Synthesis and antifungal activity of 2/3-arylthio- and 2,3-bis(arylthio)-5- hydroxy-/5-methoxy-1,4-naphthoquinones

Article abstract of DOI:10.1016/j.ejmech.2004.12.004

2/3-Arylthio- and 2,3-bis(arylthio)-5-hydroxy-/5-methoxy-1,4- naphthoquinones 5-9 were synthesized and tested for in vitro antifungal activity against Candida species and Aspergillus niger. The synthesized compounds 5-9 generally showed good activities ag

Full text of DOI:10.1016/j.ejmech.2004.12.004

European Journal of Medicinal Chemistry 40 (2005) 438–444
www.elsevier.com/locate/ejmech
Synthesis and antifungal activity of 2/3-arylthio-
and 2,3-bis(arylthio)-5-hydroxy-/5-methoxy-1,4-naphthoquinones
Chung-Kyu Ryu *, Ju-Yeon Shim, Mi Jin Chae, Ik Hwa Choi, Ja-Young Han, Ok-Jai Jung,
JungYoon Lee, Seong Hee Jeong
College of Pharmacy, Ewha Womans University, Seodaemun-ku, Seoul 120-750, South Korea
Received 10 July 2004; received in revised form 16 December 2004; accepted 21 December 2004
Available online 11 March 2005
Abstract
2/3-Arylthio- and 2,3-bis(arylthio)-5-hydroxy-/5-methoxy-1,4-naphthoquinones 5–9 were synthesized and tested for in vitro antifungal
activity against Candida species and Aspergillus niger. The synthesized compounds 5–9 generally showed good activities against Candida
albicans and C. tropicalis. The results suggest that the 1,4-naphthoquinones 5–9 would be potent antifungal agents.
© 2005 Elsevier SAS. All rights reserved.
Keywords: 1,4-Naphthoquinones; Juglone; Antifungal activity; Fungi
1. Introduction
5-hydroxy-/5-methoxy-1,4-naphthoquinone, as bioisosteres
of the quinones 1–4, were synthesized and their antifungal
activity was evaluated (Fig. 1).
Naphthoquinone compounds possess various biological
activities [1–9]. An interesting sub-group of naphthoquino-
nes is the 5-hydroxy-1,4-naphthoquinone. A number of
5-hydroxy-1,4-naphthoquinones such as juglone (1) and
plumbagin (2) display potent biological properties including
antimalarial activity [1] as well as antibacterial [2], cytotoxic
properties [3], inhibitory effect of dihydroorotate dehydroge-
nase [4], trypanothione reductase [5] and topoisomerase [6].
The 2-(n-dodecyl)thio-3-hydroxy-1,4-naphthoquinone (3)
blockades mitochondrial electron transport in Saccaromyces
cerevisiae, as an inhibitor of mitochondrial cytochrome com-
plex in yeast [7]. In previous reports [8,9], 2,3-disubstituted-
1,4-naphthoquinones (4), which are derivatives of com-
pounds 1–3, had demonstrated antifungal activity against
pathogenic fungi (Fig. 1). A variety of quinones with differ-
ent substituents could exhibit the antifungal activity through
different action and sometimes improves the activity. We
assumed that incorporating an additional arylamino, arylthio
or halo moiety to quinones would contribute to the improve-
ment in biological efficacy [9,10]. Based on this speculation,
2/3-arylthio- and 2,3-bis(arylthio)-derivatives 5–9 of
There have been a few reports on 5-hydroxy-1,4-
naphthoquinone derivatives, exhibiting potent biological prop-
erties including antimalarial activity as well as antibacterial
[1], and antiparasitic properties [5]. However, the antifungal
activity of 5-hydroxy-/5-methoxy-1,4-naphthoquinone classes
against Candida and Aspergillus species has not been reported
to the best of our knowledge. Therefore, we synthesized a
series of quinones 5–9 to elucidate their contribution to the
antifungal activity. The in vitro antifungal activity of quino-
nes 5–9 against pathogenic fungi was determined by the two-
fold broth dilution method. Additional data for the antifungal
activity of another 1,4-naphthoquinone derivative are pro-
vided.
2. Chemistry
A method for the synthesis of 1,4-naphthoquinones 5–9
(Table 1) is shown in Scheme 1. 5-Methoxy-1,4-naphtho-
quinone (10) and 2-bromo-5-hydroxy-1,4-naphthoquinone
(11) were prepared from 5-hydroxy-1,4-naphthoquinone (1,
juglone) according to the known method [11] with minor
modification. Compound 10 was formed by methylation of
compound 1 with CH3I and Ag2O in CHCl3.
