Syntheses and biological properties of brefeldin analogues

Article abstract of DOI:10.1002/ejoc.201001297

Total and partial syntheses of brefeldin analogues are described. (6R)-Hydroxy-BFA (5) was obtained through a total synthesis from (1S,2R)-2-[(trityloxy)methyl]cyclopent-3-ene-1-carbonitrile (cis-8) in 13 steps. The BFA lactam analogue 6 was prepared via

Full text of DOI:10.1002/ejoc.201001297

FULL PAPER
DOI: 10.1002/ejoc.201001297
Syntheses and Biological Properties of Brefeldin Analogues
Sebastian Förster,[a] Elke Persch,[a] Olena Tverskoy,[a] Frank Rominger,[a]
Günter Helmchen,*[a] Christian Klein,[b] Basak Gönen,[c] and Britta Brügger[c]
Keywords: Brefeldin A analogues / Total synthesis / Structure–activity relationships / Molecular modeling / Nozaki–
Hiyama–Kishi reaction / Macrolactonization
Total and partial syntheses of brefeldin analogues are de-
scribed. (6R)-Hydroxy-BFA (5) was obtained through a total
synthesis from (1S,2R)-2-[(trityloxy)methyl]cyclopent-3-ene-
1-carbonitrile (cis-8) in 13 steps. The BFA lactam analogue 6
The structures of the brefeldin analogues were determined
by X-ray analyses. The activities of the brefeldin analogues
in blocking protein traffic between the endoplasmatic reticu-
lum and the Golgi apparatus were determined both for mam-
was prepared via the key building block 25, which was ac- malian cells and for plant cells. Molecular mechanics calcula-
cessed in four steps from BFA (1). (7S)-Amino-BFC (7) was
obtained from 7-dehydro-BFA (3) by reductive amination.
tions and docking into the Arf1-GEF protein complex, an es-
tablished receptor of BFA, were carried out.
Another interesting and well-investigated effect of BFA
is its ability to block protein traffic from the endoplasmatic
reticulum (ER) to the Golgi apparatus. Because of this ef-
fect, treatment with BFA leads to dramatic morphological
changes of the Golgi apparatus in eukaryotic cells.[9] In par-
ticular, the cis-Golgi redistributes into the endoplasmatic
reticulum and becomes part of it. Investigation of this mode
of action revealed that the molecular target of BFA is a
protein complex of a GDP-bound adenosine ribosylation
factor (Arf1) and a guanidine exchange factor (GEF).[10]
Arf1 belongs to the family of small G-proteins and is re-
sponsible for vesicle budding at the donor membrane at the
ER.[11] Generally, G-proteins are switched between active
and inactive states by exchange of the nucleotide GDP for
GTP. This exchange is catalyzed by GEFs. In 2003, Cherfils
et al.[12] and Goldberg et al.[13] independently published X-
ray crystal structures of a GDP-bound Arf1/Sec7 protein
complex. The Sec7 domain is the catalytically active part of
the GEF, and BFA binds at the interface of the interacting
proteins. It thus appears that BFA is a noncompetitive in-
hibitor of the Arf1 activation.
Introduction
Brefeldin A (BFA, Figure 1) is a secondary metabolite of
several Ascomycetes and was first isolated in 1958 from
Penicillium decumbens.[1] It exhibits antifungal,[2] antiviral,[3]
nematodic,[4] and cytostatic activity against numerous hu-
man cancer cell lines.[5] The cytostatic effect is caused by
the induction of apoptosis in a p-53-independent manner.[6]
After the discovery of this quite unusual effect in the 1990s,
many research groups became interested in BFA. The full
mechanism of apoptosis induction still remains to be eluci-
dated, however, and so the molecular target responsible for
that mode of action is not known. Although BFA exhibits
strong cytostatic activity accompanied by low toxicity,[2,7]
which is important for the development of drugs, BFA
could not be established as an anticancer agent because of
its low bioavailability and poor pharmacokinetics.[8]
The two modes of action displayed by BFA are indicative
of several molecular targets. A first hint of this was found
by Nojiri et al.,[6c] who showed that the induction of
apoptosis is caused by a modulatory effect of BFA on the
biosynthesis of gangliosides. They demonstrated that for-
skolin did not affect the terminal differentiation of cancer
cells treated with BFA, whereas it antagonizes the effects of
BFA on protein traffic and the Golgi apparatus. Further-
more, Cushman et al.[14] developed BFA analogues that
evoked the same morphologic changes to the Golgi appara-
tus as BFA but did not induce apoptosis. On the other
hand, it was found by Ellerby, Bredesen et al. that con-
tinuous BFA-induced stress on the endoplasmatic reticulum
Figure 1. Various brefeldins.
[a] Organisch-Chemisches Institut der Universität Heidelberg
Im Neuenheimer Feld 270, 69120 Heidelberg, Germany
Fax: +49-6221-544205
E-mail: g.helmchen@oci.uni-heidelberg.de
[b] Institute for Pharmacy and Molecular Biology der Universität
Heidelberg
Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
Fax: +49-6221-546430
[c] Biochemie Zentrum der Universität Heidelberg
Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
Fax: +49-6221-54-4366
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201001297.
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Syntheses and Biological Properties of Brefeldin Analogues
leads to cell death through caspase activation.[15] Accord- E, obtained by Ir-catalyzed allylic substitution as already
ingly, the induction of apoptosis might be related to the described.[16g] The protecting group scheme was based on
perturbation of protein traffic.
the use of a trityl group because of convenience in the allylic
Our aim was to search for brefeldin analogues[16] with substitution. The trityl group can be removed by a Birch-
cytostatic activity similar to that of BFA but improved in type reduction; as further orthogonal protecting groups, an
vivo stability and solubility in aqueous systems. The new O-silyl group (Σ2) and an acetonide (Σ1) were chosen.
brefeldin analogues were submitted to the National Cancer
Institute (Maryland, USA) for tests of their cytostatic ac-
tivities in the Developmental Therapeutics Program
(dtp).[17] Furthermore, the effects of the brefeldin analogues
on the Golgi apparatus were investigated. Both plant and
mammalian cells were used. To gain a deeper understand-
ing of the interactions of the brefeldin analogues with the
Arf1-GEF protein complex, the structures of the brefeldin
analogues were modeled and docked into the BFA binding
site.
The following brefeldin analogues were prepared. The
first target was (6R)-hydroxy-BFA (5, cf. Figure 2). This
compound was expected to have a higher solubility in water
Scheme 1. Retrosynthetic analysis for (6R)-hydroxy-BFA (5). Σ:
than BFA. Furthermore, previous work by Nojiri et al.[16a]
Protecting group.
and Cushman et al.[14] had indicated that structural alter-
ation in the vicinity of the 7-OH group without loss of ac-
The starting point of the synthesis was the dihydrox-
tivity might be possible.[18] Remarkably, 8-hydroxy-BFA has
ylation of the nitrile cis-8 with N-methylmorpholine N-ox-
ide (NMO)/(K2OsO4)cat (Scheme 2). The reaction pro-
ceeded with high diastereoselectivity and in high yield. The
resulting diol 9 was protected as an acetonide, and the cy-
ano group of 10 was reduced with DIBAL-H at low tem-
perature to give the aldehyde 11, which was epimerized as
the crude product under basic conditions in order to estab-
been isolated from the broth of an unknown fungus,[19] al-
though no information on its biologic properties is avail-
able. Another target was the BFA lactam analogue 6.[20]
Obviously, lactone hydrolysis would be expected to be a
major path of deactivation for BFA. Accordingly, the corre-
sponding lactam 6 was a logical target (Figure 2).[21]
lish the desired trans configuration. The diastereomeric ra-
1
tio (84:16) was determined by H NMR spectroscopy. The
isomers could not be separated, and the further synthesis
was carried out with the mixture; the products resulting
from the minor epimer were removed at a later stage.
Figure 2. Brefeldin analogues described in this article.
Another possibility was the replacement of a hydroxy
group by an amino group, and so the synthesis of (7S)-
amino-BFC (7) was carried out. Apparently, the compound
has also been prepared elsewhere, because test results are
recorded in the NCI database.[17] Nevertheless, we want to
present the synthesis and the X-ray crystal structure of this
compound, because no report on it has yet been published.
Results and Discussion
Total Synthesis of (6R)-Hydroxybrefeldin A
(6R)-Hydroxy-BFA (5) was synthesized according to a
Scheme 2. Synthesis of the five-membered ring compound 11.
strategy previously applied by us in a synthesis of the struc-
turally simpler BFC lactam analogue 4 (Scheme 1).[16g] The
building blocks A–C were combined in a convergent man-
The lower side chain was introduced through a Julia–
ner. Of these, the tetrazolyl sulfones C are established com- Kocien´ski olefination, with use of the aldehyde 11 and the
pounds.[16f,16g,22] As the cyclopentanoid building block, the tetrazolyl sulfone 12 as starting materials (Scheme 3). The
nitrile D with a cis configuration was chosen in order to product 13 was a mixture of C-5 epimers (dr = 84:16),
allow a diastereoselective dihydroxylation. Epimerization to which we could not separate; the double bond was exclu-
the required trans configuration was carried out at the stage sively formed in the (E) configuration according to 13C
of the aldehyde A. The nitrile was prepared from compound NMR spectroscopy. The trityl protecting group was re-
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Scheme 3. Assembly of the carbon skeleton of (6R)-hydroxy-BFA through a Julia–Kocien´ski olefination and a Nozaki–Hiyama–Kishi
reaction.
ductively removed (LiC10H8 in DME) to give the alcohol
In order to find a method that would avoid this reaction,
14 in high yield. Swern oxidation gave the aldehyde 15. At we used (2E)-4-hydroxyundec-2-enoic acid as a model sub-
this stage the minor epimer (5R)-15 was removed by col- strate and screened several macrolactonization methods;[27]
umn chromatography. The enoate moiety was introduced the corresponding γ-lactone – 5-heptylfuran-2(5H)-one –
through a Nozaki–Hiyama–Kishi reaction[23] between the was formed in high yield on use of the methods of Yamagu-
isomerically pure aldehyde (5S)-15 and methyl iodoacrylate. chi, Corey/Nicolaou,[28] and Mukaiyama[29] and upon acti-
No diastereoselectivity was observed in this step, but the vation with HBTU. As pointed out previously,[25,26] nucleo-
mixture 16 was used for further synthesis, and the stereo- philic reaction conditions must be avoided in order to pre-
chemical problem was rectified at a later stage.
vent lactone formation. There are only a few corresponding
Attempts to protect the 4ЈЈ-OH group of 16 were unsuc- methods, developed by Trost et al.[30] and by Gais et al.[31]
cessful, presumably because of steric shielding around the We investigated the latter method, for which a push-pull-
4ЈЈ-OH group. It was decided to make positive use of this substituted alkyne – 4-(dimethylamino)but-3-yn-2-one – is
effect by carrying out the macrolactonization without an used as reagent (Scheme 5), reacting with a carboxylic acid
O-protecting group in this position. Accordingly, the silyl to give an enol ester of N,N-dimethyl-3-oxobutanamide.
protecting group was removed by treatment with NEt3- This rather stable intermediate can react with an alcohol
(HF)3 in CH3CN at reflux, and the methyl ester 17 was under acid catalysis.
saponified to give the macrocyclization precursor 18 in
Application of the Gais method to our problem led to
quantitative yield. Initially, we tested the Yamaguchi the clean formation of the macrocycle in 78% yield.
method[24] for macrolactonization, but the desired cycliza- Furthermore, the C-4-epimeric macrolactones 21a and 21b
tion product was not formed. Instead, the γ-lactone 19 was could readily be separated by column chromatography. The
obtained (Scheme 4). Obviously, a double-bond isomeriza- assignment of the configuration is based on an X-ray crys-
tion of the enoate moiety, induced by conjugate addition of tal structure of 21b. In addition, complete epimerization of
a nucleophile, and a ring closure had occurred. Analogous 21a was possible by oxidation with MnO2 followed by re-
reactions have been observed by Trost et al.[25] and Fürstner duction of the crude ketone with NaBH4 at –78 °C. Finally,
et al.[26]
the acetonide protecting group was removed under acidic
conditions to give (6R)-hydroxy-BFA (5) in 78% yield.
