Synthesis and antifungal evaluation of 7-arylamino-5,8-dioxo-5,8- dihydroisoquinoline-4-carboxylates

Article abstract of DOI:10.1016/j.bmcl.2013.01.130

7-Arylamino-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylates were synthesized and tested for in vitro antifungal activity against two pathogenic strains of fungi. Most of tested compounds showed good antifungal activity. The results suggest that those 5,8

Full text of DOI:10.1016/j.bmcl.2013.01.130

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2065–2068
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antifungal evaluation of 7-arylamino-5,8-dioxo-5,8-
dihydroisoquinoline-4-carboxylates
⇑
Chung-Kyu Ryu , Ae Li Song, Jung An Hong
College of Pharmacy & Division of Life and Pharmaceutical Sciences, Ewha Womans University, 52 Ewhayeodae-gil, 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:
7-Arylamino-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylates were synthesized and tested for in vitro
antifungal activity against two pathogenic strains of fungi. Most of tested compounds showed good anti-
fungal activity. The results suggest that those 5,8-dioxo-5,8-dihydroisoquinolines would be potent anti-
fungal agents.
Received 17 December 2012
Revised 25 January 2013
Accepted 31 January 2013
Available online 13 February 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
5,8-Dioxo-5,8-dihydroisoquinoline-4-
carboxylates
Antimicrobial compounds
Antifungal
Fungi
Substitution effects
Heterocyclic quinonoid scaffolds represent an important class
of biologically active molecules.1 5,8-Quinolinedione derivative, a
heterocyclic quinone, inhibits cytochrome B-complex by the block-
ade of mitochondrial electron transport in Saccaromyces cerevisiae,
which is different from commonly used antifungal drugs.2 In our
previous reports,3,4 6-arylamino-quinoline-5,8-diones 1 and 6-
arylamino-phthalazine-5,8-diones 2 have demonstrated potent
antifungal activity against pathogenic fungi (Fig. 1).
genic fungi has not been reported to the best of our knowledge.
Therefore, 5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylates
with various substituents were designed and synthesized to eluci-
date their contribution to the antifungal activity.
3
O
O
O
R1
H
N
H
N
R1
R2
Structure–activity relationship studies from quinonoid com-
pounds indicated that the position and number of nitrogen (N)
atoms substituted in the heterocyclic ring with various substitu-
ents were considerably important factors to affect the biological
activities.4,5 We speculated that incorporation of nitrogen atom
into the ring of the quinone skeleton with various substituents
would change the physicochemical properties and lead to a new
pharmacophore with a different biological profile from quinone
compounds. The presence of arlyamino, arylthio or halo moiety
to the quinones affects their antifungal activity.6–8 Based on this
speculation, 7-arylamino- and 7-arylthio-1,3-dimethyl-5,8-dioxo-
5,8-dihydroisoquinolines 3 which would be bioisosteres or ana-
logues of quinones 1 and 2, were synthesized and evaluated for
their antifungal activity.
R2
R3
N
N
X
N
X
O
1
2
3
1
2
1 : R , R , R = H, F, ..
2 : R , R = H, F, ..
X = Cl, Br or H
X = Cl or H
O
Y
R1
R2
N
X
O
O
O
There have been a few reports9,10 on 5,8-dioxo-5,8-dihydroiso-
quinolines exhibiting cytotoxic activity10 against cancer cell lines.
However, the antifungal activity of compounds 3 against patho-
1
2
3 : R , R = H, F, ..
X = Cl, Br or H
Y = NH or S
⇑
Corresponding author.
E-mail address: ckryu@ewha.ac.kr (C.-K. Ryu).
