Pochonins A-F, new antiviral and antiparasitic resorcylic acid lactones from Pochonia chlamydosporia var. catenulata

Article abstract of DOI:10.1021/np020556v

Monorden (1) and the novel resorcylic acid lactones pochonins A (2), B (4), C (6), D (7), and E (8) as well as tetrahydromonorden (5) and pseurotin A (22) were isolated from cultures of the clavicipitaceous hyphomycete Pochonia chlamydosporia var. catenulata strain P 0297. Fermentation of P 0297 in bromide-containing culture media led to a shift in secondary metabolite production and yielded monocillins III (3) and II (9) as major metabolites besides monorden (1) as well as the novel compounds pochonin F (10) and a monocillin II glycoside (11) as minor metabolites. Most of these compounds showed moderate activities in a cellular replication assay against Herpes Simplex Virus 1 (HSV1) and against the parasitic protozoan Eimeria tenella. In contrast to the structurally related zearalenone derivatives none of the metabolites of strain P 0297 were found to be active in a fluorescence polarization assay for determination of modulatory activities on the human estrogenic receptor ERβ. β-Zearalenol (17), but not zearalenone (15) and α-zearalenol (16), showed antiherpetic effects. We report the production, isolation, and structure elucidation of compounds 1-11 and their biological characterization.

Full text of DOI:10.1021/np020556v

J . Nat. Prod. 2003, 66, 829-837
829
P och on in s A-F , New An tivir a l a n d An tip a r a sitic Resor cylic Acid La cton es fr om
P och on ia ch la m yd ospor ia va r . ca ten u la ta
,
†
†
†
‡
§,|
Veronika Hellwig,* Anke Mayer-Bartschmid, Hartwig M u¨ ller, Gisela Greif, Gerald Kleymann,
†
⊥
†
Werner Zitzmann, Hans-Volker Tichy, and Marc Stadler
Bayer Health Care, Pharma Research, Life Science Center Natural Products, Bayer Health Care, Animal Health, Research &
Development, and Bayer Health Care, Pharma Research, Antiviral Research, P.O. Box 101709, D-42096 Wuppertal, Germany,
and T U¨ V ISB, Engesserstrasse 4b, D-79108 Freiburg, Germany
Received December 4, 2002
Monorden (1) and the novel resorcylic acid lactones pochonins A (2), B (4), C (6), D (7), and E (8) as well
as tetrahydromonorden (5) and pseurotin A (22) were isolated from cultures of the clavicipitaceous
hyphomycete Pochonia chlamydosporia var. catenulata strain P 0297. Fermentation of P 0297 in bromide-
containing culture media led to a shift in secondary metabolite production and yielded monocillins III (3)
and II (9) as major metabolites besides monorden (1) as well as the novel compounds pochonin F (10)
and a monocillin II glycoside (11) as minor metabolites. Most of these compounds showed moderate
activities in a cellular replication assay against Herpes Simplex Virus 1 (HSV1) and against the parasitic
protozoan Eimeria tenella. In contrast to the structurally related zearalenone derivatives none of the
metabolites of strain P 0297 were found to be active in a fluorescence polarization assay for determination
of modulatory activities on the human estrogenic receptor ERâ. â-Zearalenol (17), but not zearalenone
(
15) and R-zearalenol (16), showed antiherpetic effects. We report the production, isolation, and structure
elucidation of compounds 1-11 and their biological characterization.
The vast majority of the world population is infected by
at least one member of the human herpes virus family.
These viruses remain latent in their hosts after primary
infection and may be reactivated from a pool of latently
infected cells upon diverse internal and external stimuli,
thus causing serious symptoms. Since the late 1970s,
herpes virus infections have been mostly treated with
nucleoside antibiotics such as acyclovir (Zovirax). Develop-
ing resistance to these drugs and increasing numbers of
immunocompromised patients have created medical need
for novel antivirals. This has led to high-throughput based
screening (HTS) approaches, which recently resulted in the
development of nonnucleosidic antiviral agents that act on
1
the helicase-primase in Herpes Simplex Virus (HSV). With
the same HTS system, microbial extracts were screened
for potent antiherpetic metabolites. In the following we
wish to report the isolation and biological and physico-
chemical characterization of a new group of biologically
active metabolites 1-11 from the fungal strain P 0297.
Resu lts a n d Discu ssion
Shake flask fermentations of P 0297, Pochonia chlamy-
dosporia var. catenulata (Kamyschko ex Barron & Onions)
Zare & W. Gams (see Figure 1), were carried out in the
culture media YMG, Q6/2, MGP, and MGP-Br. Aliquots of
the culture broth were analyzed during the fermentation
course for determination of growth parameters, estimation
of secondary metabolite production, and biological activities
(Figure 2) in order to harvest the fermentors at the optimal
production rate and pH value regarding the instability of
the bioactive metabolites in alkaline medium. MGP (and
later MGP-Br) were chosen for scale-up in 10 L fermentors
due to slightly higher production rates in this medium
compared with Q6/2 and YMG. The isolation procedures
using reversed-phase (RP) HPLC and, if necessary, sub-
sequent high-performance gel-permeation chromatography
(HPGPC) are summarized in Table 1. From stirring fer-
mentors and corresponding shake flask fermentation car-
ried out in the same media, similar production rates of the
resorcylic acid lactones (RAL) 1-11 were obtained.
*
Corresponding author. Tel: +49-202-368092. Fax: +49-202-365312.
E-mail: Veronika.Hellwig.VH@bayer-ag.de.
†
Bayer Health Care, Pharma Research, Life Science Center Natural
Products.
‡
Bayer Health Care, Animal Health, Research & Development.
Bayer Health Care, Pharma Research, Antiviral Research.
Current address: Leopoldsh o¨ herstrasse 7, D-32107 Bad Salzuflen,
§
|
Germany.
⊥
T U¨ V ISB.
1
0.1021/np020556v CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/03/2003

8
30 J ournal of Natural Products, 2003, Vol. 66, No. 6
Hellwig et al.
monorden (1). This well-known RAL, also named as radici-
col in the literature, had previously been reported from
various ascomycetes,2 including Pochonia chlamydospo-
ria (albeit under the synonym “Verticillium chlamydospo-
rium”).6
-5
The bioassay-guided isolation procedure yielded several
bioactive co-metabolites of monorden (1), which were
produced in minor quantities (1-2 mg/L, less than 1 mg/L
for 5) and differ in the substitution pattern of the 14-
membered macrocyclic lactone ring.
Pochonin A (2) represents a dihydro derivative of monor-
den (1) due to its molecular formula C18
H
19ClO
6
from
HREIMS. Indeed, the H NMR signals (Table 2) for only
one double bond in conjugation to the carbonyl moiety (δ
1
H
1
1
6
.06, δ
COSY correlation signals, the two additional methylene
groups (δ 1.20-1.25, 2.20-2.29 and δ 2.20-2.29, 2.38)
H
6.92) are detected. Due to corresponding H- H
H
H
F igu r e 1. Dictyochlamydospores of Pochonia chlamydosporia var.
catenulata. SEM micrograph of strain P 0297 (YMG, 500×).
are located between the epoxide and the R,â-unsaturated
carbonyl moiety. Pochonin A (2) differs only in the halogen
substitution from monocillin III (3).7
Ta ble 1. Isolation of 1-11 and 22
retention time tR [min]
Pochonin B (4), C18
oxygen atom in comparison to pochonin A (2). The H NMR
signals (Table 2) for a secondary alcohol function at δ 4.26
ca. 5.3) as well as its 1H- H COSY
7
H19ClO , contains an additional
1
system A
RP-HPLC
small scale
system B
RP-HPLC
large scale
system C
HPGPC
H
1
C H
(δ 68.04; OH: δ
preparationa
correlation to the â-H atom of the R,â-unsaturated carbonyl
b
1
2
3
4
5
6
7
8
9
1
1
2
all media
78-80
84-86
74-75
59-60
-
60-72
74-76
77-79
42-45
80-85
58-59
125-129
52-58
101-111
69-74
60-62
-
36-39
1
indicate formula 4 for pochonin B. H NMR signals for a
c
c
d
all media
-
H C
diastereotopic methylene group at δ 1.15, 2.36 (δ 39.98)
are assigned to position 6 between the secondary alcohol
function and the epoxide ring.