* Corresponding author. Tel.: +82 2 3277 2027; fax: +82 2 3277 3052.
E-mail address: ckryu@mm.ewha.ac.kr (C.-K. Ryu).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.12.004

C.-K. Ryu et al. / European Journal of Medicinal Chemistry 40 (2005) 438–444
439
Fig. 1. 1,4-Naphthoquinone derivatives: juglone (1), plumbagin (2), 2-(n-dodecyl)thio-3-hydroxy-1,4-naphthoquinone (3) and 2,3-disubstituted-1,4-
naphthoquinones (4).
Compounds 11 and 12 were prepared by bromination of
compound 1 or 10. The 2-arylthio-5-hydroxy-1,4-naphtho-
quinones (5a–e) were synthesized by regioselective nucleo-
philic substitutions of the compound 11 with appropriate
arylthiols via palladium(0)-catalyzed reaction. The reaction
of the compound 11 with the arylthiols smoothly proceeded
in the presence of palladium catalyst, which might play a cru-
cial role in the reactivity. Otherwise, the reaction in the
absence of the palladium catalyst produced with very poor
yields.
On the other hand, 2-arylthio-5-methoxy-1,4-naphtho-
quinones (6a–d) were prepared by regioselective nucleo-
philic substitutions of the compound 12 with arylthiols at 0 °C
[12]. For the substitutions, the palladium catalyst is not essen-
tial. Without the catalyst, the substitution smoothly pro-
ceeded and had overall high yields of 65–80%.
Table 1
Structures and in vitro antifungal activities for 1,4-naphthoquinone series 5–9
Compounds
R1
R2
R3
MICa (µg ml–1
)
C. albicansb
4.0
C. tropicalis
4.0
C. krusei
>64.0
>64.0
2.0
A. niger
>64.0
8.0
5a
5b
5c
5d
5e
6a
6b
6c
6d
7a
7b
7c
8a
8b
8c
9a
9b
9c
9d
9e
1
4a
10
H
H
F
H
H
H
H
H
H
H
H
F
CH3
Cl
H
2.0
32.0
4.0
2.0
16.0
4.0
H
H
H
H
F
F
2.0
2.0
8.0
H
4.0
32.0
4.0
4.0
8.0
CH3
Cl
H
32.0
16.0
8.0
>64.0
16.0
4.0
>64.0
>64.0
>64.0
>64.0
8.0
64.0
4.0
H
H
H
F
H
32.0
4.0
32.0
3.0
4.0
H
H
H
H
H
F
CH3
Cl
H
>64.0
>64.0
64.0
2.0
4.0
0.5
4.0
64.0
2.0
1.0
>64.0
8.0
H
H
H
H
H
H
F
CH3
Cl
H
4.0
2.0
0.5
8.0
8.0
16.0
32.0
4.0
32.0
0.5
16.0
64.0
4.0
>64.0
32.0
>64.0
2.0
H
H
H
H
H
CH3
Cl
F
0.5
2.0
0.5
4.0
F
8.0
0.5
>64.0
8.0
4.0
H
H
64.0
16.0
16.0
64.0
0.25
4.0
8.0
8.0
64.0
32.0
32.0
1.0
16.0
16.0
8.0
H
H
F
32.0
2.0
Amphotericin B
0.5
2.0
a The MIC values of the compounds against Candida spp. and A. niger were determined by broth micro dilution testing in accordance with the guidelines in
NCCLS document M27-A and M38-P [14].
b Fungi tested: C. albicans Berkout KCCM 50235, C. tropicalis Berkout KCCM 50662, C. krusei Berkout KCCM 11655 and A. niger KCTC 1231.

440
C.-K. Ryu et al. / European Journal of Medicinal Chemistry 40 (2005) 438–444
Scheme 1. Synthesis of juglone derivatives 5–9.
It is well known that substitution of arylthiols to juglone
(1) proceeds regioselectively, the substitutions giving mainly
the 3-substituted juglone product along with the 2-substituted
juglone as by-product [13]. 3-Arylthio-5-hydroxy-/5-
methoxy-1,4-naphthoquinones (7a–c) and (8a–c) were pre-
pared by the substitution on compound 1 or 10 with the
arylthiols. The products 7a–c and 8a–c were separated by
silica gel column chromatography with CHCl3.