Figure 1. Heterocyclic quinone derivatives.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.130

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C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2065–2068
Table 1
Structures and in vitro antifungal activity for 5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylates
O
X
R1
R2
N
Y
O
O
O
Compound
X
Y
R1
R2
MICa
(l
g/mL)
C. neoformans
b
C. albicans
C. tropicalis
C. krusei
A. niger
A. flavus
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
S
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
H
H
H
H
H
H
H
H
F
H
H
H
H
H
F
H
H
H
H
H
CH3
—
—
—
—
CH3
CH3O
F
CN
Cl
Br
I
F
CH3
CH3O
F
Cl
Br
F
CH3
I
1.6
1.6
1.6
1.6
1.6
1.6
6.3
1.6
6.3
0.8
6.3
25.0
1.6
6.3
25.0
12.5
12.5
6.3
25.0
25.0
50.0
25.0
50.0
3.2
0.8
12.5
1.6
12.5
6.3
12.5
6.3
25.0
1.6
12.5
6.3
6.3
1.6
1.6
25.0
12.5
12.5
6.3
25.0
12.5
50.0
12.5
6.3
3.2
12.5
6.3
12.5
12.5
6.3
12.5
6.3
6.3
6.3
3.2
6.3
1.6
12.5
3.2
3.2
12.5
6.3
1.6
6.3
12.5
12.5
12.5
12.5
6.3
12.5
1.6
3.2
1.6
3.2
1.6
6.3
3.2
1.6
1.6
1.6
6.3
1.6
6.3
12.5
1.6
6.3
25.0
12.5
6.3
6.3
6.3
25.0
6.3
6.3
6.3
1.6
6.3
25.0
6.3
25.0
12.5
12.5
12.5
3.2
12.5
25.0
12.5
12.5
3.2
6.3
6.3
100
50
25.0
6.3
1.6
12.5
25.0
12.5
25.0
25.0
12.5
12.5
12.5
6.3
12.5
100.0
50.0
25.0
6.3
H
H
H
H
3q
3r
3s
3t
4
5
Br
CH3
OH
CH3
—
—
—
S
S
—
—
—
—
H
6.3
6.3
—
—
—
—
50.0
50.0
6.3
>100
>100
25.0
6.3
Fluconazole
5-FCc
—
3.2
12.5
a
The MIC value is defined as lowest concentration of the antifungal agent exhibiting no fungal growth. MIC values were read after 1 day for Candida species and C.
neoformans, and 2 days for A. niger, A. flavus in 37 °C. The inoculum sizes contained approximately 1 Â 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, Aspergillus
niger KCTC 1231 and A. flavus KCCM 11899
c
5-FC: 5-fluorocytosine
O
O
O
O
O
OH
X
X
NH O
2
N
O
b or c
a
N
O
+
O
OH
O
O
6a : X = Cl
6b : X = Br
4
5
1
1
R
R
2
2
d
d or e
H N
R
HX
R
2
O
H
O
1
2
1
2
N
R
X
R
N
N
O
X
R
R
O
O
O
O
O
3a-n
X = Cl or Br
R , R =H, Cl, ...
3o-t
X = NH or S
R , R = H, Cl, ...
1
2
1
2
Scheme 1. Synthesis of 5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylates. Reagents and conditions: (a) methyl 3-aminocrotonate (1 equiv)/MgSO4/CH2Cl2/Ag2O (4 equiv)/
rt/3 h/72%; (b) 6a: c-HCl/c-HNO3/reflux/0.5 h/79%; (c) 6b: NaAc/AcOH/Br2/rt/24 h/85%; (d) arylamine (1 equiv)/EtOH/50 °C/30 min/49–91%; (e) arylthiol (1 equiv)/EtOH/24
h/rt/59–76%.
A method for the synthesis of 5,8-dioxo-5,8-dihydroisoquino-
line-4-carboxylates 3a–t (Table 1) is shown in Scheme 1. Methyl
1,3-dimethyl-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylate (5)
was prepared from commercially available 1-(2,5-dihydroxy-
phenyl)ethanone (4) and methyl 3-aminocrotonate through its
cyclization to methyl 5,8-dihydroxy-1,3-dimethylisoquinoline-4-
carboxylate followed by the oxidation with Ag2O according to
the reported method10 with minor modification.
