Acremines H-N, novel prenylated polyketide metabolites produced by a strain of Acremonium byssoides

Article abstract of DOI:10.1016/j.tet.2008.11.058

Five novel metabolites, acremines H-N, have been isolated from malt extract-peptone-glucose agar cultures of a strain of Acremonium byssoides. Their structures and stereochemistry were elucidated using a combination of ¹³C and ¹H hom

Full text of DOI:10.1016/j.tet.2008.11.058

Tetrahedron 65 (2009) 786–791
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Acremines H–N, novel prenylated polyketide metabolites produced by a strain
of Acremonium byssoidesq
,
a,
*
Alberto Arnone a, Gemma Assante b, Adriana Bava a, Sabrina Dallavalle c , Gianluca Nasini
*
a Dipartimento di Chimica, Materiali ed Ingegneria Chimica ‘Giulio Natta’ del Politecnico, CNR-Istituto di Chimica del Riconoscimento Molecolare, Sezione ‘Adolfo Quilico’;
via Mancinelli 7, 20131 Milano, Italy
b
`
Istituto di Patologia Vegetale, Universita degli Studi, via Celoria 2, I 20133 Milano, Italy
c
`
Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, via Celoria 2, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Five novel metabolites, acremines H–N, have been isolated from malt extract–peptone–glucose agar
cultures of a strain of Acremonium byssoides. Their structures and stereochemistry were elucidated using
a combination of 13C and 1H homo and heteronuclear 2D NMR experiments. Acremines H–N inhibited
the germination of sporangia of Plasmopara viticola.
Received 26 September 2008
Received in revised form 5 November 2008
Accepted 20 November 2008
Available online 25 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Acremonium
Plasmopara viticola
Acremines
Isolation
Natural products
NMR
1. Introduction
by a Diels–Alder reaction and successive oxidative coupling, was
isolated, although in a small amount.1
Endophytic fungi are a source of intelligent screening, as they
grow within the plant hosts in a continuum of interactions with
respect to physiological status, colonization pattern and secondary
metabolism.2
We describe herein the isolation of new acremines (H–N) pro-
duced by the fungus on a different culture medium, composed by
malt extract–peptone and glucose agar.
Many endophytic species of the genus Acremonium have been
proved to be a rich source of biologically active metabolites, i.e.,
prenylated phenol inhibitors of N-SMase.3 In a recent investigation,
Acremonium byssoides was isolated as a residential endophyte in
the grapevines of a Sicilian vineyard, never treated with fungicides,
and the fungus was found to parasitize Plasmopara viticola, growing
and sporulating into sporangiophores and sporangia.4 As a part of
a program carried out to study new bioactive metabolites produced
by this species, we have recently isolated from A. byssoides strain A
20, cultured on corn-step-agar (CSA), a series of structurally related
metabolites, acremines A (1), B (2), C (6), D–F, biosynthetically
derived from a monoterpene unit and a polyketide moiety.4 Suc-
cessively, from the same cultures on CSA medium, acremine G,
a new dimeric metabolite generated from acremines A (1) and B (2)
2. Results and discussion
A. byssoides A 20 was cultured on malt–peptone–glucose (MPG)
agar for 3 weeks and the metabolites were extracted with EtOAc.
The crude extracts were submitted to successive chromatographic
fractionation and purification, yielding a family of five novel com-
pounds, named acremines H (3), I (4), L (5a), M (7) and N (8), be-
sides the known acremines B (2) and C (6) (Scheme 1).
Structure assignments of the isolated compounds were based on
spectroscopic data, especially those from NMR and MS analysis, and
chemical reactions.
Acremine H (3) was isolated as a colourless solid, mp 110–
112 ꢀC; [
a
]
D þ22 (c 0.06, CHCl3); the CIMS showed an [MH]þ at m/z
243 corresponding to a molecular formula C12H18O5, confirmed by
analysis. Based on 1H and 13C NMR spectral data (Tables 1 and 2),
the structural features of 3 were remarkably similar to those of
acremine A (1), the only difference being the presence in 3 of an
epoxy ring in place of the C(10)H]C(20)H double bond.
q
See Ref. 1.
* Corresponding authors. Tel.: þ39 2 50316818; fax: þ39 2 50316801.
E-mail addresses: sabrina.dallavalle@unimi.it (S. Dallavalle), gianluca.nasini@
polimi.it (G. Nasini).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.11.058

A. Arnone et al. / Tetrahedron 65 (2009) 786–791
787
OH
CH3
This assignment is supported by the analysis of the NOESY NMR
spectra.
