Isolation and structure elucidation of cruentarens A and B - Novel members of the benzolactone class of ATPase inhibitors from the myxobacterium Byssovorax cruenta

Article abstract of DOI:10.1002/ejoc.200600421

Two novel secondary metabolites, namely cruentaren A (1) and B (2), have been isolated from the myxobacterium Byssovorax cruenta. Their structures have been elucidated by detailed NMR spectroscopic analysis. Both compounds are isomers of a C₂₂ polyketide with a 2-hydroxy-4-methoxybenzoic acid terminus, which forms a 12-membered lactone in 1 and a six-membered lactone in 2. An amino group at C-22 is acylated by a 3-hydroxy-2-methylhexanoic acid group whose absolute configuration has been determined by GC analysis after hydrolysis and stereoselective synthesis. Degradation of 2 by olefin cross-metathesis in the presence of ethylene yields the C-12 to C-21 fragment 8, whose relative configuration has been predicted applying Kishi's ¹³C NMR spectroscopic data base approach, and whose absolute configuration has been proven by comparison with two synthetic stereoisomers. The configuration of C-9 and C-10 has been determined by Mosher's method and NMR spectroscopy. Degradation of cruentaren A (1) by olefin cross-metathesis in the presence of ethylene gives crystalline 21,22-seco-cruentaren (14). Its crystal structure analysis reveals the relative configuration and conformation of the macrocycle and enables solution conformation analysis. The high cytotoxic activity of 1 is essentially lost on derivatisation or rearrangement to 2. Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejoc.200600421

FULL PAPER
DOI: 10.1002/ejoc.200600421
Isolation and Structure Elucidation of Cruentarens A and B − Novel Members
of the Benzolactone Class of ATPase Inhibitors from the Myxobacterium
Byssovorax cruenta[‡]
Larissa Jundt,[a] Heinrich Steinmetz,[a] Peter Luger,[b] Manuela Weber,[b] Brigitte Kunze,[a]
Hans Reichenbach,[a] and Gerhard Höfle*[a]
Keywords: Natural products / Polyketides / Inhibitors / Medium-ring compounds / Configuration determination
Two novel secondary metabolites, namely cruentaren A (1)
and B (2), have been isolated from the myxobacterium Bysso-
vorax cruenta. Their structures have been elucidated by de-
tailed NMR spectroscopic analysis. Both compounds are iso-
mers of a C22 polyketide with a 2-hydroxy-4-methoxybenzoic
acid terminus, which forms a 12-membered lactone in 1 and
a six-membered lactone in 2. An amino group at C-22 is acyl-
ated by a 3-hydroxy-2-methylhexanoic acid group whose ab-
solute configuration has been determined by GC analysis af-
ter hydrolysis and stereoselective synthesis. Degradation of
2 by olefin cross-metathesis in the presence of ethylene
yields the C-12 to C-21 fragment 8, whose relative configura-
tion has been predicted applying Kishi’s 13C NMR spectro-
scopic data base approach, and whose absolute configuration
has been proven by comparison with two synthetic stereoiso-
mers. The configuration of C-9 and C-10 has been deter-
mined by Mosher’s method and NMR spectroscopy. Degrada-
tion of cruentaren A (1) by olefin cross-metathesis in the
presence of ethylene gives crystalline 21,22-seco-cruentaren
(14). Its crystal structure analysis reveals the relative configu-
ration and conformation of the macrocycle and enables solu-
tion conformation analysis. The high cytotoxic activity of 1 is
essentially lost on derivatisation or rearrangement to 2.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
its structural resemblance to apicularen it was hypothesized
that it might also be an ATPase inhibitor. Indeed, prelimi-
nary results indicate that it efficiently inhibits mito-
chondrial F-ATPase from baker’s yeast.[8]
Introduction
Myxobacteria are a particularly rich source of cytotoxic
secondary metabolites.[1] In addition to 12 novel inhibitors
of electron transport and seven structurally diverse com-
pounds that interact with different components of the cyto-
skeleton like the epothilones[2] and tubulysins,[3] we have
also isolated specific inhibitors of functional proteins. Thus,
ratjadon inhibits the nuclear transporter CRM1,[4] and ap-
icularen[5] and archazolid[5] inhibit mammalian V-ATPase,
while for other highly toxic compounds like the spirangi-
ens[6] the target has not yet been identified. Here, we de-
scribe the isolation and structural elucidation of cruentaren
A and B (1 and 2) from the myxobacterium Byssovorax
cruenta.[7] With an IC50 of 1.2 ngmL–1 for our standard
mouse fibroblast cell line L929, cruentaren A (1) is one of
the most cytotoxic metabolites from myxobacteria.[8] From
Results and Discussion
Byssovorax cruenta strain Ha r1 was first identified in a
screening for antifungal activity, and cytotoxic activity was
later also observed.[8] HPLC analysis of the culture extracts
indicated the presence of several different types of metabo-
lites, of which only one turned out to be responsible for the
biological activity. A 90-L bioreactor was run in the pres-
ence of 1% of amberlite XAD adsorber resin to bind ex-
creted secondary metabolites. Adsorber resin and cell mass
were harvested by centrifugation and extracted with ace-
tone to give 10 g of crude extract. Consecutive chromatog-
raphy on silica gel and RP-18 material yielded 270 mg of
cruentaren A (1) and 7 mg of cruentaren B (2) as colourless
amorphous solids. HR-MS characterisation indicated that
both compounds are isomers with the elemental composi-
tion C33H51NO8. Their UV spectra, with strong bands at
216, 265, and 302 nm, are also essentially identical and indi-
cate the presence of a substituted aromatic system. In the
1H and 13C NMR spectra of cruentaren A signals for a
[‡] Antibiotics from gliding bacteria, 105. Part 104: B. Kunze, H.
Steinmetz, G. Höfle, H. Reichenbach J. Antibiot., submitted.
[a] Bereich Naturstoffe, Gesellschaft für Biotechnologische For-
schung mbH,
Mascheroder Weg 1, 38124 Braunschweig, Germany
E-mail: gho@gbf.de
[b] Institut für Chemie und Biochemie/Kristallographie, Freie Uni-
versität Berlin,
Takustr. 6, 14195 Berlin, Germany
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 5036–5044

Isolation and Structural Elucidation of Cruentarens A and B
FULL PAPER
2,4,6-substituted benzoyl residue were identified. A hydroxy
group, which forms a strong hydrogen bond (δ = 11.48 pm),
is situated next to the carbonyl group and shifts the IR
carbonyl band to 1646 cm–1. A methoxy substituent at C-5
is indicated by correlation of this carbon with the methoxy
protons in the HMBC spectrum. Starting from the benzylic
methylene group at C-8, an extended substituted carbon
chain could be derived in a straightforward manner from
the combined NMR spectroscopic data in Table 1. The
chain terminates with an amide group at C-22. Both the
C-22 methylene and the amide protons are coupled to the
carbonyl carbon of a 3-hydroxy-2-methylhexanoic acid resi-
due. Finally, a three-bond coupling between 15-H and the
benzoyl carbonyl carbon proves the presence of a 12-mem-
bered lactone in cruentaren A (1).