Methyl
6,7-dichloro-1,3-dimethyl-5,8-dioxo-5,8-dihydroiso-
quinoline-4-carboxylate (6a) was synthesized by chlorination of

C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2065–2068
2067
compound 5 with HNO3/HCl variation. Also, 6,7-dibromo-1,3-di-
methyl-5,8-dioxo-5,8-dihydroisoquinoline 6b was synthesized by
bromination of compound 5 with Br2/NaAc in AcOH.
7-Arylamino-6-halo-5,8-dioxo-5,8-dihydroisoquinoline-4-car-
boxylates 3a–n were synthesized by regioselective nucleophilic
addition of compound 6a or 6b with appropriate arylamines and
subsequent oxidation. When compound 6a or 6b with 1 equiv
amount of arylamines in EtOH was refluxed for 4–12 h, compounds
3a–n were formed. Most of these additions went as expected and
had overall high yields of 49–91%.
line (6b) with equivalent of appropriate arylamines. 7-Arylthio-
5,8-dioxo-5,8-dihydroisoquinolines 3r–t were synthesized by
nucleophilic substitution on 5,8-dioxo-5,8-dihydroisoquinolinate
5 with equivalent of arylthiols. All tested compounds 3a–t showed
potent antifungal activity against tested fungi. These 5,8-dioxo-
5,8-dihydroisoquinoline scaffolds may thus be a promising lead
for the development of antifungal agents. Moreover, the results
would encourage the synthesis of 5,8-dioxo-5,8-dihydroisoquino-
line analogs for improving antifungal properties.
In a similar manner, 7-arylamino-5,8-dioxo-5,8-dihydroiso-
quinoline-4-carboxylates 3o–q were synthesized by nucleophilic
addition of compound 5 with arylamines in overall high yields of
63–86%.
Acknowledgment
This work was supported by Seoul R&BD Program (10688).
To prepare 7-arylthio-5,8-dioxo-5,8-dihydroisoquinoline-4-car-
boxylates 3r–t by nucleophilic addition of arylthiol and to avoid a
further nucleophilic addition of arylthiol to 5, 1 equiv amount of
appropriate arylthiol was treated with compound 5 in overall high
yields of 59–76%.
References and notes
1. Middleton, R. W.; Parrick, J. In The Chemistry of the Quinonoid Compounds; Patak,
S., Rappoport, Z., Eds.; John Wiley & Sons: London, 1988; p 1019.
2. Roberts, H.; Choo, W. M.; Smith, S. C.; Mrzuki, S.; Linnane, A. W.; Porter, T. H.;
Folkers, K. Arch. Biochem. Biophys. 1978, 191, 306.
3. Ryu, C.-K.; Sun, Y. J.; Shim, J. Y.; You, H. J.; Choi, K. U.; Lee, C. O. Arch. Pharm. Res.
2002, 25, 784.
4. Ryu, C.-K.; Park, R.-E.; Ma, M.-Y.; Nho, J.-H. Bioorg. Med. Chem. Lett. 2007, 17,
2577.
5. Shaikh, I. A.; Johnson, F.; Grollman, A. P. J. Med. Chem. 1986, 29, 1333.
6. Tandon, V. K.; Maurya, H. K.; Tripathi, A.; ShivaKesva, G. B.; Shukla, P. K.;
Srivastava, A.; Panda, D. Eur. J. Med. Chem. 2009, 44, 1086.
7. Tandon, V. K.; Chhor, R. B.; Singh, R. V.; Rai, S.; Yadav, D. B. Bioorg. Med. Chem.
Lett. 2004, 14, 1079.
8. Ryu, C.-K.; Choi, K. U.; Shim, J.-Y.; Chae, M. J.; Choi, I. H.; Han, J.-Y.; Jung, O.-J.;
Lee, J. Y.; Jeong, S. H. Eur. J. Med. Chem. 2005, 40, 438.