Partial Synthesis of the BFA Lactam Analogue 6
The BFA lactam analogue 6 was prepared from BFA (1,
Scheme 6). The strategy was aimed at an intermediate, the
aldehyde 25, that would be expected to allow introduction
of modified southern side chains through Julia–Kocien´ski
olefinations. Compounds 22–25 are known intermediates of
various total syntheses of BFA, but had not so far been
derived from BFA itself.[32] Firstly, BFA was transformed
Scheme 4. Formation of the γ-lactone 19 under Yamaguchi condi-
tions.
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Syntheses and Biological Properties of Brefeldin Analogues
Scheme 5. Final steps of the synthesis of (6R)-hydroxy-BFA (5).
into the MEM derivative 22[33] in 87% yield. Saponification was examined. This reaction was very clean and gave the
and treatment of the resulting carboxylic acid with diazo- BFA lactam analogue 6 in excellent yield.
methane then gave the methyl ester 24[33a] in 69% yield. The
southern side chain was cleaved by dihydroxylation/oxi-
dation. As anticipated, treatment with NMO/(K2OsO4)cat
preferentially led to dihydroxylation at the electron-rich
double bond. The oxidation product was treated with
NaIO4 in a stirred mixture of water and diethyl ether. Epi-
merization (ca. 10%) occurred at C-2Ј of the aldehyde 25,
despite careful control of the pH during aqueous workup.
Scheme 7. Synthesis of the BFA lactam analogue 6.
Partial Synthesis of (7S)-Amino-BFC
7-Dehydro-BFA (3) was used as the starting material for
Scheme 6. Synthesis of the key building block 25.
the synthesis of (7S)-amino-BFC (7, Scheme 8). A reductive
The aldehyde 25 was subjected to a Julia–Kocien´ski ole- amination protocol developed by Ellman et al.[34] was em-
fination (Scheme 7) with the tetrazolyl sulfone 26[16g] to give ployed: a solution of 3, tert-butyl sulfinamide, and Ti-
the (E) olefin 27 exclusively according to 13C NMR spec- (OEt)4 in THF was heated at reflux. The resulting sulfin-
troscopy. The MEM and Boc protecting groups were then imine was reduced with NaBH4 without isolation at –15 °C.
removed under acidic conditions, and the methyl ester was As anticipated, the reaction proceeded with very low selec-
saponified to an amino acid, which was subjected to macro- tivity to give a mixture of the diastereoisomers 28a and 28b,
lactamization. A method that had been successful in the from which the latter was separated by column chromatog-
synthesis of the BFC lactam analogue, was used first.[16g] raphy (46% yield). Finally, treatment with methanolic HCl
However, the macrolactamization product 6 was formed in gave the amine 7. The configuration at C-7 was assigned
very low yield. Next, amide formation with HBTU/NEtiPr2 through an X-ray crystal structure analysis of 7.
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Anticancer Activity
The brefeldin analogues were submitted to the National
Cancer Institute (NCI, Maryland, USA) of the U.S.
National Institutes of Health and tested for their cytostatic
activities in the Developmental Therapeutics Program
(dtp). Modest cytostatic activities were found. The detailed
screening results are given in the Supporting Information;
the data will also soon be published in the database of the
NCI.[35]
X-ray Crystal Structures of the Brefeldin Analogues
X-ray crystal structures of the brefeldin analogues 3, 5,
6, 7, and 21b were determined (Table 1). Structural devia-
tions from BFA are negligible for (6R)-hydroxy-BFA (5) in
the 13-membered ring moiety (Figure 4A). Conformational
differences for the five-membered ring, however, are sub-
stantial. Whereas the 7-OH group is found in an axial dis-
position in the crystal of BFA, in 5 it occupies an equatorial
position. Similarly to 5, (7S)-amino-BFC (7) only shows a
deviation from BFA with respect to the five-membered ring.
The amino group at C-7 is found in an equatorial disposi-
tion (Figure 4B).
Scheme 8. Reductive amination of 7-dehydro-BFA (3).
Biologic Properties of the Brefeldin Analogues
Golgi Disruption
HeLa cells were treated with solutions of the brefeldin
analogues (compounds 4,[16g] 5, 6, 7, and 21b) in DMSO
(1 μgmL–1), and Golgi disrupting effects were determined
after 30 min and 60 min. Furthermore, the reversibility of
the morphologic changes was confirmed by washout of the
brefeldin analogues after 2 h. Immunofluorescence (IF)
with the Golgi marker GM130 was performed in order to
monitor the Golgi disruption. Representative images were
taken with a spinning disk confocal microscope (Figure 3).
Activity was observed for (6R)-hydroxy-BFA (5) and the
BFA lactam analogue 6. In the case of 5, Golgi reassembly
is already starting at an incubation time of 60 min (without
washout). This inactivation is likely caused by secretion
from cells or metabolization. (7S)-Amino-BFC (7) showed
a very weak effect, whereas the BFC lactam analogue and
21b did not show activity.
Figure 4. Superposition of the X-ray crystal structures of the brefel-
din analogues (gray) and BFA (black). A: (6R)-hydroxy-BFA (5);
B: (7S)-amino-BFC (7); C: BFA lactam analogue 6.
A closer look at the crystal lattice of BFA indicates that
the axial orientation of the 7-OH group in the solid state is
caused by hydrogen bonds. This assumption is supported
by two observations. (a) The BFA conformation of lowest
energy obtained by force-field calculations (see below)
shows an equatorial position of the 7-OH. (b) The X-ray
crystal structure of the aminoacetyl derivative of BFA at 7-
OH (Breflat) also exhibits an equatorial position of the O-
acyl group at C-7. In this case a hydrogen bond is not pos-
sible because of the esterification.[36] It thus appears that
the axial orientation in the solid state is a crystal-packing
effect and that the five-membered ring is flexible, with a
small energy difference for the axial and equatorial disposi-
tion of the 7-OH group.
Figure 3. Treatment of HeLa cells with BFA analogues. Cells were
incubated with (6R)-hydroxy-BFA (5), the BFA lactam analogue
6, or BFA or were mock-treated (control) for the times indicated.
Washout experiments were performed after incubation (60 min) in
the presence of BFA or BFA analogues, followed by incubation
(2 h) in fresh growth medium. Golgi structures were analyzed with
the aid of an anti-GM130 antibody.
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Syntheses and Biological Properties of Brefeldin Analogues
The conformation of the BFA lactam analogue 6 differs formation. These conformers are structurally very similar
considerably from that of BFA (Figure 4C). The main dif- to the low-energy structure of BFA. Overall, the molecular
ference is found for the enoate moiety, which possesses an mechanics calculations reproduced the structures of brefel-
s-trans conformation [τ(O=C–C=C) = 177°] in the case of din A and its analogues very well.
BFA and an s-cis conformation [τ(O=C–C=C) = 26°] in the
case of 6.
Ligand Docking of the Brefeldin Analogues into the Binding
Site of BFA
Molecular Modeling of the Brefeldin Analogues
Methods
The structures of the brefeldin analogues were also inves-
tigated by molecular modeling, with use of the Macromodel
software. Conformational searches based on the MMFFS
The starting geometries of the ligands were built by the
method described above. Docking was performed with
GOLD v. 4.1,[39] and the results were visualized with Chi-
mera.[40] The X-ray structure of the protein complex was
downloaded from the PDB (code 1RE0) and prepared for
docking with the auxiliary programs of the GOLD package.
The BFA ligand was removed from the structure, and the
resulting cavity was defined as binding pocket in the dock-
ing runs. Water molecules near the binding pocket were in-
cluded in the definition of the binding site and were defined
as flexible. Different rotamers were allowed for three amino
acids (Ser198, Tyr256, and Met260; numbering from
1RE0), which appeared to have an increased degree of flexi-
bility and were thought to be relevant for the binding of
the brefeldin analogues. Otherwise, the default settings of
the GOLD software were used for the docking runs.
force field and
a Monte Carlo Multiple Minimum
(MMCM) search were performed. This procedure was de-
veloped by Still et al., who used 7-dehydro-BFA for its vali-
dation.[37,38] The geometric parameters of the low-energy
structures were used as starting geometries for ligand dock-
ing (see below).
The (6R)-hydroxy-BFA (5) conformer of lowest energy
fits the X-ray structure well with regard to the macrocyclic
ring. In the case of the five-membered ring, an envelope
conformation with an axial disposition of the 7-OH group
was identified by a conformational search for the conformer
of lowest energy, although a conformer with an equatorial
orientation of the 7-OH group was only less stable by
3 kJmol–1. It is the latter that is present in the X-ray crystal
structure of 5. As pointed out above for BFA, the confor-
mational situation of the five-membered ring is strongly in-
fluenced by crystal packing effects, and in solution an equi-
librium of both conformers is likely. The coupling constant
Results
All ligands were docked in a position and orientation
(pose) that resembles the pose of the brefeldin molecule.
This is demonstrated in Figure 6, in which the docked pose
of (6R)-hydroxy-BFA is overlaid with the brefeldin X-ray
structure 1RE0. High docking scores, which indicate a good
geometric fit and favorable interactions in the binding
pocket, were obtained for all analogues except for the acet-
onide 21b of hydroxy-BFA. This compound fits into the
pocket, but has a significantly lower hydrogen-bonding
score than the other compounds.
1
3J(5-H/6-H) = 8.8 Hz was determined by H NMR spec-
troscopy. This value is in good agreement with the value
calculated [3J(5-H/6-H) = 9.2 Hz] for the conformer of low-
est energy.
The X-ray crystal structure of (7S)-amino-BFC (7) was
almost perfectly reproduced by the force-field calculations.
Reproduction of the structure of the BFA lactam analogue
6 was also satisfactory. All the conformers with energies of
up to 10 kJmol–1 were calculated. Two sets of conformers
were found, differing mainly in the enoate moiety (Fig-
ure 5), one exhibiting the s-cis and the other the s-trans con-
Figure 6. Close-up view of BFA in the binding pocket between Arf1
(red ribbon) and Sec7 (yellow ribbon), based on the X-ray structure
1RE0. The key hydrophilic residues Tyr256, Ser198, and Trp78 are
represented as sticks. Overlaid is the docked pose of (6R)-hydroxy-
BFA (5). This compound can, depending on the conformation of
the cyclopentane ring, enter into hydrogen-bonding interactions
with Tyr256 and Trp78. A rotation of the Ser198 side chain results
in additional interactions with the 6-hydroxy group of 5.
Figure 5. Conformational plot for the BFA lactam analogue 6 cal-
culated by molecular modeling.
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G. Helmchen et al.
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or Bruker Avance III 600 (600.13 MHz) spectrometers. Chemical
shifts are reported in δ units relative to the solvent residual peak
Discussion of Structure–Activity Relationships and Docking
Significant activity is observed for BFA and its hydroxyl-
ated analogues [i.e., (6R)-hydroxy-BFA (5) and the BFA lac-
tam analogue 6]. The binding pocket of BFA is charac-
terized by a large lipophilic area, occupied by the macrolac-
tone ring, and a smaller hydrophilic region. The latter is
formed by the residues Tyr256 and Ser198 from Sec7 and
Trp78 from ARF1 (numbering from 1RE0). Although
Ser198 is not directly involved in the binding of BFA, a
rotation of the side chain of this residue results in an ad-
ditional hydrogen-bonding interaction to ligands in the
pocket. In Figure 6, the X-ray structure of BFA bound to
Sec7 and Arf1 is compared with the docked pose of (6R)-
hydroxy-BFA (5). The conformations of the two ligands are
clearly very similar. It is also obvious that the additional
hydroxy group of 5 has the potential to enter into ad-
ditional interactions with Ser198 if the side chain is rotated
towards the ligand, which can easily explain the high ac-
tivity of this analogue.
