Pseudoanguillosporin A and B: Two new isochromans isolated from the endophytic fungus Pseudoanguiliospom sp.

Article abstract of DOI:10.1002/ejoc.200801083

Two new isochromans named pseudoanguillosporin A (2a) and B (3), together with the known cephalochromin (1), were isolated from Pseudoanguiliospom sp. The C-2 absolute configuration of 2a and 3 was deduced from its CD spectrum by the isochroman helicity r

Full text of DOI:10.1002/ejoc.200801083

FULL PAPER
DOI: 10.1002/ejoc.200801083
Pseudoanguillosporin A and B: Two New Isochromans Isolated from the
Endophytic Fungus Pseudoanguillospora sp.[‡]
Ines Kock,[a] Siegfried Draeger,[b] Barbara Schulz,[b] Brigitta Elsässer,[a] Tibor Kurtán,[c]
Ágnes Kenéz,[c] Sándor Antus,[c,d] Gennaro Pescitelli,[e] Piero Salvadori,[e]
John-Bryan Speakman,[f] Joachim Rheinheimer,[f] and Karsten Krohn*[a]
Keywords: Biological activity / Isochromans / Natural products / Circular dichroism / NMR spectroscopy
Two new isochromans named pseudoanguillosporin A (2a)
and B (3), together with the known cephalochromin (1), were
MPA esters of its methylated derivative. The axial chirality
of 1 was assigned through exciton analysis of its CD spec-
isolated from Pseudoanguillospora sp. The C-2 absolute con- trum and confirmed by ZINDO CD calculations. The metabo-
figuration of 2a and 3 was deduced from its CD spectrum by
the isochroman helicity rule, supported by TDDFT CD calcu-
lations. The absolute configuration of the sec-hydroxyl group
of 3 was determined by the Mosher NMR method from the
lites showed broad antimicrobial activities.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
3-Alkylisochromans are quite rare as natural products;
the known derivatives include (3S)-6-hydroxy-8-methoxy-
3,5-dimethylisochroman,[5] the anticoccidial arohynapene,[6]
the toxic 8-hydroxy-6-methoxy-3,7-dimethylisochroman,[7]
and the antibacterial bruguierol C.[8] Moreover, the isolated
1-hydroxy-3-heptylisochroman derivative CJ-12,373 was re-
ported to have topoisomerase II inhibitor activity.[9] The
absolute configurations of these isochroman derivatives
have not been determined or are only tentatively proposed
on the basis of biogenetic origin.[5b]
Introduction
In connection with our ongoing search for biologically
active metabolites from fungi,[1] we investigated the con-
stituents of an endophytic fungus, Pseudoanguillospora sp,
internal strain number 6577, isolated from the red algae Po-
lyides rotundus and found on the shore of the Baltic See
near Ahrenshoop. The crude culture extract of the fungus
was found to have good antifungal activity against Micro-
botryum violaceum and antibacterial activity against Bacil-
lus megaterium. Extensive column and preparative thin-
layer chromatography of the ethyl acetate culture extract
afforded two new isochromans, named pseudoanguillospo-
rin A (2a) and B (3), together with the known cephalochro-
min (1).[2–4] Cephalochromin (1) was recently identified as
the first selective FabI [bacterial enoyl–acyl carrier protein
(ACP) reductase] inhibitor of Staphylococcus aureus and
Escherichia coli.[2]
Results and Discussion
The first compound was crystallized from dichlorometh-
ane by dropwise addition of hexane as a yellow, amorphous
powder. It was active against Phytophthora infestans, Micro-
botryum violaceum, Botrytis cinerea, Pyricularia oryzae,
Septoria tritici and Chlorella fusca (see Tables 1 and 2). In
the 13C NMR spectrum, 10 aromatic carbon atoms, 1 car-
bonyl group, and 3 aliphatic carbon atoms were identified.
With the use of HMQC and HMBC spectra, fragment A
shown in Figure 1 was constructed.
[‡] Biologically Active Secondary Metabolites from Fungi, 41. Part
40: Ref.[1]
[a] Department of Chemistry, University of Paderborn,
Warburger Straβe 100, 33098 Paderborn, Germany
Fax: +49-5251-60-3245
E-mail: k.krohn@uni-paderborn.de
[b] Institut für Mikrobiologie, Technische Universität
Braunschweig,
The EI-MS with a molecular ion of 518.3 indicated that
compound 1 is a dimer of fragment A, and the natural
product was identified as the well-known cephalochromin[2]
(1, Figure 2), first described by Tertzakian et al.[3] The
structure was confirmed by comparison with the published
Spielmannstraße 7, 31806 Braunschweig, Germany
[c] Department of Organic Chemistry, University of Debrecen,
P.O.B. 20, H-4010 Debrecen, Hungary
[d] Research Group for Carbohydrates of the Hungarian Academy
of Sciences,
1
data of the H NMR spectrum.[4]
4010 Debrecen, Hungary
The axial chirality of cephalochromin (1) was previously
determined as (aS) from its positive exciton couplet around
280 nm by comparison of its CD with those of other natu-
ral bis(naphtha-γ-pyrones) of known configuration.[10] Our
[e] Università di Pisa, Dipartimento di Chimica e Chimica Indus-
triale,
Via Risorgimento 35, 56126 Pisa, Italy
[f] BASF AG, GVA/G – A 030,
67056 Ludwigshafen, Germany
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K. Krohn et al.
