl
O
CH3
S
O
O
3
OH
4
R1
R2
c
R = H, Me,or X
NH2
2b-f
O
O
CH3
H3C
H3C
O
Cl
OH
2a
Scheme 1. Synthesis of benzofuran-5-ol derivatives. Reagents and conditions: (a) NH4OH/EtOH/rt/10 min/45%; (b) arylthiol (1 equiv)/EtOH/reflux/5 h/65–90%; (c) hydrazine
hydrate (1 equiv)/EtOH/reflux/2 h/78%.
5a, 5b, 5c, or 5d with equivalent of alkyl cyanoacetate or malono-
O
O
NH
C
R
N
2
nitrile in EtOH in the presence of K2CO3 via one pot cyclization. To
a suspension of compound 5a, 5b, 5c, or 5d in EtOH was added
drop wise equivalent of alkyl cyanoacetate and K2CO3. The reaction
mixture was stirred at rt for 4 h and monitored by TLC analysis. The
mixture was then extracted with CH2Cl2, and the organic layer was
washed with water, and concentrated. Purification of crude prod-
uct by column chromatography yielded compounds 2g–k. Most
of these reactions went as expected and had overall high yields
of 51–78%.
In a similar manner, 2-amino-5-hydroxybenzofuran-3-carbo-
nitriles 2m–n synthesized by cyclization of benzoquinone 5a or
5b with equivalent of malononitrile in the presence of K2CO3 in
EtOH.
1
R
O
O
1
O
R
O
2
O
R
a or b
2
H
2
R
OH
1
2
5a: R = Cl, R = Me
5b: R = Cl, R = H
5c: R = R = Cl
5d: R = R = Me
1
2
1
2g: R = R = Me
1
2
1
2
2h: R = Me, R = H
1
2
1
2
2i: R = Et, R = H
1
2
2j: R = Me, R = Cl
2k: R = Et, R = Cl
1
2
N
C
N
C
O
c
d
C
N
O
Et
The experimental data of representative compounds 2a, 2b, 2g,
2l, 2m, and 4 were cited in Ref. 14.
The synthesized benzofuran-5-ols 2a–n, cyclohexa-2,5-diene-
1,4-dione derivatives 4, 5d, and 6 were tested in vitro for their
growth inhibitory activity against pathogenic fungi using the stan-
dard method.15 The MIC (minimum inhibitory concentration) val-
ues were determined by comparison with fluconazole and 5-
fluorocytosine as standard agents.
As indicated in the Table 1, most benzofuran-5-ols 2g–n
showed potent antifungal activity against all tested fungi. Actually,
the activity of compounds 2h, 2i, and 2l was superior or compara-
ble to those of 5-fluorocytosine against fungi. The compounds 2h,
2i, and 2l completely inhibited the growth of all fungal species
NH
2
NH
2
O
O
1
O
R
2
C
N
R
O
1
2
R
R
OH
OH
1
2m: R = Me
1
2
2l: R = Et, R = Me
1
2n: R = H
Scheme 2. Synthesis of benzofuran-5-ol derivatives. Reagents and conditions:
(a) 5a, 5b, or 5c/methyl cyanoacetate (1 equiv)/K2CO3/EtOH/4 h/51–78%; (b) 5b or
5c/ethyl cyanoacetate (1 equiv)/K2CO3/EtOH/4 h; (c) 5d/ethyl cyanoacetate
(1 equiv)/K2CO3/EtOH/4 h/45%; (d) 5a or 5b/malononitrile (1 equiv)/K2CO3/EtOH/
4 h/55–75%.
tested at the MIC level of 1.6–12.5 lg/mL.
Many of 2-amino-4-arylthio-5-hydroxybenzofurans 2b–f also
showed potent antifungal activity against Candida krusei, Crypto-
coccus neoformans, and Aspergillus niger. Actually, the activity of
compounds 2b–e were superior to those of 5-fluorocytosine
against C. krusei and C. neoformans.