There are three inactive compounds in the data set: (7S)-
amino-BFC (7), the BFC lactam analogue, and the aceto-
nide-protected (6R)-hydroxy-BFA 21b. The hydrogen-bond-
ing capacity of the cyclopentane moiety of the last two
compounds is either nonexistent or severely restricted. The
inactivity of these compounds can be explained unproblem-
atically in accordance with the previous reasoning.
(7S)-Amino-BFC (7) is inactive in the cellular assays, al-
though it has a hydrogen-bonding potential similar to that
of BFA and consequently obtains high scores in the dock-
ing runs. It appears likely that the basic functionality, which
under physiological conditions is completely protonated
and positively charged, confers a different extra- and intra-
cellular “pharmacokinetic” profile to this compound. This
can result, through various mechanisms, in a diminished
presence of this derivative at the locus of action.
(CHCl3 in CDCl3 at δH = 7.26 ppm, CD2HOD in CD3OD at δH
=
3.31 ppm).[41] The following abbreviations are used for description
of the signal multiplicity: s (singlet), d (doublet), t (triplet), q (quar-
tet), dd (doublet of doublets), dt (doublet of triplets), ddd (doublet
of doublets of doublets), etc., br. s (broad signal), m (multiplet).
Diastereotopic hydrogen atoms are described with superscripts 1
and 2 (CH1H2). Chemical shifts are not reported for acidic protons
(NH, OH) if CD3OD was used as solvent. 13C NMR spectra were
recorded at room temperature with Bruker DRX 200 (50.27 MHz),
Bruker Avance DRX 300 (75.47 MHz), Bruker Avance DRX 500
(125.76 MHz), or Bruker Avance III 600 (150.90 MHz) spectrome-
ters. Chemical shifts are reported in δ units relative to the solvent
signal: CDCl3 [δC = 77.16 ppm (central line of the triplet)], CD3OD
[δC = 49.00 ppm (central line of the septet)].[41] The following ab-
breviations were used: s (singlet, quaternary C-atom), d (doublet,
CH group), t (triplet, CH2 group), q (quartet, CH3 group). The
multiplicity stated refers to {1H}-decoupled spectra. In every case,
the assignments of signals were confirmed through 1H,1H-COSY,
1H,13C-COSY, and DEPT spectra. High-resolution mass spectra
were recorded with a JEOL JMS-700 (EI+, FAB+) or a Bruker
ApexQe FT-ICR (ESI+) mass spectrometer. Elemental analyses
were carried out at the Organisch-Chemisches Institut, Universität
Heidelberg. Analytical thin-layer chromatography was performed
with precoated TLC plates (Polygram® SIL G/UV254, Macherey &
Nagel). Spots were visualized with UV light (λ = 254 nm) or by
dipping plates into an aqueous solution of KMnO4 [KMnO4
(1.5 g), K2CO3 (2.5 g), in H2O (250 mL)] and subsequent heating.
Flash column chromatography was carried out with silica gel
(0.032–0.062) (Macherey, Nagel and Co.). Tetrahydrofuran was
dried with benzophenone ketyl, and the water content was deter-
mined by Karl Fischer titration. The following compounds were
prepared according to published procedures: 5-{[(5S)-5-{[tert-
butyl(diphenyl)silyl]oxy}hexyl]sulfonyl}-1-phenyl-1H-tetrazole
(12),[16f] 4-(dimethylamino)but-3-yn-2-one,[42] (1S,2R)-2-[(trityl-
oxy)methyl]cyclopent-3-ene-1-carbonitrile (cis-8),[16g] methyl (2E)-
3-iodoacrylate,[43] and tert-butyl {(1S)-1-methyl-5-[(1-phenyl-1H-
tetrazol-5-yl)sulfonyl]pentyl}carbamate (27).[16g] IUPAC names
were generated with the aid of the program ACD/Labs 6.0 from
Advanced Chemistry Development Inc.
Conclusions
Golgi Disruption: HeLa cells were cultivated in DMEM medium
[supplemented with fetal calf serum (10%), penicillin
(100 unitsmL–1), streptomycin (100 μgmL–1), and l-glutamine
(2 mm)]. BFA or BFA analogues (final concentration 5 μgmL–1)
were added at 37 °C for 30 or 60 min. For reassembly studies cells
were incubated in the presence of BFA or BFA analogues for
60 min. After washout, the cells were incubated at 37 °C in DMEM
medium for an additional 2 h. Cells were processed for immuno-
fluorescence and microscopy with the aid of an anti-GM130 anti-
body (BD Transduction) as a Golgi marker as described.[44] Images
were taken with a Perkin–Elmer Ultra-view spinning disk confocal
microscope at the Nikon Imaging Center, Heidelberg.
We have synthesized (6R)-hydroxy-BFA through a total
synthesis by a route previously developed for the BFC lac-
tam analogue. The BFA lactam analogue and (7S)-amino-
BFC were prepared by partial syntheses starting from natu-
ral BFA and 7-dehydro-BFA. In biological tests, (6R)-hy-
droxy-BFA induced morphological changes to the Golgi
apparatus in mammalian cells and in Arabidopsis cells sim-
ilar to those observed with BFA. The BFA lactam analogue
6 was only active in mammalian cells. Furthermore, confor-
mation analyses by molecular mechanics calculations and
docking of the brefeldin analogues into the BFA binding
site of the Arf1-GEF protein complex were carried out.
Analytical Data for (1S,2R)-2-[(Trityloxy)methyl]cyclopent-3-ene-1-
carbonitrile (cis-8): [α]2D0 = –61.0 (c = 0.435, CHCl3, 97% ee). 1H
NMR (300.13 MHz, CDCl3): δ = 2.69–2.81 (m, 2 H, 5-H), 3.02–
3.15 (m, 1 H, 2-H), 3.17–3.40 (m, 3 H, 1-H, CH2O), 5.66–5.75 (m,
1 H, 3-H or 4-H), 5.75–5.84 (m, 1 H, 3-H or 4-H), 7.15–7.36 (m,
9 H, Ar-H), 7.48–7.53 (m, 6 H, Ar-H) ppm. 13C NMR (75.48 MHz,
CDCl3): δ = 29.79 (d, C-1), 37.55 (t, C-5), 47.73 (d, C-2), 64.12 (t,
CH2O), 87.22 (s, CPh3), 120.83 (s, CN), 127.16, 127.90, 128.91
(3ϫd, C-Ar), 129.84, 131.34 (2ϫd, C-3, C-4), 144.02 (s, C-Ar)
ppm. HR-MS (FAB+): m/z calcd. for C26H23NO+ 365.1774 [M]+;
Experimental Section
General Information: Melting points are uncorrected. Optical rota-
tions were measured with a Perkin–Elmer 341 Polarimeter and a
1
mercury lamp. H NMR spectra were recorded at room tempera-
ture with Bruker DRX 200 (199.92 MHz), Bruker Avance
DRX 300 (300.13 MHz), Bruker Avance DRX 500 (500.13 MHz),
884
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 878–891

Syntheses and Biological Properties of Brefeldin Analogues
found 365.1779. C26H23NO (365.47): calcd. C 85.45, H 6.34, N
3.83; found C 85.37, H 6.37, N 3.77. HPLC [Chiralpak AD-H, n-
hexane/iPrOH (99:1), flow 0.5 mLmin–1, room temp., λ = 210 nm]:
tR[(+)-(1R,2S)-8] = 24.8 min, tR[(–)-(1S,2R)-8] = 32.8 min.
(–78 °C) solution of 10 (647 mg, 1.47 mmol) in dry CH2Cl2 (6 mL).
After the mixture had been stirred for 2 h, MeOH (4.4 mL) was
added, and stirring was continued at –78 °C for further 10 min.
The mixture was then added to a saturated aqueous solution of
sodium potassium tartrate. The aqueous layer was extracted with
CH2Cl2 (3ϫ20 mL). The combined organic layers were dried with
Na2SO4, filtered, and concentrated in vacuo. The crude product
was dissolved in CH2Cl2 (15 mL), and DBU was added (0.11 mL,
0.74 mmol). After 2 h of stirring, the solvent was evaporated in
vacuo. The 1H NMR spectrum of the crude product showed incom-
plete isomerization. More DBU (0.08 mL, 0.05 mmol) was added
to a solution of the crude product in CH2Cl2 (4.5 mL). After 2 h
of stirring, the solvent was evaporated in vacuo, and the residue
was subjected to flash column chromatography on silica gel [petro-
leum ether/ethyl acetate (5:1); UV detection; Rf(11) = 0.38] to yield
11 (473 mg, 1.069 mmol, 73%) as a colorless oil. The ratio of dia-
stereoisomers was determined by H NMR to be 84:16. H NMR
(300.13 MHz, CDCl3): δ = 1.22, 1.28 [2ϫs, 6 H, C(CH3)2], 1.74
(ddd, J = 13.9, J = 12.2, J = 5.2 Hz, 1 H, 6-H1), 1.96 (dd, J = 14.0,
J = 6.1 Hz, 1 H, 6-H1), 2.07–2.20 (m, 1 H, 4-H), 2.64–2.78 (m, 1
H, 5-H), 3.22 (dd, J = 9.0, J = 9.0 Hz, 1 H, CH2O), 3.55 (dd, J =
9.5, J = 5.7 Hz, 1 H, CH2O), 4.56–4.68 (m, 2 H, 3a-H, 6a-H), 7.18–
7.34 (m, 9 H, Ar-H), 7.36–7.44 (m, 6 H, Ar-H), 9.67 (d, J = 3.1 Hz,
1 H, CHO) ppm. 13C NMR (75.48 MHz, CDCl3): δ = 24.04, 26.00
[2ϫq, C(CH3)2], 33.97 (t, C-6), 47.97 (d, C-4), 52.55 (d, C-5), 62.28
(t, CH2O), 79.74, 80.81 (2ϫd, C-3a, C-6a), 87.43 (s, CPh3), 109.80
(s, C-2), 127.09, 127.89, 128.86 (3 ϫd, C-Ar), 144.06 (s, C-Ar),
203.13 (d, CHO) ppm. HR-MS (FAB+ ): m/z calcd. for
C29H30O4Na+ 465.2036 [M + Na]+; found 465.2056 . C29H30O4
(442.55): calcd. C 78.71, H 6.83; found C 78.92, H 7.01.
(1S,2R,3R,4S)-3,4-Dihydroxy-2-[(trityloxy)methyl]cyclopentane-
carbonitrile (9): K2OsO4·2H2O (55 mg, 0.15 mmol) and NMO
[2.53 mL, 12.00 mmol (50 wt.-% solution in water)] were added
consecutively to a solution of cis-8 (2.193 g, 6.00 mmol) in acetone/
water (9:1, 30 mL). The mixture was stirred at room temperature
for 3 h [TLC monitoring: petroleum ether/ethyl acetate (3:2); Rf(9)
= 0.14]. Na2S2O4 (100 mg) was then added, and the mixture was
stirred for a further 30 min. A black precipitate was removed by
filtration through a pad of Celite, which was rinsed with diethyl
ether (100 mL). The solvent was evaporated in vacuo, and the crude
product was purified by flash chromatography on silica gel [100 g;
petroleum ether/ethyl acetate (1:1); Rf(9) = 0.22] to give 9
(2.134 mg, 89%) as a white powder. [α]2D0 = 21.0 (c = 1.42, CHCl3).
