Synthesis and antifungal activity of 6-hydroxycinnolines

Article abstract of DOI:10.1016/j.bmcl.2006.01.005

6-Hydroxycinnolines 2 and cyclohexa-2,5-diene-1,4-dione derivatives 6 were synthesized and tested for in vitro antifungal activity against Candida and Aspergillus species. 6-Hydroxycinnolines 2 showed, in general, more potent antifungal activity against Candida species than the other cyclohexa-2,5-diene- 1,4-diones. The results suggest that 6-hydroxycinnolines would be potent antifungal agents.

Full text of DOI:10.1016/j.bmcl.2006.01.005

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1850–1853
Synthesis and antifungal activity of 6-hydroxycinnolines
Chung-Kyu Ryu* and Jung Yoon Lee
College of Pharmacy, Ewha Womans University, Seodaemun-ku, Seoul 120-750, Republic of Korea
Received 8 December 2005; revised 30 December 2005; accepted 4 January 2006
Available online 24 January 2006
Abstract—6-Hydroxycinnolines 2 and cyclohexa-2,5-diene-1,4-dione derivatives 6 were synthesized and tested for in vitro antifungal
activity against Candida and Aspergillus species. 6-Hydroxycinnolines 2 showed, in general, more potent antifungal activity against
Candida species than the other cyclohexa-2,5-diene-1,4-diones. The results suggest that 6-hydroxycinnolines would be potent
antifungal agents.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Cinnoline derivatives 1 are used in pharmaceuticals and
mainly patented as bactericides1 and fungicides2 (Fig. 1).
An interesting sub-group of cinnolines is 6-hydroxycinn-
olines 2 which tautomerize to cinnolin-6(2H)-ones with
a quasi quinonoid structure.3 The compounds 2 could,
respectively, exist as a mixture of tautomers 6-hydroxy-
cinnolines and cinnolin-6(2H)-one in equilibrium shown
in Figure 1. Quinonoid compounds display potent bio-
logical properties including antimalarial, antibacterial,
and antifungal activities.4 We assumed that 6-hydroxy-
cinnolines 2 could show biological activities similar to
those of the quinonoid compounds. The antifungal
activity of 6-hydroxycinnolines 2 against pathogenic
fungi has not been reported to the best of our knowl-
edge. A variety of quinonoid compounds with different
substituents could exhibit the biological activities
through different actions and sometimes improve upon
the activities. The presence of arylthio, amino group
or halogen atoms on quinonoid compounds was a con-
siderably important factor to affect their antifungal
activity.5 Based on this speculation, 2-amino-7,8-di-
methyl-6-hydroxycinnolines 2a–f with various substitu-
ents were designed and synthesized to elucidate their
contribution to the antifungal activity (Scheme 1). The
in vitro antifungal activity of compounds 2a–f against
pathogenic fungi was determined by the twofold broth
dilution method. Additional data for properties and
antifungal activity of cyclohexa-2,5-diene-1,4-dione
derivatives are provided.
N
NH2
O
N
N
N
H3C
H3C
R1
CH3
O
R2
R
OH
R1, R2 = Ph, alkyl, X, ..
R = Cl or arylthio
2a-f
1
H
R1
R2
N
R1
R2
N
N
N
R5
R4
R5
R4
R3
R3
OH
O
6-hydroxycinnolines 2
cinnolin-6(2H)-ones
Figure 1. Cinnoline derivatives.
A method for the synthesis of 6-hydroxycinnolines 2a–f
(Table 1) is shown in Scheme 1. The 2,3-dichloro-5,6-
dimethylcyclohexa-2,5-diene-1,4-dione (4)6 was pre-
pared by oxidizing 2,3-dimethylbenzene-1,4-diol (3) with
HNO3/HCl variation.
Methyl-2-(2-chloro-4,5-dimethyl-3,6-dioxocyclohexa-
1,4-dienyl)-2-cyanoacetate (5) was synthesized by nucle-
ophilic substitution of the compound 4 with equivalent
of methyl cyanoacetate in EtOH in the presence of
ammonia. When equivalent amounts of compound 5
and hydrazine hydrate7 were mixed in EtOH and
refluxed for 1 h, cinnoline 2a was formed. Methyl-2-
Keywords: 6-Hydroxycinnoline; Antimicrobial compounds; Anti-
fungal; Fungi; Substitution effects.
*
Corresponding author. Tel.: +82 2 3277 3027; fax: +82 2 3277
3051; e-mail: ckryu@mm.ewha.ac.kr
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.005