OH5'
OH
CH3
4'
H3C
H3C
H3C
CH3
3'
2'
The NOE enhancements observed for H-10 and H-20 upon irra-
diation of H-4 are 1.5 and 1.5%, respectively, for compound 3, and
2.5 and 0.5%, respectively, for compound 9. Molecular mechanics
calculations6 showed that for compound 9 the minimal energy
conformation (Fig. 2) may be favoured by an intramolecular hy-
drogen bond between the oxirane oxygen and the proton of the
hydroxy group in position 4. The distances between H-4 and H-10
and H-20 (2.98 and 4.40 Å, respectively) are in agreement with the
NOE results. Similarly, the distances between the same hydrogens
in the minimal energy conformation of compound 3 (3.31 and
3.88 Å, respectively) are in agreement with the NOE
measurements.
O
1'
H
H
3
O
2
1
HO
HO
4
5
O
OH
O
OH
6
O
H3C
H3C
H3C
OH
1''
2
3
1
OH
CH3
OH
CH3
H3C
O
H3C
O
CH3
CH3
H3C
HO
O
H
H
R1O
HO
Acremine I (4) was isolated as an oil, [
a
]
ꢁ126 (c 0.1, CHCl3),
D
with an HREIMS consistent with the formula C12H16O5. 13C and 1
H
R2O
O
O
CH3
O
CH3
H
NMR spectra revealed its structural similarity with compounds 3
5a R1 = R2 = H
6
1
4
and 9: two signals at 53.9 and 68.8 ppm, having JCH¼181 and
5b R1 = Ac; R2 = H
174.5 Hz, respectively, provided evidence of the presence of an
epoxy ring in position 10-20. The upfield shift of the quaternary C-6
carbon and the presence of a methine carbon at 63.96 ppm, having
1JCH¼181 Hz, in place of the C-5 methylene carbon, suggested the
presence of an additional epoxy ring in position 5-6.
5c R1 = H; R2 = Ac
5d R1 = R2 = Ac
4
8 OH
1'
1'' O
H3C
5
3
HO
CH3
CH3
O
2
3
The NOE enhancements observed between H3-100, assumed as
disposed, and H-5 and between H-4 and H-5 (see Section 4) suggest
that the latter two protons are on the -side of the molecule.
Acremine L (5a) was isolated as an oil; [
D þ17 (c 0.65, MeOH);
b
-
3'
8a
2
7
CH3
OH
6
2'
H3C
O
7
6
H
1''
O
4
5
OH
CH3
b
2'
OH
a
]
8
the ESIMS showed an [MHþNa]þat m/z 265, consistent with the
formula C12H18O5. The 1H and 13C NMR spectra of 5a were similar to
those of 3, the only relevant difference being the presence in 5a of
a hydroxy group in position 5 rather than in position 6. Typically,
the H3-100 protons showed a vicinal coupling of 6.8 Hz with H-6,
which on its turn exhibited a trans diaxial coupling of 11.1 Hz with
H-5. Moreover, the magnitude of the coupling between H-4, as-
7
OH
2
CH3
H3C
3
5
OH
4
OH
O
6
8
sumed as
assumes a half-chair conformation in which the 5-OH and the 6-Me
groups are - and -disposed, respectively.
b-disposed, and H-5 suggests that the cyclohexenone ring
7
O
O
CH3
b
a
Scheme 1.
The formation of the monoacetates 5b and 5c and of the diac-
etate 5d with the low-field shift of H-4 and H-5 was in agreement
with the proposed structure.
In fact, the 13C NMR spectrum of 3 showed the presence of two
signals at 52.8 and 69.2 ppm, having one-bond 1JCH couplings of 183
and 176 Hz, respectively, characteristic of epoxide carbons. Ac-
cordingly, the 1H NMR spectrum revealed the absence of the ole-
finic protons and the presence of two new epoxy protons at 3.93
and 2.97 ppm having a trans coupling constant of 2.1 Hz.
To confirm the structure of 3, acremine A (1) was treated with 3-
chloroperoxybenzoic acid (m-CPBA) in dichloromethane. In-
terestingly, the reaction showed a high diastereoselectivity, giving
a >95:<0.5 mixture of diastereoisomers 9 and 3, in which natural
compound 3 was the minor isomer (Scheme 2).