1H NMR (300.13 MHz, CDCl3): δ = 2.08 (ddd, J = 14.4, J = 6.9,
J = 4.9 Hz, 1 H, 5-H1), 2.18 (ddd, J = 14.4, J = 8.9, J = 2.3 Hz,
1 H, 5-H1), 2.45 (ddd, J = 17.6, J = 8.5, J = 6.4 Hz, 1 H, 2-H),
2.55 (br. s, 1 H, OH), 2.85 (d, J = 2.4 Hz, 1 H, OH), 3.26 (ddd, J
= 9.2, J = 9.2, J = 7.1 Hz, 1 H, 1-H), 3.46 (mc, 1 H, CH2O), 3.55
(dd, J = 9.5, J = 6.3 Hz, 1 H, CH2O), 3.80–3.88 (m, 1 H, 3-H),
4.11–4.17 (m, 1 H, 4-H), 7.21–7.36 (m, 9 H, Ar-H), 7.39–7.50 (m,
6 H, Ar-H) ppm. 13C NMR (75.48 MHz, CDCl3): δ = 26.70 (d, C-
1), 35.76 (t, C-5), 44.08 (d, C-2), 64.24 (t, CH2O), 71.55 (d, C-4),
77.19 (d, C-3), 87.92 (s, CPh3), 120.40 (s, CN), 127.50, 128.18,
128.73 (3ϫd, C-Ar), 143.51 (s, C-Ar) ppm. HR-MS (FAB+): m/z
calcd. for C26H25O3NNa+ 422.1727 [M + Na]+; found 422.1748.
C26H25NO3 (399.48): calcd. C 78.17, H 6.31, N 3.51; found C
78.34, H 6.29, N 3.55.
1
1
tert-Butyl{[(1S,5E)-6-{(3aR,4R,5S,6aS)-2,2-dimethyl-4-[(trityloxy)-
methyl]tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-yl}-1-methylhex-
5-en-1-yl]oxy}diphenylsilane (13): A solution of KHMDS in toluene
(0.5 m, 6.66 mL, 3.33 mmol) was added dropwise to a cooled
(–78 °C) solution of 12 (1.895 g, 3.453 mmol) in dry DME
(13.3 mL, freshly dried with sodium), and the mixture was stirred
at this temperature for 30 min. A solution of 11 (1.18 g, 2.67 mmol)
in dry DME (13.3 mL) was then added dropwise. After stirring at
–78 °C for 2 h, the solution was allowed to warm to room tempera-
ture and was stirred for a further 1 h. Afterwards, water (30 mL),
brine (30 mL), and diethyl ether (60 mL) were added. The aqueous
layer was separated and extracted with diethyl ether (3ϫ50 mL).
The combined organic layers were dried with Na2SO4, filtered, and
concentrated in vacuo. The residue was subjected to flash column
chromatography on silica gel [petroleum ether/ethyl acetate (20:1);
UV detection; Rf(13) = 0.36] to yield 13 as a colorless, viscous oil
(1.658 g, 2.167 mmol, 81%). The product was a mixture of dia-
stereoisomers at C-5 (84:16) that could not be separated. 1H NMR
(300.13 MHz, CDCl3): δ = 1.03–1.10 (m, 3 H, C-1Ј-CH3), 1.08 [s,
9 H, C(CH3)3], 1.20–1.54 (m, 4 H, 2Ј-H, 3Ј-H), 1.34, 1.53 [2ϫs, 6
H, C(CH3)2], 1.65 (ddd, J = 13.8, J = 10.5, J = 5.1 Hz, 1 H, 6-H1),
1.72–1.88 (m, 2 H, 4Ј-H), 1.95 (ddd, J = 8.8, J = 8.7, J = 4.3 Hz,
1 H, 4-H), 2.17 (ddd, J = 13.2, J = 6.6, J = 6.6 Hz, 1 H, 6-H1),
2.57 (dddd, J = 9.8, J = 9.8, J = 7.3, J = 7.3 Hz, 1 H, 5-H), 3.10
(dd, J = 9.2, J = 4.5 Hz, 1 H, CH2O), 3.27 (dd, J = 9.2, J = 4.4 Hz,
1 H, CH2O), 3.76–3.92 (m, 1 H, 1Ј-H), 4.52–4.70 (m, 2 H, 3a-H,
6a-H), 5.12–5.38 (m, 2 H, 5Ј-H, 6Ј-H), 7.18–7.50 (m, 21 H, Ar-H),
7.67–7.74 (m, 4 H, Ar-H) ppm. 13C NMR (75.48 MHz, CDCl3): δ
= 19.40 [s, C(CH3)3], 23.37 (q, C-1Ј-CH3), 25.10 (t, C-3Ј), 25.21,
27.74 [2ϫq, C(CH3)2], 27.19 [q, C(CH3)3], 32.49 (t, C-4Ј), 39.10 (t,
C-2Ј), 39.15 (t, C-6), 44.41 (d, C-5), 51.86 (d, C-4), 62.11 (t, CH2O),
(3aR,4R,5S,6aS)-2,2-Dimethyl-4-[(trityloxy)methyl]tetrahydro-3aH-
cyclopenta[d][1,3]dioxole-5-carbonitrile (10): 2,2-Dimethoxypropane
(13.2 mL, 107.4 mmol) and pyridinium toluene-4-sulfonate
(134 mg, 0.533 mmol) were added at room temperature to a solu-
tion of 9 (2.134 g, 5.342 mmol) in CH2Cl2 (21 mL). After 2.5 h,
TLC monitoring showed complete conversion [petroleum ether/
ethyl acetate (1:1); Rf(9) = 0.22, Rf(10) = 0.76]. The solution was
added to a mixture of saturated aqueous NaHCO3 (50 mL) and
CH2Cl2 (50 mL). The aqueous layer was separated and extracted
with CH2Cl2 (3 ϫ 20 mL). The combined organic layers were
washed with brine, dried with Na2SO4, filtered, and concentrated
in vacuo. The crude product was subjected to flash chromatography
on silica gel [petroleum ether/ethyl acetate (5:1); UV detection;
Rf(10) = 0.35] to give 10 (2.219 g, 94%) as a glass. [α]2D0 = –25.5 (c
1
= 0.970, CHCl3). H NMR (300.13 MHz, CDCl3): δ = 1.23, 1.40
[2ϫs, 6 H, C(CH3)2], 2.12–2.35 (m, 2 H, 6-H), 2.45 (dd, J = 11.6,
J = 5.4 Hz, 1 H, 4-H), 3.20 (ddd, J = 12.1, J = 7.2, J = 7.2 Hz,
1 H, 5-H), 3.37 (dd, J = 10.2, J = 5.7 Hz, 1 H, CH2O), 3.42 (dd, J
= 10.2, J = 4.6 Hz, 1 H, CH2O), 4.32 (d, J = 5.7 Hz, 1 H, 3a-H),
4.60 (dd, J = 5.5, J = 5.5 Hz, 1 H, 6a-H), 7.21–7.36 (m, 6 H, Ar-H),
7.41–7.50 (m, 9 H, Ar-H) ppm. 13C NMR (75.48 MHz, CDCl3): δ
= 23.81, 26.41 [2ϫq, C(CH3)2], 28.60 (d, C-5), 37.36 (t, C-6), 47.85
(d, C-4), 61.88 (t, CH2O), 79.61 (d, C-6a), 82.98 (d, C-3a), 88.00
(s, CPh3), 109.88 [s, C(CH3)2], 119.92 (s, CN), 127.38, 128.10,
128.83 (3ϫd, C-Ar), 143.58 (s, C-Ar) ppm. HR-MS (ESI+): m/z
calcd. for C29H29NO3Na+ 462.2040 [M + Na]+; found 462.2043.
C29H29NO3 (439.55): calcd. C 79.24, H 6.65, N 3.19; found C
79.61, H 6.66, N 2.86.
(3aR,4R,5S,6aS)-2,2-Dimethyl-4-[(trityloxy)methyl]tetrahydro-3aH-
cyclopenta[d][1,3]dioxole-5-carbaldehyde (11): A solution of 69.60 (d, C-1Ј), 80.03, 83.24 (2ϫd, C-3a or C-6a), 86.47 (s, CPh3),
DIBAL-H in hexane (4.4 mL, 1 m) was added dropwise to a cooled 111.92 (s, C-2), 127.02, 127.51, 127.59, 127.83, 128.90, 129.51,
Eur. J. Org. Chem. 2011, 878–891
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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885

G. Helmchen et al.
FULL PAPER
129.58 (7ϫd, C-Ar), 130.57 (d, C-5Ј), 132.50 (d, C-6Ј), 134.71,
135.05 [2ϫs, OSiPh2C(CH3)3], 135.98, 136.00 (2ϫd, C-Ar), 144.29
(s, CPh3) ppm. HR-MS (ESI+): m/z calcd. for C51H60O4SiNa+
787.4153 [M + Na]+; found 787.4152. C51H60O4Si (765.11): calcd.
C 80.06, H 7.90; found C 80.22, H 8.03.
311 mg, 67 %, Rf[(5S)-15] = 0.25 [petroleum ether/ethyl acetate
(10:1)], 85% overall yield}. [α]2D0 = –39.8 (c = 1.11, CHCl3). 1H
NMR (300.13 MHz, CDCl3): δ = 1.04 [s, 9 H, C(CH3)3], 1.04 (d,
J = 6.0 Hz, 3 H, 7Ј-H), 1.32, 1.52 [2ϫs, 6 H, C(CH3)2], 1.32–1.51
(m, 4 H, 4Ј-H, 5Ј-H), 1.68–1.80 (m, 1 H, 6-H1), 1.83–1.93 (m, 2 H,
3Ј-H), 2.14 (ddd, J = 11.4, J = 6.5, J = 6.5 Hz, 1 H, 6-H1), 2.70–
2.83 (m, 2 H, 5-H, 4-H), 3.76–3.88 (m, 1 H, 6Ј-H), 4.63 (ddd, J =
6.6, J = 6.6, J = 4.5 Hz, 1 H, 6a-H), 4.82 (dd, J = 6.7, J = 4.7 Hz,
1 H, 3a-H), 5.31–5.59 (m, 2 H, 1Ј-H, 2Ј-H), 7.32–7.45 (m, 6 H, Ar-
H), 7.63–7.71 (m, 4 H, Ar-H), 9.72 (d, J = 1.1 Hz, 1 H, CHO)
ppm. 13C NMR (75.48 MHz, CDCl3): δ = 19.41 [s, C(CH3)3], 23.37
(q, C-6Ј-CH3), 24.79 [q, C(CH3)2], 24.95 (t, C-4Ј), 27.17 [3 ϫ q,
C(CH3)3], 27.29 [q, C(CH3)2] 32.38 (t, C-3Ј), 38.60 (t, C-6), 39.01
(t, C-5Ј), 43.92 (d, C-5), 63.46 (d, C-4), 69.53 (d, C-6Ј), 79.95 (d,
C-6a), 80.49 (d, C-3a), 112.63 (s, C-2), 127.52, 127.60, 129.53,
129.59 (4ϫd, C-Ar), 130.85, 131.87 (d, C-1Ј, C-2Ј), 134.70, 135.01
(2ϫs, C-Ar), 136.00, 136.01 (2ϫd, C-Ar), 201.52 (d, CHO) ppm.
HR-MS (ESI+): m/z calcd. for C32H45O4Si+ 520.3082; found
521.3085 [M + H]+. C32H44O4Si (520.77): calcd. C 73.80, H 8.52;
found C 74.05, H 8.53.
{(3aR,4R,5S,6aS)-5-[(1E,6S)-6-{[tert-Butyl(diphenyl)silyl]oxy}hept-
1-en-1-yl]-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-
yl}methanol (14): Granular lithium (152 mg, 21.9 mmol) was added
to a solution of naphthalene (1.402 g, 10.94 mmol) in dry DME
(55 mL), and the mixture was sonicated at room temperature for
1 h to give a dark green stock solution of LiC10H8 in DME (0.2 m).