2,3-Bis(arylthio)-5-hydroxy-1,4-naphthoquinones (9a–e)
were synthesized by nucleophilic substitution on 2,3-dichloro-
5-hydroxy-1,4-naphthoquinone (13) with two equivalents of
the arylthiols. Most of the substitutions went as expected and
had an overall yield of 40–54%.
8.0 µg ml–1. In addition, the compound 1 or 10 without the
arylthio group exhibited weak antifungal activity. Thus, the
arylthio moiety of compounds 5–9 appears to partially con-
tribute to biological potency.
The structure–activity relationship may not exist between
properties of substituents (R1, R2, R3: H, CH3, Cl, F, ..) for
the arylthio moieties of compounds 5–9. The compounds such
as 5d and 9c containing 4-flouro-phenylthio substituent exhib-
ited the potent antifungal activity.
The cytotoxic potential of compounds 5–9 has also been
determined in human cancer cells according to the NCI pro-
tocols [15].As represented in Table 2, the compounds 5d, 8a,
8b and 9c did not show significant cytotoxic activity but
showed the selectivity, in that they possess potent antifungal
activities.
3. Results and discussion
The 5-hydroxy-1,4-naphthoquinone series 5–9 were tested
in vitro for their growth inhibitory activity against patho-
genic fungi by the standard broth dilution method according
to the NCCLS guidelines [13,14]. The minimum inhibitory
concentration (MIC) values were determined by comparison
with amphotericin B as a standard agent. As represented in
Table 1, most of the 5-hydroxy-1,4-naphthoquinones series
5–9 generally showed potent antifungal activity against Can-
dida albicans and C. tropicalis. Actually, the activity of com-
pounds 5d, 8a, 8b and 9c was comparable to those of ampho-
4. Conclusions
2/3-Arylthio- and 2,3-bis(arylthio)-5-hydroxy-/5-methoxy-
1,4-naphthoquinones 5–9 were synthesized from 5-hydroxy-
1,4-naphthoquinones and tested for in vitro antifungal activ-
ity against Candida species and Aspergillus niger. The
synthesized compounds 5–9 generally showed good activi-
ties against C. albicans and C. tropicalis. The results of this
study suggest that 5-hydroxy-1,4-naphthoquinone series 5–9
would be potent antifungal agents. Moreover, the results
should encourage the synthesis of 5-hydroxy-1,4-naphtho-
quinone analogs for improving antifungal properties.
tericin
B against C. tropicalis. The 5-hydroxy-1,4-
naphthoquinones 5d, 8a, 8b and 9c completely inhibited the
growth of all fungal species tested at the MIC level of 0.25–

C.-K. Ryu et al. / European Journal of Medicinal Chemistry 40 (2005) 438–444
441
Table 2
heated in a capped heavy-walled pyrex tube under an argon
atmosphere for 10 h at 60 °C.After cooling, the reaction mix-
ture was filtered through celite to remove Pd catalyst, and
then the mixture was extracted with three 100 ml portions of
diethyl ether. The crude product was purified by silica gel
column chromatography with n-hexane/EtOAc. Crystalliza-
tion from aq. EtOH afforded the 2-arylthio-5-hydroxy-1,4-
naphthoquinones (5a–e).
Cytotoxic activity of 1,4-naphthoquinone series 5–9
Compounds
Cytotoxicitya IC50 (µg ml–1
)
A 549b
27.90
21.56
76.83
25.00
3.20
SK-OV-3
1.28
SK-MEL-2
0.61
5a
5b
5c
5d
5e
6a
6b
6c
6d
7a
7b
7c
8a
8b
8c
9a
16.10
13.87
36.01
>100
>100
5.89
>100
3.20
32.50
3.20
25.00
16.10
13.87
36.01
>100
>100
5.88
>100
12.50
6.30
5.1.1.1. 5-Hydroxy-2-(4-methylphenylthio)-1,4-naphtho-
quinone (5a). Light brown needle (yield; 60%); m.p. 167–
169 °C; 1H-NMR (DMSO-d6) d 11.90 (s, 1H, OH), 7.74 (t,
1H, benzene), 7.61 (d, 1H, benzene), 7.36 (m, 4H, Ph–H),
5.86 (s, 1H, H3), 2.46 (s, 3H, CH3); HRMS Anal. Calc. for
C17H12O3S, 296.0507, Found: 296.0508.