All four stereoisomers of 4 were synthesised from 3 by
Evans aldol (R3 = H)[9] and chromium Reformatzky reac-
tions (R3 = Br).[10] The (2R,3S) configuration for the degra-
dation product was determined by chiral GC co-
chromatography, and from this the (24R,25S) configuration
for cruentaren A and B.
The relative configuration of the four stereocentres in the
central part of the molecules is best analysed with cruen-
taren B. To predict the most probable of the eight possible
diastereomers we adopted the 13C NMR spectroscopic data
base approach developed by Kishi et al.[11] for two contigu-
ous propionate units in polyketide natural products. Two
diastereomers with all-syn and syn-syn-anti configurations
(A and B, Figure 1) were predicted with high significance.
Figure 1. Relative configuration of C-15 to C-18 of cruentaren B
predicted using Kishi’s 13C NMR chemical shift analysis.[11]
To decide which was the correct diastereomer and deter-
mine its absolute configuration, the central part of cruen-
taren B had to be isolated by cleavage of the adjacent
double bonds and compared with reference samples from
stereoselective synthesis.[7] To facilitate isolation of the de-
gradation products, a UV-active handle was introduced by
acetalisation with acetophenone to give mono- and dipro-
tected cruentaren B 5 and 6. Only one stereoisomer was
formed at each of the newly created benzylic positions,
however at this stage their configuration was not known.
The NMR spectroscopic data of cruentaren B (2) are
very similar to those of 1, with the exception that 9-H and
15-H are shifted by 0.72 ppm downfield and 1.44 ppm up-
field, respectively. This indicates a six-membered lactone
ring for cruentaren B (2), which is apparently formed by an
intramolecular transesterification driven by release of ring
strain. On a preparative scale, this rearrangement was
achieved in quantitative yield by acid or base catalysis.
The configuration of the 12,13 double bond in 1 and 2
is cis according to the vicinal coupling constant of 11 Hz.
Whereas the olefinic proton signals for 20-H and 21-H are
not resolved, the cis configuration follows from the rela-
tively high-field shifts of the allylic carbons C-19 and C-22.
After attempts to crystallise 1, 2 and several of its deriva-
tives failed, the relative and absolute configuration of the
eight stereocentres had to be determined from NMR spec-
troscopic data and degradation studies. Fortunately, the ste-
reocentres in 2 are clustered in the N-acyl group, the central
diol part and the isochromanone system. Total acidic hy-
drolysis of 1 and 2 yielded 3-hydroxy-2-methylhexanoic acid
(4) as a single isomer, as shown by chiral GC analysis on a
cyclodextrin column.
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G. Höfle et al.
FULL PAPER
1
[a]
Table 1. H and 13C NMR spectroscopic data of cruentaren A (1) in CDCl3 (600 and 100 MHz).
H atom
δ [ppm]
Multiplicity
J [Hz]
C atom
δ [ppm]
HMBC
Correlated H atoms
3-OH
4-H
6-H
8-Ha
8-Hb
9-H
10-H
11-Ha
11-Hb
12-H
11.48 br. s
6.35 d
6.30 d
2.20–2.30 m
3.75 dd
3.63 ddd
1.92–2.06 m
1.92–2.06 m
2.32 dt
5.50 ddd
5.44 ddd
2.20–2.30 m
2.82 dt
5.29 ddd
1.92–2.06 m
3.45 dd
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
171.5
105.0
165.8
99.7
163.6
112.4
143.7
36.7
73.1
38.2
31.7
132.2
125.8
29.9
78.1
39.3
74.8
36.9
30.7
131.0
126.8
36.6
176.5
44.9
71.9
35.8
19.3
14.1
55.4
14.2
8.6
4, 15
4, 6, 8a,b
4
2.7
2.7
6
12.8, 1.8
10.8, 3.0, 1.8
4, 6, 29
4, 8a,b
8a,b
6, 9, 11b
8a, 11b, 30
11b, 30
13, 30
11b,14b
14.3, 11.7
11.0, 2.9, 1.0
11.0, 4.5, 2.0
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
C-31
C-32
C-33
13-H
14-Ha
14-Hb
15-H
16-H
17-H
17-OH
18-H
19-H2
20-H
14.3, 11.5
11.7, 5.6, 2.0
11b,14b
12, 13, 15, 16
14b, 16, 17, 31
16, 31
15, 18, 19, 31, 32
17, 19, 32
18, 20, 21, 32
18, 19, 21, 22a,b
19, 20, 22a,b
20, 21
22a,b, 24, 25, 33
33
26a,b, 33
27a,b, 28
25-OH, 26a,b, 28
26a,b, 27a,b
–
9.2, 2.3
2.70 sbr
1.70 qddd
2.20–2.30 m
5.54–5.59 m
5.38–5.42 m
3.80–3.84 m
3.91 dddd
6.09 br. t
2.28 qd
3.80–3.84 m
1.58 br. s
1.28–1.32 m
1.42–1.50 m
1.28–1.32 m
1.42–1.50 m
0.92 t
6.8, 6.8, 2.3, 2.0
21-H
22-Ha
22-Hb
22-NH
24-H
14.8, 7.4, 5.8, 1.5
5.6
7.2, 2.8
25-H
25-OH
26-Ha
26-Hb
27-Ha
27-Hb
28-CH3
29-OCH3
30-CH3
31-CH3
32-CH3
33-CH3
9, 11b
15, 16, 17
18, 19
7.1
16.2
11.2
3.80 s
1.01 d
0.89 d
0.79 d
24
6.8
7.0
6.8
7.2
1.14 d
[a] Selected signals in [D6]DMSO: δ = 10.58 (br. s, 3-OH), 4.05 (d, J = 6.0 Hz, 9-OH), 4.67 (d, J = 7.0 Hz, 17-OH), 7.84 (d, J = 5.5 Hz,
22-NH), 4.48 ppm (d, J = 5.8 Hz, 25-OH).
