Total synthesis and antifungal activity of 9-methoxystrobilurin L as the originally proposed 1,4-benzodioxan structure

Article abstract of DOI:10.1016/S0960-894X(01)00561-3

Total synthesis of both enantiomers of 9-methoxystrobilurin L as the originally proposed 1,4-benzodioxan structure was successfully achieved. The ¹H and ¹³C NMR spectra of synthesized 9-methoxystrobilurin L were compared with those of a naturally-occurring sample. It was strongly indicated that naturally-occurring 9-methoxystrobilurin L has not the originally reported 1,4-benzodioxan structure but a 1,5-benzodioxepin structure, the same as previously reported 9-methoxystrobilurin K. Antifungal activities of the synthesized compounds toward several typical fungi were also examined, and they were less active than 9-methoxystrobilurin K.

Full text of DOI:10.1016/S0960-894X(01)00561-3

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2783–2786
Total Synthesis and Antifungal Activity of
9-Methoxystrobilurin L as the Originally Proposed 1,4-
Benzodioxan Structure
Yasuyuki Aiba, Daiju Hasegawa, Takahiro Marunouchi, Koh Nagasawa,
Hiromi Uchiro and Susumu Kobayashi*
Faculty of Pharmaceutical Sciences, Science University of Tokyo, Ichigaya-Funagawara-machi, Shinjuku-ku,
Tokyo 162-0826, Japan
Received 26 June 2001; accepted 10 August 2001
Abstract—Total synthesis of both enantiomers of 9-methoxystrobilurin L as the originally proposed 1,4-benzodioxan structure was
1
successfully achieved. The H and 13C NMR spectra of synthesized 9-methoxystrobilurin L were compared with those of a natu-
rally-occurring sample. It was strongly indicated that naturally-occurring 9-methoxystrobilurin L has not the originally reported
1,4-benzodioxan structure but a 1,5-benzodioxepin structure, the same as previously reported 9-methoxystrobilurin K. Antifungal
activities of the synthesized compounds toward several typical fungi were also examined, and they were less active than 9-methoxy-
strobilurin K. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
of 9-methoxystrobilurin K 3 was recently achieved and
its 1,5-benzodioxepin structure was synthetically
confirmed in our previous paper.6
9-methoxystrobilurin L was isolated as a new analogue
of b-methoxyacrylate antibiotics from mycelial culture
of basidomycete Favolaschia pustulosa by Xenova’s
group in 1996.1 The compound exhibits strong anti-
fungal activities toward several typical fungi by inhibit-
ing a mitochondrial respiration pathway as other b-
methoxyacrylates (MOAs) do.2 In addition, the com-
pound shows remarkable cytostatic activity toward
human Burkitt’s lymphoma derived cell lines (Jijoye) at
According to these results, 9-methoxystrobilurin L also
seemed to have the same 1,5-benzodioxepin structure;
however, it should be confirmed synthetically for future
study of structure–activity relationships. In addition,
1,4-benzodioxan structure 1 was also expected to show
strong biological activities because strobilurin E (4)
having a similar 1,4-benzodioxan structure indicated
strong antifungal and cytostatic activities (Fig. 1).7 In
this paper, we would like to report the asymmetric total
synthesis of both enantiomers of 9-methoxystrobilurin
L as the originally proposed 1,4-benzodioxan structure
and its antifungal activities.
