Remarkable influence of the aromatic substructure in 9-methoxystrobilurin derivatives on their antifungal activity

Article abstract of DOI:10.1016/S0960-894X(02)00558-9

9-Methoxystrobilurin-type β-methoxyacrylate antibiotics (MOSBs) having various aromatic substructures were synthesized. The antifungal activity of the synthesized MOSBs against pathogenic and non-pathogenic fungi was examined, and the obtained results revealed that the antifungal activity of MOSBs was highly dependent on the aromatic substructures. However, no significant correlation was observed between cytotoxicity against human fibroblasts-like cell line and their structural properties. In addition, our results suggested that the strong growth-inhibitory activity of 9-methoxystrobilurin K against human-derived cell lines should be related to its hindered ether-type substructure.

Full text of DOI:10.1016/S0960-894X(02)00558-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2699–2702
Remarkable Influence of the Aromatic Substructure in
9-Methoxystrobilurin Derivatives on their Antifungal Activity
Hiromi Uchiro,a Koh Nagasawa,a Tomohiro Sawa,a Daiju Hasegawa,a Tomoya Kotake,a
-
Yoshitsugu Sugiura,a,y Susumu Kobayashi,a,* Kazuhiko Otogurob and Satoshi Omurac
aFaculty of Pharmaceutical Sciences, Tokyo University of Science, 12 Ichigayafunagawara-machi, Shinjuku-ku, Tokyo 162-0826, Japan
bResearch Center for Tropical Diseases, Research Center for Biological Function, The Kitasato Institute, 5-9-1 Shirokane, Minato-ku,
Tokyo 108-8642, Japan
cKitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan
Received 20 May 2002; accepted 5 July 2002
Abstract—9-Methoxystrobilurin-type b-methoxyacrylate antibiotics (MOSBs) having various aromatic substructures were synthe-
sized. The antifungal activity of the synthesized MOSBs against pathogenic and non-pathogenic fungi was examined, and the
obtained results revealed that the antifungal activity of MOSBs was highly dependent on the aromatic substructures. However, no
significant correlation was observed between cytotoxicity against human fibroblasts-like cell line and their structural properties. In
addition, our results suggested that the strong growth-inhibitory activity of 9-methoxystrobilurin K against human-derived cell
lines should be related to its hindered ether-type substructure.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
remarkable influence of the aromatic substructures on
their antifungal activity (Fig. 1).
Strobilurin A (1) was isolated from mycelia of basido-
mycete Strobilurus tenacellus as the first b-methoxy-
acrylate antibiotic (MOA) by Anke et al. in 1977.1 The
compound exhibits strong antifungal activity against
various fungi by binding to cytochrome bc1 (complex
III) in a mitochondrial respiration pathway. A certain
number of the same class of compounds have been dis-
covered and several artificial analogues are effectively
applied for agricultural fungicides.2 On the other hand,
9-methoxystrobilurin-type b-methoxyacrylates (MOSBs)
such as 9-methoxystrobilurin A (2) and K (3) were iso-
lated in 1995, and 3 indicated strong growth-inhibitory
activities toward typical human-derived tumor cell lines,
along with the original antifungal activities.3ꢀ5 Recently,
total syntheses of several natural-type MOSBs were
achieved in our laboratory6 and further studies are now
in progress to clarify the structure–activity relationships
of MOSBs. In this paper, we would like to describe an
efficient synthesis of several modified MOSBs and the
Figure 1. Strobilurin A and 9-methoxystrobilurins.
Molecular Design
According to the original report, 9-methoxystrobilurin
A (2) and K (3) indicated the same level of antifungal
activities; however, there is much greater difference in
their growth-inhibitory activities toward human-derived
tumor cell lines.3 Surprisingly, the IC50 value of 3 for
cell growth of HeLaS3 cells reached the order of nM,
*Corresponding author. Tel.: +81-3-3260-8848; fax: +81-3-3260-
8848; e-mail: kobayash@ps.kagu.sut.ac.jp
yPresent address: Department of Food Chemsitry, Kobe Institute of
Health, 4-6 Minatojima-nakamachi, Chuo-ku, Kobe 650–0046, Japan
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00558-9

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H. Uchiro et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2699–2702
which is 1000-fold more potent than 2. This remarkably
high growth-inhibitory activity of 3 is probably due to
its ‘more complicated’ aromatic substructure. There-
fore, several derivatives 4a–f modified on the aromatic
substructure of 2 were designed to clarify the structure-
activity relationships in these two kinds of bioactivities
(Fig. 2).
Scheme 1. Syntheic plan for 9-methoxystrobilurin derivatives.
methanol in the presence of trimethyl orthoformate and
a catalytic amount of p-toluenesulfonic acid for 1 h.