1
All attempts to oxidatively cleave the olefinic double mer showed identical H NMR chemical shifts and an op-
bonds of 6, optionally with reductive work-up, produced tical rotation of the same magnitude but opposite sign.
intractable mixtures instead of the expected dialdehyde or Thus, the central part of cruentaren
B
has
a
diol. Therefore we turned to the cleavage of double bonds (15S,16R,17R,18R) configuration and that of cruentaren A
by olefin metathesis in the presence of ethylene, which has has a (15S,16S,17R,18R) configuration.
worked well in a similar situation with epothilone[12] and
An anti configuration for the isochromanone 7 degrada-
spirangien.[7] Using the Hoveyda–Grubbs ruthenium cata- tion product followed from comparison of the vicinal 9-H,
lyst we indeed observed cleavage of both double bonds at a 10-H coupling of 6.6 Hz with model compounds, which
similar rate. The individual fragments 7, 8 and 9 were iso- show couplings of 4.2 and 5.7 Hz for the syn and anti dia-
lated in yields of 64, 48 and 69%.
stereomers, respectively.[13] To apply Mosher’s method for
In addition, “dimers” 10 and 11 were recovered in 25% absolute configuration determination a free 9-hydroxy
and 24% yields. However, no non-natural dimers or oligo- group is required. To this end, the phenolic hydroxy group
mers were observed, which would have indicated C–C bond was methylated and the lactone reduced to the diol 12a with
formation under the cleavage conditions.
lithium aluminium hydride. When it turned out that a tetra-
Conformational analysis of 8 using proton coupling con- hydropyran was always formed on attempted acylation, the
stants and selected strong NOEs proved the syn-syn config- benzylic hydroxy group was hydrogenated with palladium
uration for C-15 to C-17, whereas a 10 Hz coupling between in acetic acid to give toluene derivative 12b. Subsequent
17-H and 18-H indicated an anti configuration (Figure 2). acylation with (R)- and (S)-Mosher’s acid chloride
At this stage the two stereoisomers of 8 in question became smoothly produced the diastereomeric MTPA esters 13a
available as side products of an ongoing total synthesis of and 13b. Although the important signals of the 10-H, 11-
cruentaren B.[7] Of these, only the (15R,16S,17S,18S) iso- H2 and 12-H2 protons were obscured by multiplets, positive
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Eur. J. Org. Chem. 2006, 5036–5044

Isolation and Structural Elucidation of Cruentarens A and B
FULL PAPER
Having observed smooth cleavage of protected cruen-
taren B (6) by olefin metathesis, the behaviour of unprotec-
ted cruentaren A (1) in the presence of ethylene and various
ruthenium catalysts was investigated. The highest conver-
sion was observed with the Hoveyda–Grubbs catalyst, with
an isolated yield of 61% of 21,22-seco-cruentaren A (14).
No cleavage of the sterically hindered 12,13 double bond,
and no dimer of 14 from cross metathesis was observed.
Fortunately, 14 crystallised from ethyl acetate and an X-
ray crystal structure analysis could be performed. This not
only proved the structure and relative configuration but
also greatly facilitated solution conformation analysis. The
conformation of the lactone ring in the crystal is character-
ised by a nearly antiperiplanar orientation of 8a-H/9-H,
11b-H/12-H, 13-H/14b-H and 14b-H/15-H. The lactone
oxygen O-25 is the acceptor of an intramolecular hydrogen
bond with the OH group OH-26 as donor [O-26···O-25 =
2.548(2), H-26···O-25 = 1.82(3) Å; O-26–H-26···O-25 =
147(2)°].
In chloroform solution the corresponding vicinal coup-
ling constants are consistently in the range of 10–12 Hz.
This, and the strong NOEs between 8b-H/11b-H and 14a-
H/15-H, prove that the lactone ring in 14 adopts the same
configuration as in the crystal (Figure 3, b). Also, the side-
chain conformation seems to be retained from the 8.5 and
8.0 Hz coupling constants for 17-H/19b-H and 19b-H/20-
H, whereas the C-15/C-16 bond, which connects the macro-
cycle with the side-chain, is rotated by about +120°, as can
be seen from the 2 Hz coupling between 15-H/16-H and
strong NOEs between 15-H/16-H/31-H3 and 14a-H/31-H3.
(Figure 3, b). On the basis of this analysis it can be con-
Figure 2. Relative configuration of 8 with selected NOEs and vici-
nal coupling constants.
∆δ values in the order of 0.02 to 0.08 ppm were observed cluded that the lactone ring in cruentaren A (1) adopts the
for 1-H, 4-H, 6-H and 8-H2, and a negative one of 0.12 ppm same conformation in chloroform, methanol and DMSO
for the 30-methyl protons. This indicated a (9R) configura- solution.
tion for 13a,b[14] and, according to the previously estab-
From a biosynthetic point of view cruentaren A is a
lished relative configuration, a (9R,10S) configuration for tetradecaketide with a glycine building block inserted after
cruentarens A and B.
the third ketide unit. From feeding of [1-13C]propionate and
Eur. J. Org. Chem. 2006, 5036–5044
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G. Höfle et al.
FULL PAPER
Figure 3. a) Crystal structure and b) proposed solution conformation with selected NOEs of 21,22-seco-cruentaren A (14).
[13CH3]methionine it follows that all four C-methyl groups
originate from incorporation of propionate. Contrary to
that, the structurally related apicularen A (15) is an unde-
caketide assembled solely from acetate.[15] The glycine
building block in apicularen is transformed into an N-acyl-
vinylamine, whereas in cruentaren it is transformed into an
N-acylallylamine. It can be speculated whether the PKS/
NRPS synthases have evolved from a common ancestor or
were derived from each other.
Both apicularen A and cruentaren A (Figure 4) are 12-
membered lactones with the difference that 9-OH in apicul-
aren is blocked in an ether ring which prevents isomeris-
ation to a six-membered lactone like cruentaren A. The bi-
cyclic lactone structure forces apicularen into a fundamen-
tally different conformation, which also prevents hydrogen-
bond formation between the lactone carbonyl and the 3-
OH.[16] As a consequence, it is very unlikely that cruentaren
A and apicularen A, in spite of their planar structural simi-
larity, occupy the same protein binding site.
Preliminary data on the structure–activity relationship
indicate that an intact macrolactone, both C=C double
bonds and the acylamino part of the side-chain are essential
for cytotoxic activity. However, it is not known what the
exact structural requirements for the side-chain are. Prelimi-
nary experiments to obtain side-chain-modified cruentarens
by olefin cross-metathesis of 14 with synthetic building
blocks have failed so far.
Figure 4. Acetate and propionate labelling patterns of apicularen
A (15) and cruentaren A (1).
Experimental Section
General Procedures: Optical rotations were determined with a Per-
kin–Elmer 241 instrument. UV spectra were recorded with a Shim-
adzu UV-2102 PC scanning spectrometer. IR spectra were mea-
sured with a Nicolet 20DXB FT-IR spectrometer. NMR spectra
were recorded in CDCl3 with a Bruker DMX-600 spectrometer.
For structural analogues only a selection of significant NMR spec-
troscopic data are given. EI and DCI mass spectra (reactant gas
ammonia) were obtained with a Finnigan MAT 95 spectrometer;
high resolution data were acquired using peak matching (M/DM
= 10000). Pure compounds were characterised by analytical HPLC
on Nucleosil C 18 (column 125×2 mm, 5 µm, flow 0.3 mLmin–1),
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Eur. J. Org. Chem. 2006, 5036–5044

Isolation and Structural Elucidation of Cruentarens A and B
acetonitrile/water, isocratic 55:45, diode array detection. Prepara-
FULL PAPER
Table S1 in the Supporting Information. HRDMS (DCI, NH3) for
tive HPLC was performed on Nucleosil (column 250×21 mm, C33H52NO8: calcd. 590.3714; found 590.3693.