There are examples in the literature of m-CPBA substrate con-
trolled epoxidation of olefins.5 A high diastereoselectivity is ach-
ieved when a hydroxyl group is present in a suitable position to
anchor the peroxyacid, thus giving stereofacial differentiation of
the diastereotopic faces of the double bond. We reasoned that the
hydroxyl group on C-4 ((S) absolute configuration) in acremine A
could play an important role in directing the epoxidation reaction,
presumably due to hydrogen bond formation with the reagent
(Fig. 1). This interaction should preferentially stabilize the transi-
tion state related to the electrophilic addition to the Re,Re face of
the double bond.
Acremine M (7) is an oil. The EIMS showed an [MH]þ at m/z
257 and the HREIMS confirmed the formula C12H16O6. The 13C
NMR spectrum of 7 showed some similarities with that of com-
pound 4. The presence of a methyl-substituted epoxy ring was
inferred from signals at 58.0, 65.6 and 14.7 ppm in 13C NMR
spectrum. Further, three signals at 194.8, 156.5 and 118.0 ppm
were attributable to an a,b-conjugated ketone moiety. Finally, the
13C spectrum showed the presence of two methyl groups and four
oxygen-bearing carbons, one of which, resonating at 93.6 ppm,
was assigned to a hemiketalic carbon. This led us to hypothesize
that compound 7 derived from compound 4 by an oxidation,
followed by a ring closure between C-4 and the oxygen on C-30, to
form a tetrahydrochromen-6-one ring. (In this case, the number-
ing of the carbon and hydrogen atoms throughout the rings was
assigned according to chemical nomenclature and is different
from the numbering of the previous acremines.) The 1H NMR
spectrum confirmed the above findings. An allylic coupling be-
tween H-4 and H-5 indicated a linkage between C-4 and C-4a,
whereas the presence of a hemiketalic OH was inferred from an
exchangeable singlet at 6.02 ppm. Two exchanging hydrogens
coupled with H-3 and H-4 revealed the presence of a diol. The
above observations allowed to establish the structure as depicted
in Scheme 1.
On the basis of these mechanistic considerations, the configu-
ration of the stereogenic centres in the oxirane ring of the major
isomer should be 10(S),20(R). Consequently, the natural compound 3
should have the absolute configuration 10(R),20(S).
The NOE enhancements observed between H3-100, assumed as
disposed, and H-8, between H-8 and OH-8a, between H3-10 and H-4
and OH-8a place all these protons on the -side of the molecule.
b-
b

788
A. Arnone et al. / Tetrahedron 65 (2009) 786–791
Table 1
1H NMR spectroscopic data for new compounds (acetone-d6)
Position
Compound
3
4
5a
7
8
9
d
(ppm), J (Hz)
2
3
5.85 (1H, dd,
J¼1.7 and 1.0)
5.91 (1H, br d, J¼1.0)
5.78 (1H, br dd, J¼2.1 and 1.1)
4.49 (1H,
dd, J¼9.2 and 8.1)
5.80 (1H,
dd, J¼1.9
and 1.0)
3.24 (1H, dd,
3.12 (1H, br
J¼10.2 and 4.0)
dd, J¼15.8 and 8.1),
3.02 (1H, br dd,
J¼15.8, 9.2)
4
5
4.59 (1H,
4.51 (1H, br d, J¼1.8)
3.70 (1H, d, J¼1.8)
4.40 (1H, ddd,
J¼8.5, 6.8
4.60 (1H, ddd,