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Table 1. Biological activity of pure metabolites 1, 2a, and 3 against pounds.[13,14] Torsional PM3-energy scans around the C-9/
microbial test organisms in an agar diffusion assay; results as mm
C-9Ј aryl linkage revealed the existence of an energy mini-
mum for a ωC-8,C-9,C-9Ј,C-8Ј dihedral ≈ 90°. Subsequent DFT
of radius of zone of inhibition.[a]
Concentration
[mg]
Test organism
optimization [B3LYP/6-31G(d)] of that structure led to a
slightly larger value ≈94°. The rest of the geometry (Fig-
ure 3, right) is dictated by a network of intramolecular hy-
drogen bonds and favorable OH–π interactions (see the
conformation of 8- and 8Ј-OH). The pyrone ring assumes
a half-chair conformation determined by the favored equa-
torial position of the C-2 methyl group. For the arbitrarily
chosen (2S) configuration, the pyrone chirality is M (i.e.,
dihedral ωC-10a,O-1,C-2,C-3 Ͻ 0).[15]
Metabolites
or Standards
Bacillus
megaterium
Microbotryum
violaceum
Chlorella
fusca
1
1
2a
2a
3
3
0.05
0.25
0.05
0.25
0.05
0.25
0.05
0.05
0.05
0.05
0
0
0
5
5 gi
9
0
7 gi
0
0
20
50
5 gi
5
7
13
0
0
0
10 gi
0
7 gi
7 + 6 gi
0
6 gi
18
18
0
Penicillin
Tetracycline
Nystatin
Actidione
0
35
[a] gi = growth inhibition, that is, there was some growth within
the zone of inhibition.
Figure 3. Measured (MeCN) and ZINDO-calculated CD spectra
for cephalochromin [(aS)-1]. The calculation was run on the lowest-
energy DFT structure for (aS)-1 shown on the right.
Figure 1. HMBC couplings of fragment A of compound 1.
In consideration of the large molecular size, a semiempir-
ical NDO method was devised as the most proper to predict
the CD spectrum of 1, and ZINDO[16] was chosen for its
good performance on systems with multiple aromatic chro-
mophores.[14,17] A good agreement was obtained (Figure 3)
between the experimental CD spectrum of 1 in acetonitrile
and the ZINDO-calculated spectrum on the DFT-opti-
mized structure with (aS) configuration, which confirms the
axial chirality assumed above. Interestingly enough, MO
analysis revealed that the couplet between 240–320 nm
comes from the exciton coupling between three distinct
transitions. In each naphtha-γ-pyrone chromophore, the
first two (around 275 nm) are polarized along the C-4/C-9
direction, whereas the third (around 255 nm) is perpendicu-
lar to the first two.
Figure 2. Structures of cephalochromin (1), pseudoanguillosporins
A and B (2a, 3), synthetic isochroman derivative 2b, and model 2c
used in the calculations.
The strong exciton coupled interaction between the two
naphtha-γ-pyrones dominates the CD spectrum of their di-
cephalochromin sample also showed a positive CD couplet mer 1, even in the region of the n–π* transition, where each
around 280 nm [292 (137.1), 264 (–129), Figure 3], suggest- naphtha-γ-pyrone should show an intrinsic Cotton effect.
ing the same (aS) axial chirality. This CD couplet is con- In fact, the CD calculated for 2-methylnaphtha-γ-pyrone
ceivably due to the exciton coupling[11] between the two with the TDDFT method[18] [B3LYP/TZVP//B3LYP/6-
electric-dipole allowed 1Bb-like transitions, each of them 31G(d)] is two orders of magnitude smaller than the exci-
having long-axis polarization in each aromatic unit.[10] In ton-coupled spectra shown in Figure 3. Therefore, the abso-
addition to the strong positive CD couplet, a weaker nega- lute configuration of the C-2 and C-2Ј stereogenic centers
tive couplet above 300 nm and a positive one below 225 nm could not be determined from the present CD data. A sim-
also appeared in the CD spectrum of 1. Definitive and inde- ilar predominance of the axial chirality was previously ob-
pendent confirmation of the axial chirality of 1 came from served in the phomoxanthone A CD spectrum[19] and seems
comparison between experimental and calculated CD spec- to be of general importance in chiral biaryl systems. Cur-
tra. A conformational analysis was performed with the rently, vibrational circular dichroism is being investigated
semiempirical PM3 method,[12] which offers accurate pre- to solve this problem, which has been waiting for a solution
dictions of geometries and torsional energies of biaryl com- for more than 20 years.[10]
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Isochromans Isolated from the Endophytic Fungus Pseudoanguillospora sp.
The second compound was obtained as a yellow optically
active solid with a melting point of 46–48 °C. It was par-
ticularly active against Pyricularia oryzae, but also against
Phytophthora infestans, Botrytis cinerea, Septoria tritici, Ba-
cillus megaterium and Chlorella fusca (see Tables 1 and 2).
1
The H and 13C NMR spectra revealed a pentasubstituted
benzene ring, two methyl and eight CH2 groups, as well as
one CH unit. The HRMS spectrum gave a molecular ion
at 278.18869, which is indicative of the molecular formula
C17H26O3. The chemical shift of the aromatic proton at δ =
6.21 ppm as well as that of the corresponding carbon atom
at δ = 99.7 ppm suggested oxygenation at both ortho posi-
Figure 4. Left: measured CD spectra of (3R)-2a, (3R, 6ЈR)-3, and
tions. Furthermore, the signal at δ = 1.98 ppm, and the two
signals at δ = 2.33 and 2.57 ppm, can be assigned to a
methyl and a methylene group attached to the benzene ring.