1
very low concentration. The H and 13C NMR spectra
of 9-methoxystrobilurin L were very similar to those of
9-methoxystrobilurin K (2) previously reported by Anke
et al.;3 however, it was assigned as an original 1,4-ben-
zodioxepin structure 1. On the other hand, Blunt and
Munro isolated a compound showing almost the same
1H and 13C NMR spectra as 1 or 2 from another culture
in 1996, and claimed the 1,5-benzodioxepin structure 3
based on the assignment of the HMBC spectrum in
CDCl3 for these compounds.4 Recently, Anke and Ste-
glich formally revised the structure of 9-methoxy-
strobilurin K to 3 by comparison of a synthesized 1,5-
benzodioxepin substructure with the degradation pro-
duct of natural strobilurin K.5 Further, a total synthesis
*Corresponding author. Tel./fax: +81-3-3260-8848; e-mail:
kobayash@ps.kagu.sut.ac.jp
Figure 1. Cytostatic strobilurin analogues.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00561-3

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Y. Aiba et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2783–2786
Synthetic Plan
of methylmagnesium bromide gave a tertiary alcohol 12
in good yield. The optical purity of 12 was determined
by HPLC analysis as 93% ee.9 A direct ether synthesis
using the thus-obtained tertiary alcohol 12 with iso-
prene under acidic conditions failed due to the exclusive
polymerization of the diene. Therefore, stepwise synth-
esis was next carried out. The a-alkoxy-a-methyl ester
13 was prepared by treatment of the alcohol 12 with
sodium hydride and alkylation of the resulting alkoxide
with methyl a-bromopropionate. Further, an enolate
anion generated from 13 and sodium bis(tri-
methylsilyl)amide was methylated by the addition of
methyl iodide. The prepared a,a-dimethyl-a-alkoxy
ester 14 was reduced to the alcohol and subsequently
oxidized to the corresponding aldehyde 15. Terminal
olefination of the aldehyde 15 utilizing Wittig reaction
gave the aryl bromide 16, and the desired tertiary–tertiary
ether linkage was successfully constructed.
A retrosynthesis of 9-methoxystrobilurin L is shown in
Scheme 1.
A construction of the relatively unstable conjugated tri-
ene moiety was to be performed at the last stage of total
synthesis. Therefore, an enone-type intermediate 18 was
decided as the first target molecule. Further, an optically
active 1,4-benzodioxan 10 would be constructed by 6-exo
selective intramolecular cyclization of epoxyphenol 9.
The epoxyphenol 9, in turn, would be prepared from 20-
hydroxy-50-bromoacetophenone
6
chlorohydrin via Baeyer–Villiger oxidation.
and chiral epi-
Synthesis
The reaction of 20-hydroxy-50-bromoacetophenone 6
with optically active epichlorohydrin 5 was carried out
by Augstein’s a-selective coupling method8 and the
desired ether 7 was obtained in 68% yield (Scheme 2).
The Heck reaction of the aryl bromide 16 with a vinyl
ketone 1710 proceeded smoothly to give the desired
enone-type intermediate 18 (Scheme 3). The ester 18 was
hydrolyzed to the corresponding carboxylic acid, which
led to the methylenolether 19 by treatment with potas-
sium tert-butoxide and dimethyl sulfate. Further, the b-
methoxyacrylate moiety was constructed by our pre-
viously reported method,10 and total synthesis of (R)-9-
methoxystrobilurin L (1R) was successfully achieved.
The enantiomer (1S) was also synthesized by the same
procedure.
The Baeyer–Villiger oxidation of 7 proceeded smoothly
to afford the corresponding epoxy ester 8. Interestingly,
the desired 6-exo cyclization subsequently proceeded on
hydrolysis of the epoxy ester 8 for the preparation of
cyclization precursor 9. The thus-obtained primary
alcohol 10 having a 1,4-benzodioxan-ring was oxidized
to the carboxylic acid, which led to the corresponding
methyl ester 11. Treatment of the ester 11 with an excess
Spectral Comparison
1
The H and 13C NMR spectra of synthesized 9-meth-
oxystrobilurin L (1)11 were compared with those of a
natural sample reported by Xenova’s group1,13 (Typical
1H NMR data are summarized in Table 1). The present
study made it clear that 9-methoxystrobilurin L was not
the ‘originally reported’ 1,4-benzodioxan structure
because of the obvious differences in the chemical shifts
1
on its H and 13C NMR spectra in CD3OD. On the
Scheme 1. Retrosynthesis of 9-methyoxystrobilurin L.