Further, the reaction mixtures were evaporated to
remove an excess of methanol and once more refluxed in
trimethyl orthoformate for 2 h. The desired dienolethers
8a–c, 8e and 8f were obtained in good yields except for
8d (8a: 80%; 8b: 85%; 8c: 88%; 8e: 72%; 8f: 81%) since
the methylenedioxy moiety of 7d did not survive in the
above acidic condition. Therefore, a stepwise enol ether
construction via the corresponding carboxylic acid 10d
in a similar manner to the synthesis of 36b was carried
out. The desired intermediate 8d was thus obtained in
83% yield from 7d. The prepared dienolethers 8a–f were
formylated by sodium hydride-methyl formate and suc-
cessively methylated with sodium carbonate-dimethyl
sulfate to afford a mixture of three geometrical isomers
9a–f (the mixture of 4a–f, 11a–f and 12a–f). The mixture
was effectively isomerized by ultraviolet lamp irradia-
tion (l: 365 nm), and the desired MOSBs 4a–f were iso-
lated along with an unseparable mixture of two other
isomers (11a–f, 12a–f). These undesired isomers were
again subjected to photoisomerization, and the com-
bined yields of 4a–f were as follows: (4a: 43%, 4b: 42%,
4c: 41%, 4d: 43%, 4e: 43%, 4f: 43%).7 At the same
time, the non-isomerized starting mixtures 9a–f were
recovered (9a: 43%, 9b: 42%, 9c: 41%, 9d: 43%, 9e:
43%, 9f: 43%).
Figure 2. Aromatic modified 9-methoxystrobilurin derivatives.
A comparison between their antifungal activities and
growth-inhibitory activities (as cytotoxicities) for mam-
malian normal-type cell would bring about valuable
pharmacostructural information for designing a novel
‘cell-specific’ growth inhibitor. In addition, during the
course of this study, it was also expected to clarify the
importance of hindered ether-type substituents on the
aromatic ring of 3.
Synthetic Plan
A general synthetic plan for modified MOSBs based on
our previous reports6 is shown in Scheme 1. A key step
in the efficient preparation of MOSBs is the Heck reac-
tion of aryl bromides 5 with vinylketone 6. The vinyl-
ketone 6 was already prepared in three steps from
commercially available monomethyl itaconate in high
yield.6a The resulting enone-type intermediates 7 would
lead to dienolether 8 by acetalization and successive
elimination of methanol under an acidic condition.
Further formylation at the a-position of 8 and sequen-
tial methylation of the resulting enolic hydroxyl group
would afford the corresponding b-methoxyacrylates as a
mixture of geometrical isomers 9. The isomers 9 would
be isomerized to the desired MOSBs 4 by ultraviolet
lamp irradiation.
Antifungal Activity
The antifungal activities of synthesized MOSBs against
several pathogenic and non-pathogenic fungi, which
were maintained by Tokyo University of Science, were
examined by a disk diffusion assay, and the obtained
data are summarized in Table 1. Interestingly, anti-
fungal activities of synthesized MOSBs were highly
dependent on their aromatic substructures. For exam-
ple, newly synthesized derivatives 4a–c bearing benzene-
type aromatic rings exhibited potent antifungal activ-
ities against Penicillium citrinum, while naphthalene
analogues 4e–f with larger aromatic substructures gave
less sensitivity. In addition, 9-methoxystrobilurin K (3)
having the largest aromatic moiety was almost inactive
against the same fungus. On the other hand, the com-
pounds 4a–d produced relatively large inhibition zones
toward Saccharomyces cerevisiae during 12–48 h incu-
bation; however, the zones became gradually obscure
and were lost after 96 h. In contrast, compound 4e–f
and 9-methoxystrobilurin K (3) evidently showed the
Synthesis
The Heck reaction of several aryl bromides 5a–f with
vinylketone 6 proceeded smoothly to give the corre-
sponding enone-type intermediates 7a–f in good yields
(7a: 80%; 7b: 85%; 7c: 79%; 7d: 83%; 7e: 72%; 7f:
81%). These intermediates 7a–f were refluxed with

H. Uchiro et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2699–2702
2701
inhibition against S. cerevisiae up to 96 h. A similar
observation was also found in the case of Candida albi-
cans, which is a typical yeast-type pathogenic fungus.
Further, it is noted that 9-methoxystrobilurin K (3) and
compound 4f were slightly active against Fusarium
solani which is known as one of the most drug-resistant
pathogenic fungi; however, their activity was insufficient
similarly to those of the compounds 4a–d against S.
cerevisiae. These results demonstrated that b-naphthyl-
type derivative 4f has the broadest antifungal spectrum,
and the geometrical modification of the aromatic
substructure is apparently responsible for their anti-
fungal properties (Scheme 2).