Compound 5 exhibits a molecular ion peak at m/z 368
6
for C18H21ClO and represents a tetrahydro derivative of
c
MGP-Br
-
-
all media
b
MGP or MGP-Br
34-35
29-32
39-40
-
b
all media
all media
-
b
-
64-66
c
all media
monorden (1). No signals other than the one for H-15 are
c
MGP-Br
MGP-Br
-
1
b
detected in the low-field region of the H NMR spectrum,
0
1
2
-
-
53-55
19-24
23-30
-
b
but additional signals for a (CH
2 4
) moiety appear in the
MGP-Br
c
Q6/2
aliphatic shift region. The natural product is identical with
2
,3
a
the semisynthetic tetrahydromonorden 5 (see below).
If not indicated otherwise, all culture media described in the
Experimental Section are suited to produce the respective com-
For pochonin C (6) the HRMS of the molecular peak at
m/z 400 with a characteristic isotopic pattern for two
pounds. Purification by RP-HPLC followed by HPGPC. c RP-
b
HPLC in either system A or B (see Experimental Section) yielded
chlorine atoms resulted in the molecular formula C18
Cl O . The H NMR spectrum (Table 2) contains the signals
2 6
18
H -
d
the pure compounds at the indicated tR.
employed for purification.
-: Method not
1
for the Michael system as in monorden (1), but lacks those
for the trans epoxide ring. Instead a signal for a secondary
hydroxyl function at δ 3.98 (δ 70.70; OH; δ ca. 5.45)
H C H
The main metabolite was produced in yields of 15-20
1
mg/L and identified by LC-MS and H NMR data as
F igu r e 2. Time course of production of monorden (1) and pseurotin A (22) in Q6/2 medium (150 mL shake flasks) by strain P 0297.

Antiviral Resorcylic Acid Lactones
J ournal of Natural Products, 2003, Vol. 66, No. 6 831
Ta ble 2. 1H NMR Data [500 MHz] of Pochonins A-C (2, 4, 6) in [D6]DMSO
2
4
6
proton
δH [ppm]
1.31
5.12
1.77
J HH [Hz]
6.3
δH [ppm]
J HH [Hz]
6.4
δH [ppm]
1.37
J HH [Hz]
Me-1
2
3
d
m
m
1.31
5.13
1.77
d
m
m
d
6.1
5.28
m
m
m
m
d
Ha: 1.91
Hb: 1.83
3.98
OH: 5.45
5.10
4
2.80
m
2.80
m
5.0
10.5, 5.0
10.5, 10.5
5
6
2.59
dm
m
m
m
m
8.6
2.60
dm
10.2
12.1, 11.4, 10.2
12.1
dd
dd
R: 1.20-1.25
â:2.20-2.29
Ha: 2.20-2.29
Hb: 2.38
6.92
R: 1.15
â: 2.36
4.26
ddd
dm
m
d
dd
d
5.78
7
6.26
dd
10.5, 10.5
OH: 5.34a
6.77
3.5
15.9, 8.1
15.9
8
9
ddd
d
15.6, 10.2, 5.0
15.6
7.12
6.04
dd
d
16.1, 11.2
16.1
6.06
6.10
1
1
0
1
Ha: 4.17
Hb: 4.08
OH: 10.81
6.52
OH: 10.73
d
d
s, br
s
s
17.5
17.5
Ha: 4.18
Hb: 4.07
OH: 10.81
6.52
d
d
s
s
s
17.7
17.7
Ha: 4.04
Hb:3.61
d
d
s
s
s
16.0
16.0
a
OH: 10.12b
6.56
1
1
1
4
5
6
a
OH: 10.57b
OH: 10.81
a
Signals may be interchanged. b δH dependent on concentration.
Ta ble 3. 1H NMR Data [500 MHz] of Pochonins D-E (7, 8) in
D6]DMSO
[
7
8
proton
δ
H
[ppm]
J
HH [Hz]
δ
H
[ppm]
J
HH [Hz]
6.1
Me-1 1.23
2
3
d
m
6.2
1.23
4.98
: 2.33-2.40
: 2.10-2.23
5.30
d
5.00
m
m
m
H
H
a
: 2.32
: 2.19
dm 14.4
H
H
a
a
b
b
m
m
b
F igu r e 3. Selected NOESY correlation signals of pochonin B (4) (500
MHz, [D ]DMSO).
4
5.21
ddd 15.6, 8.9,
8
.9
6
5
6
5.21
2.21-2.01
m
m
5.23
4.00
OH: 5.03
dd 15.6, 6.5
m
The three asymmetric carbon atoms in monorden (1)
were determined to have R configurations from single-
crystal X-ray diffraction through an analysis of the anoma-
d
4.1
7
8
2.21-2.01
6.61
m
H
H
a
: 2.33-2.40
: 2.06-2.17
m
m
b
9
b
lous scattering. The sample from strain P 0297 has the
ddd 15.5, 7.4, 7.4 6.60
ddd 15.8, 7.9,
same absolute configuration, as evidenced by its optical
rotation. The stereochemistry of pochonins A (2), B (4), C
(6), D (7), and E (8) is proposed to be analogous to that of
monorden (1). The relative configuration of the secondary
alcohol function in pochonin B (4) was assigned by a
NOESY experiment (see Figure 3). The trans epoxide ring
as described for monorden (1) is assumed due to the very
weak NOESY correlation signal between the two protons
H-4 and H-5. H-4 shows a NOESY correlation signal to
7
.9
9
1
5.78
d
d
d
15.5
17.1
17.1
5.78
d
d
d
15.8
17.2
17.2
1
H
H
a
: 3.86
: 3.70
H
H
a
: 3.87
: 3.71
b
b
1
1
1
1
1
2
3
4
5
6
OH: 10.64c s br
OH: 10.63c
s
s
s
6.53
s
s
6.52
OH: 10.28c
OH: 10.23c
a
b
Signal hidden by signal of 6-CH2 and 7-CH2. Overlapping
c
H-6R at δ
reotopic H-6â at δ
secondary alcohol function at C-7 shows strong NOESY
correlation signals to H-5 and to H-6â at δ 2.36, but only
a weak one to H-6R at δ 1.15. Due to the lack of the
1.15, while H-5 correlates with the diaste-
signals. Signals may be interchanged.
H
H
2.36. The methine proton of the
1
1
shows H- H COSY correlation signals to the methylene
group in position 3 at δ 1.91 and 1.83 as well as to a
secondary methine proton at δ 5.10 (δ 60.26). The latter
shows H- H COSY correlation signals to the proton δ
.78 in the δ position of the Michael system. Therefore,
H
H
H
C
H
1
1
H
epoxide ring, pochonin C (6) as well as E (8) are too flexible
to enable determination of the relative configuration of
their secondary alcohol function by NOESY correlation
spectroscopy.
The pochonins obtained by fermentation of P 0297 with
standard conditions have a chlorine substitution in the
aromatic nucleus in common. Considering previous results
5
formal ring opening of the epoxide ring yields a chlorhydrin
functionality in 6. This structure is already part of a patent
describing tyrosine kinase inhibitors, but it is described
8
there as a semisynthetic derivative of monorden (1).
1
For two further metabolites the H NMR signals (Table
3
) corresponding with the epoxide ring are missing. In-
on the influence of halogen salts to increase the metabolic
diversity in fungi by fermentation,1
0,11
the MGP medium
stead, signals for olefin protons in position H-4 and H-5 at
1
1
δ
H
5.2-5.3 and the corresponding H- H COSY correlation
signals are detected. Pochonin D (7), with the molecular
formula C18 , contains one oxygen less in compari-
son with pochonin A (2). Pochonin E (8), with the molecular
formula C18 , is a formal oxidation product of
pochonin D (7). In the H NMR spectrum there are signals
for a secondary alcohol function (δ 4.00, 5.03 (OH)) in
was thus supplemented with bromide salts in substitution
for the chloride salts in the standard protocol (see Experi-
mental Section). While monorden (1) still constituted a
main component in MGP-Br medium, some further biologi-
cally active compounds, 3, 9, 10, and 11, were isolated as
described in the Experimental Section and Table 1, which
could not be detected in bromide-free culture media.