7 µm, flow 18 mLmin–1), acetonitrile/water, 55:45, detection by UV
13C-Labeled Cruentaren A: Two days after inoculation each of two
absorption at 254 nm. Analytical TLC [TLC aluminium sheets sil-
ica gel Si 60 F254 (Merck), solvent: ethyl acetate], detection by UV
absorption at 254 nm, dark blue spots on staining with cerium(IV)
sulfate/phosphomolybdic acid in sulfuric acid followed by charring.
Precoated silica gel Si 60 F254 plates of 0.25-mm layer thickness
were used for preparative TLC. GC analysis: Shimadzu GC-17 A
gas chromatograph with FID detection; capillary column
HYDRODEX® β-PM [heptakis(2,6-di-O-methyl-3-O-pentyl)-β-
cyclodextrin], 25 m×0.25 mm ID, temperature gradient: 5 min
80 °C, 6 °Cmin–1 to 180 °C. The Hoveyda–Grubbs catalyst [1,3-
bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(o-isoprop-
oxyphenylmethylene)ruthenium was purchased from Aldrich.
2-L batches of strain Ha r1 in the presence of Amberlite XAD16
(10 mL) was fed with [13C-CH3]methionine or sodium [1-13C]propi-
onate (1 g each, added in 5 portions, 95% enrichment) and culti-
vated for 7 and 8 d, respectively. Cell mass and adsorber resin were
collected by centrifugation and extracted with acetone. From the
crude extracts of 0.24 g and 0.12 g pure cruentaren A samples (7.7
and 2.1 mg) were isolated by RP18 HPLC as described above.
Methionine-Labelled Cruentaren A: 13C NMR (CDCl3): δ =
55.4 ppm (C-29) 2.8% 13C.
Propionate-Labelled Cruentaren A: 13C NMR (CDCl3): δ = 73.1
(C-9, 3.1%), 78.1 (C-15, 3.0%), 74.8 (C-17, 2.4%), 176.4 (C-23,
3.5% 13C).
Isolation of Cruentarens A (1) and B (2) from Byssovorax cruenta:
A 90-L fermentation batch of strain Ha r1 grown in the presence
of 0.9 L of Amberlite XAD 16 adsorber resin was harvested by
centrifugation. The wet cell mass and adsorber resin mixture was
extracted with four 3-L batches of acetone by stirring, sedimen-
tation and decanting. The extract was analysed by HPLC to con-
tain 290 mg of cruentaren A and about 10 mg of cruentaren B. It
was then concentrated on a rotary evaporator at 30 °C/200 mbar
to the water phase, which was extracted with three 0.5-L portions
of ethyl acetate. The organic phase was dried with magnesium sul-
fate and evaporated at 30 °C/150 mbar. The resulting crude extract
(10.0 g) was separated by chromatography on 180 g of silica gel Si
60 (15–40 µm, Merck) in an open glass column of 6 cm diameter.
After elution of lipophilic components with 0.8 L of ethyl acetate/
n-heptane (2:1) and 0.3 L of ethyl acetate the cruentarens were
eluted with 0.7 L of ethyl acetate/methanol (8:2) at a flow of
10 mLmin–1. Cruentaren-containing fractions were identified by
TLC, combined and the solvents evaporated in vacuo (320 mg).
This material was further purified and separated by HPLC (RP-
18, Kronlab ODS AQ 120 A, 16 µm, 3×48 cm, detection at 254 nm)
with acetonitrile/water (55:45) at 18 mLmin–1. Cruentaren A and
B eluted at 68–77 min and 100–110 min, respectively. The fractions
were concentrated in vacuo to the water phase, extracted with two
portions each of 100 mL of ethyl acetate, evaporated and dried to
give 270 mg of cruentaren A and 7 mg of cruentaren B.
Acetalisation of Cruentaren B (2): a) Cruentaren B (30 mg,
51 µmol), 1,1-dimethoxyethylbenzene (83 mg, 500 µmol) and pyr-
idinium p-toluenesulfonate (17 mg, 71 µmol) were dissolved in
1 mL of dried dichloromethane under nitrogen. After stirring over-
night saturated sodium hydrogen carbonate solution was added
and the mixture extracted three times with diethyl ether. The ether
solution was then washed with saturated sodium chloride solution,
dried with magnesium sulfate and the solvents evaporated in vacuo.
The residue was separated by chromatography on silica gel with
diethyl ether as eluent to yield monoacetal 5 (5.6 mg, 16%) and
diacetal 6 (8.7 mg, 21%).
b) Cruentaren B (2) (87 mg, 147 µmol), 1,1-(dimethoxyethyl)ben-
zene (485 mg, 2.95 mmol) and pyridinium p-toluenesulfonate
(51 mg, 206 µmol) were dissolved in 1 mL of dichloromethane. The
rest of the procedure was performed as above to give diacetal 6 as
a colourless solid (86.3 mg, 74%).
Cruentaren B Monoacetal (5): Rf = 0.58 (Et2O). UV (MeOH): λmax
(lgε) = 204 (4.5), 266 (4.2), 301 nm (3.8). IR (KBr): ν = 2961, 2874,
˜
1666 cm–1. 1H NMR (CDCl3, 400 MHz): δ = 0.83 (d, J = 6.61 Hz,
31-H3, 32-H3), 0.92 (t, J = 7.12 Hz, 28-H3), 1.04 (d, J = 7.12 Hz, -
H3), 1.11 (d, J = 7.12 Hz, 33-H3), 1.23–1.52 (m, 26-H2, 27-H2), 1.55
(s, 35-H3), 1.60 (m, 1 H, 16-H), 1.68–1.83 (m, 18-H), 2.03 (ddd, J
= 13.86, 8.39, 8.27 Hz, 10-H), 2.10–2.25 (m, 11b-H, 4b-H, 9b-H),
2.28–2.45 (m, 11a-H, 14a-H, 19a-H), 2.47–2.59 (m, 24-H), 2.71–
2.84 (m, 8b-H), 2.83–2.98 (m, 8a-H), 3.67 (dd, J = 9.92, 1.78 Hz,
17-H), 3.81 (s, 29-H3), 3.84–4.00 (m, 15-H, 22-H2), 4.01–4.09 (m,
25-H), 4.36 (ddd, J = 12.21, 6.10, 3.05 Hz, 9-H), 5.40–5.70 (m, 12-
H, 13-H, 21-H), 5.74 (br. t, 22-NH), 6.24 (d, J = 2.54 Hz, 6-H),
6.36 (d, J = 2.03 Hz, 4-H), 7.26–7.37 (m, 3 H, phenyl), 7.56 (d, J
= 8.65 Hz, 2 H, phenyl), 11.21 (s, 3-OH) ppm. 13C NMR (CDCl3,
75 MHz): δ = 4.8 (C-31), 11.0 (C-33), 14.0 (C-28), 14.2 (C-32), 15.0
(C-30), 19.2 (C-27), 21.2 (C-35), 29.9 (C-8), 30.1 (C-11), 30.9 (C-
19), 31.1 (C-14), 32.7 (C-16), 34.8 (C-18), 35.8 (C-26), 36.5 (C-22),
37.3 (C-10), 44.6 (C-24), 55.6 (C-29), 71.7 (C-25), 73.8 (C-15), 77.2
(C-17), 82.2 (C-9), 99.0 (C-34), 99.5 (C-4), 101.9 (C-2), 106.4 (C-
6), 124.6 (C-37,C-38), 125.5, 127.60, 127.62, 128.0 (2 C), 128.6,
132.4 (C-12, C-13, C-20, C-21, C-39, C-40, C-41), 141.2 (C-7),
145.9 (C-36), 164.6 (C-3), 165.8 (C-5), 170.0 (C-1), 176.4 (C-23)
ppm. EI-MS: m/z (%) 691 (16) [M+], 676 (70), 554 (55), 409 (50),
256 (83). HREI-MS for C41H57N1O8: calcd. 691.4084; found
691.4079.