C.-K. Ryu, J. Y. Lee / Bioorg. Med. Chem. Lett. 16 (2006) 1850–1853
1851
O
O
N
NH2
O
CH3
OH
O
N
O
H3C
H3C
H3C
H3C
H3C
H3C
H3C
H3C
Cl
Cl
a
CH3
CN
c
b
O
Cl
Cl
OH
O
OH
2a
O
5
4
3
d
N
NH2
O
N
O
O
CH3
O
O
H3C
H3C
CH3
H3C
H3C
CN
O
c
S
OH
R1
S
R1
R2
R2
R3
R3
6a-e
2b-f
Scheme 1. Synthesis of 6-hydroxycinnolines. Reagents and conditions: (a) HNO3/HCl/90 ꢁC/10 min; (b) methyl cyanoacetate/EtOH/NH4OH/rt/
10 min; (c) hydrazine hydrate/EtOH/reflux/1 h; (d) arylthiol/EtOH/reflux/5 h.
Table 1. Structures and antifungal activity for 6-hydroxycinnolines and cyclohexa-2,5-diene-1,4-diones
Compound
R1
R2
R3
MICa (lg/mL)
C. albicansb
C. tropicalis
C. krusei
C. neoformans
A. niger
2a
2b
—
H
H
H
H
H
H
H
H
H
H
—
H
—
H
>50
>50
>50
>50
>50
>50
50
50
25
25
6.3
3.2
3.2
25
25
1.6
6.3
1.6
6.3
1.6
12.5
50
6.3
12.5
25
2c
2d
H
CH3
CH3
H
25
25
CH3
F
12.5
1.6
2e
50
2f
6a
CH3
H
H
>50
50
>50
50
>50
50
H
6b
6c
H
CH3
CH3
H
>50
50
>50
50
50
>50
50
CH3
F
>50
25
>50
50
6d
6e
50
50
50
CH3
H
>50
25
>50
25
1.6
12.5
25
50
50
>50
25.0
25.0
12.5
3
5
5-Fluorocytosine
50
50
12.5
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 day for Candida species and 2 days for
A. niger 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 100 lg/mL.
b Fungi tested: Candida albicans Berkout KCCM 50235, C. tropicalis Berkout KCCM 50662, C. krusei Berkout KCCM 11655, Cryptococcus
neoformans KCCM 50564, and Aspergillus niger KCTC 1231.
cyano-2-(2-arylthio-4,5-dimethyl-3,6-dioxocyclohexa-
1,4-dienyl)acetates 6a–e were synthesized by nucleophil-
ic substitution of the compound 5 with appropriate
arylthiols in boiling EtOH. When equivalent amounts
of compounds 6a–e and hydrazine hydrate were mixed
in EtOH and refluxed for 1 h, cinnolines 2b–f were
formed. Experimental details and data for this proce-
dure are cited in the References and notes.8–12
As indicated in Table 1, most of the 2-amino-7,8-dimeth-
yl-6-hydroxycinnolines 2a–f showed potent antifungal
activity against C. krusei, C. neoformans, and A. niger.
The antifungal activity against C. neoformans was prom-
inent. In contrast, the methyl-2-cyano-2-(2-arylthio-4,5-
dimethyl-3,6-dioxocyclohexa-1,4-dienyl)acetates 6a–e
did not show significant antifungal activity against all
tested fungi, although some compounds of them exhibit-
ed poor activity. The 2-amino-7,8-dimethyl-6-hydroxyc-
innoline compounds 2a–f exhibited the greatest activity,
indicating a correlation that may offer insight into the
mode of action of these compounds. Actually, the
activities of compounds 2b–d were superior or compara-
ble to those of 5-fluorocytosine against C. krusei,
C. neoformans, and A. niger.
The synthesized 6-hydroxycinnolines 2a–f, cyclohexa-
2,5-diene-1,4-dione derivatives 3, 5, and 6a–e were tested
in vitro for their growth inhibitory activity against path-
ogenic fungi by the standard method.13 The minimum
inhibitory concentration (MIC) values were determined
by comparison with 5-fluorocytosine as a standard agent.