Part of this solution (38.3 mL, 7.62 mmol of LiC10H8) was then
added dropwise by syringe to a cooled (0 °C) solution of 13
(1.340 g, 1.751 mmol) in dry DME (7.7 mL). After 10 min of stir-
ring, TLC monitoring showed complete conversion of 13 [petro-
leum ether/ethyl acetate (10:1); UV detection; Rf(13) = 0.47, Rf(14)
= 0.10]. Brine (50 mL), water (50 mL), and diethyl ether (50 mL)
were then added. The aqueous layer was separated and extracted
with diethyl ether (3ϫ80 mL). The combined organic layers were
dried with Na2SO4, filtered, and concentrated in vacuo. The residue
was subjected to flash column chromatography on silica gel (petro-
Methyl (2E)-4-{(3aR,4R,5S,6aS)-5-[(1E,6S)-6-{[tert-Butyl(diphen-
leum ether/ethyl acetate (10:1 to 1:1)] to yield 14 (826.5 mg, yl)silyl]oxy}hept-1-en-1-yl]-2,2-dimethyl-tetrahydro-3aH-cyclopenta-
1.581 mmol, 90%) as a colorless oil. The product was a mixture
of diastereoisomers (84:16), which were not separated by column
chromatography. However, pure fractions could be obtained for an-
alytical evaluation. [α]2D0 = –20.2 (c = 1.35, CHCl3). 1H NMR
[d][1,3]dioxol-4-yl}-4-hydroxybut-2-enoate (16a/b): CrCl2 (350 mg,
2.85 mmol) was added to a cooled (0 °C) mixture of methyl (2E)-
3-iodoacrylate (253 mg, 1.19 mmol), a catalytic amount of NiCl2,
and 15 (311 mg, 0.597 mmol) in dry THF (6.0 mL). After 10 min
(300.13 MHz, CDCl3): δ = 1.05 [s, 9 H, C(CH3)3], 1.01–1.10 (m, 3 at 0 °C, the ice bath was removed, and stirring was continued at
H, C-6Ј-CH3), 1.20–1.52 (m, 4 H, 4Ј-H, 5Ј-H), 1.32, 1.52 [2ϫs, 6 room temperature for 1 h. TLC monitoring [petroleum ether/ethyl
H, C(CH3)2], 1.62 (ddd, J = 13.0, J = 11.2, J = 5.6 Hz, 1 H, 6-H1), acetate (5:1)] showed complete conversion. The solution was
1.80–1.98 (m, 3 H, 3Ј-H1, 4-H), 2.17 (ddd, J = 13.4, J = 6.8, J = treated with diethyl ether (10 mL) and water (2 mL), and, after
6.8 Hz, 1 H, 6-H1), 2.30 (dddd, J = 10.7, J = 10.7, J = 7.0, J = 5 min of stirring, the mixture was filtered through a pad of Celite,
7.0 Hz, 1 H, 5-H), 3.55 (dd, J = 10.7, J = 6.7 Hz, 1 H, CH2O),
3.75 (dd, J = 10.7, J = 4.6 Hz, 1 H, CH2O), 3.79–3.89 (m, 1 H, 6Ј- vacuo, and the residue was subjected to flash column chromatog-
H), 4.42 (dd, J = 6.5, J = 6.5 Hz, 1 H, 3a-H), 4.58 (dd, J = 12.8, raphy on silica gel {petroleum ether/ethyl acetate (5:1); detection
which was washed with Et2O. The solution was concentrated in
J = 6.5 Hz, 1 H, 6a-H), 5.25–5.45 (m, 2 H, 1Ј-H, 2Ј-H), 7.32–7.47 with KMnO4; Rf(16) = 0.07, Rf[dimethyl (2E,4E)-hexa-2,4-dien-
(m, 6 H, Ar-H), 7.64–7.74 (m, 4 H, Ar-H) ppm. 13C NMR edioate] = 0.30} to yield 16 (328 mg, 91%) as a colorless oil. The
(75.48 MHz, CDCl3): δ = 19.40 [s, C(CH3)3], 23.37 (q, C-6Ј-CH3),
product was a 1:1 mixture of diastereoisomers, which could not be
25.11 (t, C-4Ј), 25.13 [q, C(CH3)2], 27.17 [q, C(CH3)3], 27.67 [q, separated. 1H NMR (300.13 MHz, CDCl3): δ = 1.05 [s, 9 H,
C(CH3)2], 32.42 (t, C-3Ј), 38.92 (t, C-6), 39.02 (t, C-5Ј), 44.64 (d, C(CH3)3], 1.03–1.08 (m, 3 H, C-6Ј-CH3), 1.28, 1.29, 1.49, 1.51
C-5), 53.35 (d, C-4), 63.17 (t, CH2O), 69.54 (d, C-6Ј), 79.53 (d, C- [4ϫs, 6 H, C(CH3)2], 1.22–1.51 (m, 4 H, 5Ј-H, 4Ј-H), 1.54–1.66 (m,
6a), 83.57 (d, C-3a), 112.73 [s, C(CH3)2], 127.51, 127.59, 129.52, 1 H, 6-H1), 1.80–1.93 (m, 2 H, 3Ј-H), 1.93–2.00 (m, 1 H, 4-H),
129.58 (4ϫd, C-Ar), 131.47, 132.06 (2ϫd, C-1Ј, C-2Ј), 134.71,
2.04–2.23 (m, 1 H, 6-H1), 2.29–2.58 (m, 1 H, 5-H), 3.73, 3.74 (2ϫs,
3 H, OCH3), 3.77–3.88 (m, 1 H, 6Ј-H), 4.28–4.58 (m, 3 H, 4ЈЈ-H,
135.00 (2ϫs, C-Ar), 135.99 (d, C-Ar) ppm. HR-MS (ESI+) calcd.
for C32H46O4SiNa+ 545.3058 [M + Na]+; found 545.3066. 3a-H, 6a-H), 5.20–5.46 (m, 2 H, 1Ј-H, 2Ј-H), 6.08, 6.15 (2ϫdd, J
C32H46O4Si (522.79): calcd. C 73.52, H 8.87; found C 73.30, H
8.89.
= 15.6, J = 1.7, J = 15.6, J = 1.8 Hz, 1 H, 2ЈЈ-H), 6.97, 7.02 (2ϫdd,
J = 15.3, J = 4.6, J = 15.5, J = 4.7 Hz, 1 H, 3ЈЈ-H), 7.32–7.45 (m,
6 H, Ar-H), 7.66–7.70 (m, 4 H, Ar-H) ppm. 13C NMR (75.48 MHz,
CDCl3): δ = 19.40 [s, C(CH3)3], 23.37 (q, C-6Ј-CH3), 25.01, 25.04
(2ϫt, C-4Ј), 25.21 [q, C(CH3)2], 27.16 [q, C(CH3)3], 27.72, 27.75
[2 ϫ q, C(CH3)2], 32.38, 32.42 (2 ϫ t, C-3Ј), 38.83, 39.07, 39.18
(3ϫt, C-6, C-5Ј), 44.44, 44.61 (2 ϫd, C-5), 51.72, 51.77 (2ϫ q,
OCH3), 56.07, 56.20 (2ϫd, C-4), 69.51, 69.53 (2ϫd, C-6Ј), 70.50,
71.29 (d, C-4ЈЈ), 79.25, 79.44, 81.00, 81.74 (4 ϫd, C-3a, C-6a),
112.60, 113.05 (2ϫs, C-2), 120.49, 121.57 (2ϫd, C-2ЈЈ), 127.52,
127.60, 129.52, 129.59 (4ϫd, C-Ar), 131.84, 132.08, 132.11, 132.20
(4ϫd, C-1Ј, C-2Ј), 134.70, 134.72, 134.99 (3ϫs, C-Ar), 135.99 (d,
C-Ar), 147.59, 149.43 (2ϫd, C-3ЈЈ), 166.88 (s, CO2) ppm. HR-MS
(ESI+): m/z calcd. for C36H51O6Si+ 607.3449 [M + H]+; found
607.3459. C36H50O6Si (606.86): calcd. C 71.25, H 8.30; found C
71.03, H 8.44.
(3aR,4S,5S,6aS)-5-[(1E,6S)-6-{[tert-Butyl(diphenyl)silyl]oxy}hept-1-
en-1-yl]-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxole-4-carb-
aldehyde (15): DMSO (0.51 mL, 7.12 mmol) was added to a cooled
(–78 °C) solution of oxalyl chloride (0.31 mL, 3.56 mmol) in dry
CH2Cl2 (4.5 mL). After 10 min of stirring, a solution of 14
(465 mg, 0.89 mmol) in dry CH2Cl2 (4.5 mL) was added to the mix-
ture. After further 20 min of stirring, NEt3 (1.98 mL, 14.2 mmol)
was added dropwise. After 45 min, TLC monitoring [petroleum
ether/ethyl acetate (3:1)] showed complete conversion. The solution
was treated with phosphate buffer solution (10 mL, 0.5 m solution
of NaH2PO4/Na2HPO4), and the mixture was extracted with
CH2Cl2 (3ϫ25 mL). The combined organic layers were dried with
Na2SO4, filtered, and concentrated in vacuo. The residue was sub-
jected to flash column chromatography on silica gel [petroleum
ether/ethyl acetate (10:1); UV detection] to yield (5R)-15 {minor
diastereoisomer: 77.5 mg, 17 %, Rf[(5R)-15] = 0.35 [petroleum
ether/ethyl acetate (10:1)]} and (5S)-15 {major diastereoisomer:
Methyl (2E)-4-Hydroxy-4-{(3aR,4R,5S,6aS)-5-[(1E,6S)-6-hy-
droxyhept-1-en-1-yl]-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]-
dioxol-4-yl}but-2-enoate (17): A solution of 16 (194 mg,
886
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Eur. J. Org. Chem. 2011, 878–891

Syntheses and Biological Properties of Brefeldin Analogues
0.319 mmol) and NEt3(HF)3 (208 μL, 1.275 mmol) in dry CH3CN appropriate for X-ray analysis. Analytical data for 21a: [α]2D0 = 59.2
(3.2 mL) was heated at reflux under argon for 21 h. TLC monitor-
ing showed complete conversion [petroleum ether/ethyl acetate
(c = 1.105, CHCl3). 1H NMR (500.13 MHz, CDCl3): δ = 0.96–1.06
(m, 1 H, 11-H1), 1.23 (d, J = 6.4 Hz, 3 H, CH3), 1.30, 1.50 [2ϫs,
(1:1); Rf(16) = 0.64/0.56; Rf(17) = 0.14]. The reaction mixture was 6 H, C(CH3)2], 1.46–1.61 (m, 2 H, 15-H1, 10-H1), 1.65–1.88 (m, 3
allowed to cool to room temperature and subjected directly to flash
column chromatography on silica gel [petroleum ether/ethyl acetate
(3:1 to 0:1)] to yield 17 (109 mg, 93%) as a colorless oil and as a
mixture of diastereoisomers that could not be separated. 1H NMR
(300.13 MHz, CDCl3): δ = 1.16 (d, J = 6.2 Hz, 3 H, 7Ј-H), 1.26,
1.27 [2ϫs, 3 H, C(CH3)2], 1.32–1.52 (m, 4 H, 5Ј-H, 4Ј-H), 1.47,
H, 12-H1, 11-H1, 10-H1), 1.91–2.00 (m, 2 H, 3b-H, 12-H1), 2.16
(mc, 1 H, 15-H1), 2.24 (br. s, 1 H, OH), 2.87 (mc, 1 H, 14a-H), 4.50
(ddd, J = 6.7, J = 6.7, J = 6.7 Hz, 1 H, 15a-H), 4.64–4.72 (m, 2 H,
4-H, 3a-H), 4.93 (dqd, J = 12.5, J = 6.3, J = 1.5 Hz, 1 H, 9-H),
5.22 (mc, 1 H, 14-H), 5.64 (ddd, J = 14.9, J = 9.7, J = 4.9 Hz, 1
H, 13-H), 5.79 (dd, J = 15.8, J = 0.6 Hz, 1 H, 6-H), 7.03 (dd, J =
1.49 [2ϫs, 3 H, C(CH3)2], 1.54–1.68 (m, 1 H, 6-H1), 1.82–2.23 (m, 15.9, J = 8.3 Hz, 1 H, 5-H) ppm. 13C NMR (125.48 MHz, CDCl3):
5 H, 4-H, 3Ј-H2, 6-H1, OH), 2.34–2.78 (m, 2 H, 5-H, OH), 3.72,
3.73 (2ϫs, 3 H, OCH3), 3.70–3.83 (m, 1 H, 6Ј-H), 4.24–4.34 (br. s,
0.5 H, 4ЈЈ-H), 4.38 (dd, J = 6.5, J = 6.5 Hz, 0.5 H, 3a-H), 4.42–
4.58 (m, 2 H, 4ЈЈ-H, 3a-H, 6a-H), 5.27–5.57 (m, 2 H, 1Ј-H, 2Ј-H),
6.07 (dd, J = 15.6, J = 1.6 Hz, 0.5 H, 2ЈЈ-H), 6.10 (dd, J = 15.6, J
= 1.6 Hz, 0.5 H, 2ЈЈ-H), 6.96 (dd, J = 15.5, J = 4.4 Hz, 0.5 H, 3ЈЈ-
δ = 20.69 (q, CH3), 25.66 [q, C(CH3)2], 26.17 (t, C-11), 28.21 [q,
C(CH3)2], 31.76 (t, C-12), 34.69 (t, C-10), 40.41 (t, C-15), 41.47 (d,
C-14a), 56.29 (d, C-3b), 70.73 (d, C-4), 71.98 (d, C-9), 80.02 (d, C-
15a), 83.11 (d, C-3a), 112.13 [s, C(CH3)2], 119.97 (d, C-6), 130.84
(d, C-13), 136.83 (d, C-14), 150.58 (d, C-5), 166.53 (s, CO2) ppm.