Preparation of Cruentaren B (2) from Cruentaren A (1): Compound
1 (100 mg) was dissolved in 1 mL of methanol, 0.8 mL of 1  HCl
was added and the mixture kept overnight at 45 °C. After dilution
with water the mixture was adjusted to pH 7 with 1  NaOH, the
methanol evaporated in vacuo, the water phase extracted with ethyl
acetate, dried with magnesium sulfate, and the solvents evaporated
to dryness. Yield: 99 mg (99%) of a slightly yellow oil.
Cruentaren A (1): Colourless amorphous solid; tR = 3.9 min (Nu-
cleosil C18, 125×2 mm, 5 µm, acetonitrile/water 0.3 mLmin–1, di-
ode array detection); Rf = 0.53 (silica gel Si 60 aluminium sheets,
ethyl acetate). [α]2D2 = –3.4 (c = 13.5 mgmL–1, MeOH). UV
(MeOH): λmax (lgε) = 216 (4.26), 265 (3.98), 302 (3.66), 346 nm
(1.94). IR (KBr): ν
= 3396, 2959, 2932, 2863, 1646, 1614 cm–1.
˜
max
NMR see Table 1. HRMS (DCI, NH3) for C33H51NO8: calcd.
589.3615; found 589.3645.
Cruentaren B (2): Colourless amorphous solid; tR = 5.1 min (Nu-
cleosil C18, 125×2 mm, 5 µm, acetonitrile/water 0.3 mLmin–1, di-
ode array detection); Rf = 0.40 (silica gel Si 60 aluminium sheets,
ethyl acetate). [α]2D2 = –9.1 (c = 6.6 mgmL–1, MeOH). UV (MeOH)
λmax (lgε) = 210 (4.36), 215 (4.38), 267 (4.14), 301 nm (3.77). IR
Cruentaren B Bisacetal (6): Rf = 0.75 (Et2O). UV (MeOH): λmax
(lgε) = 204 (4.5), 266 (4.2), 301 nm (3.8). [α]2D2 = –0.2 (c = 0.5,
MeOH). IR (KBr): ν
= 2962, 2874, 1665 cm–1. 1H NMR
˜
max
(CDCl3, 600 MHz): δ = 0.73 (d, J = 6.76 Hz, 32-H3), 0.78 (d, J =
7.03 Hz, 3-H3), 0.91 (t, J = 7.17 Hz, 28-H3), 1.02 (d, J = 6.76 Hz,
(KBr): ν
= 3416, 2963, 2930, 2869, 1661 cm–1. NMR see
˜
max
Eur. J. Org. Chem. 2006, 5036–5044
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5041

G. Höfle et al.
FULL PAPER
30-H3), 1.25–1.33 (m, 27-H2), 1.36 (d, J = 7.30 Hz, 33-H3), 1.43–
1.48 (m, 26b-H), 1.48–1.55 (m, 16-H, 26a-H), 1.56 (s, 43-H3), 1.62–
1.72 (m, 18-H), 1.80 (ddd, J = 14.00, 8.25, 8.05 Hz, 19b-H), 1.92
(C-5), 21.3 (C-14), 32.6 (C-2), 33.9 (C-4), 37.4 (C-6), 37.5 (C-10),
73.7 (C-9), 77.3 (C-3), 98.9 (C-13), 116. (C-8), 116.8 (C-12), 124.7
(C-16, C-17), 127.4 (C-20), 127.9 (C-18,C-19), 134.9 (C-11), 136.9
(s, 35-H3), 1.99–2.05 (m, 10-H), 2.12–2.23 (m, 11b-H, 14bH), 2.28– (C-7), 146.2 (C-15) ppm. MS (DCI): m/z (%) 301 (100), [M + H+]
2.39 (m, 11a-H, 14a-H, 19a-H), 2.50 (dq, J = 7.30, 2.71 Hz, 1 H,
198 (20). HRMS (DCI) for C20H28O2: calcd. 300.2089; found
H24), 2.74 (dd, J = 16.23, 2.98 Hz, 1 H, H8b), 2.91 (dd, J = 16.10, 300.1949.
12.31 Hz, 1 H, H8a), 3.17 (dd, J = 15.15, 5.68 Hz, 1 H, H22b),
Amide Fragment 9: UV (MeOH): λmax (lgε) = 209 (3.9), 250 nm
3.56 (dd, J = 9.87, 2.03 Hz, 1 H, H17), 3.81 (s, 3 H, H29), 3.96–
4.04 (m, 2 H, H15, H22a), 4.19 (ddd, J = 8.25, 4.87, 2.84 Hz, 1 H,
H25), 4.35 (ddd, J = 12.38, 6.29, 2.98 Hz, 1 H, H9), 5.32–5.58 (m,
12-H, 13-H, 20-H, 21-H), 6.23 (m, 6-H), 6.35 (d, J = 2.16 Hz, 4-
H), 7.22–7.37 (m, 6 H, phenyl) 7.41–7.52 (m, 4 H, phenyl), 11.21
(s, 3-OH) ppm. 13C NMR (CDCl3, 75 MHz): δ = 4.8 (C-31), 12.0
(C-33), 13.9 (C-28), 14.0 (C-32), 14.9 (C-30), 19.0 (C-27), 21.1; 21.7
(C-43, C-35), 30.0 (C-8), 30.4 (C-11), 30.5 (C-19), 31.1 (C-14), 32.6
(C-16), 33.4 (C-26), 34.5 (C-18), 37.3 (C-10), 41.2 (C-24), 41.7 (C-
22), 55.6 (C-29), 71.1 (C-25), 73.8 (C-15), 77.2 (C-17), 82.3 (C-9),
91.4; 98.9 (C-34, C-42), 99.5 (C-4), 101.9 (C-2), 106.3 (C-6), 124.6,
126.5 (4 C, phenyl), 127.5, 127.6, 127.7, 128.6, 128.9 (6 C, phenyl),
127.9 (C-13, C-20, C-21), 129.5 (C-12), 141.2 (C-7), 142.3, 145.9
(C-36, C-44), 164.6 (C-3), 165.8 (C-5), 170.0 (C-1), 172.2 (C-23)
ppm. DCI-MS: m/z (%) = 811 (100) [M + NH4+]. HRDCI-MS for
C49H64NO8: calcd. 794.4614; found 794.4631.