The second CH2 group gives rise to the two proton signals
at δ = 4.51 and 4.85 ppm, suggesting both oxygenation and
connection to the benzene ring. Both CH2 groups are prob-
ably part of a ring system as the proton signals for each
couple of C–H units are not isochronous. The chemical
shift of 3.50 ppm for a CH group suggested further oxygen-
ation, and the remaining six CH2 groups together with the
second methyl group form an aliphatic chain. Analysis of
(3S)-2b in acetonitrile. Right: P- and M-helicity of the isochromane
ring; pseudoanguillosporin A [(3R)-2a] and B [(3R,6ЈR)-3] have M-
helicity.
various functionals (B3LYP, BH&HLYP, BP86) and TZVP
basis set led to very similar CD spectra for the four struc-
tures. Moreover, their Boltzmann-weighted averages for
(3R) absolute configuration reproduced well the pattern of
bands discussed above for (R)-2a. The best match in terms
of transition energies was obtained with the pure GGA
functional BP86, as shown in Figure 5; hybrid functionals
led to the same pattern of CD bands (–/+/– from the red),
with some blueshift with respect to BP86 (20 nm for
B3LYP, 50 nm for BH&HLYP). The first, long-wavelength
negative CD band, experimentally observed for 2a around
280 nm, is due to the 1Lb transition, whereas the 1La transi-
tion contributes to the second positive band observed
around 240 nm. Both π–π* transitions, and especially the
second one, are, however, superimposed to several valence-
to-Rydberg transitions.
1
the 2D NMR spectra and the H–1H COSY spectrum led
to isochroman structure 2a (Figure 2) and this new natural
product was named pseudoanguillosporin A after the fun-
gus that produced the substance.
The absolute configuration of 2a could be deduced from
its CD spectrum (Figure 4). Pseudoanguillosporin A (2a) is
a 3-substituted isochroman derivative, the characteristic 1Lb
band Cotton effects (CE) of which had been correlated with
the P/M-helicity of the heteroring (ωC-8a,C-1,O-2,C-3 Ͼ or Ͻ
0) and hence the absolute configuration of C-3.[20] It had
been shown that P/M-helicity of the isochroman heteroring
1
results in positive/negative Lb band CE, regardless of the
nature(s) and position(s) of substituents on the phenyl ring,
even those having large spectroscopic moments (i.e., OMe,
OH).[20] Because pseudoanguillosporin A (2a) shows a
1
negative Lb band CE [284 nm (∆ε = –0.4 nm)], its hetero-
ring adopts M-helicity (Figure 4, right), which implies a (3R)
absolute configuration with an equatorial C-3 substituent.
In contrast, (3S)-2b, a synthetic compound with known ab-
1
solute configuration,[20] had positive Lb band CE and P-
helicity. Both (3R)-2a and (3S)-2b showed positive CE
around 240 nm in the 1La region, which is known to be
Figure 5. Experimental CD spectrum of pseudoanguillosporin A
more sensitive to the effect of achiral substituents of the (2a) compared with the CD calculated on model compound (R)-2c
with TDBP86/TZVP as Boltzmann average over four DFT-opti-
aromatic ring.
mized structures [B3LYP/6-31G(d)]; the two most stable ones are
shown on the right. The calculated spectrum has been scaled to
50% for better comparison.
The interpretation of the CD spectrum of compounds 2a
and 3 (vide infra) is supported by the calculation of the CD
spectrum[21] of model compound (R)-2c. Its geometry was
investigated with MMFF conformational searches and
The third compound was obtained as a yellow optically
DFT [B3LYP/6-31G(d)] optimizations and revealed a active oil that moderately inhibited Bacillus megaterium,
strong prevalence (Ͼ98% at 300 K) of conformers with the Microbotryum violaceum, Botrytis cinerea and Phytophthora
C-3 substituent occupying the equatorial position. Four mi- infestans (see Tables 1 and 2). The NMR spectra had great
nima with populations Ͼ1% at 300 K (DFT energies) were similarity to those of pseudoanguillosporin A (2a, Fig-
found, differing only in the orientation of the two OH ure 2). The main differences are seen in the chemical shift
bonds; two of them, shown in Figure 5, contribute ca. 90% and coupling patterns of the terminal methyl group of the
to the overall population. TDDFT calculations[22] using aliphatic chain and the CH group at δ = 3.73 ppm. The
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K. Krohn et al.
FULL PAPER
HRMS spectrum suggested the molecular formula porin A and B were also quite similar both in sign and
C17H26O4. This result, in conjunction with the dif- magnitude (A: [α]2D0 = –72.5, B: [α]2D0 = –72.8). The absolute
ferences in the NMR spectra, suggested structure 3 (Fig- configuration of the C-6Ј stereogenic center on the side
ure 2) and the new metabolite was named pseudoanguillos- chain of pseudoanguillosporin B was determined by Mosh-
porin B.
er’s NMR method.[23] In order to check the reliability of
The absolute configuration of pseudoanguillosporin B the method, the (R)-MPA esters of readily available rac-2-
(3) at C-3 was deduced as (R) from its CD spectrum. It was heptanol (4) were prepared as model compounds by using
nearly identical to that of 2a; the additional C-6Ј ste- (R)-MPA (5), and the resultant diastereomers were sepa-
reogenic center, very distant from the chromophore, had rated by preparative TLC (Scheme 1). In accordance with
1
practically no effect on the chiroptical properties (Figure 4). the conditions of Mosher’s rule,[23] the consistent H and
Not surprisingly, the optical rotations of pseudoanguillos- 13C chemical shift differences (∆δ2R,2S) allowed the configu-
Scheme 1. (a) i: EDC, DMAP, dry CH2Cl2, room temp. DMAP: 4-(N,N-dimethylamino)pyridine, EDC: 1-(3-dimethylaminopropyl)-3-
1
ethylcarbodiimide, MPA: α-methoxyphenylacetic acid. (b) H and 13C (in brackets) chemical shift differences (∆δ2R,2S) of (2R,3ЈR)- and
(2S,3ЈR)-6.