21.56
76.83
16.10
13.87
36.01
>100
>100
5.88
16.21
27.90
21.56
76.83
23.24
16.10
13.87
36.01
>100
>100
5.88
27.90
21.56
76.83
16.10
13.87
36.01
>100
>100
0.07
5.1.1.2. 2-(4-Chlorophenylthio)-5-hydroxy-1,4-naphtho-
quinone (5b). Brown needle (yield; 89%); m.p. 172–174 °C;
1H-NMR (DMSO-d6) d 11.87 (s, 1H, OH), 7.75 (m, 1H, ben-
zene), 7.68 (m, 4H, Ph–H), 7.61 (d, 1H, benzene), 5.91 (S,
1H, H3); HRMS Anal. Calc. for C16H9ClO3S, 315.9961,
Found: 315.9960.
27.90
21.56
76.83
0.80
9b
9c
9d
9e
13.9
12.7
Adriamycin
0.28
0.02
a Cytotoxicity evaluation: SRB assay according to NCI protocols [14].
b Human tumor cell lines: A 549 (non-small cell lung), SK-OV-3 (ova-
rian), SK-MEL-2 (melanoma), HCT-15 (colon) and XF 498 (CNS) from
National Cancer Institute (NCI) in USA.
5.1.1.3. 2-(2-Fluorophenylthio)-5-hydroxy-1,4-naphtho-
quinone (5c). Light brown needle (yield; 32%); m.p. 148–
149 °C; 1H-NMR (DMSO-d6) d 11.82 (s, 1H, OH), 7.72 (m,
3H, benzene), 7.63 (d, 1H, benzene), 7.54 (t, 1H, benzene),
7.45 (t, 1H, benzene), 7.38 (d, 1H, benzene), 5.75 (s, 1H,
H2); HRMS Anal. Calc. for C16H9FO3S, 300.0257, Found:
300.0256.
5. Experimental protocols
5.1. Chemistry
5.1.1.4. 2-(4-Fluorophenylthio)-5-hydroxy-1,4-naphtho-
quinone (5d). Light red needle (yield; 82%); m.p. 141–
142 °C; 1H-NMR (DMSO-d6) d 11.86 (s, 1H, OH), 7.75 (t,
1H, benzene), 7.70 (m, 2H, Ph–H), 7.62 (d, 1H, benzene),
7.48 (m, 2H, Ph–H), 7.38 (d, 2H, Ph–H), 5.87 (s, 1H, H3);
HRMS Anal. Calc. for C16H9FO3S, 300.0257, Found:
300.0257.
All melting points (m.p.) were measured in open capillary
tubes with a Büchi m.p. B-545 and were uncorrected. TLC
was performed on precoated silica gel (60G 254, Merck) using
chloroform as a solvent. The compounds were detected under
UV light (254 nm) or by heating to 110 °C after spraying
with a 30% H2SO4-vanillin solution. Column chromatogra-
phy was performed on silica gel G60 (70–230 mesh, ASTM,
Merck). IR spectra were taken with Perkin Elmer 1420r IR
5.1.1.5. 5-Hydroxy-2-(phenylthio)-1,4-naphthoquinone
(5e). Red-brown needle (yield; 60%); m.p. 142–143 °C,
1H-NMR (DMSO-d6) d 11.88 (s, 1H, OH), 7.74 (t, 1H, ben-
zene), 7.66–7.60 (m, 5H, Ph–H), 7.37 (d. 1H, benzene), 5.84
(s, 1H, H3); HRMS Anal. Calc. for C16H10O3S, 282.0351,
Found: 282.0350.
1
spectrometer with KBr pellets. H-NMR spectra were re-
corded on UnityVarian INOVA 400 MHz FT-NMR spectrom-
eter using DMSO-d6 as a solvent, and chemical shifts were
given in ppm with TMS as a standard. High-resolution mass
spectra (HRMS EI) were obtained on a Jeol JMSAX505 WA.
5-Hydroxy-1,4-naphthoquinone, arylamines, DMSO-d6 and
other reagents were obtained from Aldrich Chemical Co.
Reagents for biological screening was obtained from Sigma
Co.