9. Valderrama, J. A.; González, M. F.; Pessoa-Mahana, D.; Tapia, R. A.; Fillion, H.;
Pautet, F.; Rodriguez, J. A.; Theoduloz, C.; Schmeda-Hirschmann, G. Bioorg. Med.
Chem. 2006, 14, 5003.
10. Vallderrama, J. A.; Arancibia, V.; Rodriguez, J.; Theoduloz, C. Bioorg. Med. Chem.
2009, 17, 2894.
The experimental details and data of representative compounds
3 were cited in Refs. 11–13.
The synthesized 5,8-dioxo-5,8-dihydroisoquinolines 3a–t were
tested in vitro for their growth inhibitory activity against patho-
genic fungi by the twofold broth dilution method.14 The MIC (min-
imum inhibitory concentration) values were determined by
comparison with fluconazole and 5-fluorocytosine as standard
agents. 5,8-Dioxo-5,8-dihydroisoquinoline derivative 3 as a hetero-
cyclic quinone could be an inhibitor of cytochrome B-complex by
the blockade of mitochondrial electron transport in fungi. How-
ever, 5-fluorocytosine is an inhibitor of biosynthesis of both RNA
and DNA, incorporation of 5-fluorouracil into RNA, inhibition of
ribosomal protein synthesis.15
11. Experimental: All melting points were measured with Büchi melting point B-
545 and were uncorrected. 1H NMR spectra or 13NMR spectra were recorded on
Varian Unity INOVA 400 MHz FT-NMR spectrometer using CDCl3 with TMS.
HRMS spectra were recorded with a Agilent 6220 accurate-mass TOF/LC-MS
equipped with an electrospray ionisation ion source used. Mass spectra were
taken with Jeol JMS AX505 WA. Methyl 1,3-dimethyl-5,8-dioxo-5,8-
dihydroisoquinoline-4-carboxylate (5) was prepared from commercially
As indicated in the Table 1, most of 5,8-dioxo-5,8-dihydroiso-
quinolines 3a–t generally showed potent antifungal activity
against all tested fungi. The compounds 3a–t completely inhibited
the growth of all fungal species tested at the MIC level of 0.8–25
lg/mL and were superior or comparable to those of fluconazole
available 1-(2,5-dihydroxyphenyl)ethanone (4) and methyl 3-aminocrotonate
according to the reported method
against all tested fungi.
10
.
Actually, the activity of compounds 3a, 3c and 3m was superior
or comparable to those of 5-fluorocytosine against all tested fungi.
The compounds 3a, 3c and 3m completely inhibited the growth of
12. Synthesis of methyl 6,7-dichloro-5,8-dihydro-1,3-dimethyl-5,8-dioxoisoquinoline-
4-carboxylate (6a): 2.5 mL of c-HNO3 was added dropwise to compound 5
(0.50 g, 1.9 mmol) dissolved in 50 mL of c-HCl at 80–90 °C for 30 min. The
solution was extracted with 100 mL of CH2Cl2 for three times. The organic layer
was concentrated in vacuo. The residue was purified by column
chromatography (silica gel, EtOAc/hexane = 1:5) to afford the compound 6a:
pale yellow crystal (50%); mp 139–140 °C; 1H NMR (CDCl3) d 3.03 (s, 3H, CH3),
3.04 (s, 3H, CH3), 4.02 (s, 3H, OCH3); HRMS Calcd for chemical formula:
Candida albicans and Aspergillus niger at the MIC level of 1.6 lg/mL.