6.65 (1H, br s)
4.74 (1H,
dddddd, J¼8.9, 5.2, 3.8, 1.7, 0.7
J¼10.2, 4.5 and 2.1)
ddddd, J¼9.8,
7.2, 5.3 and 0.7)
2.42 (1H, dd,
J¼12.6 and 5.3),
1.98 (1H, dd,
J¼12.6 and 9.8)
and 0.6)
and 2.1)
2.36 (1H, dd, J¼12.9, 5.2), 2.08
(1H, ddd, J¼12.9, 8.9 and 0.7)
3.51 (1H, ddd,
11.2, 8.5 and 5.5)
5.99 (1H, d, J¼2.1)
6
2.32 (1H, dq, J¼
11.2 and 6.8)
7
6.43 (1H, br s)
8
10
3.56 (1H, s)
1.45 (3H, s)
3.93 (1H, ddd, J¼2.1,
3.71(1H, dd,
J¼2.1, 1.0)
3.99 (1H, ddd,
J¼2.1, 1.1 and 0.7)
3.72 (1H, ddd,
J¼2.1, 1.0
1.0 and 0.6)
and 0.7)
20
2.97 (1H, d, J¼2.1)
3.12 (1H, d, J¼2.1)
2.83 (1H, d, J¼2.1)
1.26 (3H, s)
1.20 (3H, s)
1.18 (3H, s)
2.82 (1H, d,
J¼2.1)
30
40
1.27 (3H, s)
1.25 (3H, s)
1.24 (3H, s)
1.25 (3H, s)
1.22 (3H, s)
1.41 (3H, s)
1.26 (3H, s)
1.22 (3H, s)
1.15 (3H, d, J¼6.8)
1.27 (3H, s)
1.27 (3H, s)
1.21 (3H, s)
50
100
1.38 (3H, s)
2.11 (3H, br s)
3-OR
4-OR
4.76 (1H, d, J¼4.0)
4.85 (1H, br d, J¼3.8)
4.45 (1H, s)
5.17 (1H, br s)
4.98 (1H, d, J¼6.8)
4.71 (1H, d, J¼5.5)
4.79 (1H, d, J¼4.5)
4.98 (1H, d,
J¼7.2)
5-OH
6-OH
8a-OH
10-OH
30-OH
7.56 (1H, br s)
3.55 (1H, br s)
4.28 (1H, s)
3.83 (1H, s)
6.02 (1H, br s)
3.68 (1H, s)
3.65 (1H, br s)
3.63 (1H, br s)
Finally, the vicinal coupling of 10.2 Hz observed between H-3 and
H-4 indicates that these protons are trans diaxially disposed (Fig. 3).
The 13C NMR spectrum of 8 exhibited signals due to six sp2 and
six sp3 carbon atoms. The sp2 resonances indicated the presence of
a tetrasubstituted benzene ring while the sp3 resonances were
assigned to three methyl, one methylene, one methine and one
quaternary carbon atom, the last two bearing an oxygen atom.
Acremine N (8) was obtained as a solid, mp 150–153 ꢀC, [
a]
D þ35
(c 0.2, MeOH) and analyzed for C12H16O3; no carbonyl stretching
band was present in the IR spectrum.
Table 2
13C NMR spectroscopic data for new compounds (acetone-d6)
Position
Compound
3
4
5a
7
8
9
d
(ppm), J (Hz)
1
200.4 (s)
194.5 (s)
198.5 (s)
201.3 (s)
2
3
3a
4
4a
5
6
7
7a
8
8a
10
20
30
40
50
100
120.3 (d, J¼163)
122.2 (d, J¼163.5)
122.2 (d, J¼163)
78.07 (s)
89.8 (d, J¼147.5)
31.4 (t, J¼133)
125.9a (s)
112.2 (d, J¼156.5)
119.0 (d, J¼164)
163.0 (s)
155.7 (s)
160.4 (s)
79.9a (d, 141.5)
164.9 (s)
66.1 (d, J¼142)
63.5 (d, J¼144)
78.3a (d, J¼143)
68.9a (d, J¼142)
156.5 (s)
67.0 (d, J¼143)
46.1 (t, J¼131)
64.0 (d, J¼181)
73.8a (d, J¼142)
48.5 (d, J¼125)
118.0 (d, J¼168)
149.7b (s)
46.6 (t, J¼131)
73.4 (s)
58.2 (s)
194.8 (s)
123.6a (s)
73.6 (s)
58.0 (s)
111.1 (d, J¼157.5)
154.1b (s)
65.6 (d, J¼181.5)
93.6 (s)
52.8 (d, J¼183)
69.2 (d, J¼176)
67.9 (s)
26.6 (q, J¼126)
25.5 (q, J¼126)
24.6 (q, J¼127)
53.9 (d, J¼181)
68.8 (d, J¼174.5)
68.3 (s)
26.8 (q, J¼126.5)
26.0 (q, J¼126.5)
15.0 (q, J¼127.5)
52.3 (d, J¼184)
69.6 (d, J¼175)
67.9 (s)
26.6 (q, J¼126)
25.4 (q, J¼126)
10.9 (q, J¼127)
29.6 (q, J¼127.5)
71.5 (s)
53.8 (d, J¼181)
69.7 (d, J¼175)
68.2 (s)
27.0 (q, J¼127)
25.1 (q, J¼127)
25.0 (q, J¼127)
22.7 (q, J¼127.5)
26.0 (q, J¼125.5)
25.5 (q, J¼125.5
14.7 (q, J¼129.5)
16.6(q, J¼126.5)
a
May be interchanged.