(2.5). [α]2D2 = –40.1 (c = 4.2, MeOH). 1H NMR (CDCl3, 300 MHz):
δ = 0.92 (t, J = 7.06 Hz, 6-H3), 1.26–1.48 (m, 5-H2), 1.36 (d, J =
7.35 Hz, 7-H3), 1.48–1.64 (m, 4-H2), 1.92 (s, 12-H3), 2.51 (ddd, J =
14.55, 7.30, 2.83 Hz, 2-H), 2.98–3.20 (m, J = 15.59, 6.55, 1.27 Hz,
8b-H), 3.90–4.05 (m, J = 15.61, 5.02, 1.55 Hz, 8a-H), 4.16–4.26 (m,
3-H), 4.85–5.03 (m, 10-H2), 5.60–5.80 (m, J = 17.07, 10.38, 6.45,
5.09 Hz, 9-H), 7.29–7.51 (m, 5 H, phenyl) ppm. 13C NMR (CDCl3,
75 MHz): δ = 12.0 (C-7), 14.0 (C-6), 19.0 (C-5), 21.9 (C-12), 33.3
(C-4), 41.2 (C-2), 46.8 (C-8), 71.1 (C-3), 91.5 (C-11), 116.3 (C-10),
126.5 (2-C)/128.6 (2-C)/128.9 (1-C) (C14–C-18), 134.0 (C-9), 142.2
(C-13), 172.2 (C-1) ppm. MS (EI): m/z (%) 287.0 (100) [M+].
HRMS for C18H25NO2: calcd. 287.1885; found 287.1905.
O-Methylation and Reduction of 7: Isochromanone 7 (6 mg,
23 µmol), silver(I) oxide (5.6 mg, 240 mmol) and 1.5 mL of methyl
iodide were stirred under an atmosphere of nitrogen at room tem-
perature overnight. The reaction mixture was filtered through a bed
of Celite, the Celite washed with dichloromethane and the com-
bined solutions evaporated. The residue (6.1 mg) was dissolved in
dried THF (200 µL) and a 1  solution of lithium aluminium hy-
dride in THF (43 µL, 43 µmol) was added with stirring at 0 °C
under an atmosphere of nitrogen. After two hours the reaction was
quenched with water. The mixture was then filtered through a bed
of Celite, the Celite washed with THF and the combined solutions
evaporated. The residue was purified by preparative TLC (silica
gel, diethyl ether) to yield 12a (5.2 mg, 85%) as a light-yellow oil.
Rf = 0.62 (PE/Et2O, 1:1). UV (MeOH): λmax (lgε) = 210 (4.2), 229
Degradation of Cruentaren B Bisacetal (6) by Cross Metathesis with
Ethylene: Cruentaren B Bisacetal (6; 66 mg, 83 µmol) and Hov-
eyda–Grubbbs catalyst (11 mg, 17 µmol) were dissolved in dried
dichloromethane (10 mL). The atmosphere above the light-green
solution was then replaced with ethylene. After stirring for four
hours the solvent was evaporated in vacuo and the residue sepa-
rated by preparative RP-18 HPLC (MeCN/H2O, 57:43). The fol-
lowing compounds were obtained as black oils: 7 (13.8 mg, 63%),
8 (11.9 mg, 48%), 9 (16.4 mg, 69%), 10 (11.0 mg, 25%) and 11
(11.1 mg, 24%). Filtration through a bed of silica gel (PE/Et2O,
1:1) gave light-yellow oils without loss of material.
(3.6), 283 nm (3.0). [α]2D2 = +78.1 (c = 0.8, MeOH). IR (KBr): ν =
˜
1
Isochromanone 7: Rf = 0.69 (PE/Et2O, 1:1). UV (MeOH): λmax (lgε)
= 215 (4.3), 267 (4.1), 301 nm (3.7). [α]2D2 = +32.6 (c = 0.7, MeOH).
3417 (w), 2959 (w), 2934 (w), 1749 (w), 1606 cm–1 (m). H NMR
(CDCl3, 300 MHz): δ = 1.02 (d, J = 6.78 Hz, 14-H3), 1.64–1.89 (m,
IR (KBr): ν = 3430 (w), 2922 (m), 2851 (w), 1667 (s), 1627 cm–1 10-H), 1.95–2.18 (m, 11b-H), 2.23–2.51 (m, 11a-H), 2.67–3.03 (m,
˜
(s). 1H NMR (CDCl3, 600 MHz): δ = 1.02 (d, J = 6.42 Hz, 14-H3),
2.02 (td, J = 13.12, 6.61 Hz, 10-H), 2.05–2.12 (m, 11b-H), 2.33–
2.46 (m, 11a-H), 2.77 (dd, J = 16.24, 3.02 Hz, 8b-H), 2.92 (dd, J =
16.05, 12.28 Hz, 8a-H), 3.81 (s, 15-H3), 4.35 (ddd, J = 12.18, 6.33,
8-H2), 3.62 (ddd, J = 9.04, 5.37, 4.05 Hz, 9-H), 3.79, 3.82 (s, 15-
H3, 16-H3), 4.54 (d, J = 11.49 Hz, 1b-H), 4.82 (d, J = 11.87 Hz,
1a-H), 4.97–5.15 (m, 13-H2), 5.84 (td, J = 17.14, 7.16 Hz, 12-H),
6.32 (d, J = 2.26 Hz, 6-H), 6.36 (d, J = 2.45 Hz, 4-H) ppm. 13C
3.02 Hz, 9-H), 4.97–5.18 (m, 13-H2), 5.64–5.92 (m, J = 17.14, 10.06, NMR (CDCl3, 75 MHz): δ = 14.7 (C-14), 31.3 (C-8), 37.3 (C-11),
6.99 Hz, 12-H), 6.25 (s, 6-H), 6.35 (d, J = 2.27 Hz, 4-H), 11.20 (d, 37.7 (C-10), 55.2, 55.4 (C-15, C-16), 77.7 (C-9), 96.1 (C-4), 104.4
3-OH) ppm. 13C NMR (CDCl3, 75 MHz): δ = 14.7 (C-14), 29.9 (C-6), 116.1 (C-13), 116.4 (C-2), 135.7 (C-7), 137.1 (C-12), 156.5,
(C-8), 36.5; 36.7 (C-11, C-10), 55.6 (C-15), 82.1 (C-9), 99.5 (C-4), 159.1 (C-3, C-5) ppm. MS (EI): m/z (%) 281 (70) [M + H+], 280.1
101.9 (C-2), 106.3 (C-6), 117.1 (C-13), 135.8 (C-12), 141.2 (C-7),
165.8 (C-5), 169.9 (C-1) ppm. MS (DCI): m/z (%) 280.9 (100) [M
+ NH4+], 102 (65). HRMS (DCI) for C15H18O4: calcd. 262.1205;
found 262.1199.