Scheme 2. (a) i: MeI, K2CO3, dry acetone, ∆. ii: EDC, DMAP, dry CH2Cl2. (b) 1H and 13C (in brackets) chemical shift differences
(∆δ3ЈЈR,3ЈЈS) of (3R,6ЈR,3ЈЈR)- and (3R,6ЈR,3ЈЈS)-8.
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Isochromans Isolated from the Endophytic Fungus Pseudoanguillospora sp.
ration of the diastereomers at C-2 to be assigned as
(2R,3ЈR)- and (2S,3ЈR)-6 (see Experimental Section for de-
tails).
Experimental Section
General Experimental Procedures: Melting points were determined
in open capillaries with a Büchi melting point apparatus and are
uncorrected. Optical rotations were measured with a Perkin–Elmer
241 MC polarimeter. The IR spectra were taken with a NICOLET–
510P spectrometer. NMR spectra were run with a Bruker ARX 200
or Bruker Avance 500 NMR spectrometer with TMS as internal
standard. EI-MS were obtained with a MAT 8200 mass spectrome-
ter. CD spectra were recorded with a J-810 spectropolarimeter by
using 0.2, 1.0, 5.0, and 10.0 mm cells. Silica gel (230–400 mesh) was
used for CC. Spots were detected on TLC under UV light or by
heating after spraying with a reagent composed of 0.5 mL of anisal-
dehyde in 50 mL of HOAc/1 mL H2SO4.
Subsequently, the (R)-MPA ester of pseudoanguillospo-
rin B [(3R,6ЈR,3ЈЈR)-8] was prepared by the same procedure
after protecting the phenolic hydroxy groups of 3 as its di-
methyl ether 7 (Scheme 2). Comparison of its chemical
shifts with those of (2R,3ЈR)- and (2S,3ЈR)-6 (Scheme 1b),
especially the similar H-7Ј (H-1 in 6) chemical shifts, sug-
gested the (6ЈR) absolute configuration of pseudoanguillos-
porin B. This was unambiguously confirmed by the prepa-
ration of the diastereomeric (S)-MPA ester [(3R,6ЈR,3ЈЈS)-
8], whose NMR spectroscopic data were used to calculate
the ∆δ3ЈЈR,3ЈЈS chemical shift differences by Mosher’s proto-
Extraction and Isolation: The fungus Pseudoanguillospora sp. (6577)
was isolated from the red alga Polyides rotundus from the shores of
the Baltic Sea near Ahrenshoop and was cultivated for 21 d at room
temperature on 5 L of biomalt solid agar. The agar plates were
frozen for 3 d and filtered after thawing. Filtrate and mycelium
were then separately extracted with ethyl acetate. After removal of
the solvent under reduced pressure, 18 g of crude extract was ob-
tained. The crude extract was then washed with hexane and dis-
solved in dichloromethane. The insoluble part was separated by
filtration. Cephalochromin (1, 14.9 g) crystallized from the dichlo-
romethane phase. The mother liquor was then separated by column
chromatography with silica gel and a gradient of dichloromethane/
methanol (0–10% methanol in 1% steps) in 5 fractions. From frac-
tion 3, pseudoanguillosporin A (2) (1.5 g) was purified by prepara-
tive TLC on silica gel by using dichloromethane/methanol (93:7).
Part of the extract was insoluble in dichloromethane; this part was
dissolved in methanol and filtered. The methanol-soluble portion
was then separated into 4 fractions by silica gel column chromatog-
raphy by using the same gradient as before. Fraction 4 contained
pseudoanguillosporin B (3, 222 mg), which was purified by prepar-
ative TLC on reverse-phase silica gel by using methanol/water (1:1).
1
col (Scheme 2).[23] The H and 13C differences showed con-
sistent positive and negative values for the two sides flank-
ing the stereogenic center in accordance with the previous
(6ЈR) assignment.
Biological Activity
The antibacterial, fungicidal, and algicidal properties of
the three compounds in comparison to four standard anti-
biotics were tested in an agar diffusion assay (Table 1), and
for inhibitions of Oomycete and fungal test organisms in a
microtiter assay in liquid media (Table 2). Although all
three compounds were biologically active, the strongest in-
hibitions were caused by 2a. Substances 2a and 3 were anti-
bacterial against the Gram-positive bacterium Bacillus me-
gaterium. All three compounds were antifungal in both as-
says against the fungal and/or Oomycete test organisms.
The best inhibitions of P. infestans were attained with com-
pound 1 and 2a. These substances were also antialgal
against Chlorella fusca.
Cephalochromin (1): Yellow, amorphous powder (CH2Cl2), m.p.