May be interchanged.
b

A. Arnone et al. / Tetrahedron 65 (2009) 786–791
789
OH
CH3
OH
H
CH3
H3C
H3C
O
H
O
O
CH3
OH
O
H
CH2Cl2
MCPBA
HO
1
HO
H
O
OH
Figure 3. Selected NOE correlations for compound 7.
H3C
9
Scheme 2.
to an intramolecular cyclization of the isoprenyl side chain. The
presence of two signals at 3.02 and 3.06 ppm, showing a geminal
coupling constant (J¼15.8 ppm) and a further coupling with a hy-
drogen resonating at 4.49 ppm, along with the absence of the
olefinic proton, led us to assume a reduction of the double bond in
the benzofuran ring with respect to acremine D.
The absolute configuration determination was based on analogy
to other natural compounds. It is well known that for structurally
similar compounds, similarity in sign of the specific rotation and CD
curves indicates a similarity in configuration at the stereogenic
centre. Ramadas and co-workers7 reported the specific rotation and
the CD spectra of a series of substituted 2-hydroxymethyl-2,3-
dihydrobenzofurans with known (S) absolute configuration; the
compounds showed a (þ) sign of specific rotation. The reported CD
of compound 10 (Scheme 3) showed a positive Cotton effect with
Accordingly, the 1H NMR spectrum showed the presence of two
para aromatic protons at 6.65 and 6.43 ppm, assigned to H-4 and H-
7, two methylene protons at 3.02 and 3.06 ppm (H2-3) and three
methyl protons at 2.11 ppm (H3-100), presenting benzylic couplings
with H-4 and H-7, respectively. Moreover, the spectrum showed
H3C
HO
H3C
HO
O
OH
CH3
H
H
O
O
O
maxima at 238 nm (
q
¼þ18.52ꢂ10ꢁ3 deg cm2 dmolꢁ1) and 292 nm
Cl
(q
¼þ6.74ꢂ10ꢁ3 deg cm2 dmolꢁ1).
Pfefferle and co-workers8 reported the CD of (ꢁ) (R)-arthrog-
Figure 1. Model of the possible diastereoselective epoxidation of acremine A.
raphol (11), which showed a (ꢁ) sign of specific rotation and the
mirror image CD curve with respect to the previous 2-hydroxy-
methyl-2,3-dihydrobenzofurans.
one methine proton, having vicinal couplings with H2-3, two ter-
tiary methyl groups, a phenolic and aliphatic hydroxy protons at
7.56 and 3.55 ppm, respectively. The NOE enhancements observed
between the phenolic OH and H-4 and H3-100, together with those
observed between H-4 and H2-3 and H-7 and H3-100, permitted us to
locate the H2-3, the OH and the H3-100groups at C-3, -5 and -6,
respectively.
CH3
OH
CH3
OH
H
H
H
O
O
O
H3C
HO
OH
HO
(-) (R)-11
8
(+) (S)-10
Scheme 3.
The above observations suggested for compound 8 a bicyclic
structure correlated to the previously described acremine D,4 due
Since, compound 8 showed a (þ) sign of specific rotation (þ35)
as well as a similar CD curve (see Section 4) with respect to com-
pound 10, we may safely deduce that the absolute configuration of
compound 8 is S.
Acremines H, I, L and N were tested for their capability to inhibit
germination of P. viticola sporangia.
The results of the biological assays are summarized in Table 3.
All the new acremines affected germinability of sporangia more or
less efficiently.
Table 3
Mean values (%) of the inhibition of P. viticola sporangia germination by acremines H,
I, L and N
Germinability % inhibition
Control
Water
DMSO
7.7
8.9
Metabolite concentration
0.5 mM
1 mM
Acremine H
Acremine I
Acremine L
Acremine N
15.6
23.2
49.6
54.6
30.4
32.8
55.2
69.4
Figure 2. Model of compounds 3 (right) and 9 (left).

790
A. Arnone et al. / Tetrahedron 65 (2009) 786–791
Selected compounds were tested for their cytotoxicity against
non-small cell lung tumour cell line H460, and showed modest
activity. [IC50 M): for 2: 35.7; for 3 >100 and 4: 56].