5.1.2. General procedure for the synthesis of 5-arylthio-5-
methoxy-1,4-naphthoquinones (6)
A solution of arylthiol (5 mmol) and KOH (5 mmol) in
10 ml of MeOH was added dropwise, over 15 min, to a solu-
tion of 2-bromo-5-methoxy-1,4-naphthoquinone (12)
(5 mmol) in 12 ml of THF and 2 ml of MeOH at 0 °C. The
reaction mixture was stirred at this temperature for 2 h and
then concentrated under reduced pressure. The residue was
taken up in 100 ml of CH2Cl2 and washed with 30 ml of water
and 30 ml of brine. The crude product was purified by silica
5.1.1. General procedure for the synthesis of 2-arylthio-5-
hydroxy-1,4-naphthoquinones (5)
A mixture of 2-bromo-5-hydroxy-1,4-naphthoquinone (11)
(2 g, 0.8 mmol), (Ph3P)4Pd (92 mg, 0.08 mmol), triethy-
lamine (110 µl) and arylthiol (0.8 mmol) in 10 ml THF was

442
C.-K. Ryu et al. / European Journal of Medicinal Chemistry 40 (2005) 438–444
gel column chromatography with n-hexane/EtOAc. Crystal-
lization from aq. EtOH afforded the 5-arylthio-5-methoxy-
1,4-naphthoquinones (6a–d).
1527, 1263 (s), 1094 (s) cm–1; 1H-NMR (DMSO-d6) d 11.39
(s, 1H, OH), 7.76 (t, 1H, benzene), 7.67 (s, 4H, Ph–H), 7.48
(d, 1H, benzene), 7.35 (d, 1H, benzene), 5.9 (s, 1H, H2);
HRMS Anal. Calc. for C16H9ClO3S, 315.9961, Found:
315.9960.
5.1.2.1. 5-Methoxy-2-(4-methylphenylthio)-1,4-naphtho-
quinone (6a). Brown plate (yield; 80%); m.p. 132–134 °C;
1H-NMR (DMSO-d6) d 7.79 (t, 1H, benzene), 7.67 (d, 1H,
benzene), 7.57 (d, 1H, benzene), 7.49 (d, 2H, Ph–H), 7.41 (d,
2H, Ph–H), 5.72 (s, 1H, H1), 3.88 (s, 3H, OCH3), 2.41 (s,
3H, CH3); HRMS Anal. Calc. for C18H14O3S, 310.0664,
Found: 310.0663.
5.1.3.3. 3-(3-Fluorophenylthio)-5-hydroxy-1,4-naphtho-
quinone (7c). Dark brown powder (yield; 50%); m.p. 169–
171 °C; IR (KBr) 3072 (w), 1631 (s, C=O), 1459–1567, 1287
(s) cm–1; 1H-NMR (DMSO-d6) d 11.4 (s, 1H, OH), 7.76 (t,
3H, benzene), 7.68 (q, 1H, benzene), 7.59 (dt, 1H, benzene),
7.49 (t, 3H, benzene), 7.59 (d, 1H, benzene), 5.89 (s, 1H,
H2); HRMS Anal. Calc. for C16H9FO3S, 315.9961, Found:
315.9962.
5.1.2.2. 2-(4-Chlorophenylthio)-5-methoxy-1,4-naphtho-
quinone (6b). Yellow powder (yield; 73%), m.p. 132–
134 °C, 1H-NMR (DMSO-d6) d 7.81 (t, 1H, benzene), 7.68
(d, 1H, benzene), 7.65 (s, 4H, Ph–H), 7.59 (d, 1H, benzene),
5.78 (s, 1H, H1), 3.89 (s, 3H, OCH3). HRMS Anal. Calc. for
C17H11ClO3S, 330.0118, Found: 330.0117.
5.1.4. General procedure for the synthesis of 3-arylthio-5-
methoxy-1,4-naphthoquinones (8)
A solution of 5-methoxy-1,4-naphthoquinone (10) (0.1 g,
0.57 mmol) in 100 ml of 95% EtOH was added to the solu-
tion of the arylamine (0.57 mmol) in 5 ml of 95% EtOH and
stirred at r.t. for 2 h and then refluxed for 5 h. After the mix-
ture was kept overnight in the refrigerator or poured into 20 ml
of ice water, the precipitate was collected by filtration. The
filtered crude product was purified by silica gel column chro-
matography with CHCl3 and crystallized from 95% EtOH.
5.1.2.3. 2-(2-Fluorophenylthio)-5-methoxy-1,4-naphtho-
quinone (6c). Brown needle (yield; 68%); m.p. 181–182 °C;
1H-NMR (DMSO-d6) d 7.80 (d, 1H, Ph–H), 7.70 (m, 3H,
benzene), 7.60 (d, 1H, Ph–H), 7.52 (t, 1H, Ph–H), 7.43 (t,
1H, Ph–H), 5.72 (s, 1H, H2), 3.89 (s, 3H, OCH3); HRMS
Anal. Calc. for C17H11FO3S, 314.0413, Found: 314.0414.