NMR and MS data (see Experimental Section) identified
dechloro analogues of monorden and the pochonins, some
H
19ClO
5
H
19ClO
6
1
H
1
1
position 6, in agreement with a H- H COSY correlation
signal from δ 4.00 to δ 5.23 of the isolated double bond.
H
H

8
32 J ournal of Natural Products, 2003, Vol. 66, No. 6
Hellwig et al.
Ta ble 4. 13C NMR Data [125 MHz] of Monorden (1), Pochonins
A-D (2, 4, 6, 7), and Tetra- (5) and Hexahydromonorden (12)
in [D6]DMSO
NMR signal for a phenolic proton is detected at δ
which is located at position 16 due to its HMBC signals to
C-15 (δ 102.62), C-16 (δ 158.18), and C-17 (δ 114.00).
The remaining NMR signals (Table 5) belong to a aldofura-
nose moiety linked to position 14, as evidenced by its
H
10.25,
C
C
C
δ
C
[ppm]a
carbon
1
2
4
5
12
6
7
HMBC signal from the anomeric proton H-1′ (δ
H
5.47) to
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
18.52
69.82
36.46
55.08
55.08
136.13
129.78
17.76
70.66
36.63
54.40
55.84
29.90
27.81
17.51
70.98
36.31
54.58
53.74
39.98
68.04
19.97
70.30
37.36
54.95
56.72
29.15
23.18
23.18
39.77
20.49
69.12
43.66
63.77
32.95
18.91
69.11
37.30
70.70 127.74
60.26 131.47
19.01
71.48
37.89
the quaternary carbon C-14 (δ 159.27). Exact determina-
C
tion of the sugar was not performed due to the limited
b
b
amount of sample available.
Monorden (1) has been shown to be unstable in alkali.3
22.84 136.51
25.28 129.51
23.06 139.09 147.49
40.91 132.04 128.69
30.57
30.57
Hydrogenation of monorden (1) with H /Pd-C under
2
138.02 148.70 149.75
130.67 129.95 127.08
atmospheric pressure yielded two products: tetrahy-
dromonorden (5), with an intact oxirane ring, and hexahy-
dromonorden (12), with a secondary alcohol function from
oxirane ring opening.2 Microscale epoxide ring opening
of monorden (1) with HCl in methanol yielded 13 and 14
as main products.
0
1
2
3
4
5
6
7
8
195.92 195.66 195.88 206.65 205.54 196.98 195.25
45.11 42.90 43.84 44.90 43.76 44.38 44.44
131.93 134.33 134.11 132.37 132.72 133.11 133.59
,3
115.43b 113.61 113.37 115.04 114.64 115.02 114.41
b
b
b
b
b
b
b
b
b
b
b
b
155.19 157.97 158.38 154.93 154.69 155.27 156.74
102.85 102.70 102.80 102.51 102.47 102.59 103.00
b
b
b
b
b
b
b
155.19 156.73 156.69 155.24 155.48 155.39 155.71
111.98b 111.55 111.12 112.28 112.57 112.10 112.90
b
b
b
b
166.61 168.09 167.94 167.56 167.36 166.39 167.90
a
The 13C NMR shifts are deduced from the HSQC and HMBC
b
correlation spectra. Signals may be interchanged.
of which were described as monocillins from Monocillium
nordinii.7 Monocillin III (3) is the dechloro analogue of
pochonin A (2) and monocillin II (9) that of pochonin D (7).
The LCMS data of the dechloro analogue 10 of pochonin
+
1
E (8) show a [M + H] peak at m/z 333, and its H NMR
data (Table 5) are in good agreement with that of pochonin
E (8) concerning the 14-membered lactone ring. As in the
other monocillins, two aromatic signals for protons in meta
H
position to each other (δ 6.07 and 6.20) are detected. The
new compound is called pochonin F (10).
Compound 11 constitutes a novel glycoside of monocillin
II, as the spectroscopic data of the aglycone moiety are
almost identical to that of monocillin II (9). Only one H
1
In the HSV1 replication assay, monorden (1) showed
1
inhibitory activity in the nM range accompanied by cyto-
Ta ble 5. 1H and 13C NMR Data [500 and 125 MHz] of 10 and 11
1
0
11
pos
δC [ppm]a
δH [ppm]
1.25
5,11
Ha: 2.39-2.47
Hb: 2.20
5.42
5.30
4.01
OH: 5.01
Ha: 2.39-2.47
Hb: 2.12
6.54
J HH [Hz]
6.1
δC [ppm]a
δH [ppm]
J HH [Hz]
6.6
1
2
3
19.39
70.79
37.90
d
m
m
ddd
ddd
dd
m
d
m
19.34
71.29
38.06
1.28
5.08
d
m
dm
m
m
m
m
m
m
m
ddd
d
Ha: 2.39
Hb: 2.21
5.35c
14.9
b
16.1, 7.0, 7.0
15.6, 9.9, 4.8
15.6, 6.9
4
5
6
126.87
136.47
131.08c
128.47c
30.34
b
5.33c
71.01
40.03
Ha: 2.23b
Hb: 2.02
3.9
b
7
30.05
Ha: 2.23 Hb: 2.13
m
ddd
d
8
9
146.03
131.20
197.12
44.90
15.7, 7.6, 7.6
15.7
n.d.
129.92
196.43
44.38
6.68
5.91
15.7, 7.4, 7.4
15.7
5.88
1
1
0
1
Ha: 3.94
Hb: 3.45
d
d
14.9
14.9
Ha: 3.94b
Hb: 3.47b
m
m
1
1
1
1
1
1
1
1
2
2
3
4
5
6
7
8
′
136.47b
108.35
160.33
101.56
159.40
111.16
168.75
135.53
108.82
159.27
102.62
158.18
114.00
n.d.
6.07
OH: 9.84
6.20
s
s
s
s
6.31
d
2.0
2.0
6.50
OH: 10.25
d
s
OH: 10.19
100.09
71.50
5.47
4.04
OH: 4.70
3.89 OH: 4.91
d
ddd
d
m
d
m
m
t
4.8
9.1, 5.0, 5.0
9.1
′
b
3
′
69.31
5.05
4
5
′
′
86.39
61.41
3.93b
3.45 OH: 4.80
b
5.5
a
The 13C NMR shifts are deduced from the HSQC and HMBC correlation spectra. b Overlapped signals. c Signals may be interchanged.

Antiviral Resorcylic Acid Lactones
J ournal of Natural Products, 2003, Vol. 66, No. 6 833
Ta ble 6. Antiviral (HSV1-F) Effects and Antiparasitic (versus
E. tenella) Activities of 1-11 and 22
Ta ble 7. Antiviral (HSV1-F) Effects and Agonistic Effects
against the the ERâ Receptor of Zearalenone (15) and
Analogues 16-21
antiviral (HSV 1)a
antiparasitic (E. tenella)b
IC50 [µM]
SI
10 ppm
1 ppm
1
2
3
4
5
6
7
8
9
1
1
2
0.2-0.8
n.d. (T)
8
SI 7.5
8
12
15
T (2.5 µM)
6
T (5 µM)
SI 10
2/T
2
1
T
2
T
T
T
0
1
2
0.4
10
1.5
6
not active
1.5
not active
2
n.d.
n.d.
0
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0
2/(T)
2/(T)
T
0
1
2
T (10 µM)
50
(SI 6)
2
a
Concentrations tested in cellular replication assay using Vero
cells:1 0.1 nM to 125 µM; SI: selectivity index ) Tox50/IC50; T:
cytotoxic. Concentrations tested against E. tenella grown in
PCKC cells:
b
3
7,38
a
200, 10, 1, and 0.1 ppm; 2: full activity, 1: weak
None of the compounds were active against E. tenella and N.
caninum. b Concentrations tested in cellular replication assay
using Vero cells: 0.1 nM to 125 µM. Concentrations tested: 10
nM to 10 µM.
activity; 0: no activity, T: cytotoxic. N. caninum was not sensitive.