(100) [M+]. HRMS for C16H24O4: calcd. 280.1675; found 280.1666.
2,4-Dimethoxytoluene Derivative 12b: mixture of diol 12a
A
(15 mg), Pd/C (15 mg) and acetic acid (25 µL) in methanol (1 mL)
was stirred under an atmosphere of hydrogen overnight. The mix-
ture was then filtered through a bed of Celite, evaporated and sepa-
rated by TLC on silica gel plates (PE/Et2O, 1:1) to yield 12b as a
colourless oil (9.5 mg, 67%). Rf = 0.63 (PE/Et2O, 1:1). UV
(MeOH): λmax (lgε) = 209 (4.3), 220 (sh, 3.9), 280 nm (3.5). IR
Diol Fragment 8: Rf = 0.79 (PE/Et2O, 10:1). UV (MeOH): λmax
(lgε) = 209 (4.1), 249 (2.9), 255 (2.9), 262 nm (2.8). [α]2D2 = –4.0 (c
= 0.2, MeOH). 1H NMR (CDCl3, 600 MHz): δ = 0.82 (d, J =
6.80 Hz, 1-H3, 5-H3), 1.57 (qt, J = 6.85, 2.28 Hz, 2-H), 1.63 (s, 14-
H3), 1.75–1.84 (m, J = 10.09, 7.94, 6.79, 3.38 Hz, 4-H), 2.03 (dt, J
(KBr): ν = 3464 (w), 2956 (s), 2933 (m), 2872 (m), 1607 cm–1 (s).
˜
= 13.70, 8.26 Hz, 6b-H), 2.16–2.22 (m, 10b-H), 2.38–2.45 (m, 10a- 1H NMR (CDCl3, 300 MHz): δ = 0.93 (t, J = 7.12 Hz, 13-H3), 0.99
H), 2.47–2.54 (m, J = 13.69, 6.14, 3.32, 1.60, 6a-H), 3.67 (dd, J =
9.96, 2.22 Hz, 3-H), 4.10 (ddd, J = 7.91, 6.02, 2.31 Hz, 9-H), 5.04
(ddd, J = 9.89, 2.22, 1.25 Hz, 8b-H), 5.06–5.10 (m, J = 9.02, 2.28,
1.17, 1.17 Hz, 8a-H), 5.15 (ddd, J = 17.15, 3.43, 1.65 Hz, 12-H),
5.70–6.02 (m, 7-H, 11-H), 7.27 (ddd, J = 7.25, 1.79, 1.68 Hz, 20-
H), 7.33 (td, J = 7.29, 1.09 Hz, 16-H, 17-H), 7.58 (d, J = 7.03 Hz,
18-H, 19-H) ppm. 13C NMR (CDCl3, 75 MHz): δ = 4.8 (C-1), 13.9
(d, J = 6.61 Hz, 14-H), 1.16–1.35 (m, 11b-H, 12b-H12b), 1.43–1.60
(m, 11a-H, 12a-H), 1.66–1.74 (m, 10-H) 2.10 (s, 1-H3), 2.54 (dd, J
= 13.73, 10.17 Hz, 8b-H), 2.88 (dd, J = 13.73, 2.54 Hz, 8a-H), 3.62
(ddd, J = 9.92, 5.09, 2.80 Hz, 9-H), 3.78, 2.79 (s, 15-H3, 16-H3),
6.35 (s, 6-H), 6.36 (s, 4-H) ppm. 13C NMR (CDCl3, 75 MHz): δ =
11.1 (C-1), 14.4 (C-13), 15.1 (C-14), 20.5 (C-11), 34.6 (C-12), 37.9
(C-8), 38.5 (C-10), 55.4, 55.6 (C-15, C-16), 75.6 (C-9), 96.9 (C-4),
5042
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 5036–5044

Isolation and Structural Elucidation of Cruentarens A and B
FULL PAPER
106.6 (C-6), 117.6 (C-2), 139.1 (C-7), 158.4, 159.0 (C-3, C-5) ppm. (ddd, J = 11.7, 5.2, 2.0 Hz, 15-H), 5.42 (ddd, J = 11.1, 4.7, 2.1 Hz,
MS (DCI): m/z (%) 284 (100) [M + NH4+], 267 (82) [M + H+]. 13-H), 5.49 (ddd, J = 11.2, 2.9, 1.3 Hz, 12-H), 5.72–5.80 (m, 20-
HRMS for C16H26O3: calcd. 267.196; found 267.192.
H2), 6.30 (d, J = 2.6 Hz, 6-H), 6.36 (d, J = 2.6 Hz, 4-H), 11.46 (s,
3-OH) ppm. 13C NMR (CDCl3, 75 MHz): δ = 8.7 (C-31), 14.1 (C-
30), 16.0 (C-32), 29.8 (C-14), 31.7 (C-11), 36.7 (C-18), 36.7 (C-8),
37.5 (C-19), 38.3 (C-10), 39.2 (C-16), 55.4 (C-29), 73.0 (C-9), 75.8
(C-17), 77.8 (C-15), 99.7 (C-4), 104.8 (C-2), 112.4 (C-6), 116.4 (C-
21), 125.6 (C-13), 132.3 (C-12), 137.1 (C-20), 143.7 (C-7), 163.6 (C-
5), 165.9 (C-3), 171.5 (C-1) ppm. EI-MS: m/z (%) 433.1 (32) [M +
H]+, 432.1 (100) [M+]. HRMS (EI) for C25H35O6: calcd. 432.2505;
found 432.2511.
Mosher Esters 13a and 13b: Alcohol 12b (3.5 mg, 13 µmol), (S)-
MTPA-chloride [or (R)-MTPA-chloride] (6.2 µL, 33 µmol), DMAP
(3 mg,26 µmol) and pyridine (15 µL) were mixed in dichlorometh-
ane and stirred at room temperature for 2 h. The solvents were
evaporated in vacuo and the residue separated by preparative TLC
on silica gel plates (PE/Et2O, 1:1). (R)-MTPA-ester 13a and (S)-
MTPA-ester 13b were obtained as a colourless oils (5.4 mg, 87%
and 5.0 mg, 80%, respectively).
