Synthesis and evaluation of novel antifungal agents-quinoline and pyridine amide derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.06.005

Quinoline amide, azaindole amide and pyridine amides were synthesized and tested for in vitro antifungal activity against fungi. These synthesized amides have potent antifungal activity against Candida albicans and Aspergillus fumigatus. Our results sugge

Full text of DOI:10.1016/j.bmcl.2010.06.005

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4624–4626
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of novel antifungal agents-quinoline and
pyridine amide derivatives
a
a
a
a
Kazutaka Nakamoto a, , Itaru Tsukada , Keigo Tanaka , Masayuki Matsukura , Toru Haneda ,
Satoshi Inoue a, Norio Murai a, Shinya Abe a, Norihiro Ueda a, Mamiko Miyazaki a, Naoaki Watanabe a,
Makoto Asada b, Kentaro Yoshimatsu b, Katsura Hata a
*
a Discovery Research Laboratories, Eisai Co., Ltd, 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
b Eisai R&D Management Co., Ltd, 4-6-10 Koishikawa Bunkyo-ku, Tokyo 112-8088, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Quinoline amide, azaindole amide and pyridine amides were synthesized and tested for in vitro anti-
fungal activity against fungi. These synthesized amides have potent antifungal activity against Candida
albicans and Aspergillus fumigatus. Our results suggest that hetero ring amides may be potent antifungal
agents that operate by inhibiting the function of Gwt1 protein in the GPI biosynthetic pathway.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 4 March 2010
Revised 28 May 2010
Accepted 1 June 2010
Available online 8 June 2010
Keywords:
GPI-anchored protein
GWT1
Candia albicans
Aspergillus fumigatus
The overall frequency of fungal infections has continued to in-
crease in immunocompromised patients receiving immunosup-
pressive or anticancer therapy; the most common opportunistic
pathogen causing these infections continues to be Candida albicans.
The occurrence of infections due to non-C. albicans species is also
increasing in frequency and invasive pulmonary aspergillosis is a
leading cause of mortality in bone marrow transplant recipients.
Amphotericin B has been the most common treatment for infec-
tions due to these species, but newer therapies including azoles
and candins that are active against Candida and Aspergillus species
have recently been reported. Seven of these newer treatments
(fluconazole, itraconazole, voriconazole, posaconazole, caspofun-
gin, micafungin, and anidulafungin) are already in clinical use.
While these agents offer potential advantages, including reduced
toxicity and a broad therapeutic index, their activities are still lim-
ited. Resistance to the azole-class of drugs is increasing and can-
dins are not intrinsically active against Cryptococcus spp. as well
as some clinically important filamentous fungi. Thus, the develop-
ment of new drugs is still necessary in order to ensure successful
clinical treatment of opportunistic fungal infections.1–11
utilize their cell wall glucan-anchored mannoproteins to initiate
the binding to target tissues and begin the process of invasion.
GPI-anchored proteins are part of a highly conserved pathway that
exports, assembles, and anchors surface structures. In yeast and
other fungi, GPI-anchored proteins play important roles in cell wall
biosynthesis and maintenance of homeostasis. As GPI-anchored
proteins are one of the major cell wall components of eukaryotic
microorganisms, it may be possible to design drugs that target this
biosynthetic pathway in fungi without causing adverse effects in
human cells. Previously, we have reported that the GWT1 gene
product is a target of the novel antifungal derivative, 1-[4-butyl-
benzyl-isoquinoline] (1, BIQ), which inhibits cell wall localization
of GPI-anchored mannoproteins in Saccharomyces cerevisiae
(Fig. 1). Moreover, we have shown that compound 1 has moderate
antifungal activity against C. albicans (minimum inhibitory concen-
tration (MIC): 1.56 lg/mL). However, BIQ was not active against
Aspergillus fumigatus, one of the clinically important pathogenic
Glycosylphosphatidylinositols (GPIs) are glycolipids that play a
role in attaching cell surface proteins to eukaryotic plasma mem-
branes, including those in fungi. Fungal pathogens are thought to
* Corresponding author. Tel.: +81 029 847 5895; fax: +81 029 847 4012.
E-mail address: k3-nakamoto@hhc.eisai.co.jp (K. Nakamoto).
Figure 1. Antifungal 1-[butylbenzyl]isoquinoline.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.005