Cytotoxicity
The cytotoxicity of synthesized MOSBs toward a
human diploid embryotic cell line (MRC-5) was mea-
sured by the previously reported method8 and their IC50
Scheme 2. Synthesis of 9-methoxystrobilurin derivatives. Reaction conditions: (i) 10 mol% Pd(OAc)2, PPh3, Et3N, 100 ꢂC; (ii) 10 mol% p-TsOH,
HC(OMe)2, MeOH, reflux; (iii) 10 mol% p-TsOH, HC(OMe)3, reflux; (iv) NaOHaq–MeOH, rt then HCl; (v) KOtBu, Me2SO4, DMF, ꢀ45 to
ꢀ15 ꢂC; (vi) NaH, HCOOMe, rt; (vii) K2CO3, Me2SO4, HCOOMe, rt; (viii) hn (l: 365 nm), acetone-benzene, rt.
Table 1. Biologocal activities of 9-methoxystrobilurins
Antifungal activity
Cytotoxicity
IC50 (mM)
MRC-5
Compd
Diameter of inhibition zone (mm)a,b
Concn
(mg/disk)
Pencillium citrinum
(R-3703)
Aspergillus
fumigatus
(R-1301)
Fusarium solani
(R-2800)
Candida
albicans
(IFO1594)
Saccharomyces
cerevisiae
(IAM4861)
Nystatin
(positive control)
2
10
12
14
—
16
18
34
34
10
1
0.1
10
1
0.1
10
1
0.1
10
1
0.1
10
1
0.1
10
1
0.1
10
1
0.1
10
1
28
18
4
—
—
—
25
19
8
31
15
ꢁ
—
—
—
25i
19i
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
13i
14i
—
36i
28i
ꢁ
49i
40i
17i
25
20
ꢁ
3
10
8
22
17
—
32i
27i
ꢁ
0.36
27
ꢁ
4a
4b
4c
4d
4e
4f
23
9
35i
29i
16i
42i
36i
28i
43i
38i
20i
42i
36i
8i
—
29
18
4
29
23
9
33i
27i
ꢁ
35
22
13
ꢁ
20
ꢁ
34i
26i
ꢁ
12
—
22
ꢁ
18
10
ꢁ
32i
22i
ꢁ
>100
>100
>100
—
14
10
—
18
13
4
17
12
ꢁ
15
5
22
18
5
37
30
12
—
26
16
ꢁ
19
14
ꢁ
0.1
aThe diameter of each inhibition zone (mean value of two samples) was measured after 48 h incubation.
b—, not effective, ꢁ, slightly effective, i, incomplete inhibition.

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H. Uchiro et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2699–2702
values are listed in Table 1. No significant correlation
was observed between cytotoxicity and the structural
properties of synthesized MOSBs. It is noteworthy that,
among the examined MOSBs, b-naphthyl-type deriva-
tive 4f with the broadest antifungal spectrum showed
the lowest cytotoxicity. In contrast, 9-methoxy-
strobilurin K (3) inhibited the growth of MRC-5
cell in 30–500-fold lower concentration than other
MOSBs; however, the IC50 value was still 40–100-fold
higher than those previously reported for several
human-derived tumor cell lines. The strong growth-
inhibitory activity is probably due to the specific bind-
ing affinity of 3 to a target protein in the mammalian
cell, which is based on its hindered ether-type sidechain
moiety.
2. Sauter, H.; Steglich, W.; Anke, T. Angew. Chem., Int. Ed.
Engl. 1999, 38, 1328 and references cited therein.
3. Zapf, S.; Werle, A.; Anke, T.; Klostermeyer, D.; Steffan,
B.; Steglich, W. Angew. Chem., Int. Ed. Engl. 1995, 34, 196.
4. Wood, K. A.; Kau, D. A.; Wrigley, S. K.; Beneyto, R.;
Renno, D. V.; Ainsworth, A. M.; Penn, J.; Hill, D.; Killacky,
J.; Depledge, P. J. Nat. Prod. 1996, 59, 646.
5. Nicholas, G. M.; Blunt, J. W.; Cole, A. L. J.; Munro,
M. H. G. Tetrahedron Lett. 1997, 38, 7465.
6. (a) Uchiro, H.; Nagasawa, K.; Aiba, Y.; Kobayashi, S.
Tetrahedron Lett. 2000, 41, 4165. (b) Uchiro, H.; Nagasawa,
K.; Aiba, Y.; Kotake, T.; Hasegawa, D.; Kobayashi, S. Tet-
rahedron Lett. 2001, 42, 4531. (c) Aiba, Y.; Hasegawa, D.;
Marunouchi, T.; Nagasawa, K.; Uchiro, H.; Kobayashi, S.