1
c
static effects. The pochonins (2-5) with epoxide moieties
exhibited bioactivities in the low µM range, while hexahy-
dromonorden (12), with a secondary alcohol function, was
found devoid of activity (Table 6). Probably, the chlorine
substituent is not essential for the antiviral activity, as
monocillin III (3) showed inhibitory activity in the nM
range. Pochonin D (7) and monocillin II glycoside (11),
which contain a double bond instead of the epoxide ring,
showed only cytostatic effects. For those pochonins with
an allyl alcohol moiety (8, 10) moderate inhibitory activity
was determined. Inhibition of HSV was generally ac-
companied by weak cytostatic effects on the host cell, as
observed microscopically (Table 6). This results in rather
low tolerability in vitro described by the selectivity indices
of monorden and derivatives on cytokine (particularly
interleukin 1) release were described.20 Recently, the
biochemical mode of action of the antagonistic effects of
monorden against the heat shock protein HSP90 was
elucidated.21 The use of monorden (1) as an antiinflamma-
tory agent was claimed as well. These previous results
indicated the potential usefulness of monorden and related
metabolites as anticancer agents and in a wide range of
further pharmaceutical indications. Its antiviral activities
and effects on coccidial parasites other than Plasmodium,
however, had not been noted previously.
2
2
Various other resorcylic acid lactones had previously
been obtained from fungi; that is, the zearalenones are
well-known estrogenic mycotoxins from several Fusarium
spp.23 These compounds act on the human estrogen recep-
(SI, i.e., the ratio of Tox50/IC50 with IC50 or Tox50 for the
concentration at which a drug reduced 50% of the cyto-
pathic effects of viral replication or cell viability).
2
4
tor in competition with 17-estradiol. In our own studies,
zearalenone (15), both stereoisomers of zearalenol (16, 17),
and semisynthetic derivatives from hydrogenation, methy-
Tanaka et al. had described antimalarial in vivo effects
for monorden (1) and discussed the heme-dependent radical
generation in Plasmodium falciparum as a possible mode
of action.1
2-14
lation, and acetylation were studied in comparison with
Therefore we evaluated the antiparasitic
the metabolites of P 0297.2
5-27
Zearalenone (15) and
potential of the pochonins and the effectiveness of monor-
den (1) against economically important animal parasites.
Compounds 1-11 were tested against Eimeria tenella and
Neospora caninum, both of which are taxonomically related
to Plasmodium. While N. caninum was not susceptible, the
partly hydrogenated pochonins containing an epoxide func-
tion showed moderate activities against E. tenella. Among
these, pochonin A (2) and tetrahydromonorden (5) exhibited
the best selectivity toward the parasite (Table 6). Recently,
Isaka et al. reported moderate antimalarial activity of the
resorcylic acid derivative hypothemycin and the related
R-zearalenol (16) and their derivatives did not exhibit any
activity against HSV1, while â-zearalenol (17) was the most
potent among all compounds examined in this study (Table
7
). Hydrogenation of â-zearalenol (17) to â-zearalanol (21)
resulted in decreased activities, while the methylation and
acetylation products of 17 were inactive. However, the
HSV1 inhibition of these zearalenone type compounds was
not accompanied by any cytostatic effects. The structure-
activity relationships (SAR) of the antiviral properties of
these zearalenols are not parallel to their estrogenic
properties (Table 7). However, neither of the zearalenone
derivatives were found effective against the parasites.
On the other hand, all pochonins, the derivatives of
monorden, and the monocillins were inactive against the
ERâ receptor up to 10 µM, suggesting that other determi-
nants are also relevant for the estrogenic activity, e.g., the
lactone chain of RAL and not only the aromatic substitution
pattern,23 which is identical at least in the monocillins and
zearalenone derivatives.
aigialomycins from the marine mangrove fungus Aigialus
parvus.15
Among the several other biological effects reported for
monorden (1) are cytotoxic activities. As a result, the use
of monorden (1) and semisynthetic derivatives as antican-
cer agents was therefore evaluated extensively in past
years. Compound 1 is known to inhibit tyrosin kinases16
and to induce the differentiation of HL-60 cells in the 100
nM range, probably addressing this target.17 Recently, a
semisynthetic derivative of monorden was reported to
exhibit tyrosin kinase inhibition at 20 nM (as compared to
In conclusion, these results suggest that neither the
antiviral nor the antiparasitic modes of action of RAL are
related to their effects on the estrogenic receptor. The
antiherpetic activity of â-zearalenol derivatives may thus
be coincidental. Unfortunately, â-zearalenol (17) seems to
be an inappropriate antiviral lead structure because of the
1
8
3
70 nM for 1) and was patented as an anticancer agent.
Monorden and related compounds were also proven to
modulate MAP kinase activity, thus acting as promoters
of nerve regeneration in vitro.19 Moreover, inhibitory effects

8
34 J ournal of Natural Products, 2003, Vol. 66, No. 6
Hellwig et al.
estrogenic side effects and the rather narrow SAR observed
upon preliminary studies. Concerning monorden (1) and
the pochonins, it was not possible to retain the strong
bioactivities of monorden and reduce the cytostatic effects
at the same time. The antiparasitic and antiviral target
sites of the metabolites of P 0297 may be different from
one another as well. Multiple targets possibly are ad-
dressed by these fungal metabolites.
temperature, 220 °C. System 5: HP 1100 liquid chromatograph
directly coupled with a Micromass-LCT mass spectrometer in
ESIpos. and ESIneg. mode with the following instrumental
conditions: column, Symmetry-C18, 3.5 µm; 2.1 × 50 mm
(
2
Waters); eluant A, H O + 0.1% HCOOH; eluant B, acetonitrile
+
0.1% HCOOH; gradient 0 min 0% B; 1 min 0% B 10% B; 5
min 90% B; 6 min 90% B; 6.1 min 100% B; flow 0.5 mL/min;
temperature, 40 °C. MS parameters: source temperature, 100
°C; desolvation temperature, 200 °C; capillary voltage, 3.2 kV;
During our investigations, the spirocyclic alkaloid pseuro-
tin A (22) was found to be a main metabolite in most of
the P. chlamydosporia isolates examined when propagated
in Q6-medium. Compound 22 was isolated and also sub-
jected to all biological investigations performed with the
pochonins. Pseurotin A (22) had been obtained previously
gas flow (total), 550l/h; nebulizer gas, N ; flow, 3l/h; resolution,
4000; scan time, 1 s; scan range, 100-1200.
NMR sp ectr a were recorded at 300 K with a Bruker DRX
2
5
00 spectrometer (500 MHz) with the solvent peak as internal
reference (CD
3
1
OD: δ
H
3.35, δ
C
49.0; [D
6
]DMSO: δ
H
2.50, δ
H
1
13
3
9.8). Direct ( J CH) and long-range H- C connectivity were
determined by inverse experiments using gradient pulses. The
HMBC experiment was optimized for a coupling constant of
2
8
from other fungi such as Pseudeurotium ovalis and,
repeatedly, from Aspergillus fumigatus.29
1
0 Hz (D
Biologica l Ma ter ia l. P 0297 was isolated from a sample
of plant debris collected in Germany. A voucher specimen of
the fungal strain is kept under liquid N in 10% glycerol at
6
) 0.05 s).
The distribution of the pochonins in submerged cultures
of several Pochonia species and further conidial fungi was
studied with HPLCUV and LCMS. Until recently, these
fungal species with verticillium-like anamorphs were all
included in Verticillium sect. Prostrata. The results from
2
the Bayer Pharma Research Center, Wuppertal, Germany.
Morphological studies, DNA extraction, and sequencing of the
nuclear ribosomal DNA (nrDNA) were carried out according
to the literature,30 while SEM was performed according to the
previously published procedure.34 Morphological studies showed
the production of chlamydospores (Figure 1) in abundance
besides typical verticillate conidiophores producing phialio-
conidia, corresponding to species of Verticillium sect. Pros-
these chemotaxonomical evaluations support a recent
generic segregation by Zare et al.,30 as the pochonins were
found to occur exclusively in species of the genus Pochonia.