X-ray Crystal Structure Determination of 14:[16] Diffraction data
were collected at 293 K on a Bruker SMART 1000 diffractometer
with Mo-Kα radiation (graphite monochromator, λ = 0.7107 Å) and
CCD area detector.[17] The crystal lattice is orthorhombic, space
group P212121, with unit cell parameters a = 9.216(1), b =
13a: Rf = 0.79 (PE/Et2O, 1:1) UV (MeOH): λmax (lgε) = 209 (4.5),
225 (4.1), 282 nm (3.6). [α]2D2 = +13.2 (c = 0.56, MeOH). IR (KBr):
ν = 3439 (m), 2959 (m), 2874 (w), 1741 cm–1(s). 1H NMR (CDCl ,
˜
3
300 MHz): δ = 0.93 (t, J = 7.12 Hz, 14-H3), 1.00 (d, J = 6.78 Hz,
13-H3), 1.15–1.37 (m, 11b-H, 12b-H), 1.41–1.64 (m, 11a-H, 12a-
H), 1.79–1.95 (m, 10-H), 2.06 (s, 1-H3), 2.86 (d, J = 6.97 Hz, 8-
H2), 3.34 (s, 17-H3), 3.65 (s, 15-H3), 3.76 (s, 16-H3), 5.34 (td, J =
7.02, 3.67 Hz, 9-H), 6.22 (d, J = 2.45 Hz, 6-H), 6.29 (d, J = 2.45 Hz,
4-H), 7.25–7.42 (m, 5 H, phenyl) ppm. 13C NMR (CDCl3,
75 MHz): δ = 11.0 (C-1), 14.3 (C-13), 15.2 (C-14), 20.5 (C-12), 33.9,
34.2 (C-8, C-11), 36.2 (C-10), 55.1 (C-15, C-16), 55.5 (C-17), 80.7
(C-9), 85.0 (C-19), 97.1 (C-4), 106.4 (C-6), 117.7 (C-2), 121.5, 125.3
(C-26), 127.5/128.2/129.3 (5 C, C-21 to C-25), 132.1 (C-20), 137.1
(C-7), 158.0 (C-3), 166.2 (C-18) ppm. MS (EI): m/z (%) 483.1 (30)
[M + H+], 482.0 (100) [M+]. HRMS for C26H33F3O5: calcd.
482.2280; found 482.2274.
13.524(1), c = 19.278(1) Å, V = 2402.8(2) Å3, Z = 4, Dcalc
=
1.196 gcm–3, µ = 0.084 mm–1. 11599 Reflections were collected up
to 2θmax = 56.5° and reduced to 5527 independent reflections (Rint
= 0.028). The phase problem was solved by direct methods
(SHELXS-97)[18] and the structure was refined on F2 with
SHELXL-97 with anisotropic displacement parameters for the
non-H atoms.[19] The hydrogen atoms located from difference syn-
theses were given isotropic displacement parameters. After conver-
gence of the refinements discrepancy factors were R1 = 0.0406, wR2
= 0.0852 for 4268 reflections with Fo Ͼ 4σ(Fo) and R1 = 0.0553,
wR2 = 0.0900 for all data; Gof = 0.930.
Supporting Information (see also the footnote on the first page of
this article): The synthesis and GC analysis of 3-hydroxy-2-methyl-
hexanoic acids 4, Kishis’s 13C-NMR chemical shift analysis of
cruentaren B (2), and spectroscopic data of 12,13,20,21-tetra-
hydrocruentaren A, 20,21-dihydrocruentaren A and cruentaren B
(2).
13b: Rf = 0.79 (PE/Et2O, 1:1). UV (MeOH): λmax (lgε) = 209 (4.5),
224 (4.0), 282 nm (3.5). [α]2D2 = –19.0 (c = 0.56, MeOH). IR (KBr):
˜
ν = 3444 (m), 2959 (m), 2874 (w), 1743 cm–1 (s). 1H NMR (CDCl3,
300 MHz): δ = 0.88 (d, J = 6.78 Hz, 14-H3), 0.93 (t, J = 6.97 Hz,
13-H3), 1.14–1.36 (m, 11b-H, 12b-H), 1.40–1.57 (m, 11a-H, 12a-
H), 1.83–2.01 (m, 10-H), 2.09 (s, 1-H3), 2.80–2.93 (m, 8-H2), 3.35
(d, J = 0.75 Hz, 17-H), 3.66 (s, 15-H), 3.79 (s, 16-H3), 5.31 (ddd, J
= 8.43, 5.04, 3.11 Hz, 9-H), 6.30 (d, J = 2.45 Hz, 6-H), 6.36 (d, J =
2.45 Hz, 4-H), 7.08–7.38 (m, 5 H, phenyl) ppm. 13C NMR (CDCl3,
75 MHz): δ = 11.0 (C-1), 14.3, 14.4 (C-13, C-14), 20.6 (C-12), 33.0
(C-8), 34.5 (C-11), 35.9 (C-10), 55.1 (C-15, C-16), 55.6 (C-17), 81.1
(C-9), 84.8 (C-19), 97.1 (C-4), 106.4 (C-6), 121.5, 125.4 (C-26),
127.5/128.2/129.3 (5C, C-21 to C-25), 132.3 (C-20), 137.3 (C-7),
158.2 (C-5), 158.8 (C-3), 166.4 (C-18) ppm. MS (EI): m/z (%):
483.01 (47) [M + H+], 482.0 (100) [M+]. HRMS for C26H33F3O5:
calcd. 482.2280; found 482.2296.
Acknowledgments
We thank I. Schleicher and S. Reinecke for technical assistance,
A. Roß, H. Schüler, R. Krützfeld and co-workers for large scale
fermentation, C. Kakoschke and B. Jaschok-Kentner and R. Christ
for recording NMR and mass spectra, respectively, and Dr. V. Wray
and Dr. H.-J. Hecht for helpful discussions.
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21,22-seco-Cruentaren A (14): Cruentaren A (1; 100 mg, 170 µmol)
and the Hoveyda–Grubbs catalyst (21 mg, 34 µmol) were dissolved
in dried dichloromethane (50 mL). This solution was stirred under
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residue separated by preparative HPLC (MeOH/H2O, 70:30).
21,22-seco-Cruentaren A was obtained as a light-yellow viscous oil
(42 mg, 61%). Thin needles formed on slow evaporation of an
NMR sample in CDCl3, and these were recrystallised from ethyl
acetate by slow evaporation at 4 °C. M.p. 170 °C (from ethyl ace-
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1
IR (KBr): ν
= = 3438, 2960, 2929, 1646, 1614 cm–1. H NMR
˜
max
(CDCl3, 600 MHz): δ = 0.77 (d, J = 6.8 Hz, 32-H3), 0.91 (d, J =
7.1 Hz, 31-H3), (d, J = 6.9 Hz, 30-H3), 1.62–1.69 (m, 18-H), 1.90
(dt, J = 14.0, 8.0 Hz, 19a-H), 1.95 (d, J = 14.8 Hz, 11a-H), 1.99–
2.05 (m, 16-H, 10-H), 2.17–2.23 (m, 14a-H, 8a-H), 2.29–2.34 (m,
19b-H, 11b-H), 2.83 (dt, J = 14.3, 11.5 Hz, 14b-H), 3.45 (dd, J =
8.5, 2.5 Hz, 17-H), 3.63 (ddd, J = 10.8, 3.1, 1.7 Hz, 9-H), 3.74 (dd,
J = 12.8, 1.7 Hz, 8b-H), 3.79 (s, 29-H3), 5.02–5.04 (m, 21-H2), 5.26
Eur. J. Org. Chem. 2006, 5036–5044
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5043

G. Höfle et al.
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Received: May 13, 2006
Published Online: September 7, 2006
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 5036–5044