Bioorg. Med. Chem. Lett. 2001, 11, 2783.
7. Physical data of synthesized compounds: 4a: 1H NMR
(CDCl3, 300MHz) d 1.91 (s, 3H), 3.68 (s, 3H), 3.72 (s, 3H),
3.82 (s, 3H), 3.82 (s, 3H), 6.51 (d, 1H, J=16.0 Hz), 6.68 (d,
1H, J=16.0 Hz, H-7), 6.76 (d, 1H, J=7.9 Hz), 6.90 (s, 1H),
6.98 (d, 1H, J=7.8 Hz), 7.22 (dd, 1H, J=7.8, 7.9 Hz), 7.40 (s,
1H); EI–MS 318 (M+); 4b: 1H NMR (CDCl3, 300MHz) d 1.90
(s, 3H), 3.67 (s, 3H), 3.71 (s, 3H), 3.80 (s, 3H), 3.81 (s, 3H),
6.38 (d, 1H, J=15.8 Hz), 6.66 (d, 1H, J=15.8 Hz), 6.84 (d,
2H, J=8.6 Hz), 7.31 (d, 2H, J=8.6 Hz), 7.39 (s, 1H); EI–MS
318 (M+); 4c: 1H NMR (CDCl3, 300 MHz) d 1.91 (s, 3H), 3.68
(s, 3H), 3.72 (s, 3H), 3.82 (s, 3H), 3.88 (s, 3H), 3.90 (s, 3H),
6.37 (d, 1H, J=16.0 Hz), 6.66 (d, 1H, J=16.0 Hz), 6.81 (d,
1H, J=8.3 Hz), 6.89 (d, 1H, J=1.7 Hz), 6.96 (dd, 1H, J=1.7,
8.3 Hz), 7.40 (s, 1H); EI–MS 348 (M+); 4d: 1H NMR (CDCl3,
300 MHz) d 1.90 (s, 3H), 3.66 (s, 3H), 3.71 (s, 3H), 3.81 (s,
3H), 5.94 (s, 2H), 6.52 (d, 1H, J=16.0 Hz), 6.71 (d, 1H,
J=16.0 Hz), 6.81 (d, 1H, J=8.3 Hz), 6.89 (d, 1H, J=1.7 Hz),
6.96 (dd, 1H, J=1.7, 8.3 Hz), 7.18–7.40 (m, 3H); 7.40 (s, 1H);
Conclusion
Several 9-methoxystrobilurin-type b-methoxyacrylate
antibiotics (MOSBs) having variotus aromatic sub-
structures were synthesized. The antifungal activity of
synthesized MOSBs is closely related to their aromatic
substructures. The b-naphthyl-type derivative 4f showed
the broadest antifungal spectrum and the lowest cyto-
toxicity. This derivative is expected to be a lead com-
pound as a new and effective disinfectant with a broad
antifungal spectrum. Due to lack of significant corre-
lation between cytotoxicity against MRC-5 and the
structural properties of MOSBs, the strong growth-
inhibitory activity of human-derived tumor cell lines
to 9-methoxystrobilurin K should be related to its
hindered ether-type substructure. Further extensive
studies are now in progress for the design and develop-
ment of a new ‘cell-specific’ growth inhibitor.
1
EI–MS 332 (M+); 4e: H NMR (CDCl3, 300 MHz) d 1.96 (s,
3H), 3.72 (s, 3H), 3.79 (s, 3H), 3.82 (s, 3H), 6.58 (d, 1H,
J=15.6 Hz), 7.40 (s, 1H), 7.50 (d, 1H, J=15.6 Hz), 7.41–7.56
(m, 3H), 7.74 (d, 2H, J=8.1 Hz), 7.83 (dd, 1H, J=2.4, 7.1
1
Hz), 8.18 (dd, 1H, J=2.0, 7.1 Hz); EI–MS 338 (M+); 4f: H
NMR (CDCl3, 300 MHz) d 1.94 (s, 3H), 3.72 (s, 3H), 3.74 (s,
3H), 3.83 (s, 3H), 6.65 (d, 1H, J=15.8 Hz), 6.88 (d, 1H,
J=15.8 Hz), 7.38–7.48 (m, 2H), 7.44 (s, 1H), 7.56 (dd, 1H,
J=1.8, 8.5 Hz), 7.71–7.82 (m, 4H); EI–MS 338 (M+).
References and Notes
8. Otoguro, K.; Kohana, A.; Manabe, C.; Ishiyama, A.; Ui,
H.; Shiomi, K.; Yamada, H.; Omura, S. J. Antibiotics 2001,
54, 658.
-
1. Anke, T.; Oberwinkler, F.; Steglich, W.; Scharmm, G. J.
Antibiotics 1977, 30, 806.