With few exceptions, the production of pochonins and
monorden appears to be a rather constant feature in
cultures of P. chlamydosporia from around the world. The
chemotaxomical investigations as well as results from
minisatellite PCR fingerprinting studies on Pochonia and
35
trata. According to the currently valid taxonomy, P 0297
keyed out with Pochonia chlamydosporia var. catenulata
(Kamyschko ex Barron & Onions) Zare & W. Gams. Sequence
data of the ITS/5.8S nrDNA region of P 0297 showed 100%
identity to the sequence AJ 292398 (deposited as Verticillium
other conidial fungi with verticillium-like anamorphs are
reported concurrently.31
3
6
chlamydosporium var. catenulatum in EMBL ) of CBS 504.66,
the ex-type strain of this variety. The correspondence of P 0297
with this taxon was further confirmed by extensive morpho-
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Unless stated oth-
erwise, culture media ingredients and solvents for chroma-
tography and spectroscopy were obtained from Merck (Darm-
stadt, Germany) and all solid chemicals, including zearalenone
and zearalenols, from Sigma-Aldrich (Deisenhofen, Germany).
HPLCUV characterization of the natural products and
semisynthetic derivatives was performed with system 13 and
system 2, respectively. System 2 was carried out using a HP
31
logical, PCR, and HPLC profiling studies on related strains.
F er m en ta tion , Extr a ction , a n d Isola tion . Small-scale
fermentations of P 0297 were carried out by adding 2 mL of a
1
0% glycerol culture to 500 mL Erlenmeyer flasks containing
150 mL of Q6/2 (D-glucose 0.2%, glycerol 1%, cotton seed meal
0.5%; tap water 1 L, pH 7.2), YMG medium (yeast extract
0.4%, malt extract 1%, glucose 0.4%, tap water 1 L, pH 6.3),
or MGP.10 In further experiments MGP was also supplemented
2,33
with 100 mM CaBr
2
as previously described.11 Free glucose in
1
090 (Agilent, Waldbronn, Germany) unit with automated
sample injector, diode array detector, and Sedex 55 light
scattering detector (ERC, Alteglofsheim, Germany), employing
the following instrumental conditions: column, Eurospher-100
the culture media was estimated by “Diastix Harnzuckerstre-
ifen” (Bayer Diagnostics, Munich, Germany), and the pH was
determined. The flasks were sterilized at 1 bar and 121 °C for
30 min. The cultures were propagated in the dark on a rotary
shaker at 140 rpm. Daily samples (25 mL) of the broth were
withdrawn under sterile conditions and extracted with ethyl
acetate (EtOAc) for 30 min in an ultrasonic bath. The organic
C
18, 5 µm; 2 × 125 mm (Knauer); eluant A, water + 0.05%
TFA; eluant B, ACN + 0.05% TFA; gradient 0 min 10% B; 1
min 10% B; 15 min 100% B; 17 min 100% B; 18 min 0% B;
flow 0.4 mL/min; temperature, 40 °C. UV spectra were
recorded from 200 to 400 nm.
phases were dried over Na
2 4
SO and evaporated to dryness
HPLCMS was performed using the following conditions.
System 3: HP 1100 liquid chromatograph (Agilent, Waldbronn,
Germany) directly coupled with a Micromass Quattro LCZ
mass spectrometer (Micromass, Manchester, UK) in the posi-
tive electrospray ionization (ESIpos) mode with the following
instrumental conditions: column, Symmetry-C18, 3.5 µm; 2.1
under reduced pressure, and aliquots thereof were used for
biological assays and HPLC analyses.37 The shake cultures
were harvested after 7 or 8 days, freeze-dried, and extracted
with MeOH in an ultrasonic bath. The residue was resus-
pended in 1 L 1:1 H
dried over Na SO and evaporated in vacuo. Fermentations
2
O/EtOAc, and the organic layers were
2
4
×
50 mm (Waters); eluant A, H
2
O + 0.1% HCOOH; eluant B,
in 10 flasks each yielded 500 mg of crude product from Q6/2,
450 mg from YMG, and 600 mg from MGP medium. These
crude extracts were subjected in portions of 100-200 mg to
preparative HPLC (system A) as described below.
ACN + 0.1% HCOOH; gradient 0 min 10% B; 4 min 90% B; 6
min 90% B; 6.1 min 10% B, 7.5 min 10% B; flow 0.5 mL/min;
temperature, 40 °C. MS parameters: scan speed, 1.0 s/decade
against scan; scan range, 100-1200; heated capillary temper-
ature, 120 °C. System 4: TSP liquid chromatograph directly
coupled with a MAT 900S mass spectrometer (Finnigan,
Bremen, Germany) in the ESIpos mode with the following
instrumental conditions: column, Symmetry-C18, 5 µm; 2.1 ×
Fermentations in 10 L scale were carried out using a 10 L
Braun (Melsungen, Germany) Biostat E stirring fermentor
with aeration (3 L/min) and agitation (100 rpm) at 23 °C. The
culture media were sterilized in situ for 30 min at 121 °C with
steam. The cultures were inoculated with 300 mL of well-
grown YMG shake cultures propagated for 72-96 h, and 3 mL
of Sigma Antifoam A was added to the medium to avoid
extensive foaming. Daily samples were taken and analyzed
as described above. The cultures were harvested after 168 h.
After separation from the fluid by filtration, the wet mycelium
was extracted with acetone in an ultrasonic bath. The aqueous
1
50 mm (Waters); eluant A, 0.01 M HCl, eluant B, ACN;
gradient 0 min 10% B; 9 min 90% B; 18 min 90% B; flow 0.6
mL/min; temperature, 50 °C. MS parameters: scan speed, 1.5
s/decade against scan; scan range, 150-1200; resolution, 2000;
capillary voltage, 4.750 V; identity of nebulizer gas, N
9.999%; nebulizer gas pressure, 5 bar; heated capillary
2
9

Antiviral Resorcylic Acid Lactones
J ournal of Natural Products, 2003, Vol. 66, No. 6 835
residue was diluted and extracted with EtOAc, and the organic
does not discriminate between ERâ agonists and antagonists.
The assays were performed as described in the PanVera assay
protocol40 in 96-well microtiter plates. 13-â-Hydroxyestradiol
served as control (EC50 ) 19 nM). The plates were incubated
for 2 h in a Thermo Labsystems (Cambridge, UK) iEMS
incubator without shaking. After incubation, the fluorescence
polarization of the sample was determined using a Tecan
(Crailsheim, Germany) Polarion polarization reader. The
analysis was performed at λex ) 485 nm (excitation wave-
length) and λem ) 535 nm (emission wavelength). Low mP
values (low polarization) indicated release of fluorescent tracer
by an ERâ ligand. EC50 values of compounds were calculated
using GraphPad Prism 3.0 (GraphPad Software, Inc., San
Diego, CA).
layer was dried over Na
0 L of culture fluid). The culture filtrate was applied onto a
column packed with 500 g of Bayer Lewapol CA 9225 resin.
The resin was rinsed with H O (5 L) and subsequently eluted
2 4
SO to yield an oily residue (5 g from
1
2
with acetone (6 L). The organic eluates were extracted with
EtOAc to yield 4.5 g of crude product. The crude products were
subjected to preparative HPLC (system B, see below) in
portions of 1 g each.
HPLC system A was used for small scale: column, Li-
chroSorb RP18, 7 µm; 25 × 250 mm (Merck Darmstadt,
2
Germany); eluant A, H O; eluant B, acetonitrile; gradient, 0
min 20% B; 15 min 20% B; 35 min 55% B; 50 min 55% B; 70
min 100% B; flow 9 mL/min. System B was used for large
scale: column, Kromasil RP 18, 7 µm; 40 × 250 mm (MZ
Mon or d en (1): colorless crystals; HPLC, t
R
7.1 min (system
6
1); [R]20
+108° (c 0.05, CHCl
); H NMR ([D ]DMSO, 500
1
Analysentechnik, Mainz, Germany); eluant A, H
2
O, eluant B,
D
3
acetonitrile; gradient, 0 min 20% B; 30 min 40% B; 70 min
5% B; 100 min 55% B; 150 min 100% B; flow 9 mL/min.