4.2.2. Synthesis of compound (9) from acremine A (1)
Acremine A (50 mg) was dissolved in dry CH2Cl2 (5 mL) and
treated with m-CPBA (60 mg) for 3 h at rt; the solution was washed
with a solution of NaHCO3, dried and evaporated. The residue was
purified on silica gel (plates 1 mm) with CH2Cl2–MeOH (9:1) as
eluant to obtain 35 mg of the epoxide 9 as an oil and 1.5 mg of
acremine H; ESIMS m/z 265 (MþNa)þ. The 1H and 13C NMR data are
listed on Tables 1 and 2. NOEs (acetone-d6þD2O): {H-2} enhanced
H-10 (0.5%) and H-20 (1.5%), {H-4} enhanced H-5a (3%), H-10 (2.5%),
H-20 (0.5%) and H3-100(1%), {H-10} enhanced H-4 (2%), H-20 (0.5%),
H3-40 (0.5%) and H3-50 (1%), {H-20} enhanced H-2 (2%), H-10 (0.5%),
H3-40 (1%) and H3-50 (1%), {H3-40} enhanced H-10 (1.5%) and H-20
(4.5%), {H3-50} enhanced H-10 (3%) and H-20 (4.5%), {H3-100} en-
hanced H-4 (5%) and H-5a (2.5%).
(
m
3. Conclusion
We have established the structure and stereochemistry of
a further group of acremines (H–N), produced by a strain of
A. byssoides on a culture medium composed by malt extract–
peptone and glucose agar. The oxygenation pattern of these
compounds may suggest that they are formed by bioconversion
of the main metabolite acremine A (1) first to the epoxide 3 and
subsequently to compounds 4, 5a and 7 by the monooxygenase
enzymes of the fungus, grown in particular conditions (MPGA
cultures). It is well known that these enzymes are able to acti-
vate molecular oxygen in order to transfer it to an organic
compound.9
4.2.3. Acremine I (4)
CIMS, m/z 241 (MH)þ (100%), 223 (MHꢁ18)þ (78) and 183
(65); HREIMS, m/z 240.0972 (calcd for C12H16O5 240.0997). The
1H and 13C NMR data are listed in Tables 1 and 2. NOEs
(CDCl3þD2O): {H-2} enhanced H-10 (4.5%) and H-20 (2%), {H-4}
enhanced H-5 (7.5%), H-10 (4%) and H-20 (2.5%), {H-5} enhanced
H-4 (5.5%) and H3-100 (1%), {H-10} enhanced H-2 (5.5%), H-4
(3.5%) and H-20 (1%), {H-20} enhanced H-2 (3.5%), H-10 (2.5%),
H3-40 (1%) and H3-50 (1%), {H3-40} enhanced H-10 (3%) and H-20
(6%), {H3-50} enhanced H-4 (1%), H-10 (3.5%) and H-20 (5%), {H3-
100} enhanced H-5 (7%).
4. Experimental
4.1. General
Flash column chromatography was performed with Merck
silica gel (0.040–0.63 mm); thin and preparative layer chroma-
tography (TLC and PLC) were performed on precoated Merck
silica gel 60 F254 plates. The IR spectra were measured on a Per-
kin–Elmer 177 spectrophotometer. MS spectra were recorded
4.2.4. Acremine L (5a)
HREIMS, m/z 242.1136 (calcd for C12H18O5 242.1154). The 1H and
13C NMR data are listed in Tables 1 and 2.
with
a Bruker Esquire 3000 Plus instrument, HRMS with
a Bruker APEX-QZT ICR. The NMR spectra were recorded with
a Bruker AMX-600 spectrometer, at 600.13 MHz for 1H and
150.92 MHz for 13C.
4.2.5. Acetylation of compound 5a
Compound 5a (30 mg) was dissolved in dry pyridine (0.2 mL)
and treated with Ac2O (0.5 mL) overnight at 0 ꢀC. Standard work-up
followed by PLC on silica gel in hexane–EtOAc (2:1) gave mainly the
diacetate 5d (18 mg) as a solid, mp 120–125 ꢀC; ESIMS, m/z 349
(MþNa)þ and 675 (2MþNa)þ and the monoacetates 5b and 5c in
the ratio 3:1. Compound 5b. Rf (hexane–ethyl acetate 2:1) 0.4; 1H
4.2. Culture of A. byssoides, extraction and isolation
of acremines H–N
The fungal strain A20 was isolated in pure culture from grape-
vine leaves infected by P. viticola and identified as A. byssoides, by
conventional taxonomy.1 For chemical investigations, the fungus
was grown in batches of 40 Roux flasks containing 100 mL MPGA
(malt extract–peptone–glucose agar, 20, 2, 20 and 15 g Lꢁ1). After
two-week-growth period at 24 ꢀC, the cultures were extracted
twice with EtOAc–MeOH (100:1). The extracts (1.8 g) were dried on
Na2SO4, evaporated to dryness and chromatographed on a silica gel
flash column eluted with hexane–EtOAc at increasing polarity.