5.1.2.4. 2-(3-Fluorophenylthio)-5-methoxy-1,4-naphtho-
quinone (6d). Yellow powder (yield; 65%); m.p. 127–
128 °C; 1H-NMR (DMSO-d6) d 7.81 (t, 1H, benzene), 7.69
(m, 1H, benzene), 7.64 (m, 1H, benzene), 7.58 (m, 2H, Ph–H),
7.47 (m, 2H, Ph–H), 5.83 (s, 1H, H2), 3.89 (s, 3H, OCH3);
HRMS Anal. Calc. for C17H11FO3S, 314.0413, Found:
314.0413.
5.1.4.1. 5-Methoxy-3-(4-methylphenylthio)-1,4-naphtho-
quinone (8a). Orange powder (yield; 26%); m.p. 146–
148 °C; IR (KBr) 3090 (w), 1663 (s, C=O), 1444–1584 cm–1;
1H-NMR (DMSO-d6) d 7.81 (q, 1H, benzene), 7.54 (dd, 2H,
benzene), 7.52 (d, 1H, benzene), 7.41 (d, 1H, benzene), 6.94
(dd, 1H, benzene), 6.82 (dd, 1H, benzene), 5.77 (s, 1H, H2),
3.95 (s, 3H, OCH3), 2.40 (s, 3H, CH3); HRMS Anal. Calc.
for C18H14O3S, 310.0664, Found: 310.0663.
5.1.3. General procedure for the synthesis of 3-arylthio-5-
hydroxy-1,4-naphthoquinones (7)
5.1.4.2. 3-(4-Chlorophenylthio)-5-methoxy-1,4-naphtho-
quinone (8b). Orange powder (yield; 27%); m.p. 135–
138 °C; IR (KBr) 3041 (w), 1653 (s, C=O), 1461–1587 cm–1;
1H-NMR (DMSO-d6) d 7.80 (q, 1H, benzene), 7.72 (dd, 2H,
benzene), 7.66 (dd, 2H, benzene), 7.41 (d, 1H, benzene), 6.91
(dd, 1H, benzene), 6.80 (dd, 1H, benzene), 5.76 (s, 1H, H2),
3.98 (s, 3H, OCH3); HRMS Anal. Calc. for C16H9ClO3S,
330.0118, Found: 330.0117.
A solution of 5-hydroxy-1,4-naphthoquinone (1, 0.1 g,
0.57 mmol) in 100 ml of 95% EtOH was added to the solu-
tion of the arylamine (0.57 mmol) in 5 ml of 95% EtOH and
stirred at r.t. for 2 h and then refluxed for 5 h After the mix-
ture was kept overnight in a refrigerator or poured into 20 ml
of ice water, the precipitate was collected by filtration. The
filtered crude product was purified by silica gel column chro-
matography with CHCl3 and crystallized from 95% EtOH.
5.1.4.3. 3-(3-Fluorophenylthio)-5-methoxy-1,4-naphtho-
quinone (8c). Yellow powder (yield; 38%); m.p. 196–
198 °C; IR (KBr) 3063 (w), 2838 (m), 1651 (s, C=O), 1470–
5.1.3.1. 5-Hydroxy-3-(4-methylphenylthio)-1,4-naphtho-
quinone (7a). Black powder (yield; 32%); m.p. 142–147 °C;
IR (KBr) 3061 (w), 1650 (s, C=O), 1630 (s), 1452–1561, 1263
(s), 1094 (s) cm–1; 1H-NMR (DMSO-d6) d 11.42 (s, 1H, OH),
7.75 (t, 2H, Ph–H), 7.52 (d, 2H, Ph–H), 7.45 (q, 3H, ben-
zene), 7.34 (d, 1H, benzene), 5.8 (s, 1H, H2), 2.4 (s, 3H, CH3);
HRMS Anal. Calc. for C17H12O3S, 296.0507, Found:
296.0509.
1
1583, 1213 (m) cm–1; H-NMR (DMSO-d6) d 7.82 (t, 1H,
benzene), 7.66 (m, 2H, benzene), 7.55 (dd, 3H, benzene), 7.47
(d, 1H, benzene), 5.87 (s, 1H, H2), 3.98 (s, 3H, OCH3); HRMS
Anal. Calc. for C17H11FO3S, 314.0413, Found: 314.0413.
