Semisynthesis and antibacterial activities of nidulin derivatives

Article abstract of DOI:10.1038/s41429-018-0133-0

Derivatives of the fungal depsidone, nidulin, have been synthesized in order to evaluate the potential of the chemical skeleton as antibacterial agents. Alkylation, acylation, and arylation reactions of nornidulin underwent in a regioselective manner to p

Full text of DOI:10.1038/s41429-018-0133-0

The Journal of Antibiotics
https://doi.org/10.1038/s41429-018-0133-0
BRIEF COMMUNICATION
Semisynthesis and antibacterial activities of nidulin derivatives
Masahiko Isaka1 Arunrat Yangchum Sumalee Supothina Sukitaya Veeranondha Somjit Komwijit
1 ●
1 ●
1 ●
1 ●
●
2
Souwalak Phongpaichit
Received: 5 October 2018 / Revised: 22 November 2018 / Accepted: 23 November 2018
©
The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2018
Abstract
Derivatives of the fungal depsidone, nidulin, have been synthesized in order to evaluate the potential of the chemical
skeleton as antibacterial agents. Alkylation, acylation, and arylation reactions of nornidulin underwent in a regioselective
manner to predominantly produce 8-O-substituted derivatives. Many of the semisynthetic derivatives showed more potent
antibacterial activities than nidulin, In particular, 8-O-aryl ether derivatives displayed significant activities against Gram-
positive bacteria, including Methicillin-resistant Staphylococcus aureus.
Nidulin (1), nornidulin (2), and related depsidones are
secondary metabolites specifically produced by fermenta-
tion of the common fungi Aspergillus unguis and Asper-
gillus nidulans (Fig. 1). There have been several reports
regarding biological activities of nidulin and closely related
Aspergillus depsidones. Unguinol (2,4,7-trisdechloro-nor-
nidulin) was reported to be a potential herbicide candidate
Several years ago, we isolated nidulin, nornidulin and
three related depsidones from cultures of a mangrove-
derived fungus Halosarpheia kandeliae BCC 16551.
However, based on the checking of the culture collection
deposit of the strain, and the failure of the reproduction by
new fermentations, it was later concluded that these depsi-
dones were most likely produced due to the contamination
of Aspergillus unguis into a seed culture in the large-scale
fermentation process. Evaluation of biological activities of
the isolated depsidones revealed that nidulin exhibits
activity against Gram-positive bacteria, Bacillus cereus
due to its inhibitory activity of the C plant enzyme pyr-
4
uvate phosphate dikinase [1]. Unguinol and its derivatives
are claimed to be animal growth permittants in a US patent
[
2]. Kittakoop and co-workers reported aromatase inhibi-
−
1
tory, radical scavenging, and cytotoxic activities of nidulin
and co-metabolites, aspergillusidones, from A. unguis CRI-
(MIC 1.56 μg ml ) and Enterococcus faecium (MIC 3.13
−
1
μg ml ). Nornidulin showed weaker activity when com-
pared with nidulin. As preliminary study on structure-
activity relationship (SAR), we examined preparation of
3-O-methyl-nornidulin by alkylation of 2 with one equiva-
lent of MeI (K CO , DMF, room temperature) expecting the
2
82-03 [3, 4]. Recently, antibacterial, antifungal, anti-
malarial, and cytotoxic activities of depsidones and dep-
sides from A. unguis have been reported [5, 6]. During the
preparation of this paper, a report on isolation of many
depsidones from A. unguis and evaluations of their activities
against Gram-positive bacteria was published [7].
2
3
production of a mixture of 3-O-methyl-2 and 8-O-methyl-2
(nidulin, 1). Interestingly, the O-methylation underwent in a
highly regioselective manner giving only nidulin (1). 3-O-
Methyl-nidulin, prepared by O-methylation of 1 with excess
MeI, was inactive against these bacteria, which suggested
that 3-OH group is crucial for antibacterial activity. On the
basis of these preliminary results on SAR and regioselec-
tivity of O-alkylation, we had planned semisynthesis of
nidulin derivatives by selective alkylation of nornidulin 8-
OH to evaluate the potential of the skeleton as antibacterial
agents. We report here synthesis of various nidulin deriva-
tives and evaluation of their antibacterial activity.
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-018-0133-0) contains supplementary
material, which is available to authorized users.
*
Masahiko Isaka
isaka@biotec.or.th
1
National Center for Genetic Engineering and Biotechnology
(
BIOTEC), 113 Thailand Science Park, Phaholyothin Road,
Klong Luang, Pathumthani 12120, Thailand
2
As substrate for the semisynthesis, nornidulin was pro-
duced by fermentation of the commercial strain, A. unguis
ATCC 10032, which is a known to producer of nidulin
Department of Microbiology and Natural Products Research
Center of Excellence, Faculty of Science, Prince of Songkla
University, Hat Yai, Songkhla 90112, Thailand