Collected fractions were further purified by means of PLC with
CH2Cl2–MeOH 9:1 to give the pure metabolites in order of de-
creasing Rf value: acremine N (8) (75 mg, Rf 0.5), acremine B (2)
(60 mg), acremine I (4) (140 mg, Rf 0.4), acremine C (6) (65 mg),
acremine H (3) (186 mg, Rf 0.3), acremine L (5a) (220 mg, Rf 0.3) and
acremine M (7) (15 mg, Rf 0.2).
NMR (acetone-d6) d: 1.17 (3H, s, Me), 1.24 (3H, s, Me), 1.19 (3H, d, J
6.8 Hz, MeCH), 2.16 (3H, s, OCOMe), 2.51 (1H, dq, J 6.8, 11.1 Hz,
CHMe), 2.84 (1H, d, J 2.1 Hz, CHO), 3.43 (1H, s, OH), 3.57 (1H, ddd, J
0.8, 1.0, 2.1 Hz, CHO), 3.77 (1H, ddd, J 5.5, 8.2, 11.1 Hz, CHOH), 4.83
(1H, d, J 5.5 Hz, OH), 5.80 (1H, ddd, J 0.8, 2.3, 8.2 Hz, CHOCO), 5.92
(1H, dd, J 1.0, 2.3 Hz, CH]C).
4.2.6. Compound 5c
Rf (hexane–ethyl acetate 2:1) 0.3; 1H NMR (acetone-d6)
d: 1.06
(3H, d, J 6.8 Hz, MeCH), 1.22 (3H, s, Me), 1.26 (3H, s, Me), 2.09
(3H, s, OCOMe), 2.56 (1H, dq, J 6.8, 11.2 Hz, CHMe), 2.88 (1H, d, J
2.1 Hz, CHO), 3.51 (1H, s, OH), 3.98 (1H, ddd, J 0.8, 1.1, 2.1 Hz,
CHO), 4.65 (1H, dddd, J 0.8, 2.2, 6.8, 8.3 Hz, CHOH), 5.05 (1H, dd, J
8.3 11.2 Hz, CHOCO), 5.13 (1H, d, J 6.8 Hz, OH), 5.88 (1H, dd, J 1.1,
2.2 Hz, CH]C).
4.2.1. Acremine H (3)
UV: lmax 220 and 280 nm (3 1875 and 17.620); IR: nmax (KBr)
1685 cmꢁ1, conj. CO group; CIMS, m/z 243 (MH)þ (22%), 227
(100) and 209 (30). (Found: C, 59.6; H, 7.6; C12H18O5 requires C,
59.49; H, 7.48.) The 1H and 13C NMR data are listed in Tables 1
and 2. NOEs (acetone-d6þD2O): {H-2} enhanced H-10 (2%) and H-
20 (2.5%), {H-4} enhanced H-5a (4.5%), H-5b (0.5%), H-10 (1.5%),
H-20 (1.5%) and H3-100 (1%), {5a} enhanced H-4 (3%), H-5b (7%)
and H3-100 (1%), {H-5b} enhanced H-4 (0.5%), H-5a (9%) and H3-
100 (0.5%), {H-10} enhanced H-2 (2.5%), H-4 (1%), H-20 (1%), H3-40
(0.5%) and H3-50 (0.5%), {H-20} enhanced H-2 (3%), H-4 (1%), H-10
(1%), H3-40 (0.5%) and H3-50 (0.5%), {H3-40 and H3-50} enhanced
H-10 (8%) and H-20 (11), {H3-100} enhanced H-4 (8%), H-5a (3%)
and H-5b (0.5%).