HR-MS (ESI+): m/z calcd. for C19H29O5 337.2010 [M + H]+;
+
H), 7.02 (dd, J = 15.7, J = 4.6 Hz, 0.5 H, 3ЈЈ-H) ppm. 13C NMR found 337.2019.
(75.48 MHz, CDCl3): δ = 23.54, 23.55 (2ϫq, C-7Ј), 25.18, 25.18
Inversion of the Stereocenter at C-4. (3aR,3bR,4R,9S,14aS,15aS)-4-
[2 ϫ q, C(CH3)2], 25.50, 25.56 (2 ϫ t, C-4Ј), 27.69, 27.70 [2 ϫ q,
C(CH3)2], 32.33, 32.35 (2 ϫ t, C-3Ј), 38.80, 38.83 (2 ϫ t, C-5Ј),
38.93, 39.20 (2 ϫt, C-6), 43.97, 44.71 (2ϫ d, C-5), 51.76, 51.80
(2ϫq, OCH3), 56.11, 56.21 (2ϫd, C-4), 67.97, 67.97 (2ϫd, C-6Ј),
70.72, 71.07 (2ϫd, C-4ЈЈ), 79.40, 79.34 (2ϫd, C-6a), 81.98, 81.46
(2ϫd, C-3a), 112.58, 112.88 [2ϫs, C(CH3)2], 121.27, 120.28 (2ϫd,
C-2ЈЈ), 131.74, 131.74, 132.43, 132.50 (4ϫd, C-1Ј, C-2Ј), 148.18,
149.80 (2ϫd, C-3ЈЈ), 166.99, 167.08 (2 ϫs, CO2) ppm. HR-MS
Hydroxy-2,2,9-trimethyl-3a,3b,4,9,10,11,12,14a,15,15a-decahydro-
7H-[1,3]dioxolo[4,5]cyclopenta[1,2-f]oxacyclotridecin-7-one (21b):
MnO2 (491 mg) was added to a solution of 21a (22.3 mg,
0.066 mmol) in CH2Cl2 (1.3 mL), and the resulting suspension was
stirred at room temperature for 2 h [TLC monitoring; petroleum
ether/ethyl acetate (3:1); Rf(21a) = 0.16]. The mixture was then fil-
tered through a pad of Celite, which was washed with ethyl acetate,
and the solvent was evaporated in vacuo. A solution of the crude
product (9.5 mg) in dry methanol (1.3 mL) was cooled to –78 °C
and treated with NaBH4 (5 mg, 0.132 mmol). After the mixture
had been stirred at –78 °C for 30 min, TLC monitoring showed
complete conversion [petroleum ether/ethyl acetate (3:1); Rf(21b) =
0.10]. Water (5 mL) was then added, and the mixture was allowed
to warm to room temperature. After a further 10 min of stirring,
ethyl acetate (10 mL) was added, and the aqueous layer was sepa-
rated and extracted with ethyl acetate (3ϫ10 mL). The combined
organic layers were dried with Na2SO4 and filtered, and the solvent
was removed in vacuo. The crude product was purified by flash
column chromatography on silica gel [petroleum ether/ethyl acetate
(3:1 to 1:1)] to yield 21b (8.7 mg, 39%) as small white needles. [α]
(ESI+): m/z calcd. for C20H33O6 369.2272 [M + H]+; found
+
369.2269.
(3aR,3bR,9S,14aS,15aS)-4-Hydroxy-2,2,9-trimethyl-3a,3b,4,9,10,
11,12,14a,15,15a-decahydro-7H-[1,3]dioxolo[4,5]cyclopenta[1,2-f]-
oxacyclotridecin-7-one (21): A solution of 17 (53 mg, 0.144 mmol)
in a mixture of THF (1.5 mL) and water (1.2 mL) was treated with
an aqueous solution of LiOH (290 μL, 1 m), and the mixture was
stirred at room temperature for 2 h. The mixture was then neutral-
ized with the strongly acidic ion exchange resin Amberlite IR-120
and filtered through a pad of Celite, and the filtrate was concen-
trated in vacuo. The crude acid 18 was placed in a Schlenk tube
and dissolved in dry THF (2.9 mL). The solution was cooled to
–50 °C and treated with 4-(dimethylamino)but-3-yn-2-one (29 μL,
0.261 mmol). After 1 h at –50 °C, the solution was allowed to warm
to room temperature, and the solvent was evaporated in vacuo. The
crude product was dissolved in ethyl acetate and filtered through a
short column of silica gel, which was eluted with ethyl acetate/
methanol (20:1) [Rf(20) = 0.23]. After evaporation of the solvent in
vacuo, 20 was dissolved in dry toluene (20 mL), and the solution
was added dropwise by syringe at 80 °C to a solution of cam-
phorsulfonic acid (6.7 mg, 0.029 mmol) in dry toluene (20 mL) over
a period of 40 min. After the addition, the syringe was flushed with
dry toluene (4 mL). After 2 h, the solution was allowed to cool to
room temperature, and a saturated aqueous solution of NaHCO3
(20 mL) and ethyl acetate (20 mL) were added. The aqueous layer
was separated and extracted with ethyl acetate (3ϫ20 mL). The
combined organic layers were dried with Na2SO4 and filtered, and
the solvent was removed in vacuo. A mixture of the product 21 and
unreacted 20 was obtained and separated by flash column
chromatography {petroleum ether/ethyl acetate (3:1) to ethyl acet-
ate/MeOH (10:1); Rf(21) = 0.16; Rf(20) = 0.10 [petroleum ether/
= 71.9 (c = 0.755, CHCl3). 1H NMR (300.13 MHz, CDCl3): δ =
20
D
0.93–1.07 (m, 1 H, 11-H1), 1.24 (d, J = 6.2 Hz, 3 H, CH3), 1.32,
1.52 [2ϫs, 6 H, C(CH3)2], 1.46–1.90 (m, 7 H, 3b-H, 15-H1, 12-H1,
10-H2, 11-H1, OH), 1.92–2.08 (m, 1 H, 12-H1), 2.21 (ddd, J = 13.9,
J = 6.9, J = 6.9 Hz, 1 H, 15-H1), 2.32–2.50 (m, 1 H, 14a-H), 4.23
(d, J = 10.1 Hz, 1 H, 4-H), 4.47–4.55 (m, 1 H, 3a-H), 4.55–4.62
(m, 1 H, 15a-H), 4.87 (mc, 1 H, 9-H), 5.23 (mc, 1 H, 14-H), 5.69
(ddd, J = 14.9, J = 9.5, J = 5.1 Hz, 1 H, 13-H), 5.95 (dd, J = 15.7,
J = 1.8 Hz, 1 H, 6-H), 7.23 (dd, J = 15.8, J = 3.4 Hz, 1 H, 5-H)
ppm. 13C NMR (75.48 MHz, CDCl3): δ = 20.85 (q, CH3), 25.52
[q, C(CH3)2], 26.72 (t, C-11), 27.97 [q, C(CH3)2], 31.53 (t, C-12),
34.45 (t, C-10), 39.73 (t, C-15), 45.45 (d, C-14a), 58.77 (d, C-3b),
71.64 (d, C-9), 74.72 (d, C-4), 79.50 (d, C-15a), 87.27 (d, C-3a),
113.19 [s, C(CH3)2], 118.21 (d, C-6), 131.33 (d, C-13), 135.78 (d,
C-14), 150.79 (d, C-5), 166.29 (s, CO2) ppm. HR-MS (ESI+): m/z
+
calcd. for C19H29O5 337.2010 [M + H]+; found 337.2025.
(1R,6S,11aS,13S,14R,14aR)-1,13,14-Trihydroxy-6-methyl-
1,6,7,8,9,11a,12,13,14,14a-decahydro-4H-cyclopenta[f]oxacyclotri-
decin-4-one (5): A solution of 21b (15.7 mg, 0.047 mmol) in meth-
ethyl acetate (3:1)]}. Recovered 20 (24.9 mg, 0.053 mmol) was anol (930 μL) was treated with an aqueous solution of HCl (94 μL,
treated in the same way as described above (18 mL of toluene,
6.7 mg of CSA). The total yield of 21a (22.3 mg), as a colorless oil,
1 m) and stirred at room temperature for 15 min. More HCl solu-
tion (24 μL) was added, and the mixture was stirred for another
was 46%, and that of 21b (15.7 mg) was 32%. Crystallization of 30 min. TLC showed complete conversion [petroleum ether/ethyl
21b from ethyl acetate afforded colorless needles (m.p. 164–166 °C) acetate (1:2); Rf(21b) = 0.56, Rf(5) = 0.14]. The mixture was filtered
Eur. J. Org. Chem. 2011, 878–891
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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887

G. Helmchen et al.
FULL PAPER
through a small pad of the strongly basic anion exchange resin
DOWEX MONOSPHERE 550A (OH), and the filtrate was con-
centrated in vacuo. The crude product was purified by flash column
chromatography on silica gel [petroleum ether/ethyl acetate (1:2 to
0:1)] to give 5 (10.8 mg, 78%) as a white solid. Recrystallization
from ethyl acetate afforded colorless needles suitable for X-ray
analysis (m.p. 128–131 °C). [α]2D0 = 80.9 (c = 0.485, CHCl3). 1H
by two inseparable side products derived from the excess of MEM-
Cl. These compounds were removed in the next step. The analytical
data were consistent with reported data.[32,33] [α]2D0 = –21.8 (c =
1.23, in CHCl3). NMR data for the side products derived from the
excess of MEM-Cl: 1H NMR (300.13 MHz, CDCl3): δ = 3.38 (s,
OCH3), 3.52–3.74 (m, CH2O), 4.77, 4.83 (2ϫs, CH2O2) ppm. 13C
NMR (75.48 MHz, CDCl3): δ = 59.15 (q, OCH3), 71.87, 71.89
NMR (500.13 MHz, CDCl3): δ = 0.82–0.97 (m, 1 H, 13-H1), 1.26 (2ϫt, CH2O), 92.47, 95.79 (2ϫt, CH2O2) ppm.