Ͼ180 °C (decomp.) (ref.[4] Ͼ300 °C). [α]2D0 = +618 (c = 0.11, diox-
ane) {ref.[4] [α]2D0 = +727 (c = 0.19, dioxane)}. CD (MeCN, c =
2.84ϫ10–4): λ (∆ε) = 209 (–13.1), 224 (27.8), 238 sh. (–18.3), 264
(–129), 279 sh. (59.5), 292 (137.1), 326 (9.8), 338 (–14.6) nm. 1H
NMR (200 MHz, CD3OD): δ = 1.44 (d, J11,2 = 6.1 Hz, 6 H, 11-H,
11Ј-H), 2.70 (d, J3,2 = 7.4 Hz, 4 H, 3-H, 3Ј-H), 4.51 (m, 2 H, 2-H,
2Ј-H), 5.94 (s, 2 H, 10-H, 10Ј-H), 6.17 (br. s, 2 H, 8-OH, 8Ј-OH),
6.53 (s, 2 H, 7-H, 7Ј-H), 9.63 (s, 2 H, 6-OH, 6Ј-OH), 14.98 (s, 2 H,
5-OH, 5Ј-OH) ppm. 13C NMR (50 MHz, CD3OD): δ = 21.3 (C-
11, C-11Ј, 2 C, CH3), 43.6 (C-3, C-3Ј, 2 C, CH2), 73.7 (C-2, C-2Ј,
2 C, CH), 100.0 (C-10, C-10Ј, 2 C, CH), 100.3 (C-7, C-7Ј, 2 C,
CH), 102.7 (C-9, C-9Ј, 2 C, Cq)*, 102.8 (C-4a, C-4aЈ, 2 C, Cq)*,
105.8 (C-5a, C-5aЈ, 2 C, Cq), 142.6 (C-9a, C-9aЈ, 2 C, Cq), 156.7
(C-10a, C-10aЈ, 2 C, Cq), 160.5 (C-8, C-8Ј, 2 C, Cq), 161.1 (C-6, C-
6Ј, 2 C, Cq), 164.8 (C-5, C-5Ј, 2 C, Cq), 198.9 (C-4, C-4Ј, 2 C, Cq)
ppm. *Assignment of the signals is interchangeable. EI-MS (70 eV,
200 °C): m/z (%) = 518 (27.6) [M+], 194 (8.8), 166 (9.5), 149 (14.3),
125 (10.6), 111 (13.3), 97 (21.3), 83 (27.3), 69 (36.3), 57 (64.4), 43
(100), 28 (95.6).
Table 2. Antifungal activities (values in %-growth in comparison to
the controls).
Metabolite
c
Phytophthora
infestans
Botrytis
cinerea
Pyricularia
oryzae
Septoria
tritici
[ppm]
1
125
31
8
2.6
1.6
6.3
82.3
100
100
50.3
55.9
75.2
77.9
81.5
77.3
5.8
10.9
29.1
61.5
65.4
72.8
74
74.1
87.4
86.4
85.4
84.5
2
0.5
0.125
2a
125
31
8
5.3
2.4
58.5
100
100
100
0.2
0.2
42
83.6
82.3
83.2
0
0.3
0
0
87
100
3.9
0.4
0
68.6
100
100
2
0.5
0.125
Pseudoanguillosporin A (2a): Yellow solid, m.p. 46–48 °C. [α]2D0
=
–72.5 (c = 0.65, MeOH). CD (MeCN, c = 1.23ϫ10–3): λ (∆ε) =
198 (–9.8), 218 sh. (–1.0), 238 (0.7), 284 (–0.4) nm. UV (CH3CN):
3
125
31
8
42.6
85.1
99.5
95.2
100
90.7
85.6
76
79.8
80.7
81.4
100
92
89.5
88.9
91.2
89.7
100
100
99.3
90.4
86.1
85.3
λ (log ε) = 283 (3.42), 201 (4.72) nm. IR (film): ν = 3437, 3330,
˜
2952, 2922, 2852, 1604, 1462, 1452, 1254, 1105, 1043 cm–1. 1H
NMR (200 MHz, CD3OD): δ = 0.92 (t, J15,14 = 6.8 Hz, 3 H, 7Ј-
H), 1.33 (m, 8 H, 3Ј-H, 4Ј-H, 5Ј-H, 6Ј-H), 1.42 (m, 1 H, 2Ј-Ha),
2
0.5
0.125
99.9
1.60 (m, 3 H, 1Ј-H, 2Ј-Hb), 1.98 (s, 3 H, 5-CH3), 2.33 (dd, Jgem
=
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K. Krohn et al.
FULL PAPER
16.4 Hz, J4a,3 = 10.8 Hz, 1 H, 4-Ha), 2.57 (dd, Jgem = 16.4 Hz, J4b,3
(66 mg, 0.48 m) in dry acetone (5 mL) was added MeI (50 mg,
= 2.6 Hz, 1 H, 4-Hb), 3.50 (m, 1 H, 3-H), 4.51 (d, Jgem = 14.7 Hz, 0.34 m). The reaction mixture was heated at reflux for 24 h and
1 H, 1-Ha), 4.85 (d, Jgem = 14.7 Hz, 1 H, 1-Hb), 6.21 (s, 1 H, 7-H)
ppm. 13C NMR (125 MHz, CD3OD): δ = 9.1 (5-CH3), 13.2 (7Ј- and the residue was diluted with water and extracted with EtOAc.
then filtered. The solvent was evaporated under reduced pressure,
CH3), 22.4 (6Ј-CH), 25.4 (2Ј-CH2), 29.1 (6Ј-CH2)**, 29.5 (3Ј-
CH2)**, 31.7 (5Ј-CH2), 32.3 (CH2, C-4), 36.0 (1Ј-CH2), 64.7 (CH2,
The organic layer was dried with MgSO4 and evaporated under
reduced pressure, and the residue was purified by preparative TLC
C-1), 75.0 (CH, C-3), 99.7 (CH, C-7), 112.7 (Cq, C-5)*, 112.8 (Cq, (hexane/EtOAc, 1:7) to yield a pale-yellow oil (16 mg, 64%) that
C-8a)*, 133.4 (Cq, C-4a), 150.8 (Cq, C-8), 153.5 (Cq, C-6) ppm. crystallized during cooling to give a white, amorphous powder
*Assignments marked with * and ** are interchangeable. EI-MS
(70 eV, 200 °C): m/z (%) = 278 (20) [M+], 179 (9), 150 (78), 99 (26),
86 (58), 57 (27), 44 (100), 28 (31). HRMS (EI): calcd. for C17H26O3
278.18819; found 278.18869.