other hand, a greater spectral similarity between natu-
Scheme 2. Synthesis of 1,4-benzodioxan including tertiary–tertiary ether linkage (R-form). Reaction conditions: (i) KOh, EtOH–H2O, reflux; (ii)
mCPBA, CHCl3, reflux; (iii) NaOH aq–THF, reflux; (iv) KMnO4, KOH, H2O, rt; (v) CN2N2, Et2O, rt, 20 min; (vi) MeMGBr (excess), THF, rt, 1 h;
(vii) NaH, methyl 2-bromopropionate, 0 ꢀC; (viii) NaHMDS, MeI, THF, À78 ꢀC; (ix) DIBAH, CH2Cl2, À78 ꢀC; (x) (COCl)2, DMSO, Et3N,
CH2Cl2, À60 ꢀC; (xi) Ph3P¼CH2, DMSO–THF, rt.

Y. Aiba et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2783–2786
2785
Scheme 3. Synthesis of (R)-9-methoxystrobilurin L. Reaction conditions: (i) 10 mol% Pd(OAc)2, PPh3, Et3N, 100 ꢀC; (ii) NaOH aq–MeOH, rt then
HCl; (iii) KOtBu, Me2SO4, DMF, À45 to À15 ꢀC; (iv) NaH, HCOOMe, rt; (v) K2CO3, Me2SO4, HCOOMe, rt; (vi) hn (l: 365 nm), acetone–benzene, rt.
Table 1. 1H NMR spectra of 9-methoxystrobilurins L and Ka,b
Compound
Position
9-Methoxystrobilurin L
9-Methoxystrobilurin K
Synthesized
Natural
Synthesized
18
3.70 (m)
3.84 (dd, J=2.0, 8.8 Hz)
3.97 (dd, J=8.8, 11.2 Hz)
4.55 (dd, J=2.0, 11.2 Hz)
6.07 (dd, J=10.7, 17.6 Hz)
5.00 (dd, J=1.0, 10.7 Hz)
5.13 (dd, J=1.0, 17.6 Hz)
3.69 (dd, J=3.4, 7.6 Hz)
3.98 (dd, J=7.6, 12.5 Hz)
4.20 (dd, J=3.4, 12.5 Hz)
5.93 (dd, J=11.0, 17.7 Hz)
5.17 (dd, J=0.9, 10.7 Hz)
5.23 (dd, J=0.9, 17.7 Hz)
4.00 (dd, J=7.5, 12.4 Hz)
4.20 (dd, J=3.1, 12.3 Hz)
5.90 (dd, J=10.0, 17.4 Hz)
5.19 (dd, J=0.7, 10.0 Hz)
5.22 (dd, J=0.7, 18.0 Hz)
17a,17b
23
24a,24b
aCD3OD was used as the solvent.
bTetramethylsilane was used as the internal standard.
Table 2. Antifungal activities of synthesized (R)- and (S)-9-methoxystrobilurin L
Compound
Diameter of inhibition zone (mm)a,b
Conc.
(mg/disk)
Penicillium
citrium
Aspergillus
fumigatus
Fusarium
solani
Candida
albicans
Saccharomyces
cerevisiae
Nystatin (positive control)
10
10
1
0.1
10
1
0.1
10
1
10
—
—
—
—
—
—
—
—
—
12
8
ꢁ
ꢁ
8
—
—
—
—
—
—
—
25*
19*
—
16
10
ꢁ
17
8
(1R)
ꢁ
—
8
—
8
(1S)
(3)
8
—
—
22
17
—
—
—
25
20
ꢁ
ꢁ
10
8
ꢁ
0.1
aThe diameter of each inhibition zone (mean values of two samples) was measured after 48 h incubation.
b—, Not effective; ꢁ, slightly effective; *, incomplete inhibition.