(d, J = 6.2 Hz, 3 H, CH3), 1.46–1.56 (m, 1 H, 14-H1), 1.59 (ddd, J
Methyl (2E,4R)-4-{(1R,2S,4S)-2-[(1E,6S)-6-Hydroxyhept-1-en-1-
= 14.8, J = 5.2, J = 1.6 Hz, 1 H, 8-H1), 1.66–1.90 (m, 4 H, 12-H1,
5-H, 13-H1, 14-H1), 1.97–2.05 (m, 1 H, 12-H1), 2.14 (ddd, J = 14.7,
J = 9.9, J = 4.9 Hz, 1 H, 8-H1), 2.32 (dddd, J = 9.6, J = 9.6, J =
9.6, J = 5.0 Hz, 1 H, 9-H), 3.51, 3.30, 2.67 (3ϫbr. s, 3 H, OH),
3.97 (dd, J = 8.8, J = 4.2 Hz, 1 H, 6-H), 4.08 (br. s, 1 H, 7-H), 4.39
(d, J = 9.8 Hz, 1 H, 4-H), 4.81–4.90 (m, 1 H, 15-H), 5.29–5.37 (m,
1 H, 10-H), 5.65 (ddd, J = 14.9, J = 10.2, J = 4.6 Hz, 1 H, 11-H),
5.94 (dd, J = 15.7, J = 1.7 Hz, 1 H, 2-H), 7.25 (dd, J = 15.6, J =
3.1 Hz, 1 H, 3-H) ppm. 13C NMR (125.77 MHz, CDCl3): δ = 20.97
(q, CH3), 26.85 (t, C-13), 31.85 (t, C-12), 34.32 (t, C-14), 37.55 (t,
C-8), 41.05 (d, C-9), 55.96 (d, C-5), 72.04 (d, C-15), 72.77 (d, C-7),
75.88 (d, C-4), 80.64 (d, C-6), 117.98 (C-2), 130.42 (d, C-11), 136.93
(d, C-10), 151.13 (d, C-3), 166.44 (s, CO2) ppm. HR-MS (EI+): m/z
calcd. for C16H24O5+ 296.1618 [M]+; found 296.1591 and m/z calcd.
yl]-4-[(2-methoxyethoxy)methoxy]cyclopentyl}-4-[(2-methoxyethoxy)-
methoxy]but-2-enoate (24): An aqueous solution of KOH (10 wt.-
%, 7.4 mL) was added dropwise to a solution of 22 (598 mg,
1.31 mmol) in methanol (13.1 mL). After the mixture had been
stirred at 40 °C for 1 h, TLC monitoring showed complete conver-
sion [ethyl acetate; Rf(22) = 0.55]. The solution was allowed to cool
to room temperature and was treated with the strongly acidic ion
exchange resin Amberlite IR-120 until the mixture reached pH =
7. The solution was then filtered through a pad of Celite, which
was washed with ethyl acetate/methanol (1:1). The filtrate was con-
centrated in vacuo, and residual water was removed by co-evapora-
tion with toluene (3ϫ4 mL). The crude acid 23 was dissolved in
ethyl acetate (13 mL), and a solution of CH2N2 in diethyl ether (ca.
0.4 m, 8 mL) was added dropwise until the mixture turned yellow.
Subsequently, silica gel (ca. 1 g) was added in small portions until
+
for C16H22O4 278.1513 [M – H2O]+; found 278.1516.
Cultivation of Eupenicillium brefeldianum. Isolation of Brefeldin A bubbling of nitrogen ceased. The colorless mixture was filtered, and
(1) and 7-Dehydrobrefeldin A (3): A sample of Eupenicillium brefeld-
ianum was purchased from the ATCC® (American Type Culture
Collection, ATCC-58665) and cultivated on a potato dextrose agar
medium. The fungus grew rapidly, and BFA was produced ef-
ficiently.[45] The agar slants were extracted with ethyl acetate, and
the crude product was subjected to column chromatography on sil-
ica gel. BFA (662 mgm–2) was isolated, and the analytical data were
in accordance with reported data.[46] After 2 months, the fungus
started to produce 7-dehydrobrefeldin A (3), which is probably a
catabolite of BFA and occurs in the biosynthesis.[47] Again the ana-
lytical data were in accordance with reported data.[48] In addition,
the structure was unambiguously assigned by X-ray crystal struc-
ture analysis (Table 1). CCDC-797341 (3), -797342 (5), -797343
(6), -797344 (7), and -797345 (21b) contain the supplementary crys-
tallographic data for this paper. These data can be obtained free
of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
the filtrate was concentrated in vacuo. The crude product was sub-
jected to flash chromatography on silica gel {60 g; petroleum ether/
ethyl acetate (1:2); Rf(24) = 0.21 [petroleum ether/ethyl acetate
(1:2)]} to yield 24 (441 mg, 69%) as a colorless oil. Note that the
starting material 22 was accompanied by two side products from
the MEM protection, which could not be removed in the previous
step. Analytical data were consistent with reported data.[33] [α]2D0
–18.0 (c = 0.61, CHCl3).
=
Methyl (2E,4R)-4-{(1R,2R,4R)-2-Formyl-4-[(2-methoxyethoxy)-
methoxy]cyclopentyl}-4-[(2-methoxyethoxy)methoxy]but-2-enoate
(25): A mixture of K2OsO4·2H2O (9.2 mg, 25.0 μmol) and 4-meth-
ylmorpholine N-oxide (aqueous solution 50 wt.-%, 0.212 mL,
1.02 mmol) in water (1 mL) was added dropwise to a solution of
24 (490 mg, 1.00 mmol) in acetone (9 mL). After the mixture had
been stirred at room temperature for 1 h, TLC monitoring showed
incomplete conversion [ethyl acetate/MeOH (10:1); Rf(24) = 0.69].
Again, 4-methylmorpholine N-oxide (0.09 mL, 0.43 mmol) was
added in two portions after 0.5 h and 1 h. After an overall reaction
time of 3 h, conversion was complete. Na2S2O4 (200 mg) was then
added, and, after stirring for further 20 min, the solution was fil-
tered through a pad of Celite and then a pad of silica gel, which
was rinsed with ethyl acetate/MeOH (10:1). The filtrate was con-
centrated in vacuo to give the dihydroxylation product (436 mg,
83%) as a colorless oil and as a 1:1 mixture of diastereomers (13C
NMR), which was dissolved in a mixture of diethyl ether/water
(2:1; 8.4 mL). NaIO4 (357 mg, 1.67 mmol) was then added, and the
(1R,6S,11aS,13S,14aR)-1,13-Bis[(2-methoxyethoxy)methoxy]-6-
methyl-1,6,7,8,9,11a,12,13,14,14a-decahydro-4H-cyclopenta[f]oxa-
cyclotridecin-4-one (22): Analogously to a small-scale procedure by
Taber et al.,[33] iPr2EtN (1.66 mL, 9.70 mmol) was added dropwise
to a cooled (0 °C) suspension of BFA (672 mg, 2.397 mmol) in dry
CH2Cl2 (25 mL). After the mixture had been stirred at 0 °C for
10 min, MEM-Cl (1.10 mL, 9.56 mmol) was added dropwise. The
mixture was allowed to warm to room temperature and was stirred
for a further 3 h. The solution was then cooled (0 °C), and iPr2EtN
(1.7 mL, 9.90 mmol) and MEM-Cl (1.10 mL, 9.56 mmol) were mixture was stirred at room temperature for 30 min. TLC monitor-
added again. The resulting solution was allowed to warm to room
temperature and stirred overnight. TLC monitoring showed com-
plete conversion of BFA [petroleum ether/ethyl acetate (1:3); Rf(22)
ing showed complete conversion [ethyl acetate/MeOH (10:1); Rf(25)
= 0.65]. Phosphate buffer (10 mL, 0.5 m NaH2PO4/Na2HPO4) and
diethyl ether (10 mL) were added, and the aqueous layer was sepa-
= 0.30, Rf(BFA) = 0.10]. Water (20 mL) and brine (20 mL) were rated and extracted with diethyl ether (3ϫ10 mL). The combined
added, and the aqueous layer was separated and extracted with organic layers were dried with Na2SO4 and concentrated in vacuo.
CH2Cl2 (3ϫ20 mL). The combined organic layers were dried with The crude product was subjected to flash chromatography on silica
Na2SO4, filtered, and concentrated in vacuo. The crude product
was subjected to flash chromatography on silica gel [100 g; petro-
leum ether/ethyl acetate (1:1)] to yield fully protected 22 (1.18 g)
and a mixture of 22 and the mono-MEM-protected BFA (100 mg)
as colorless oils. Note: The major fraction of 22 was accompanied
gel [40 g; petroleum ether/ethyl acetate (1:2 to 1:1)] to yield 25
(271 mg, 67% over two steps) as a colorless oil. The compound
contained 10% of the C-2Ј epimer (13C NMR). Analytical data
were not fully reported.[32] [α]2D0 = –66.2 (c = 0.54, CHCl3). 1H
NMR (300.13 MHz, CDCl3): δ = 1.69 (ddd, J = 14.2, J = 9.6, J =
888
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 878–891

Syntheses and Biological Properties of Brefeldin Analogues
Table 1. Data for the crystal structure analyses of 3, 5, 6, 7, and 21b.