(CHCl3). M.p. 32–37 °C. [α]2D0 = –86.2 (c = 0.115, chloroform). IR
(film): ν = 3364, 2966, 2938, 2922, 2830, 2864, 2843, 1454, 1054,
˜
1032, 1016 cm–1. 1H NMR (500 MHz, CDCl3): δ = 1.12 (d, J7Ј,6Ј
=
6.5 Hz, 3 H, 7Ј-H), 1.31 (m, 2 H, 3Ј-H), 1.35 (m, 2 H, 2Ј-H), 1.42
(m, 2 H, 5Ј-H), 1.51 (m, 2 H, 4Ј-H), 1.55 (m, 2 H, 1Ј-H), 1.97 (s, 3
H, 5-H), 2.35 (dd, J = 16.5, 11 Hz, 4-Hax), 2.53 (dd, J = 16.5,
2.0 Hz, 1 H, 4-Heq), 3.42 (s, 1 H, OH), 3.47 (m, 1 H, 3-H), 3.73 (s,
3 H, 6-OMe), 3.75 (s, 3 H, 8-OMe), 3.76 (m, 1 H, 6Ј-H), 4.48 (d,
J = 15 Hz, 1 H, 1-Ha), 4.82 (d, J = 15 Hz, 1 H, 1-Hb), 6.27 (s, 1
H, 7-H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.12 (5-Me),
23.50 (C-7Ј), 25.58 (C-2Ј), 25.71 (C-4Ј), 29.67 (C-4), 32.42 (C-3Ј),
36.11 (C-1Ј), 39.30 (C-5Ј), 55.29 (6-OCH3), 55.94 (8-OCH3), 64.72
(C-1), 68.14 (C-6Ј), 74.45 (C-3), 92.83 (C-7), 115.81 (Cq-8), 121.77
(Cq-5a), 134.12 (Cq-8a), 156.46 (Cq-6), 161.12 (Cq-8) ppm. *Assign-
ment of the signals is interchangeable. HRMS (EI): calcd. for
C19H30NaO4 345.204; found 345.204.
Pseudoanguillosporin B (3): Yellow oil. [α]2D0 = –72.8 (c = 0.36,
MeOH). UV (MeOH): λ (log ε) = 427 (4.64), 338 (5.39), 286 (6.36)
nm. IR (Film): ν = 3383, 2931, 2858, 1606, 1462, 1335, 1259, 1107,
˜
1045 cm–1. 1H NMR (500 MHz, CD3OD): δ = 1.17 (d, J15,14
=
6.2 Hz, 3 H, 7Ј-H), 1.49 (m, 10 H, 1Ј-H, 2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H),
1.98 (s, 3 H, 5-H), 2.33 (dd, Jgem = 16.6 Hz, J4a,3 = 10.8 Hz, 1 H,
4-Ha), 2.59 (dd, Jgem = 16.6 Hz, J4b,3 = 2.2 Hz, 1 H, 4-Hb), 3.51
(m, 1 H, 3-H), 3.73 (m, 1 H, 14-H), 4.50 (d, Jgem = 14.7 Hz, 1 H,
1-Hb), 4.84 (d, Jgem = 14.7 Hz, 1 H, 1-Ha), 6.21 (s, 1 H, 7-H) ppm.
13C NMR (125 MHz, CD3OD): δ = 9.0 (5-CH3), 22.2 (CH3, C-7Ј),
25.3 (CH2, C-4Ј)**, 25.5 (CH2, C-2Ј)**, 29.5 (CH2, C-3Ј), 32.3
(CH2, C-4), 35.8 (CH2, C-1Ј), 38.8 (CH2, C-5Ј), 64.6 (CH2, C-1),
67.3 (CH, C-6Ј), 74.9 (CH, C-3), 99.6 (CH, C-7), 112.7 (Cq, C-5)*,
112.8 (Cq, C-8a)*, 133.4 (Cq, C-4a), 150.8 (Cq, C-8), 153.5 (Cq, C-
6) ppm. * Assignments marked with * and ** are interchangeable.
EI-MS (70 eV, 200 °C): m/z (%) = 294 (2) [M+], 279 (3), 177 (5),
143 (13), 111 (7), 97 (11), 71 (18), 56 (32), 45 (62), 31 (100). HRMS
(EI): calcd. for C17H26O4 294.18311; found 294.18316.
(R)-α-MPA Ester of 6,8-Dimethyl Ether of Pseudoanguillosporin B
[(3R,6ЈR,3ЈЈR)-8]: A stirred solution of (R)-(–)-α-MPA [(R)-5]
(13 mg, 0.08 m), EDC (15 mg, 0.08 m), and DMPA (3.8 mg,
0.03 m) in anhydrous dichloromethane (2.5 mL) was treated drop-
wise with a solution of 7 (8.6 mg, 0.03 m) in anhydrous dichloro-
methane (1 mL). Stirring was continued for 20 h at room tempera-
ture, and the reaction mixture was washed with NaHCO3 solution
and then with water. The organic layer was dried (MgSO4) and
evaporated under reduced pressure. The residue was purified by
preparative TLC (hexane/EtOAc, 2:1) to give a white, amorphous
powder (7.6 mg, 60%) (CHCl3). M.p. 64–67 °C. [α]2D0 = –94.9 (c =
(R)-α-MPA Esters of (؎)-Heptan-2-ol [(2R,3ЈR)- and (2S,3ЈR)-6]: A
stirred solution of (R)-α-MPA [(R)-5] (175 mg, 1 m), EDC
(200 mg, 1 m), and DMPA (20 mg, 0.16 m) in anhydrous dichlo-
romethane (4.5 mL) was treated dropwise with a solution of (Ϯ)-
heptane-2-ol (0.05 mL, 0.35 m) in anhydrous dichloromethane
(2 mL) and stirring was continued for 24 h at room temperature.