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Y. Aiba et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2783–2786
rally-occurring 9-methoxystrobilurin L and synthesized
9-methoxystrobilurin K12 was clearly demonstrated.
4. Nicholas, G. M.; Blunt, J. W.; Cole, A. L. J.; Munro,
M. H. G. Tetrahedron Lett. 1997, 38, 7465.
5. Hellwig, V.; Dasenbrock, J.; Klostermeyer, D.; Kroiß, S.;
Sindlinger, T.; Spiteller, P.; Steffan, B.; Steglich, W.; Engler-
Lohr, M.; Semar, S.; Anke, T. Tetrahedron 1999, 55, 10101.
6. Uchiro, H.; Nagasawa, K.; Aiba, Y.; Kotake, T.; Hase-
gawa, D.; Kobayashi, S. Tetrahedron Lett. 2001, 42, 4531.
7. Weber, W.; Anke, T.; Steffan, B.; Steglich, W. J. Antibiot.
1990, 43, 207.
8. Augstein, J.; Green, S. M.; Monro, A. M.; Potter, G. W. H.;
Worthing, C. R.; Wrigley, T. I. J. Med. Chem. 1965, 8, 446.
9. A Daicel Chiralcel OD column was used for the separation
with hexane/2-propanol (100:1) as eluent.
Biological Assay
Antifungal activities of both synthesized enantiomers of
9-methoxystrobilurin L toward several typical fungi
examined by the disk diffusion method using potato
dextrose agar are shown in Table 2.
Interestingly, both synthesized enantiomers of 9-meth-
oxystrobilurin L were unexpectedly less active than 9-
methoxystrobilurin K and nystatin (positive control)
toward all of the examined fungi.
10. Uchiro, H.; Nagasawa, K.; Aiba, Y.; Kobayashi, S. Tet-
rahedron Lett. 2000, 41, 4165.
11. Physical data of synthesized 9-methoxystrobilurin L (1):
1H NMR (d, 500 MHz, CD3OD) 1.31 (s, 3H), 1.36 (s, 6H),
1.44 (s, 3H), 1.84 (s, 3H), 3.65 (s, 3H), 3.71 (s, 3H), 3.81 (s,
3H), 3.78 (dd, 1H, J=2.0, 8.8 Hz), 3.94 (dd, 1H, J=8.8, 11.2
Hz), 4.48 (dd, 1H, J=2.0, 11.2 Hz), 5.00 (dd, 1H, J=1.0, 10.7
Hz), 5.13 (dd, 1H, J=1.0, 17.6 Hz), 6.12 (dd, 1H, J=10.7,
17.6 Hz), 6.34 (d, 1H, J=15.9 Hz), 6.54 (d, 1H, J=16.1 Hz),
6.74 (d, 1H, J=8.3 Hz), 6.81 (dd, 1H, J=2.0, 8.3 Hz), 6.84 (d,
1H, J=2.0 Hz), 7.48 (s, 1H); 13C NMR (d, 125.6 MHz,
CD3OD) 16.5, 23.8, 26.9, 30.09, 30.11, 52.0, 59.8, 62.4, 66.4,
77.2, 78.1, 80.9, 111.5, 112.1, 115.5, 118.0, 120.7, 120.9, 128.5,
132.3, 144.8, 145.3, 148.7, 154.0, 161.4, 169.8; [a]2D8=72.9 (1R),
(c=0.402, CHCl3), À71.5 (1S), (c=0.442, CHCl3); HRMS
calcd for C27H36O7 (M+) 472.2461, found 472.2446 (1R),
472.2452 (1S).