In terms of structure–activity relationship, the 7-arylamino-6-
halo-5,8-dioxo-5,8-dihydroisoquinolines 3a–n showed, in general,
a more potent antifungal activity than the other 7-arylamino-5,8-
dioxo-5,8-dihydroisoquinolines 3o–q. The activity of 7-arylthio-
5,8-dioxo-5,8-dihydroisoquinolines 3r–t was comparable to that
of 7-arylamino-5,8-dioxo-5,8-dihydroisoquinolines 3a–q. Thus,
the 7-arylamino- and 7-arylthio-moiety of compounds 3a–t appear
to be important factor to affect their antifungal activity. The 7-
arylamino-compounds 3a–n exhibited good activity, indicating a
correlation that may offer insight into the mode of action of these
compounds. The substituents (R1, R2: H, F, Cl, etc.) for the 7-aryla-
mino and 7-arylthio moieties of compounds 3a–t may contribute
partially toward biological potency.
In addition, 1-(2,5-dihydroxyphenyl)ethanone (4) and methyl
1,3-dimethyl-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylate (5)
exhibited no or poor, if any, antifungal activity. The 5,8-dioxo-
5,8-dihydroisoquinolines 3 showed, in general, more potent anti-
fungal activity than compounds 4 and 5. Thus, the 7-arylamino-
and 7-arylthio-moiety in compound 3 could be important factor
for the activity, for example, as compound 4 lost their activity.
In conclusion, 7-arylamino-5,8-dioxo-5,8-dihydroisoquinolines
3a–q were synthesized by nucleophilic substitution of 5,8-dioxo-
5,8-dihydroisoquinolinate 5, 6,7-dichloro-5,8-dioxo-5,8-dihydro-
isoquinoline (6a) and 6,7-dibromo-5,8-dioxo-5,8-dihydroisoquino-
C
13H9Cl2NO4 313.9988; Found: 313.9986 [(M+H)+]. Synthesis of methyl 6,7-
dibromo-5,8-dihydro-1,3-dimethyl-5,8-dioxoisoquinoline-4-carboxylate (6a):
Compound 5 (0.25 g, 1 mmol) and 0.66 g of NaOAc were dissolved in 8 mL of
acetic acid and stirred at rt for 10 min. The mixture was treated with 0.1 mL of
bromine and stirred at rt for 24 h. 250 mL of water was added to the reaction
mixture. After 5 min, the solid residue was filtered and purified by column
chromatography (silica gel, EtOAc/CHCl3 = 1:2.5) to afford compound 6a:
yellow powder (91%); mp 143–145 °C; 1H NMR (CDCl3) d 3.03 (s, 3H, CH3), 3.03
(s, 3H, CH3), 4.06 (s, 3H, OCH3); HRMS Calcd for chemical formula: C13H9BrNO4
401.8977; Found: 401.8979 [(M+H)+].
13. General procedure for synthesis of 7-arylamino-6-halo-5,8-dioxo-5,8-
dihydroisoquinoline-4-carboxylates 3: appropriate arylamine (1.39 mmol) was
added to a solution of compound 6a or 6b (0.05 mol) in EtOH (100 mL). The
mixture was stirred at 50 °C for 30 min. The mixture was evaporated in vacuo
and the products 3 were purified by silica gel column chromatography and
crystallized from EtOH. Methyl 7-(p-tolylamino)-6-chloro-5,8-dihydro-1,3-