MHz) δ 10.49 (1H, s, br, 14-OH or 16-OH), 10.09 (1H, s, 14-
OH or 16-OH), 7.37 (1H, dd, J ) 15.7, 9.1 Hz, H-8), 6.65 (1H,
s, H-15), 6.25 (1H, dd, J ) 10.2, 10.2 Hz, H-7), 6.10 (1H, d, J
) 15.7 Hz, H-9), 5.71 (1H, dd, J ) 10.8, 5.0 Hz, H-6), 5.20
(1H, m, H-2), 3.87 (1H, d, J ) 16.2 Hz H-11a), 3.65 (1H, d, J
) 16.2 Hz, H-11b), 3.39 (1H, m, H-5), 3.14 (1H, dm, J ) 8.8
Hz, H-4), 2.38 (1H, dm, J ) 14.4 Hz, H-3a), 1.54 (1H, m, H-3b),
5
System C was used in some cases for final purification by
isocratic high-performance gel permeation chromatography
(
HPGPC) at RT on LichroGel PS1, 10 µm; 250 × 25 mm (Merck
Darmstadt, Germany); isocratic elution with 2-propanol; flow
mL/min. The mode of isolation and their t in different
gradients are summarized in Table 1.
7
R
1.42 (3H, d, J ) 6.2 Hz, CH3-1); 13C NMR ([D
MHz), see Table 4; LCMS, t
]DMSO, 125
R
3.6 min; ESIMSpos m/z 733 (13),
6
Biologica l Assa ys. The assay for evaluation of antiviral
activities against HSV1, using strain HSV1-F, including
determination of IC50 and selectivity indices (SI) of all com-
+
+
731 (53), 729 (69) [2M + H] , 367 (34), 365 (100) [M + H] ,
+
347 (18) [M - H
2
O + H] (system 3); LCMS, t
R
5.6 min;
1
+
pounds was recently described in detail. For evaluation of
ESIMSpos m/z 733 (27), 731 (32), 729 (54) [2M + H] , 408
+
anticoccidial activities, the parasites Eimeria tenella (strain
(25), 406 (100) [M + CH CN + H] , 367 (12), 365 (88) [M +
3
3
8,39
+
+
Houghton) and Neospora caninum (strain NC-1)
were used
H] , 350 (38), 348 (45) [M - O + H] , 293 (14), 291 (31) (system
+
for primary screening and propagated at Bayer Animal Health.
Primary chicken kidney cells (PCKC, E. tenella) or African
green monkey cells (Vero, N. caninum), respectively, were used
for in vitro studies. Cells were grown to confluence in 25 cm2
flasks in DMEM or RMPI medium supplemented with 10%
fetal bovine serum, glutamine, and essential amino acids. Cell
cultures were incubated at 37 °C (E. tenella) or 41 °C (N.
4); EIMS m/z 366 (4), 364 (7) [M] , 203 (7), 201 (21), 186 (34),
184 (100), 121 (31); HREIMS m/z 364.0734 (calcd for C18
ClO , 364.0714).
P och on in A (2): brownish oil; HPLC, t
17
H -
6
R
7.3 min (system
1
13
1); H NMR ([D
DMSO, 125 MHz), see Table 4; LCMS, t
6
]DMSO, 500 MHz), see Table 2; C NMR ([D
6.4 min; ESIMSpos
m/z 369 (3), 367 (7) [M + H] , 351 (2), 349 (3) [M - H O +
H] , 292 (25), 291 (100), 157 (65), 99 (35) (system 3); LCMS,
6
]-
R
+
2
+
caninum) in a humidified atmosphere containing 5% CO
2
and
+
9
5% air. For growth of parasites, Vero cell monolayers were
tR 5.7 min; ESIMSpos m/z 408 (37) [M + CH
3
CN + H] , 369
(38), 367 (100) [M + H] (system 4); HREIMS m/z 366.0864
(calcd for C18 , 366.0870).
P och on in B (4): brownish oil; HPLC, t
+
infected with tachyzoites and examined with an inverted
microscope for the development of lesions or the presence of
extracellular tachyzoites. Once lesions were observed, the
monolayers were scraped and a small amount of tachyzoite-
containing fluid was transferred to infect new flasks of fresh
cells. Tachyzoites of N. caninum were passaged in this manner
every 3 to 4 days as infectious material for the test system. E.
tenella was maintained in 3-week-old chickens. Recovery,
sporulation, and sterilization of oocysts were accomplished
from the feces, and excystation and column fractionation
served to isolate the sporozoite stages. For determination of
the effectiveness of test samples, microtiter monolayer disrup-
tion assays (MMDA) were employed. Flat-bottomed 96-well
microtiter plates were inoculated with PCKC or Vero cells, and
the resulting monolayers were used to determine the effects
of substances on merozoite or tachyzoite production as mea-
sured by plaque formation. Monolayers were inoculated with
parasites (E. tenella or N. caninum) and inoculated with test
compounds 2 h post infection. Untreated and uninfected
monolayer wells served as parasite controls, and uninfected
agent treated host cells served as toxicity controls. The assay
was stopped either 120 h post infection (E. tenella) or when
H
19ClO
6
R
6.3 min (system
1
13
1); H NMR ([D
6
]DMSO, 500 MHz), see Table 2; C NMR ([D
6
]-
DMSO, 125 MHz), see Table 4; LCMS, t
R
5.3 min; ESIMSpos
+
+
m/z 767 (1), 765 (2) [2M + H] , 385 (38), 383 (100) [M + H]
(system 3); EIMS m/z 384 (3), 382 (7) [M] , 186 (12), 184 (39),
130 (26), 81 (58), 69 (100); HREIMS m/z 382.0809 (calcd for
+
C
18
H
19ClO
7
, 382.0819).
P och on in C (6): brownish oil; HPLC, t
R
6.6 min (system
1
13
1); H NMR ([D
6
]DMSO, 500 MHz), see Table 2; C NMR ([D
6
]-
DMSO, 125 MHz), see Table 4; LCMS, t
R
3.3 min; ESIMSpos
+
+
m/z 803 (8), 801 (5) [2M + H] , 403 (19), 401 (30) [M + H] ,
+
387 (7), 385 (41), 383 (60), [M - H O + H] , 365 (24) [M -
2
+
Cl] , 349 (43), 347 (100) (system 5); EIMS m/z 402 (7), 400
(11) [M + H] , 366 (6), 364 (6), 314 (3), 312 (7), 229 (12), 203
89), 201 (26), 186 (33), 184 (100), 157 (16), 155 (16), 121 (25),
+
96 (32); HREIMS m/z 400.0472 (calcd for C18
H
18Cl
2
O
6
, 400.0480).
P och on in D (7): brownish oil; HPLC, t
R
8.4 min (system
1
13
1); H NMR ([D
6
]DMSO, 500 MHz), see Table 3; C NMR ([D
6
]-
DMSO, 125 MHz), see Table 4; LCMS, t
R
+
7.0 min; ESIMSpos
m/z 394 (11), 392 (29) [M + CH CN + H] , 353 (30), 351 (100)
3
+
+
9
0-100% of the untreated infected cells had lysed (N. cani-
[M + H] , 335 (9), 333 (34), [M - H O + H] , 315 (6) [M -
Cl] (system 4); EIMS m/z 352 (9), 350 (27) [M] , 254 (6), 252
2
+
+
num). Determination of viability of monolayer cells was done
by microscopic control or after fixation with MeOH for 5 min
and protein staining (crystal violet solution). The plates were
evaluated microscopically or by using an ELISA plate reader
(16), 186 (13), 184 (39), 81 (100); HRESIMS m/z 349.0795 [M
-
- H] (calcd for C18
H
18ClO
5
, 349.0843).