K. Nakamoto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4624–4626
4625
fungi, and metabolically unstable as a drug. In this study we used
several techniques to attempt to synthesize compounds with
greater antifungal activity than compound 1.12,13
We first carried out chemical modifications to increase the anti-
fungal activity of compound 1; however, we found no benzyliso-
quinolines that possessed more potent antifungal activity. Next,
we screened an in-house library of compounds in order to identify
a lead compound that had antifungal activity. As a result, we suc-
cessfully obtained an active nicotine amide derivative (MIC:
After completing the synthesis of quinoline amide 8, azaindole
amide 9, and pyridine amides 10a–d and 15, we tested their inhib-
itory activity on the growth of key pathogenic fungi. The MIC val-
ues of these compounds were comparable to the most commonly
used antifungal agents fluconazole and amphotericin B in a stan-
dard in vitro growth inhibition assay (Table 1).15 The synthesized
quinoline amide 8 and azaindole amide 9, as well as compounds
10a–d and 15, showed potent antifungal activity against C. albi-
cans. In addition, compounds 8, 9, 10b–d, and 15 had more potent
antifungal activity than compound 1. The effectiveness of these
3.13 lg/mL: C. albicans) with a structure different from benzyliso-
quinoline. Finally, we synthesized quinoline amides and pyridine
amides, and tested their antifungal activities against C. albicans
and A. fumigatus.
In this study, we report new quinoline, azaindole, and pyridine
amide derivatives (8, 9, 10a–d and 15) that show potent antifungal
activity against C. albicans and A. fumigatus that act by inhibiting
the function of the Gwt1 protein in an early step of the GPI biosyn-
thetic pathway.
compounds (MIC: 0.05–0.78 lg/mL) was 2–31-fold greater than
that of amphotericin B, one of the most commonly prescribed anti-
fungal treatments; compounds 8, 10b, and 15 also had more potent
activity than fluconazole. Compounds 8, 9, 10b–d, and 15 showed
in vitro antifungal activity against A. fumigatus. The in vitro anti-
fungal activity of compounds 10b, 10d, and 15 against A. fumigatus
was the same as that of amphotericin B.
We tested the antifungal activities of most of these compounds
against a GWT1-overexpressing strain of S. cerevisiae (KE744)
(Table 2).15 The MICs of these compounds for the KE744 strain
were at least eightfold higher than those for a control strain
(KE743). Thus, the overexpression of GWT1 conferred resistance
to these compounds and was not associated with cross-resistance
to amphotericin B or fluconazole. These results support our
hypothesis that GWT1 is the target molecule for these compounds.
In conclusion, quinoline amide 8, azaindole amide 9, and pyri-
dine amides 10a–d and 15 were synthesized by coupling quinoline
carboxylic acid, azaindole carboxylic acid, and amino nicotinic acid
with benzylthiophene amine and phenoxythiophene amines.
Among those tested, compounds 8, 9, 10b–d, and 15 show potent
antifungal activity against C. albicans and A. fumigatus. This is the
first report on amides that target the biosynthetic pathway of
GPI-anchored proteins. These amide derivatives may be promising
leads for the development of new antifungal agents. Moreover,
these results suggest that the synthesis of amide analogs could
provide antifungal properties that might prove to be better than
the currently approved therapies.
We synthesized target molecules 8, 9, 10a–d, and 15 by using
coupling reactions of amines 4 and 12 with carboxylic acids 5, 6,
and 7a–c. The synthesis of amine 4 began with 5-nitro-2-thioph-
enecarbonitrile (Scheme 1).14 Subsequent treatment of com-
pound
2
with substituted phenols afforded substituted
phenoxythiophenes 3. Reduction of the nitrile functionality of
compound 3 with LAH in THF gave the alkylamine 4. We then
prepared the amidemethylene compounds 8, 9, and 10a–d by
using coupling reactions of amine 4 with carboxylic acids 5, 6,
and 7a–c by using BOP reagent as the coupling agent in DMF
at room temperature.
Alternatively, we synthesized benzylthiophene 14 from
1-bromo-5-(1,-dioxolan-2-yl)thiophene (Scheme 2). First, a cou-
pling reaction of bromothiophene 11 with benzylbromide in the
presence of n-BuLi in THF at À78 °C resulted in compound 12,
and subsequent deprotection with citric acid provided the corre-
sponding aldehyde 13. Hydrogenation of aldehyde 13 over Raney
Ni in aqueous NH3 resulted in amine 14. Finally, amide formation
between 14 and 7b resulted in amidemethylene compound 15.
Scheme 1. Synthesis of phenoxythiophene amide derivatives.