dimethyl-5,8-dioxoisoquinoline-4-carboxylate (3a): purple powder (78%); mp
163–164 °C; 1H NMR (CDCl3) d 2.37 (s, 3H, CH3), 2.39 (s, 3H, CH3), 2.99 (s, 3H,
CH3), 4.03 (s, 3H, OCH3), 7.00 (d, 1H, J = 8.4), 7.00 (d, 1H, J = 8.4), 7.18 (d, 1H,
J = 8.0), 7.18 (d, 1H, J = 8.0), 7.83 (s, 1H, NH); HRMS Calcd for chemical formula:
C
20H18ClN2O4 385.0956; Found: 385.0958 [(M+H)+]. Methyl 7-(4-
methoxyphenylamino)-6-chloro-5,8-dihydro-1,3-dimethyl-5,8-dioxoisoquinoline-
4-carboxylate (3b): purple powder (76%); mp 122–123 °C; 1H NMR (CDCl3) d
2.98 (s, 3H, CH3), 2.99 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 6.90
(d, 1H, J = 8.8), 6.90 (d, 1H, J = 8.8), 7.05 (d, 1H, J = 8.4), 7.05 (d, 1H, J = 8.4), 7.81
(s, 1H, NH); HRMS Calcd for chemical formula: C20H18ClN2O5 401.0905; Found:
401.0907 [(M+H)+]. Methyl 7-(4-fluorophenylamino)-6-chloro-5,8-dihydro-1,3-
dimethyl-5,8-dioxoisoquinoline-4-carboxylate (3c): red powder (52%); mp 181–

2068
C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2065–2068
182 °C; 1H NMR (CDCl3) d 3.00 (s, 3H, CH3), 3.10 (s, 3H, CH3), 4.03 (s, 3H, OCH3),
5.30 (s, 1H, NH), 7.08 (d, 1H, J = 8.8), 7.08 (d, 1H, J = 8.8), 7.78 (d, 1H, J = 8.4),
7.78 (d, 1H, J = 8.4); HRMS Calcd for chemical formula: 19H15ClFN2O4
3H, OCH3), 6.89 (d, 1H, J = 8.8), 6.89 (d, 1H, J = 8.8), 7.06 (d, 1H, J = 8.4), 7.06 (d,
1H, J = 8.4), 7.92 (s, 1H, NH); HRMS Calcd for chemical formula: C20H18BrN2O5
445.0400; Found: 445.0403 [(M+H)+]. Methyl 7-(4-bromophenylamino)-6-
bromo-5,8-dihydro-1,3-dimethyl-5,8-dioxoisoquinoline-4-carboxylate (3m): red
powder (78%); mp 185–186 °C; 1H NMR (CDCl3) d 3.00 (s, 3H, CH3), 3.03 (s,
3H, CH3), 4.05 (s, 3H, OCH3), 6.99 (m, 1H), 6.99 (m, 1H), 7.50 (m, 1H), 7.50 (m,
C
389.0705; Found: 389.0706 [(M+H)+]. Methyl 7-(p-tolylamino)-6-bromo-5,8-
dihydro-1,3-dimethyl-5,8-dioxoisoquinoline-4-carboxylate (3i): red powder
(80%); mp 153–156 °C; 1H NMR (CDCl3) d 2.37 (s, 3H, CH3), 2.38 (s, 3H, CH3),
2.99 (s, 3H, CH3), 4.05 (s, 3H, OCH3), 4.56 (s, 1H, NH), 7.00 (d, 1H, J = 8.4), 7.00
(d, 1H, J = 8.4), 7.17 (d, 1H, J = 8.0), 7.17 (d, 1H, J = 8.0); HRMS Calcd for
chemical formula: C20H18BrN2O4 429.0451; Found: 429.0450 [(M+H)+]. Methyl
7-(4-methoxyphenylamino)-6-bromo-5,8-dihydro-1,3-dimethyl-5,8-
1H), 7.84 (s, 1H, NH); HRMS Calcd for chemical formula:
C19H15Br2N2O4
492.9399; Found: 492.9397 [(M+H)+].
14. Mcginnis, M. R.; Rindali, M. G. In Antibiotics in Laboratory Medicine; Lorian, V.,
Ed., 4th ed.; Williams and Wilkins: Baltimore, 1996; pp 176–211.
15. Waldorf, A. R.; Polak, A. Antimicrob. Agents Chemother. 1983, 23, 79.
dioxoisoquinoline-4-carboxylate (3j): red powder (83%); mp 136–138 °C; 1H
NMR (CDCl3) d 2.98 (s, 3H, CH3), 2.99 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 4.05 (s,