M. Isaka et al.
Fig. 1 Structures of nidulin and nornidulin
derivatives. Fermentation using a common media, potato
dextrose broth (PDB), under static condition gave norni-
dulin but as a minor metabolite with unguinol as the major
product. This result was probably due to the lack of chloride
ion source in the liquid culture media. Similarly to the
fermentation of a marine-derived A. unguis CRI-282-03
[
3, 4], feeding NaCl worked fruitfully for ATCC 10032.
Thus, addition of 2% (w/v) NaCl in PDB resulted in the
enhanced production of nornidulin and nidulin as two major
metabolites, while production of unguinol and other
dechloro derivatives were reduced. Higher composition of
NaCl (5%) resulted in serious inhibition of the mycelial
growth. Gram-quantity of nornidulin was obtained using
this simply optimized fermentation conditions and chro-
matographic separation/purification.
Reaction of nornidulin with one equivalent of alkyl,
allylic, or benzylic halide (R-X; X = I, Br, Cl) and K CO
2
3
(
2 equiv) in DMF at room temperature underwent in highly
regioselective manner to give 8-O-substituted derivatives
3–17) (Fig. 2). Regioselectivity of the O-alkylation was
confirmed by the NOESY correlation between 9-CH and
(
3
1
13
H -1ʺ for compound 5, and by comparison of the H and
C
2
NMR spectroscopic data of the alkylated compounds with
those of nidulin and nornidulin. A dimeric nidulin derivative
1
8 was synthesized by the reaction of 2 with a half
equivalent of 1,4-diiodobutane. Nidulin derivatives with a
pyridine ring (19 and 20) or a hydroxyl group (21) were also
synthesized using the same procedure. A tert-butyl ester
derivative 22 was converted into a carboxylic acid deriva-
tive 23 by deprotection with trifluoroacetic acid. Two 8-O-
acylated derivatives, 24 and 25, were obtained by the
Fig. 2 Structures of the semisynthetic nidulin derivatives
reaction of 2 with acetic anhydride and benzoyl chloride,
respectively. Several nornidulin 8-O-aryl ethers (26–31)

Semisynthesis and antibacterial activities of nidulin derivatives
Table 1 Antibacterial activities and cytotoxicity of nidulin derivatives
Compound
Antibacterial activity (MIC90, μg ml−1)
Cytotoxicity (IC50, μg ml−1) Vero cellsa
B. cereusb
E. faeciumc
S. aureusd
MRSAe
Nidulin (1)
1.56
6.25
1.56
0.781
0.391
1.56
1.56
6.25
>25
3.13
6.25
3.13
1.56
3.13
0.781
1.56
25
6.25
12.5
3.13
3.13
3.13
6.25
25
4
26.5
19.2
32.8
6.8
Nornidulin (2)
NT
NT
NT
2
3
4
5
6
7
8
9
10.5
3.3
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
0.5
NT
1
9.0
>50
>50
6.25
3.13
3.13
3.13
6.25
3.13
6.25
25
10.3
48.6
3.4
>50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
0.781
0.781
0.781
1.56
0.781
1.56
0.781
0.781
6.25
1.56
6.25
6.25
0.781
>25
0.781
3.13
1.56
1.56
1.56
0.781
6.25
1.56
>50
7.1
8.1
5.3
5.9
5.1
2.2
9.0
50
32.9
7.4
6.25
50
6.25
50
36.9
41.6
3.1
12.5
3.13
>50
12.5
6.25
>50
12.5
>50
1.56
0.781
0.781
1.56
0.781
0.781
1.56
1.00
0.0781
–
>50
18.1
19.7
16.3
4.0
3.13
>25
6.25
>50
0.391
0.781
0.781
0.781
0.781
0.781
1.56
2.00
–
0.781
0.781
0.391
0.781
3.13
0.781
1.56
–
4.5
NT
0.5
0.5
NT
0.5
1.6
12.1
3.8
6.5
Vancomycin·HCl
Rifampicin
–
1.56
0.195
Tetracycline·HCl
Ellipticine
–
0.89
NT not tested
aAfrican green monkey kidney fibroblasts
bBacillus cereus ATCC 11778
cEnterococcus faecium ATCC 51559
dStaphylococcus aureus ATCC 29213
eMethicillin-resistant Staphylococcus aureus SK1
were synthesized by the reactions of 2 with 1 equivalent of
fluorobenzenes bearing an electron-withdrawing group
NOESY correlation between 9-CH3 and H-2ʺ/H-6ʺ for
1
13
compound 28, and by comparison of the H and C NMR
spectroscopic data of the O-arylated compounds with those
of nidulin and nornidulin. Similarly, a pyridyl ether
(
–NO or –CN) and K CO (2 equiv), heating at 60 °C.
2 2 3
Regioselectivity of the O-arylation was confirmed by the

M. Isaka et al.
derivative 32 was synthesized from 2 and 2-chloro-5-
nitropyridine.
discussions and valuable suggestions on antibacterial activities of
Aspergillus depsidones.
The semisynthetic nidulin derivatives were tested for
activities against Gram-positive bacteria, B. cereus ATCC
Compliance with ethical standards
1
1778, E. faecium ATCC 51559, and Staphylococcus aur-
Conflict of interest The authors declare that they have no conflict of
interest.
eus ATCC 29213 [8], and cytotoxicity to nonmalignant
Vero cells (African green monkey kidney fibroblasts) [9]
(
Table 1). Most of the 8-O-alkyl derivatives with short side-
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1
Acknowledgements Financial support from the Thailand Research
Fund (grant No. DBG5980002) is gratefully acknowledged. We thank
Professor Vatcharin Rukachaisirikul, Prince of Songkla University, for