In addition, compounds 4 and 5d did not show significant anti-
fungal activity against tested fungi, although they exhibited good
activity against C. neoformans and Aspergillus flavus. The 2,3-
dimethylbenzene-1,4-diol 6 exhibited no or poor, if any, antifungal
activity. Thus, benzofuran-5-ols moiety is important for the anti-
fungal activity. The structure activity relationship may not exist
between properties of substituents (R1, R2: H, Me, Et, X, CN, . . .)
for the 4-arylthio moieties of benzofuran-5-ols 2b–f and for the
compounds 2g–l. 4-Arylthio-moieties of compounds 2b–f did not
improve their antifungal activity in comparison to compounds
2g–l. Generally, the benzofuran-5-ol scaffolds exhibited the
ybenzofurans 2b–f were synthesized by nucleophilic substitution
and cyclization of compound 4 with appropriate arylthiols in EtOH.
To a solution of compound 4 in EtOH, equivalent of arylthiol was
added. The mixture was refluxed for 5 h, and was concentrated
in vacuo. Purification of residual crude product by column chroma-
tography yielded compounds 2b–f. Most of these reactions went as
expected and had overall high yields of 65–90%. The compounds
2b–f were identified by 1H NMR, 13C NMR, or Mass spectra. The
mechanism of formation of compounds 2 were cited in Ref. 12.
A method for the synthesis of benzofuran-5-ols 2g–l is shown in
Scheme 2 and Table 1. Compounds 2g–l were synthesized by
nucleophilic substitution of commercially available benzoquinone

C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6777–6780
6779
Table 1
Structures and antifungal activity for benzofuran-5-ols
NH
2
NH
2
NH
NH
NH
2
2
2
O
O
O
O
O
1
O
1
O
O
R
CH
3
O
R
CH
2
3
1
H C
R
3
H C
R
3
O
O
O
O
2
2
2
H
H C
3
R
H
R
R
H C
S
R
3
OH
2a
OH
OH
OH
OH
2g-k
2m-n
2l
2b-f
1
R
2
Compound
R1
R2
MICa
(lg/mL)
C. albicansb
C. tropicalis
C. krusei
C. neoformans
A. niger
A. flavus
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
4
5d
—
H
H
CH3
F
CH3
CH3
CH3
CH3CH2
CH3
CH3CH2
CH3CH2
CN
—
H
CH3
CH3
H
H
CH3
H
H
Cl
Cl
CH3
CH3
H
—
—
50.0
25.0
25.0
25.0
>50.0
>50.0
25.0
1.6
50.0
25.0
25.0
25.0
50.0
>50.0
25.0
3.2
25.0
6.3
3.2
25.0
1.6
6.3
1.6
6.3
6.3
25.0
25.0
25.0
3.2
12.5
25.0
12.5
1.6
>50.0
12.5
3.2
3.2
25.0
>50.0
3.2
3.2
1.6
>50.0
25.0
12.5
6.3
12.5
12.5
12.5
12.5
50.0
12.5
3.2
12.5
6.3
12.5
12.5
6.3
6.3
1.6
3.2
12.5
25.0
25.0
3.2
3.2
6.3
25.0
12.5
6.3
50.0
12.5
50.0
25.0
50.0
50.0
6.3
12.5
12.5
6.3
>50.0
6.3
50.0
25.0
>50.0
6.3
6.3
25.0
12.5
>50.0
6.3
25.0
25.0
50.0
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
25.0
6.3
0.8
CN
—
—
—
25.0
25.0
25.0
25.0
12.5
12.5
6
—
Fluconazole
5-Fluorocytosine
6.3
12.5
a
The MIC value was defined as the lowest concentration of the antifungal agent. MIC values were read after 1 day for Candida species and Cryptococcus neoformans, and
2 days 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 lg/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.
greatest activity, indicating a correlation that may offer insight into
the mode of action of these compounds.
References and notes
1. Masubuchi, M.; Kawasaki, K.; Ebiike, H.; Ikeda, Y.; Tsujii, S.; Sogabe, S.; Fujii, T.;
Sakata, K.; Shiratori, Y.; Aoki, Y.; Ohtsuka, T.; Shimma, N. Bioorg. Med. Chem.
Lett. 2001, 11, 1833.