5.1.5. General procedure for the synthesis of 2.3-
bisarylthio-5-hydroxy-1,4-naphthoquinones (9)
5.1.3.2. 3-(4-Chlorophenylthio)-5-hydroxy-1,4-naphtho-
quinone (7b). Brown powder (yield; 26%); m.p. 164–
166 °C; IR (KBr) 3061 (w), 1650 (s, C=O), 1630 (s), 1452–
A solution of 2,3-dichloro-5-hydroxy-1,4-naphthoquinone
(13) (0.57 mmol), arylthiol (1.14 mmol) in 100 ml of 95%

C.-K. Ryu et al. / European Journal of Medicinal Chemistry 40 (2005) 438–444
443
EtOH was refluxed for 5 h. After the mixture was kept over-
night in the refrigerator or poured into 20 ml of ice water, the
precipitate was collected by filtration. The filtered crude prod-
uct was purified by silica gel column chromatography with
CHCl3 or crystallized from 95% EtOH.
guidelines in NCCLS document M27-A and M38-P [13,14].
Briefly, stock solutions were prepared in water for flucona-
zole and DMSO for the compounds of the present study. Serial
twofold dilution of all the compound and standard drug were
made in RPMI1640 medium buffered to pH 7.0 with 0.165 M
4-morpholinepropanesulfonic acid (MOPS) buffer as out-
lined in NCCLS M27-A document. Aliquots of (0.1 ml) of
each compound at a 2× final concentration were dispensed
into the wells of micro titer plates. The final concentration of
solvent did not exceed 1% in any well. An inoculum concen-
tration of (1.5 1.0) × 103 cells per ml was prepared by spec-
trometric method of inoculum preparation for each organism
tested. Hundred microliters on individual fungal inoculum
were added to each well of micro titer plate containing the
compounds. The final concentration of all the compounds and
drug were 64–0.12 µg ml–1. The plates were incubated at
35 °C. MIC endpoints were read after 48 h incubation for
Candida spp. and after 72 h for Aspergillus spp. After the
completion of incubation, the growth in each well was com-
pared with that of the growth control well. The MIC of each
compound was defined as the lowest concentration that pro-
duced 90% inhibition in the growth of the organism com-
pared with that of the drug free control.
5.1.5.1. 5-Hydroxy-2,3-bis(4-methylphenylthio)-1,4-naphtho-
quinone (9a). Violet powder (yield; 54%); m.p. 154–157 °C;
IR (KBr) 2927 (s), 1905 (m), 1670 (s, C=O), 1615 (s), 1454–
1520, 1268 (s), 1015 (m) cm–1; 1H-NMR (DMSO-d6) d 11.62
(s, 1H, OH), 7.54–7.48 (m, 3H, benzene), 7.36 (d, 1H, ben-
zene), 7.28 (s, 2H, benzene), 7.20 (dd, 1H, benzene), 7.16 (d,
1H, benzene), 7.12 (d, 5H, benzene), 2.45 (s, 6H, 2CH3);
HRMS Anal. Calc. for C24H18O3S2, 418.0697, Found:
418.0698.
5.1.5.2. 2,3-Bis(4-chlorophenylthio)-5-hydroxy-1,4-naphtho-
quinone (9b). Dark brown powder (yield; 43%); m.p. 186–
189 °C; IR (KBr) 3077 (s), 1665 (s, C=O), 1626 (s), 1474–
1510, 1267 (s), 1091 (s) cm–1; 1H-NMR (DMSO-d6) d 11.42
(s, 1H, OH), 7.70 (t, 2H, benzene), 7.47 (m, 4H, benzene),
7.38 (m, 3H, benzene), 7.34 (d, 2H, benzene); HRMS Anal.
Calc. for C22H12Cl2O3S2, 457.9605, Found: 457.9606.
5.1.5.3. 2,3-Bis(4-fluorophenylthio)-5-hydroxy-1,4-naphtho-
quinone (9c). Brown powder (yield; 40%); m.p. 129–
132 °C; IR (KBr) 3083 (s), 1652 (s, C=O), 1623 (s), 1453–
1568 cm–1; 1H-NMR (DMSO-d6) d 11.4 (s, 1H, OH), 7.70 (t,
1H, benzene), 7.51 (t, 2H, benzene), 7.45 (d, 1H, benzene),
7.40–7.26 (m, 5H, benzene), 7.19–7.15 (m, 2H, benzene);
HRMS Anal. Calc. for C22H12F2O3S2, 426.0196, Found:
426.0196.
Acknowledgements
This study was supported by a grant of the Korea Science
and Engineering Foundation (KOSEF R06-2002-011-01004).
We thanks to Dr. B.H. Lee of Wonkwang University for the
SRB assay.