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K. Nakamoto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4624–4626
Scheme 2. Synthesis of benzylthiophene amide derivative.
Hata, K.; Kimura, J.; Miki, H.; Toyosawa, T.; Moriyama, M.; Katsu, K. Antimicrob.
Table 1
Agents Chemother. 1996, 40, 2243.
Antifungal activity of 8, 9, 10a–d and 15 against C. albicans and A. fumigatus
9. (a) Tawara, S.; Ikeda, F.; Maki, K.; Morishita, Y.; Otomo, K.; Teratani, N.; Goto, T.;
Tomishima, M.; Ohki, H.; Yamada, A.; Kawabata, K.; Takasugi, H.; Sakane, K.;
Tanaka, H.; Matsumoto, F.; Kuwahara, S. Antimicrob. Agents Chemother. 2000,
44, 57; (b) Ikeda, F.; Wakai, Y.; Matsumoto, S.; Maki, K.; Watanabe, E.; Tawara,
S.; Goto, T.; Watanabe, Y.; Matsumoto, F.; Kuwahara, S. Antimicrob. Agents
Chemother. 2000, 44, 614.
Compd
MIC (lg/ml)
C. albicans
A. fumigatus
8
9
10a
10b
10c
10d
15
1
0.1
1.56
3.13
N.T.
0.78
1.56
0.78
0.78
N.T.
N.T.
0.78
0.39
1.56
0.05
0.78
0.39
0.05
1.56
0.39
1.56
10. Kapteyn, J. C.; Van Den Ende, H.; Klis, F. M. Biochim. Biophys. Acta 1999, 1426,
373.
11. Mayor, S.; Riezman, H. Nat. Rev. Mol. Cell Biol. 2004, 5, 110.
12. Tsukahara, K.; Hata, K.; Nakamoto, K.; Sagane, K.; Watanabe, N.; Kuromitsu, J.;
Kai, J.; Tsuchiya, M.; Ohba, F.; Jigami, Y.; Yoshimatsu, K.; Nagasu, T. Mol.
Microbiol. 2003, 48, 1029; Tsukahara, K.; Hata, K.; Sagane, K.; Nakamoto, K.;
Tsuchiya, M.; Watanabe, N.; Oba, F.; Tsukada, I.; Ueda, N.; Tanaka, K.; Kai, J. WO
02/04626, 2002.
Fluconazole
Amphotericin B
13. Umemura, M.; Okamoto, M.; Nakayama, K.; Sagane, K.; Tsukahara, K.; Hata, K.;
Jigami, Y. J. Biol. Chem. 2003, 278, 23639.
14. (a) Rivaille, P.; Gautron, J. P.; Castro, B.; Milhaud, G. Tetrahedron 1980, 36, 3413;
(b) Hudson, D. J. Org. Chem. 1988, 53, 617; (c) Wong, B. B.; Magahmi, N.;
Goodlin, V. L.; Smith, P. J. Bioorg. Med. Chem. Lett. 2004, 14, 3221; (d) Nakamoto,
K.; Tsukada, I.; Tanaka, K.; Matsukura, M.; Haneda, T.; Inoue, S.; Ueda, N.; Abe,
S.; Hata, K.; Watanabe, N. WO 05/033079, 2005.; (e) Nakamoto, K.; Inoue, S.;
Tanaka, K.; Haneda, T. WO 06/106711, 2006.
15. In vitro susceptibility tests: Candida albicans E81022, Aspergillus fumigatus
Tsukuba and Saccharomyces cerevisiae W303-1A were used in this study. S.
cerevisiae W303-1A was transformed with empty vector (strain KE743) or
multi-copy vector Yep352GAPII-GWT1, in which S. cerevisiae GWT1 was
overexpressed in the presence of GAPDH promoter (strain KE744). The MICs
were determined with the broth-microdilution method developed by the
Clinical and Laboratory Standards Institute and reported in documents M27-A3
and M38-A2 with slight modification. The test compounds were dissolved in
dimethyl sulfoxide (DMSO) to yield a concentration of 20 mg/mL and further
serially twofold diluted with DMSO and test media to achieve a range of final
Table 2
Antifungal activity of 8,
S. cervisiae
9 and 10a–d against GWT1-overexpressing strain of
Compd
MIC (
l
g/ml)
Degree of
resistance
S. cerevisiae KE743
S. cerevisiae KE744
8
9
10a
10b
10c
10d
0.025
0.2
1.56
0.1
1.56
0.78
6.25
0.78
3.13
128
>64
>8
128
>8
16
1
>12.5
>12.5
12.5
>12.5
12.5
Fluconazole
Amphotericin B
6.25
0.78
concentrations from 200 to 0.05 lg/mL. The C. albicans strain was subcultured
1
in Sabouraud dextrose broth medium (SDB; Becton, Dickinson and Company,
Sparks, MD) at 35 °C for 1–2 days. The A. fumigatus strain was subcultured on
potato dextrose agar (PDA; Eiken Chemical Co., Tokyo, Japan) at 35 °C for one
week and the collected conidia was stored at À80 °C before testing. The strains
of S. cerevisiae KE743 and S. cerevisiae KE744 were subcultured in yeast
nitrogen base medium (SD ura-; Becton, Dickinson and Company) at 30 °C for
References and notes
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