4.2.7. Compound 5d
Rf (hexane–ethyl acetate 2:1) 0.6; 1H NMR (acetone-d6)
d: 1.10
(3H, d, J 6.8 Hz, MeCH), 1.17 (3H, s, Me), 1.24 (3H, s, Me), 2.05 (3H, s,
OCOMe), 2.12 (3H, s, OCOMe), 2.76 (1H, dq, J 6.8,11.1 Hz, CHMe), 2.87
(1H, d, J 2.2 Hz, CHO), 3.49 (1H, s, OH), 3.61 (1H, ddd, J 0.7,1.2, 2.2 Hz,
CHO), 5.22 (1H, dd, J 8.2, 11.1 Hz, CHOCO), 5.98 (1H, ddd, J 0.7, 2.2,
8.2 Hz, CHOCO), 6.01 (1H, dd, J 1.2, 2.2 Hz, H-2).
4.2.8. Acremine M (7)
Oil; UV: lmax 204, 245 and 290 sh (3 9720, 10,000 and 6350); IR:
nmax 1682 cmꢁ1, conj. CO group; EIMS, m/z 257 (MH)þ (10%), 239
(40), 221 (18) and 59 (100); HREIMS: 242.1146 (calcd for C12H18O5

A. Arnone et al. / Tetrahedron 65 (2009) 786–791
791
242.1154). The 1H and 13C NMR data are listed in Tables 1 and 2;
NOEs (acetone-d6): {H-3} enhanced H3-20 (1.5%), {H-4} enhanced
H-5 (2.5%) and H3-10 (1.5%), {H-5} enhanced H-4 (1.5%) and H-8
(1%), {H-8} enhanced H3-100 (1.5%), {H3-10} enhanced H-4 (10%) and
OH-8a (1%), {H3-20} enhanced H-3 (8%), {H3-100} enhanced H-8 (7%),
{OH-4 and OH-8a} enhanced H-3 (9%), H-5 (3%), H-8 (5.5%), H3-10
(1%), H3-20 (0.5%).
independent experiments, comprising readings on 1200 sporangia
for each treatment and the results are presented in Table 3.
Acknowledgements
We thank G. Pratesi (Istituto Nazionale dei Tumori, Milano) for
the cytotoxicity tests, and the University of Milano (FIRST funds) for
financial support.
4.2.9. Acremine N (8)
UV: lmax 206 and 304 nm (3 13.830 and 3500); CD (EtOH) D3:
References and notes
216.4, 238.0, 301.4 and 322 nm (ꢁ3.02, þ2.82, ꢁ1.49 and þ0.1).
(Foud: C, 69.3; H, 7.6; C12H16O3 requires C, 69.20; H, 7.74%.) CISM, m/
z 209 (MH)þ, 208, 191, 156 and 100. The 1H and 13C NMR data are in
Tables 1 and 2. NOEs (acetone-d6): {H-2} enhanced H-3a (0.5%),
H-3b (3.5%), H-7 (0.5%), H3-20 (1%) and H3-30 (1%), {H-4} enhanced
H2-3 (1%), and OH-5 (1.5%), {H-6} enhanced H3-100 (2%), {H3-20 and
H3-30} enhanced H-2 (6.5%), H2-3 (3%), H-7 (0.5%) and OH-5 (2.5%),
{H3-100} enhanced H-7 (7%).
1. Part 67 in the series, ‘Secondary Mould Metabolites’; for part 66, see Nasini, G.;
Arnone,A.;Panzeri, W.;VajnadePava,O.;Malpezzi, L. J. Nat. Prod.2008, 71,146–149.
2. Wilson, D. Oikos 1995, 73, 274–276.
3. Lindsey, C. C.; Gomes-Diaz, C.; Villalba, J. M.; Pettus, T. R. R. Tetrahedron 2002, 58,
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hedron 2005, 6, 7686–7692.
5. (a) Vega-Perez, J. M.; Vega, M.; Blanco, E.; Iglesias-Guerra, F. Tetrahedron:
ˇ `
Asymmetry 2007, 18, 1850–1867; (b) Charette, A. B.; Cote, B. Tetrahedron:
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6. Three-dimensional molecular models of compounds 3 and 9 were built on
a Silicon Graphics O2, using the programs Insight II and Discover (Accelrys Inc.,
San Diego, CA). Minimizations were performed with the cvff all-atom forcefield
and the conjugate gradients algorithm.
4.3. Bioassay on germination of P. viticola sporangia
7. Ramadas, S.; Krupadaman, G. L. D. Tetrahedron: Asymmetry 2000, 11, 3375–3393.
8. Pfefferle, W.; Anke, H.; Bross, M.; Steffan, B. J. Antibiot. 1990, 43, 648–653.
9. Furstoss, R. In Microbial Reagents in Organic Synthesis; Servi, S., Ed.; Kluwer
Academic: Dordrecht, 1992; pp 333–346.
The test of P. viticola sporangia germination inhibition was
performed as described previously.4 The analysis of the germina-
tion percentages was performed on data collected from two