P och on in E (8): brownish oil; HPLC, t
R
6.6 min (system
]DMSO, 500 MHz), see Table 3; LCMS t 5.6
min; ESIMSpos m/z 369 (5), 367 (14) [M + H] , 233 (100), 157
(72), 99 (50), 97 (31) (system 3). LCMS, t 4.8 min; ESIMSpos
1
(Alpha Diagnostic, San Antonio, TX) to quantify the protein
1); H NMR ([D
6
R
+
dye incorporation and to determine the concentration of test
compounds that inhibited monolayer destruction.
A commercially available fluorescence polarization assay
R
+
+
m/z 408 (9) [M + CH
351 (5), 349 (14) [M - H O + H] (system 4); EIMS m/z 368
(4), 366 (9) [M] , 252 (20), 224 (8), 203 (3), 201 (9), 186 (16),
184 (42), 175 (12), 157 (4), 155 (9), 117 (40), 97 (34), 91 (12),
81 (8), 69 (17), 59 (100); HREIMS m/z 366.0867 (calcd for
3
CN + H] , 369 (32), 367 (100) [M + H] ,
+
(Pan Vera Inc., Madison, WI) was used for the estrogen
2
+
receptor-â (ERâ) assays. In this competition type assay, a
fluorescent tracer (fluorescein derivative of estrogen) is dis-
placed from an estrogen receptor â/tracer complex by active
compounds resulting in fluorescence depolarization. This assay
18 6
C H19ClO , 366.0870).

8
36 J ournal of Natural Products, 2003, Vol. 66, No. 6
Hellwig et al.
Mon ocillin III (3): colorless solid; HPLC, t
R
6.9 min (system
186 (30), 184 (100); HREIMS m/z 368.1043 (calcd for C18
ClO , 368.1027).
Hexa h yd r om on or d en (12): yield: 3.5 mg (0.009 mmol,
21
H -
1
1
1
); H NMR ([D
6
]DMSO, 500 MHz) δ 11.36 (1H, s, 16-OH),
6
0.23 (1H, s, 14-OH), 6.94 (1H, ddd, J ) 16.0, 10.0, 4.8 Hz,
H-8), 6.23 (1H, d, J ) 2.3 Hz, H-15), 6.20 (1H, d, J ) 2.3 Hz,
1
1
6
6%); H NMR ([D ]DMSO, 500 MHz) δ 10.37 (1H, s, br, 14-
H-13), 6.01 (1H, d, J ) 16.0 Hz, H-9), 5.13 (1H, m, H-2), 4.42
OH or 16-OH), 9.85 (1H, s, 14-OH or 16-OH), 6.49 (1H, s,
H-15), 5.03 (1H, m, H-2), 4.37 (1H, d, J ) 5.2 Hz, 4-OH), 4.12
(
2
1H, d, J ) 17.0 Hz, H-11a), 3.78 (1H, d, J ) 17.0 Hz, H-11b),
.85 (1H, m, H-4), 2.59 (1H, dm, J ) 9.3 Hz, H-5), 2.42 (1H,
(1H, d, J ) 18.5 Hz, H-11a), 3.85 (1H, d, J ) 18.5 Hz, H-11b),
m, H-7a), 2.30-2.22 (2H, m, H-6â and H-7b), 1.85 (1H, m,
H-3a) 1.68 (1H, ddd, J ) 15.9, 4.9, 4.9 Hz, H-3b), 1.34 (3H, d,
3
2
1
1
.39 (1H, m, H-4), 2.62 (1H, ddd, J ) 13.0, 9.0, 3.2 Hz, H-9a),
.12 (1H, ddd, J ) 13.0, 9.4, 3.4 Hz, H-9b), 1.78 (1H, ddd, J )
4.1, 10.2, 3.7 Hz, H-3a), 1.56 (2H, m, H-8a and H-3b), 1.50-
.36 (3H, m, H-8b, H-6a, and H-5a), 1.32 (1H, m, H-7b), 1.25
1
3
J ) 6.4 Hz, Me-1), 1.22 (1H, m, H-6R); C NMR ([D
6
]DMSO,
1
1
(
1
(
25 MHz), shifts from HSQC/HMBC dataset, δ 197.45 (C-10),
70.55 (C-18), 163.94 (C-16), 162.34 (C-14), 149.63 (C-8), 139.49
(
3H, d, J ) 6.2 Hz, Me-1), 1.21-1.11 (3H, m, H-7b, H-6b, and
C-12), 131.00 (C-9), 112.38 (C-13), 107.09 (C-17), 101.83 (C-
5), 71.17 (C-2), 55.98 (C-5), 54.75 (C-4), 46.20 (C-11), 36.27
C-3), 30.43 (C-6), 28.47 (C-7), 18.14 (C-1); LCMS, t 5.2 min;
ESIMSpos m/z 665 (5) [2M + H] , 374 (16) [M + CH CN +
13
6
H-5b); C NMR ([D ]DMSO, 125 MHz), see Table 4; LCMS,
t
(
3
+
R
5.5 min; ESIMS pos m/z 743 (9), 741 (12) [2M + H] , 373
R
+
+
35), 371 (100) [M + H] , 355 (9), 353 (30) [M - H
37 (9), 335 (19) (system 4); HREIMS m/z 370.1167 (calcd for
, 370.1183).
Ep oxid e Rin g Op en in g. To a solution of 5 mg (0.014
2
O + H] ,
+
3
+
+
+
H] , 334 (21), 333 (100) [M + H] , 315 (7) [M - H
2
O + H]
18 6
C H23ClO
+
(
3
system 4); EIMS m/z 332 (56) [M] , 150 (100); HREIMS m/z
32.1252 (calcd for C18
20 6
H O , 332.1260).
mmol) of monorden (1) in methanol is added 1 N HCl. After
stirring for 1 day at room temperature, the reaction mixture
is evaporated and purified by preparative reversed-phase
HPLC using the following conditions: Eurospher C18, 7 µm;
Mon ocillin II (9): colorless solid; HPLC, t
); H NMR ([D
6
R
8.1 min (system
]DMSO, 500 MHz) δ 10.29 (1H, s, 16-OH),
.91 (1H, s, br, 14-OH), 6.67 (1H, m, H-8), 6.21 (1H, d, J ) 2.2
1
1
9
Hz, H-15), 6.10 (1H, d, J ) 2.2 Hz, H-13), 5.88 (1H, d, J )
5.9 Hz, H-9), 5.31 (2H, m, H-4 and H-5), 5.11 (1H, m, H-2),
1
6 × 125 mm (Knauer); eluant A, water + 0.05% TFA; eluant
B, ACN + 0.05% TFA; gradient, 0 min 0% B; 60 min 100% B;
flow 7 mL/min. Retention time of the products: t (14) ) 30
min and t (13) ) 34 min.
1
3
.92 (1H, d, J ) 15.5 Hz, H-11a), 3.50 (1H, d, J ) 15.5 Hz,
R
H-11b), 2.40 (1H, dm, J ) 14.1 Hz, H-3a), 2.27-2.17 (3H, m,
R
H-3b, H-7a, and H-7b), 2.12 (1H, m, H-6a), 2.06 (1H, m, H-6b),
.25 (3H, d, J ) 6.3 Hz, Me-1); 1 C NMR ([D
3
6,13-Dichloro-5,9,14,16-tetrahydroxy-3-methyl-3,4,5,6,9,10-
1
6
]DMSO, 125
hexahydro-1H-2-benzoxacyclotetradecine-1,11(12H)-dione
MHz), shifts from HSQC/HMBC dataset, δ 196.51 (C-10),
68.32 (C-18), 159.93 (C-14), 159.21 (C-16), 148.39 (C-8), 136.72
C-12), 131.65 (C-5), 130.03 (C-9), 128.36 (C-4), 110.70 (C-17),
08.66 (C-13), 101.57 (C-15), 71.08 (C-2), 45.81 (C-11), 37.65
C-3). 30.41 (C-7), 30.28 (C-6),19.24 (C-1); LC-MS, t 6.8 min;
ESIMSpos m/z 633 (6) [2M + H] , 358 (33) [M + CH CN +
1
(
13): yield 0.6 mg (0.0014 mmol, 10%); H NMR ([D
6
]DMSO,
1
(
1
(
5
00 MHz) δ 10.31 (1H, s, 14-OH or 16-OH), 9.91 (1H, s, 14-
OH or 16-OH), 6.49 (1H, s, H-15), 5.91 (1H, dm, J ) 10.1 Hz,
H-7), 5.84 (1H, dm, J ) 10.1 Hz, H-6), 5.12 (1H, m, H-2), 4.62
(1H, dm, J ) 12.3 Hz, H-8), 4.40 (1H, dm, J ) 6.7 Hz, H-5),
3.96 (2H, m, H2-11), 3.83 (1H, dd, J ) 8.0, 8.0 Hz, H-4), 3.12
(1H, dd, J ) 13.4, 12.3 Hz, H-9a), 2.51 (H-9b, under solvent
signal), 2.02 (1H, dm, J ) 14.7 Hz, H-3a), 1.75 (1H, m, H-3b),
R
+
3
+
+
+
H] , 318 (20), 317 (100) [M + H] , 299 (6) [M - H
2
+
O + H]
+
(
2
system 4); EIMS m/z 317 (8) [M + H] , 316 (36) [M] , 288 (5),
70 (6), 236 (9), 218 (25), 190 (13), 150 (70), 81 (100); HREIMS
, calcd 316.1311).