After washing the reaction mixture with a solution of NaHCO3
and then with water, the organic layer was dried with MgSO4 and
evaporated under reduced pressure. The raw material was purified
by preparative TLC on silica gel (hexane/EtOAc, 6:1) to give two
products, (R)- and (S)-ester of the starting material as oils (38 and
32 mg, 41 and 34%, respectively). Data for (R)-α-MPA-ester of
(2R)-heptan-2-ol: [α]2D0 = –48.9 (c = 1.27, chloroform). 1H NMR
(360 MHz, CDCl3): δ = 0.77 (t, 3 H, 7-H), 0.86 (m, 2 H, 6-H)*,
1.00 (m, 4 H, 4-H, 5-H)*, 1.21 (d, J = 6.5 Hz, 3 H, 1-H), 1.42 (m,
2 H, 3-H), 3.41 (s, 3 H, 3Ј-OMe), 4.71 (s, 1 H, 2Ј-H), 4.93 (m, 1 H,
2-H), 7.32–7.44 (m, 5 H, Ph) ppm. 13C NMR (90 MHz, CDCl3): δ
= 13.85 (C-7), 20.02 (C-1), 22.37 (C-6), 24.56 (C-4), 31.33 (C-5),
35.67 (C-3), 57.21 (2Ј-OMe), 71.98 (C-2), 82.68 (C-3Ј), 127.1 (C-6Ј,
C-7Ј, C-8Ј), 128.51 (C-5Ј, C-9Ј), 136.52 (Cq-4Ј), 170.36 (Cq-2Ј) ppm.
*Assignment of the signals is interchangeable. Data for (R)-MPA-
0.305, chloroform). IR (film): ν = 3033, 2992, 2968, 2933, 2859,
˜
2835, 1737, 1596, 1492, 1452, 1378, 1319, 1214, 1125, 1095 cm–1.
1H NMR (500 MHz, CDCl3): δ = 1.02 (m, 2 H, 4Ј-H), 1.16 (m, 2
H, 3Ј-H), 1.22 (d, J7Ј,6Ј = 6.5 Hz, 3 H, 7Ј-H), 1.25 (m, 1 H, 2Ј-Ha),
1.36 (m, 1 H, 2Ј-Hb), 1.39 (m, 1 H, 5Ј-Ha), 1.47 (m, 2 H, 1Ј-Ha, 5Ј-
Hb), 1.57 (m, 1 H, 1Ј-Hb), 2.05 (s, 3 H, 5-Me), 2.39 (dd, J = 16.5,
11 Hz, 1 H, 4-Hax), 2.57 (dd, J = 16.5, 2.5 Hz, 1 H, 4-Heq), 3.41 (s,
3 H, 3ЈЈ-OMe), 3.48 (m, 1 H, 3-H), 3.80 (s, 3 H, 6-OMe), 3.83 (s,
3 H, 8-OMe), 4.54 (d, J = 15.5 Hz, 1-Ha), 4.72 (s, 1 H, 3ЈЈ-H), 4.89
(d, J = 15.0 Hz, 1-Hb), 4.95 (m, 1 H, 6Ј-H), 6.34 (s, 1 H, 7-H),
7.31–7.45 (m, 5 H, Ph) ppm. 13C NMR (125 MHz, CDCl3): δ =
10.12 (5-Me), 20.06 (C-7Ј), 25.38 (C-2Ј), 24.91 (C-4Ј), 29.29 (C-3Ј),
32.39 (C-4), 35.70 (C-5Ј), 36.00 (C-1Ј), 55.3 (6-OMe), 55.94 (8-
OMe), 57.25 (3ЈЈ-OMe), 64.67 (C-1), 72.06 (C-6Ј), 74.39 (C-3),
82.72 (C-3ЈЈ), 92.87 (C-7), 115.82 (Cq-5)*, 115.99 (Cq-8a)*, 127.06
(C-7ЈЈ), 127.21 (C-5ЈЈ, C-9ЈЈ), 128.52 (C-6ЈЈ, C-8ЈЈ), 134.11 (C-4ЈЈ),
136.58 (Cq-5a), 153.87 (Cq-8), 156.49 (Cq-6), 170.37 (Cq-2ЈЈ) ppm.
*Assignment of the signals is interchangeable. HRMS: calcd. for
C28H38NaO6 [M+] 493.257; found 493.255.
1
ester of (2S)-heptan-2-ol: [α]2D0 = –25.4 (c = 1.08, chloroform). H
NMR (360 MHz, CDCl3): δ = 0.85 (t, 3 H, 7-H), 1.08 (d, J =
6.1 Hz, 3 H, 1-H), 1.24–1.53 (m, 8 H, 4-H, 5-H, 6-H)*, 1.53 (m, 2
H, 3-H), 3.42 (s, 3 H, 3Ј-OMe), 4.74 (s, 1 H, 2Ј-H), 4.97 (m, 1 H,
CH, 2-H), 7.31–7.46 (m, 5 H, Ph) ppm. 13C NMR (90 MHz,
CDCl3): δ = 13.92 (C-7), 19.59 (C-1), 22.46 (C-6), 24.92 (C-4),
31.38 (C-5), 35.69 (C-3), 57.28 (2Ј-OCH3), 72.06 (C-2), 82.79 (C-
3Ј), 127.01 (C-6Ј, C-7Ј, C-8Ј), 128.49 (C-5Ј, C-9Ј), 136.27 (Cq-4Ј),
170.37 (Cq-2Ј) ppm, *Assignment of the signals is interchangeable.