5.1.5.4. 2,3-Bis(2,4-difluorophenylthio)-5-hydroxy-1,4-
naphthoquinone (9d). Red brown powder (yield; 42%); m.p.
176–178 °C. IR (KBr) 3083 (s), 1672 (s, C=O), 1631 (s),
1421–1507, 1219 (m) cm–1; 1H-NMR (DMSO-d6) d 11.4 (s,
1H, OH), 7.69 (t, 1H, benzene), 7.63 (qd, 2H, benzene), 7.44
(dd, 1H, benzene), 7.42–7.36 (m, 2H, benzene), 7.33 (dd, 2H,
benzene), 7.14–7.08 (m, 2H, benzene); HRMS Anal. Calc.
for C22H10F4O3S2, 462.0007, Found: 462.0008.
References
[1] J. Krungkrai, R. Kanchanarithisak, S.R. Krungkrai, S. Rochanakij,
Exp. Parasitol. 100 (2002) 54–61.
[2] L. Cai, G.X. Wei, P. van der Bijl, C.D. Wu, J. Agric. Food Chem. 48
(2000) 909–914.
[3] J.J. Inbaraj, C.F. Chignell, Chem. Res. Toxicol. 17 (1) (2004) 55–62.
[4] W. Knecht, J. Henseling, M. Löffler, Chem. Biol. Interact. 124 (2002)
61–76.
5.1.5.5. 5-Hydroxy-2,3-bis(phenylthio)-1,4-naphthoquinone
(9e). Dark brown powder (yield; 45%); m.p. 125–126 °C; IR
(KBr) 3051 (s), 1669 (s, C=O), 1631 (s), 1453–1502, 1263
(s) cm–1; 1H-NMR (DMSO-d6) d 11.51 (s, 1H, OH), 7.69 (q,
2H, benzene), 7.51 (t, 1H, benzene), 7.42–7.25 (m, 10H,
Ph–H); HRMSAnal. Calc. for C22H14O3S2 390.0384, Found:
390.0387.
[5] L. Salmon-Chemin, E. Buisine, V. Yardley, S. Kohler, M.-A. Debreu,
V. Landry, et al., J. Med. Chem. 44 (2001) 548–565.
[6] H. Wang, Y. Mao, A.Y. Chen, N. Zhou, E.J. LaVoie, L.F. Liu, Bio-
chemistry 40 (2001) 3316–3323.
[7] H. Roberts, W.M. Choo, S.C. Smith, S. Marzuki, A.W. Linnane,
T.H. Porter, et al., Arch. Biochem. Biophys. 191 (1978) 306–315.
[8] C.-K. Ryu, D.Y. Kim, Arch. Pharm. Res. 16 (1993) 161–166.
5.2. Antifungal in vitro susceptibility testing
[9] V.K. Tandon, R.B. Chhor, R.V. Singh, S.J. Rai, D.B. Yadav, Bioorg.
Med. Chem. Lett. 14 (2004) 1079–1083.
The MIC values of compounds 5–9 were determined by
the standard broth dilution method [13,14]. MIC of the com-
pounds against Candida spp. and Aspergillus spp. was deter-
mined by Broth micro dilution testing in accordance with the
[10] C.-K. Ryu, K.U. Choi, J.-Y. Shim, H.-J. You, I.H. Choi, M.J. Chae,
Bioorg. Med. Chem. 11 (2003) 40039–44008.
[11] J. Grunwell, A. Karipides, C. Wigal, S. Heinzman, J. Parlow, J. Surso,
et al., J. Org. Chem. 56 (1991) 91–95.

444
C.-K. Ryu et al. / European Journal of Medicinal Chemistry 40 (2005) 438–444
[12] S. Laugraud, A. Guingant, C. Chassagnard, J. D’Angelo, J. Org.
Chem. 53 (1988) 1557–1560.
[14] National Committee for Clinical Laboratory Standards (NCCLS), Meth-
ods for Broth Dilution Antifungal Susceptibility Testing of Conidium
Forming Filamentous Fungi. Proposed Standard (M38-P), National
Committee for Clinical Laboratory Standards, Wayne, PA, 1998.
[13] National Committee for Clinical Laboratory Standards, Reference
Method for Broth Dilution Antifungal Susceptibility Testing for
Yeasts. Approval Standard. Document M27-A, National Committee
for Clinical Laboratory Standards, Wayne, PA, 1997.
[15] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vis-
tica, et al., J. Natl. Cancer Inst. 82 (1990) 1107–1112.