1852
C.-K. Ryu, J. Y. Lee / Bioorg. Med. Chem. Lett. 16 (2006) 1850–1853
9. Synthesis of 2,3-dichloro-5,6-dimethylcyclohexa-2,5-diene-
1,4-dione (4): 5 mL of concd HNO3 was added over a
period of 1 h to a stirred suspension of compound 3
(10 mmol) in 15 mL of concd HCl at 80–90 ꢁC. The
mixture was stirred at rt for 2 h and was extracted twice
with ether. The extract was evaporated and crystallization
from EtOH afforded compound 4: orange powder (25%);
In addition, the 2,3-dimethylbenzene-1,4-diol (3) and
methyl-2-(2-chloro-4,5-dimethyl-3,6-dioxocyclohexa-1,4-
dienyl)-2-cyanoacetate (5) exhibited poor antifungal
activity. Compounds 6a–e exhibited no or poor, if any,
antifungal activity. Thus, 6-hydroxycinnoline moiety is
essential for the antifungal activity. The structure–activ-
ity relationship may not exist between properties of sub-
stituents (R1, R2, R3: H, CH3, F, . . .) for the 5-arylthio
moieties of the 6-hydroxycinnolines 2a–f.
1
mp 248 ꢁC6; H NMR (DMSO-d6) d 2.03 (s, 6H, CH3).
10. Synthesis of methyl-2-(2-chloro-4,5-dimethyl-3,6-dioxocy-
clohexa-1,4-dienyl)-2-cyanoacetate (5): To a solution of
compound 4 (1.295 g, 2.66 mmol) and methylcyanoacetate
(0.26358 mL, 2.66 mmol) in 100 mL of EtOH, ammonia
solution (2 mL) was added dropwise. The mixture was
stirred at rt for 10 min, dil 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. The product 5 was separated by
silica gel column chromatography with n-hexane/EtOAc:
brown oil (31%); 1H NMR (CDCl3) d 2.01 (s, 3H, CH3), 2.04
(s, 3H, CH3), 3.76 (s, 3H, OCH3), 5.25 (s, 1H, CH2). HRMS
calcd for C12H10ClNO4: 267.0299. Found: 267.0298.
11. General procedure for synthesis of methyl-2-cyano-2-(-2-
arylthio-4,5-dimethyl-3,6-dioxocyclohexa-1,4-dienyl)ace-
tates. 6a–e: arylthiol (0.448 mmol) was added to the
solution of compound 5 (120 mg, 0.448 mmol) in 100 mL
of 95% EtOH and the mixture was stirred at rt or refluxed
for 4-5 h. The oily products were purified by silica gel
column chromatography.
In conclusion, methyl-2-(2-chloro-4,5-dimethyl-3,6-
dioxocyclohexa-1,4-dienyl)-2-cyanoacetate (5) was syn-
thesized by nucleophilic substitution of 2,3-dichloro-5,
6-dimethylcyclohexa-2,5-diene-1,4-dione (4) with equiva-
lent of methylcyanoacetate in the presence of ammonia.
Methyl-2-cyano-2-(2-arylthio-4,5-dimethyl-3,6-dioxocy-
clohexa-1,4-dienyl)acetates 6a–e were synthesized by
nucleophilic substitution of the compound 5 with
appropriate arylthiols. 2-Amino-7,8-dimethyl-6-hydrox-
ycinnolines 2a–f were synthesized by cyclization of
compound 5 or 6a–e with hydrazine hydrate. The 6-
hydroxycinnolines 2a–f showed, in general, a more
potent antifungal activity than the other compounds
3, 5, and 6. The results suggest that the 6-hydroxycinno-
lines would be potent antifungal agents. Moreover, the
results should encourage the synthesis of 6-hydroxycinn-
oline analogs for improving antifungal properties.
12. General procedure for synthesis of methyl-3-amino-5-chlo-
ro-7,8-dimethyl-6-hydroxycinnoline-4-carboxylates. 2a–f:
equivalent weight of hydrazine hydrate was added to the
solution of compounds 5 or 6a–e 300 mg in 100 mL of
95% EtOH and heated under reflux for 1 h. The product
was purified by silica gel column chromatography and
crystalized from 95% EtOH. Methyl-3-amino-5-chloro-7,8-
dimethyl-6-hydroxycinnoline-4-carboxylate. (2a): beige nee-
dle (46%); mp 220–221 ꢁ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); HRMS calcd for