(S)-α-MPA Ester of 6,8-Dimethyl Ether of Pseudoanguillosporin B
[(3R,6ЈR,3ЈЈS)-8]: A solution of 7 (7 mg, 0.02 m) in anhydrous
dichloromethane (1 mL) was added dropwise to a stirred solution
of (S)-α-MPA [(S)-5] (12 mg, 0.07 m), EDC (13 mg, 0.07 m),
and DMPA (1.3 mg, 0.01 m) in dry dichloromethane (2.5 mL)
and stirring was continued for 24 h at room temperature. After
washing the reaction mixture with a solution of NaHCO3 and then
with water, the organic layer was dried with MgSO4 and the sol-
vents evaporated. The raw material was purified by preparative
6,8-Dimethyl Ether of Pseudoanguillosporin B (7): To a suspension
of pseudoanguillosporin B (3) (23 mg, 0.08 m) and K2CO3
1432
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1427–1434

Isochromans Isolated from the Endophytic Fungus Pseudoanguillospora sp.
TLC (hexane/EtOAc, 2:1) to give a colorless oily material (5 mg,
search Fund (OTKA), National Office for Research and Technol-
50%) (CHCl3). [α]2D0 = –38.6 (c = 0.27, chloroform). IR (film): ν =
ogy (NKTH), and Bolyai János Foundation for financial support
(T-049436, NI-61336, and K-68429). We thank K. E. Kövér for the
NMR measurements of the MPA esters, and Qunxiu Hu and Vera
˜
3060, 3027, 2998 sh., 2969 sh., 2931, 1740, 1599, 1490, 1455, 1364,
1318, 1216, 1123, 1091 cm–1. 1H NMR (500 MHz, CDCl3): δ =
1.09 (d, J7Ј,6Ј = 6 Hz, 3 H, 7Ј-H), 1.31 (m, 4 H, 3Ј-H, 4Ј-H), 1.39 Diekmann for excellent technical assistance.
(m, 1 H, 2Ј-Ha), 1.50 (m, 2 H, 2Ј-Hb, 5Ј-Ha), 1.58 (m, 1 H, 1Ј-Ha),
1.59 (m, 1 H, 5Ј-Hb), 1.64 (m, 1 H, 1Ј-Hb), 2.04 (s, 3 H, 5-Me),
2.41 (dd, J = 16.5, 10.5 Hz, 1 H, 4-Hax), 2.59 (dd, J = 16.5, 2.5 Hz,
1 H, 4-Heq), 3.42 (s, 3 H, 3ЈЈ-OMe), 3.52 (m, 1 H, 3-H), 3.80 (s, 3
H, 6-OMe), 3.82 (s, 3 H, 8-OMe), 4.54 (d, J = 15 Hz, 1 H, 1-Ha),
4.74 (s, 1 H, 3ЈЈ-H), 4.89 (d, J = 15 Hz, 1 H, 1-Hb), 4.97 (6Ј-H),
6.34 (s, 1 H, 7-H), 7.36 (m, 3 H, 6ЈЈ, 7ЈЈ, 8ЈЈ-H), 7.42 (m, 2 H, 5ЈЈ,
9ЈЈ-H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.12 (5-Me), 19.63
(C-7Ј), 25.27 (C-2Ј)*, 25.48 (C-4Ј)*, 29.45 (C-3Ј), 32.41 (C-4), 35.73
(C-5Ј), 36.09 (C-1Ј), 55.30 (6-OMe), 55.93 (8-OMe), 57.30 (3ЈЈ-
OMe), 64.71 (C-1), 72.06 (C-6Ј), 74.41 (C-3), 82.77 (C-3ЈЈ), 92.85
(C-7), 115.81 (Cq-5)*, 115.99 (Cq-8a)*, 127.06 (C-4ЈЈ, C-9ЈЈ), 127.27
(C-7ЈЈ), 128.53 (C-6ЈЈ, C-8ЈЈ), 134.1 (C-4ЈЈ), 136.43 (C-5a), 153.86
(Cq-8), 156.48 (Cq-6), 170.38 (Cq-2ЈЈ) ppm. *Assignment of the sig-
nals is interchangeable. HRMS: calcd. for C28H38NaO6 [M+]
493.257; found 493.254.
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[12]
[13]
Computational Section: Conformational searches (MMFF and
PM3 methods) and geometry optimizations [DFT method, B3LYP/
6-31G(d) level] were run with Spartan06, Wavefunction, Inc., Irvine
CA, with default parameters and convergence criteria. CD calcula-
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spectra are available upon request to the authors. Rotational
strengths were computed with the semiempirical ZINDO-S/CI
method[16] including all possible configurational interactions (full
CI), or with the TDDFT method by using various functionals
(B3LYP,[26] BH&HLYP,[27] BP86[28]) and the TZVP basis set.[29] CD
spectra were generated as the sum of Gaussians with 1500 and
2500 cm–1 half-height widths, respectively for 1 and 2c, by using
dipole–velocity computed rotational strengths. For 2c, the first 4–
6 states, depending on the functional, had transition energies below
the estimated ionization potential (5.85 eV, B3LYP/TZVP).[30] They
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1
1
include the aromatic Lb and La transitions that contribute to the
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Acknowledgements
K. K., I. K., B. S., and S. D. thank BASF AG and the Bundesmini-
sterium für Bildung und Forschung (BMBF) (project no.
03F0360A); S. A. and T. K. thank the Hungarian Scientific Re-
Eur. J. Org. Chem. 2009, 1427–1434
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1433

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FULL PAPER
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Received: November 4, 2008
Published Online: February 4, 2009
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