2. Masubuchi, M.; Ebiike, H.; Kawasaki, K.; Sogabe, S.; Morikami, K.; Shiratori, Y.;
Tsujii, S.; Fujii, T.; Sakata, K.; Hayase, M.; Shindoh, H.; Aoki, Y.; Ohtsuka, T.;
Shimma, N. Bioorg. Med. Chem. 2003, 11, 4463.
3. Abdel-Aziz, H. A.; Mekawey, A. A. I.; Dawood, K. M. Eur. J. Med. Chem. 2009, 44,
3637.
4. Chen, Y.; Chen, S.; Lu, X.; Cheng, H.; Ou, Y.; Cheng, H.; Zhou, G.-C. Bioorg. Med.
Chem. Lett. 2009, 19, 1851.
5. Weinberg, R. A.; McWherter, C. A.; Freeman, S. K.; Wood, D. C.; Gordon, J. I.; Lee,
S. C. Mol. Microbiol. 1995, 16, 241.
6. Lodge, J. K.; Jackson-Machelski, E.; Toffaletti, D. L.; Perfect, J. R.; Gordon, J. I.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12008.
7. Lodge, J. K.; Jackson-Machelski, E.; Higgins, M.; McWherter, C. A.; Sikorski, J. A.;
Devadas, B.; Gordon, J. I. J. Biol. Chem. 1998, 273, 12482.
8. Middleton, R. W.; Parrick, J. In The Chemistry of the Quinonoid Compounds; Patak,
S., Rappoport, Z., Eds.; John Wiley & Sons: London, 1988; pp 1019–1066.
9. Ryu, C.-K.; Choi, K. U.; Shim, J.-Y.; You, H.-J.; Choi, I. H.; Chae, M. J. Bioorg. Med.
Chem. 2003, 11, 4003.
In conclusion, the compound 4 were synthesized by nucleo-
philic substitution of 2,3-dichloro-5,6-dimethylcyclohexa-2,5-
diene-1,4-dione (3) with equivalent of methyl cyanoacetate in
the presence of NH4OH. 2-Amino-4-arylthio-5-hydroxybenzofu-
rans 2b–f were synthesized by cyclization of compound 4 with
appropriate arylthiols in overall high yields. Benzofuran-5-ol 2a
was synthesized by cyclization of compound 4 with hydrazine. 5-
Benzofuran-5-ols 2g–m were synthesized by one pot cyclization
of benzoquinone 5a, 5b, 5c, or 5d with alkyl cyanoacetate or mal-
ononitrile in the presence of K2CO3. Most of these reactions went as
expected and had overall high yields. We have identified a lead
compound that has antifungal activity by screening of our benzo-
furan-5-ols 2a–n. Among them tested, many benzofuran-5-ols
showed potent antifungal activity. The results suggest that benzo-
furan-5-ol scaffolds would be promising leads for the development
of antifungal agents. Moreover, the results should encourage the
synthesis of benzofuran-5-ol analogs for improving antifungal
properties.
10. Van Allan, J. A.; Priest, W. J.; Marshall, A. S.; Reynolds, G. A. J. Org. Chem. 1968,
33, 1100.
11. Ryu, C.-K.; Lee, J. Y.; Jeong, S. H.; Nho, J.-H. Bioorg. Med. Chem. Lett. 2009, 19, 146.
12. The mechanism of formation of compounds 2 from compound 3: A 1,4-
benzoquinone derivative 4 was synthesized by nucleophilic substitution of the
compound 3 with methyl cyanoacetate in EtOH. The product 4 was formed by a
Acknowledgment
Michael-type addition of methyl cyanoacetate to
3 and subsequent
dechlorination.11 The substitution of 4 with nucleophilic thiols resulted in
the formation of aromatic hydroquinone system as intermediates 20 and
subsequent cyclization to compounds 2. The substitution was similar to the
formation of stable aromatic hydroquinone system by the substitution of thiols
on 1,4-benzoquinones.13
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).

6780
C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6777–6780
N
C
13. Kutyrev, A. A. Tetrahedron 1991, 47, 8043.
O
O
O
O
14. Experimental: All melting points were measured with Büchi melting point
B-545 and were uncorrected. 1H NMR spectra or 13C NMR spectra were
recorded on Varian Unity INOVA 400 MHz FT-NMR spectrometer with TMS.