C12H12ClN3O3: 281.0567. Found: 281.0568. Methyl-3-
amino-7,8-dimethyl-6-hydroxy-5-(phenylthio)-cinnoline-4-
Acknowledgment
This work was supported by Seoul Metropolitan
Government Grant 2005.
References and notes
1
1. Inoue, S.; Yasaki, A.; Mochizuki, H.; Tutsumi, H.,
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930824; Chem. Abstr. 120, 134503w; Tutsumi, H.; Teras-
awa, T.; Barret, D.; Murata, M.; Sakane, K.; Yazaki, A.;
Inoue, S. Jpn. Patent 9215584 A1 920217; Chem. Abstr.
118, 254944w; Yokomoto, M.; Yazaki, A.; Hayashi, N.;
Hatono, S.; Ioue, S.; Kuramoto, Y. Eur. Patent 4700578
A1 920212; Chem. Abstr. 117, 7943c.
carboxylate. (2b): beige needle (80%); mp 154–155 ꢁC; H
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); HRMS calcd for
C18H17N3O3S: 355.0991. Found: 355.0990. Methyl-3-ami-
no-7,8-dimethyl-6-hydroxy-5-(p-tolylthio)cinnoline-4-car-
boxylate. (2c): beige needle (71%); mp 142–143 ꢁC; 1H
NMR (CDCl3) d 2.25 (s, 3H, CH3), 2.26 (s, 3H, CH3), 2.34
(s, 3H, CH3), 3.60 (s, 3H, OCH3), 6.21 (s, 2H, NH), 6.94
(m, 2H, Ph), 6.99 (m, 2H, Ph), 7.38 (s, 1H, OH); HRMS
calcd for C19H19N3O3S: 369.1147. Found: 369.1148.
Methyl-3-amino-7,8-dimethyl-5-(3,4-dimethylphenylthio)-
6-hydroxycinnoline-4-carboxylate. (2d): beige needle (68%);
2. Coghlan, M. J.; Driekorn, B. A.; Suhr, R. G.; Jourdan, G.
P. Eur. Patent 0326328 A2 890802; Chem. Abstr. 112,
55907u.
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Quinonoid Compounds; Patak, S., Rappoport, Z., Eds.;
John Wiley & Sons: London, 1988; pp 1019–1066.
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6. Van Allan, J. A.; Priest, W. J.; Marshall, A. S.; Reynolds,
G. A. J. Org. Chem. 1968, 33, 1100.
1
mp 155–156 ꢁC; H NMR (CDCl3) d 2.15 (s, 6H, CH3),
2.26 (s, 3H, CH3), 2.34 (s, 3H, CH3), 3.61 (s, 3H, OCH3),
6.20 (s, 2H, NH), 6.74 (d, 1H, J = 8.0, Ph), 6.86 (m, 1H,
Ph), 6.93 (d, 1H, J = 8.0, Ph), 7.39 (s, 1H, OH); HRMS
calcd for C20H21N3O3S: 383.1304. Found: 383.1303.
Methyl-3-amino-7,8-dimethyl-5-(3-fluorophenylthio)-6-
hydroxycinnoline-4-carboxylate. (2e): beige needle (65%);
7. Gomaa, M. A.-M. Tetrahedron Lett. 2003, 44, 3493.
8. Experimental: All melting points were measured with
1
mp 148 ꢁC (dec); H NMR (CDCl3) d 2.26 (s, 3H, CH3),
Buchi melting point B-545 and are uncorrected. 1H
2.37 (s, 3H, CH3), 3.55 (s, 3H, OCH3), 6.18 (s, 2H, NH),
6.67 (m, 1H, Ph), 6.76 (m, 1H, Ph), 6.85 (m, 1H, Ph), 7.15
(m, 1H, Ph), 7.18 (s, 1H, OH); HRMS calcd for
C18H16FN3O3S: 369.1147. Found: 369.1148. Methyl-3-
amino-7,8-dimethyl-6-hydroxy-5-(m-tolylthio)cinnoline-4-
¨
NMR spectra were recorded on a Varian Unity INOVA
400 MHz FT-NMR spectrometer with TMS. High-
resolution mass spectra (HRMS EI) were taken with
Jeol JMS AX505 WA. 2,3-Dimethylbenzene-1,4-diol (3)
and other reagents were purchased from Aldrich
Chemical Co.
1
carboxylate. (2f): beige needle (86%); mp 142–143 ꢁC; H
NMR (CDCl3) d 2.24 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.32

C.-K. Ryu, J. Y. Lee / Bioorg. Med. Chem. Lett. 16 (2006) 1850–1853
1853
(s, 3H, CH3), 3.59 (s,3H, OCH3), 6.29 (s, 2H, NH), 6.82
(m, 1H, Ph), 6.86 (d, 2H, J = 8.0, Ph), 6.89 (m, 1H, Ph),
7.06 (t, 1H, J = 8.0, Ph), 7.38 (s, 1H, OH); HRMS calcd
for C19H19N3O3S: 369.1147. Found: 369.1149.
13. Mcginnis, M. R.; Rindali, M. G. In Antibiotics
in Laboratory Medicine; Lorian, V., Ed., 4th ed.;
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