12. Physical data of synthesized 9-methoxystrobilurin K (3):6
1H NMR (d, 500 MHz, CD3OD) 1.22 (s, 3H), 1.32 (s, 3H),
1.32 (s, 3H), 1.38 (s, 3H), 1.85 (s, 3H), 3.63 (s, 3H), 3.69 (dd,
1H, J=3.4, 7.6 Hz), 3.70 (s, 3H), 3.83 (s, 3H), 3.98 (dd, 1H,
J=7.6, 12.5 Hz), 4.20 (dd, 1H, J=3.4, 12.5 Hz), 5.17 (dd, 1H,
J=0.9, 10.7 Hz), 5.23 (dd, 1H, J=0.9, 17.7 Hz), 5.93 (dd, 1H,
J=11.0, 17.7 Hz), 6.36 (d, 1H, J=15.9 Hz), 6.54 (d, 1H,
J=15.9 Hz), 6.80 (d, 1H, J=8.2 Hz), 6.88 (d, 1H, J=2.1 Hz),
6.94 (dd, 1H, J=2.1, 8.2 Hz), 7.49 (s, 1H); 13C NMR (d,
125.6 MHz, CD3OD) 16.5, 22.6, 26.3, 26.7; [a]2D4=À8.87
(c=0.860, CHCl3); HRMS calcd for C27H36O7 (M+)
472.2461, found 472.2456.
Conclusion
The originally proposed 1,4-benzodioxan structure of 9-
methoxystrobilurin L was successfully synthesized;
however, its structural assignment was clearly ruled out
1
by obvious differences in H and 13C NMR spectra. In
conclusion, it was strongly indicated that naturally-
occurring 9-methoxystrobilurin L should be the same
compound as 9-methoxystrobilurin K previously iso-
lated by two other groups. In addition, both synthesized
enantiomers of the 1,4-benzodioxan structure were less
effective than 9-methoxystrobilurin K. This result is in
contrast to the strong antifungal activity of strobilurin
E (4) having a similar 1,4-benzodioxan structure and
indicated that the geometry of the hindered ether-type
side chain moiety on the 1,4-benzodioxan ring is very
influential in the biological activities of these com-
pounds. Further extensive studies for the synthesis and
design of novel b-methoxyacrylate antibiotics having a
more effective side chain moiety are now in progress.
13. Physical data of natural 9-methoxystrobilurin L:1 1H
NMR (d, 400 MHz, CD3OD) 1.21 (s, 3H), 1.32 (s, 3H), 1.32 (s,
3H), 1.39 (s, 3H), 1.85 (s, 3H), 3.62 (s, 3H), 3.70 (s, 3H), 3.70
(m, 1H), 3.82 (s, 3H), 4.00 (dd, 1H, J=7.5, 12.4 Hz), 4.20 (dd,
1H, J=3.1, 12.3 Hz), 5.19 (dd, 1H, J=0.7, 10.0 Hz), 5.22 (dd,
1H, J=0.7, 18.0 Hz), 5.90 (dd, 1H, J=10.0, 17.4 Hz), 6.39 (d,
1H, J=15.0 Hz), 6.54 (d, 1H, J=15.0 Hz), 6.80 (d, 1H, J=8.4
Hz), 6.84 (d, 1H, J=2.1 Hz), 6.97 (dd, 1H, J=2.1, 8.2 Hz),
7.44 (s, 1H); 13C NMR (d, 100 MHz, CD3OD) 16.5, 22.6, 26.4,
26.8, 28.2, 52.2, 59.9 62.6, 72.8, 77.4, 77.7, 82.9, 111.9, 114.9,
119.6, 121.3, 121.5, 122.9, 123.7, 128.3, 134.5, 145.3, 147.9,
152.2, 154.3, 161.6, 169.9.
References and Notes
1. 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.
2. Sauter, H.; Steglich, W.; Anke, T. Angew. Chem., Int. Ed.
Engl. 1999, 38, 1328.
3. Zapf, S.; Werle, A.; Anke, T.; Klostermeyer, D.; Steffan,
B.; Steglich, W. Angew. Chem., Int. Ed. Engl. 1995, 34, 196.