P och on in F (10): brownish oil; HPLC, t 6.3 min (system
1
.27 (3H, d, J ) 6.1 Hz, Me-1); 13C NMR ([D
6
]DMSO, 125
MHz), shifts from HSQC/HMBC data, only detected signals δ
31.72 (C-7), 127.04 (C-6), 102.21 (C-15), 72.73 (C-4), 72.29
C-2), 69.49 (C-8), 56.10 (C-5), 45.86 (C-11), 43.39 (C-9), 38.12
(C-3), 20.74 (C-1); LCMS, t 7.1 min; ESIMSpos m/z 405 (10),
404 (15), 403 (68), 402 (22), 401 (100) [M - H
4); EIMS m/z 404 (5), 403 (7), 402 (33), 401 (10), 400 (47), 367
m/z 316.1315 (calcd for C18
20 5
H O
R
1
(
1
13
1
); H NMR ([D
6
]DMSO, 500 MHz), see Table 5; C NMR ([D
6
]-
DMSO, 125 MHz), see Table 5; LCMS, t
R
4.4 min; ESIMSpos,
+
+
R
m/z 374 (11) [M + CH
3
CN + H] , 333 (100) [M + H] (system
+
+
2
O + H] (system
4
3
21 6
); HRESIMS m/z 333.2514 [M + H] (calcd for C18H O ,
-
33.1338); HRESIMS m/z 331.1167 [M - H] (calcd for
, 331.1182).
Mon ocillin II glycosid e (11): brownish oil; HPLC: t
(
(
2), 366 85), 365 (9), 364 (7), 231 (18), 229 (54), 203 (18), 201
58), 186 (34), 184 (100), 159 (30), 157 (97), 139 (45), 121 (90);
18 19 6
C H O
R
7.0
HRESIMS m/z 401.0579 [M - H
01.0559).
13-Chloro-5,6,9,14,16-pentahydroxy-3-methyl-3,4,5,6,9,10-
hexahydro-1H-2-benzoxacyclotetradecine-1,11(12H)-dione
2
2 6
O + H] (calcd for C18H19Cl O ,
1
min (system 1); H NMR ([D
6
]DMSO, 500 MHz), see Table 5;
4
1
3
C NMR ([D
6
]DMSO, 125 MHz), see Table 5; LCMS, t
R
5.4
+
min; ESIMSpos m/z 449 (18) [M + H] , 358 (15) [aglycone +
+
+
CH CN + H] , 317 (100) [aglycone + H] , 299 (98) [aglycone
3
1
+
(14): yield 0.6 mg (0.0015 mmol, 11%); H NMR ([D
6
]DMSO,
-
H
2
O + H] (system 4).
5
00 MHz) δ 10.30 (1H, s, 14-OH or 16-OH), 9.90 (1H, s, 14-
Hyd r ogen a tion of Mon or d en (1) to Tetr a - (5) a n d
Hexa h yd r om on or d en (12). To a solution of 20 mg (0.055
mmol) of monorden (1) in methanol is added 7 mg of palladium
OH or 16-OH), 6.48 (1H, s, H-15), 5.95 (1H, dm, J ) 9.6 Hz,
H-6), 5.24 (1H, dm, J ) 9.6 Hz, H-7), 5.09 (1H, m, H-2), 4.53
(
3
1H, dm, J ) 12.9 Hz, H-8), 4.00 (1H, d, J ) 17.6 Hz, H-11a),
.90 (1H, d, J ) 17.6 Hz, H-11b), 3.58 (1H, dd, J ) 8.1, 8.1
Hz, H-4), 3.35 (5-H, hidden by water signal), 3.02 (1H, dd, J
13.9, 12.8 Hz, H-9a), 2.45 (1H, d, J ) 13.9 Hz, H-9b, under
(
10% Pd/C). After hydrogenation for 1 h at room temperature
under atmospheric pressure, the catalyst is removed by
filtration and the residue of the filtrate is purified by prepara-
tive reversed-phase HPLC using the following conditions:
Nucleosil C18, 7 µm; 8 × 125 mm (MZ Analysentechnik); eluant
A, water; eluant B, ACN; gradient, 0 min 20% B; 40 min: 100%
)
solvent signal), 1.94 (1H, dm, J ) 15.7 Hz, H-3a), 1.66 (1H,
ddd, J ) 15.7, 7.5, 7.5 Hz, H-3b), 1.25 (3H, d, J ) 6.1 Hz, Me-
1
).
Zea r a len on e Der iva tives (15-21). Compounds 15-17
were obtained from Sigma-Aldrich, and 18 was obtained from
B, flow 5 mL/min. t
R
(5) ) 8-9 min and t (12) ) 10-11 min.
R
Tetr a h yd r om on or d en (5):2,3 yield 4.3 mg (0.01 mmol,
1
1
8%); HPLC: t
R
7.5 min (system 1); H NMR ([D
6
]DMSO, 500
1
1
3 2 3
7 by methylation with CH I/K CO in acetone. Compounds
9-21 were prepared from the respective educts 15-17 by
MHz) δ 10.42 (1H, s, 14-OH or 16-OH), 9.99 (1H, s, 14-OH or
1
1
6-OH), 6.51 (1H, s, H-15), 5.08 (1H, m, H-2), 4.01 (1H, d, J )
8.3 Hz, H-11a), 3.86 (1H, d, J ) 18.3 Hz, H-11b), 2.71 (1H,
hydrogenation in methanol at RT and 1 bar with Pd/C as
catalyst.
2
dm, J ) 8.3 Hz, H-4), 2.68 (1H, m, H-5), 2.41 (2H m, H -9),
2
1
1
6
.21 (1H, ddd, J ) 14.2, 10.0, 3.6 Hz, H-3a), 1.89 (1H, m, H-6â),
Ack n ow led gm en t. The authors wish to thank their col-
leagues H. Musche, P. Schmitt, K. Schmidt, and S. Seip for
recording NMR, MS, and LC-MS data, K. Weber for down-
stream processing of crude material, and K. Ide, who per-
formed SEM. Furthermore, the expert technical assistance of
C. Angenendt, K. Ostertag-Palm, T. K u¨ ppers, P. Splittgerber,
.61 (1H, m, H-8a), 1.53 (1H, m, H-8b), 1.45 (1H, m, H-7a),
.40 (1H, ddd, J ) 14.2, 8.3, 1.9 Hz, H-3b), 1.27 (3H, d, J )
.2 Hz, Me-1), 1.07 (1H, m, H-6R); 1 C NMR ([D
3
6
]DMSO, 125
R
MHz), see Table 4; LCMS, t 5.9 min; ESIMSpos m/z 410 (5)
+
+
[
-
M + CH
3
CN + H] , 371 (31), 369 (100) [M + H] , 351 (8) [M
+
+
H
2
O + H] (system 4); EIMS m/z 370 (18), 368 (53) [M] ,

Antiviral Resorcylic Acid Lactones
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NP020556V