Mass spectra were taken with Jeol JMS AX505 WA. 2-Amino-5-hydroxy-6,7-
dimethylbenzofuran-3-carboxylate (2a): mp 221–223 °C; 1H NMR (CDCl3) d 2.24
(s, 3H, CH3), 2.28 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 5.81 (s, 1H, OH), 6.13 (s, 2H,
NH); 13C NMR (CDCl3) d 12.5, 13.7, 61.5, 104.7, 117.2, 122.3, 124.4, 126.1,
147.4, 151.5, 169.4; MS (m/z) 235 (M+). Methyl 2-amino-5-hydroxy-6,7-
dimethyl-4-(phenylthio)-benzofuran-3-carboxylate (2b): mp 153–155 °C; 1H
NMR (CDCl3) d 2.26 (s, 3H, CH3), 2.37 (s, 3H, CH3), 3.54 (s, 3H, OCH3), 7.02
(m, 2H, Ph), 7.07 (m, 1H, Ph), 7.18 (m, 2H, Ph), 7.38 (s, 1H, OH); 13C NMR
(CDCl3) d 12.1, 12.9, 61.9, 109.7, 118.4, 122.6, 122.6, 128.7, 129.1, 132.8, 132.8,
132.8, 153.5, 153.5, 156.27, 167.1; MS (m/z) 343 (M+). Methyl 2-amino-5-
hydroxy-6-methylbenzofuran-3-carboxylate (2g): mp 191–192 °C; 1H NMR
(DMSO-d6) d 9.39 (s, 1H, OH), 7.79 (s, 1H), 6.23 (s, 2H, NH2), 5.71 (s, 1H),
3.82 (s, 3H, OCH3), 2.24 (s, 3H, CH3); 13C NMR (DMSO-d6) d 14.8, 61.3, 106.4,,
111.3, 116.3, 119.8, 126.2, 126.3, 140.8, 150.4, 167.9; MS (m/z) 221 (M+). Ethyl
2-amino-5-hydroxy-4,7-dimethylbenzofuran-3-carboxylate (2l): mp 249–250 °C;
1H NMR (DMSO-d6) d 9.73 (s, 1H, OH), 6.42 (s, 1H), 6.07 (s, 2H, NH2), 4.21 (q,
2H, J = 7.2, CH2), 2.25 (s, 3H, CH3), 2.23 (s, 3H, CH3), 1.29 (t, 3H, J = 7.2, CH2CH3);
MS (m/z) 249 (M+). 2-Amino-5-hydroxy-6-methylbenzofuran-3-carbonitrile
(2m): Black powder; mp 157–159 °C; 1H NMR (DMSO-d6) d 8.7 (s, 1H), 7.56
(s, 2H, NH2), 6.35 (s, 1H), 4.21 (q, 2H, J = 7.2, CH2CH3), 2.38 (s, 3H, CH3), 2.21 (s,
3H, CH3), 1.29 (t, 3H, J = 7.2, CH2CH3); MS (m/z) 188 (M+). Methyl-2-(2-chloro-
4,5-dimethyl-3,6-dioxocyclohexa-1,4-dienyl)-2-cyanoacetate (4): 1H NMR
(CDCl3) d 2.01 (s, 3H, CH3), 2.04 (s, 3H, CH3), 3.76 (s, 3H, OCH3), 5.25 (s, 1H,
CH2); MS (m/z) 267 (M+).
H3C
Cl
Cl
H3C
H3C
Cl
Cl
COOCH3
H3C
K CO
2
3
3
N
C
N
H
C
O
O
H3C
H3C
H3C
H3C
EtOH
COOCH3
COOCH3
Cl
S
HS
R2
OH
O
R1
4
R1
2'
R2
NH2
COOCH3
NH
O
C
S
O
H3C
H3C
H3C
H3C
COOCH3
S
OH
OH
R1
R1
15. Mcginnis, M. R.; Rindali, M. G. In Antibiotics in Laboratory Medicine; Lorian, V.,
Ed., 4th ed.; Williams and Wilkins: Baltimore, 1996; pp 176–211.
R2
2
R2