3
5
6
7
21b
Empirical formula
Formula mass
C16H22O4
278.34
C16H24O5
296.35
C16H25NO3
279.37
C16H26ClNO3
315.83
C19H28O5
336.41
Crystal size [mm]
Crystal system
Space group
a [Å]
b [Å]
c [Å]
α [°]
0.23ϫ0.13ϫ0.12 0.36ϫ0.18ϫ0.08 0.35ϫ0.08ϫ0.05 0.21ϫ0.15ϫ0.05 0.28ϫ0.06ϫ0.02
monoclinic
P21
monoclinic
P21
6.9802(7)
5.4898(5)
20.474(2)
90
orthorhombic
P212121
4.8652(6)
7.3722(9)
42.240(5)
90
orthorhombic
P212121
5.3107(6)
6.9620(8)
47.239(5)
90
monoclinic
C2
31.078(14)
5.811(3) Å
10.122(4)
90
6.8697(9)
5.2886(7)
20.663(3)
90
β [°]
99.090(3)
90
741.29(17)
2
1.25
0.09
99.552(2)
90
773.68(13)
2
1.27
0.09
90
90
1515.0(3)
4
1.23
90
90
1746.6(3)
4
1.20
96.306(18)
90
1817.0(14)
4
1.23
0.09
γ [°]
V [Å3]
Z
ρcalcd. [Mgm–3]
μ [mm–1]
0.08
0.23
Max/min transmission
0.99/0.98
0.99/0.97
1.00/0.97
0.99/0.95
1.00/0.98
Index ranges
h
k
l
θ [°]
T [K]
–9 to 9
–7 to 7
–27 to 27
2.0 to 28.4
200(2)
–9 to 9
–7 to 7
–26 to 27
2.0 to 28.4
200(2)
–5 to 5
–8 to 8
–50 to 49
2.8 to 25.1
200(2)
–6 to 6
–8 to 8
–55 to 56
2.6 to 25.1
200(2)
–31 to 31
–5 to 5
–10 to 10
2.3 to 21.3
200(2)
Reflections, collected
Reflections, independent (Rint
Reflections, observed [IϾ2σ(I)]
Data/restraints/parameters
GOF on F2
7739
3632 (0.0229)
3318
3632/1/269
1.09
8202
2120 (0.0302)
2034
2120/1/203
1.13
12508
1634 (0.0571)
1516
1634/0/193
1.22
14144
3099 (0.0522)
2776
3099/0/196
1.17
4560
1138 (0.0797)
981
1138/1/218
1.11
)
Final R indices [IϾ2σ(I)]
R1
wR2
0.050
0.107
0.0(12)
0.041
0.101
0.5(11)
0.33/–0.16
0.056
0.116
2(3)
0.20/–0.22
0.061
0.118
0.11(12)
0.26/–0.32
0.065
0.134
0(4)
0.21/–0.27
Absolute structural parameter
Max/min residual electron density [eÅ–3] 0.21/–0.17
4.9 Hz, 1 H, 5Ј-H1), 1.84–2.20 (m, 3 H, 3Ј-H2, 5Ј-H1), 2.63–2.85 –46.7 (c = 0.77, CHCl3). 1H NMR (300.13 MHz, CDCl3): δ = 1.10
(m, 2 H, 2Ј-H, 1Ј-H), 3.36, 3.37 (2ϫs, 6 H, OCH3), 3.48–3.83 (m,
8 H, CH2O), 3.73 (s, 3 H, CO2CH3), 4.23 (mc, 1 H, 4Ј-H), 4.33
(mc, 1 H, 4-H), 4.60–4.72 (m, 4 H, CH2O2), 6.00 (dd, J = 15.8, J
= 1.2 Hz, 1 H, 2-H), 6.79 (dd, J = 15.8, J = 6.4 Hz, 1 H, 3-H), 9.63
(d, J = 1.5 Hz, 1 H, CHO) ppm. 13C NMR (75.48 MHz, CDCl3):
δ = 33.85, 33.95 (2ϫt, C-3Ј, C-5Ј), 42.12 (d, C-1Ј), 51.82, 51.84
(d,q, C-2Ј, CO2CH3), 59.13, 59.18 (2 ϫ q, OCH3), 67.10, 67.72,
71.80, 71.87 (4ϫt, CH2O), 76.69 (d, C-4), 77.35 (d, C-4Ј), 93.85,
(d, J = 6.6 Hz, 3 H, 7ЈЈ-H), 1.30–1.54 (m, 5 H, 3Ј-H1, 4ЈЈ-H, 5ЈЈ-
H), 1.64–2.06 (m, 5 H, 1Ј-H, 5Ј-H, 3ЈЈ-H), 1.43 [s, 9 H, C(CH3)3],
2.17 (ddd, J = 14.0, J = 8.0, J = 6.2 Hz, 1 H, 3Ј-H1), 2.35 (mc, 1
H, 2Ј-H), 3.37, 3.38 (2ϫs, 6 H, OCH3), 3.50–3.82 (m, 9 H, CH2O,
6ЈЈ-H), 3.73 (s, 3 H, CO2CH3), 4.10–4.17 (m, 1 H, 4Ј-H), 4.17–4.23
(m, 1 H, 4-H), 4.37 (br. s, 1 H, NH), 4.64–4.71 (m, 4 H, CH2O2),
5.20–5.44 (m, 2 H, 1ЈЈ-H, 2ЈЈ-H), 5.95 (dd, J = 15.8, J = 1.2 Hz, 1
H, 2-H), 6.81 (dd, J = 15.8, J = 6.1 Hz, 1 H, 3-H) ppm. 13C NMR
93.89 (2ϫt, CH2O2), 123.14 (d, C-2), 145.92 (d, C-3), 166.38 (s, (75.48 MHz, CDCl3): δ = 21.45 (q, C-7ЈЈ), 26.05 (t, C-4ЈЈ), 28.57
CO2), 203.14 (d, CHO) ppm. HR-MS (ESI+): m/z calcd. for [q, C(CH3)3], 32.44 (t, C-3ЈЈ), 33.08 (t, C-5Ј), 36.95 (t, C-5ЈЈ), 40.35
C19H32O9Na+ 427.1939; found 427.1936 [M + Na]+. C19H32O9 (t, C-3Ј), 43.18 (d, C-2Ј), 46.52 (d, C-6ЈЈ), 48.33 (d, C-1Ј), 51.71 (q,
(404.45): calcd. C 56.42, H 7.97; found C 56.30, H 7.80.
CO2CH3), 59.13, 59.17 (2ϫq, OCH3), 66.94, 67.65, 71.81, 71.92
(4ϫt, CH2O), 75.80 (d, C-4), 76.81 (d, C-4Ј), 78.98 [s, C(CH3)3],
94.26, 94.39 (2 ϫ t, CH2O2), 121.37 (d, C-2), 130.93 (d, C-2ЈЈ),
133.70 (d, C-1ЈЈ), 148.36 (d, C-3), 155.51 (s, CO2tBu), 166.74 (s, C-
Methyl (2E,4R)-4-{(1R,2S,4S)-2-{(1E,6S)-6-[(tert-butoxycarbonyl)-
amino]hept-1-en-1-yl}-4-[(2-methoxyethoxy)methoxy]cyclopentyl}-4-
[(2-methoxyethoxy)methoxy]but-2-enoate (27): A solution of
KHMDS in toluene (1.5 mL, 0.5 m) was added dropwise to a
cooled solution (–78 °C) of 26 (328 mg, 0.801 mmol) in DME
(3.1 mL, freshly dried with sodium). After 30 min, a solution of 25
(244 mg, 0.603 mmol) in dry DME (3.1 mL) was added dropwise
over a period of 15 min. After 2 h at –78 °C, the solution was al-
lowed to warm to room temperature and was stirred for a further
60 min. Water (10 mL) and brine (10 mL) were then added, and
the mixture was extracted with ethyl acetate (3ϫ20 mL). The com-
bined organic layers were dried with Na2SO4, filtered, and concen-
trated in vacuo. The residue was subjected to flash column
chromatography on silica gel {40 g; petroleum ether/ethyl acetate
(3:1 to 2:1 to 1:1); Rf(27) = 0.25 [petroleum ether/ethyl acetate (1:1),
+
1) ppm. HR-MS (ESI+): m/z calcd. for C30H54NO10 588.3742 [M
+ H]+; found 588.3744. C30H53NO10 (587.74): calcd. C 61.31, H
9.09, N 2.38; found C 61.31, H 9.10, N 2.46.
(1R,6S,11aS,13S,14aR)-1,13-Dihydroxy-6-methyl-5,6,7,8,9,11a,12,
13,14,14a-decahydrocyclopenta[f]azacyclotridecin-4(1H)-one (6): A
solution of HCl in MeOH (1 m, 5.1 mL) was added to 27 (301 mg,
512 μmol), and the resulting solution was heated at 60 °C. After
2 h, the volatile components were removed under reduced pressure.
Traces of water and HCl were removed by co-evaporation of tolu-
ene (2ϫ2 mL). A solution of the residue in MeOH (2.6 mL) was
treated with an aqueous solution of LiOH (5.1 mL, 1 m), and the
mixture was heated at 40 °C. After 1 h and cooling to room tem-
perature, water was added (5 mL). The mixture was poured onto a
KMnO4]} to provide 27 (329 mg, 93%) as a colorless oil. [α]2D0
=
Eur. J. Org. Chem. 2011, 878–891
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
889

G. Helmchen et al.
FULL PAPER
short column loaded with the strongly acidic ion exchange resin
Dowex 50Wx8 and eluted with a solution of NH3 in acetonitrile/
water (3:2; 1 m to 4 m). The volatile components were removed in
vacuo, and a suspension of the crude product in dry DMF (26 mL)
was cooled to 0 °C. iPr2NEt (260 μL, 1.52 mmol) and HBTU
3 H, 12-H1, 8-H1, 6-H1), 2.30–2.46 (m, 1 H, 9-H), 3.19–3.34 (m, 1
H, 7-H), 4.03 (mc, 1 H, 4-H), 4.73–4.87 (m, 1 H, 15-H), 5.23 (dd,
J = 15.1, J = 9.5 Hz, 1 H, 10-H), 5.73–5.87 (m, 1 H, 11-H), 5.82
(dd, J = 15.6, J = 1.9 Hz, 1 H, 2-H), 7.44 (dd, J = 15.6, J = 3.0 Hz,
1 H, 3-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 21.07 (q, CH3),
(388 mg, 1.02 mmol) were then added. After 15 min at 0 °C, a clear 27.97 (t, C-13), 32.99 (t, C-12), 34.94 (t, C-14), 41.11 (t, C-6), 45.42
solution resulted. This was allowed to warm to room temperature
and stirred for 23 h. Water (3 mL) was then added, and, after
10 min of stirring, DMF and water were removed in vacuo at 60 °C.
Subsequently, a solution of the residue in MeOH was filtered
through a column of the strongly basic ion-exchange resin
DOWEX MONOSPHERE® 550 (OH). The filtrate containing the
crude product was taken up on Celite and subjected to chromatog-
raphy on silica gel {ethyl acetate/methanol (15:1 to 10:1); Rf(6) =
0.23 [ethyl acetate/MeOH (10:1)]} to yield 6 as a white powder
(125 mg, 87%). Recrystallization from MeOH/1,2-dichloroethane
gave small colorless needles (decomposition at 248 °C) suitable for
X-ray crystal structure analysis. [α]2D0 = –40.6 (c = 0.51, MeOH).
1H NMR (500.13 MHz, CD3OD): δ = 0.99–1.06 (m, 1 H, 13-H1),
1.15 (d, J = 6.8 Hz, 3 H, CH3), 1.22–1.32 (m, 1 H, 14-H1), 1.42
(mc, 1 H, 8-H1), 1.75–1.84 (m, 4 H, 6-H1, 12-H1, 13-H1, 14-H1),
1.86–2.02 (m, 3 H, 6-H1, 12-H1, 5-H), 2.11 (mc, 1 H, 8-H1), 2.28
(mc, 1 H, 9-H), 3.87–3.95 (m, 1 H, 15-H), 3.90 (ddd, J = 9.8, J =
6.1, J = 1.2 Hz, 1 H, 4-H), 4.19 (ddd, J = 10.9, J = 5.5, J = 5.5 Hz,
1 H, 7-H), 5.23 (dd, J = 15.0, J = 9.5 Hz, 1 H, 10-H), 5.61 (ddd, J
= 15.0, J = 10.7, J = 4.1 Hz, 1 H, 11-H), 5.92 (dd, J = 16.0, J =
1.2 Hz, 1 H, 2-H), 6.61 (dd, J = 16.0, J = 6.0 Hz, 1 H, 3-H) ppm.
13C NMR (125.75 MHz, CD3OD): δ = 21.26 (q, CH3), 27.63 (t, C-
13), 33.78 (t, C-12), 37.59 (t, C-14), 41.32 (t, C-6), 44.60 (t, C-8),
45.93 (d, C-9), 47.55 (d, C-15), 51.70 (d, C-5), 72.84 (d, C-7), 77.83
(d, C-4), 123.88 (d, C-2), 131.33 (d, C-11), 138.05 (d, C-10), 147.11
(d, C-3), 170.46 (s, CONH) ppm. HR-MS (ESI+): m/z calcd. for
(t, C-8), 46.91 (d, C-9), 52.60 (d, C-7), 53.11 (d, C-5), 73.18 (d, C-
15), 76.38 (d, C-4), 117.96 (d, C-2), 131.93 (d, C-11), 137.16 (d, C-
10), 154.86 (d, C-3), 168.32 (s, CO2) ppm. HR-MS (ESI+): m/z
calcd. for C16H26NO3 280.1907 [M + H]+; found 280.1908.
+
Supporting Information (see footnote on the first page of this arti-
cle): Copies of 13C NMR spectra of compounds 5, 6, 7, 17, 21a,
and 21b, X-ray crystal data for compounds 3, 5, 6, 7, and 21b, and
test results from the National Cancer Institute, Bethesda, USA, for
compounds 4, 5, and 6.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
(Graduiertenkolleg 850) and the Dr. Rainer Wild-Stiftung. We
thank Dr. Bernhard Wetterauer and Prof. Michael Wink (Institut
für Pharmazie und Molekulare Biologie, Universität Heidelberg)
for their help with cultivation of Eupenicillium brefeldianum. Ad-
ditionally, we thank Antje Bremer and Benjamin Lindner for moti-
vated and skilled experimental assistance. We also thank the Nicon
Imaging Center at the University of Heidelberg for support. B. B. is
an investigator in the CellNetworks Cluster of Excellence ECX81.
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+
C16H26NO3 280.1907 [M + H]+; found 280.1913.
(1R,6S,11aS,13S,14aR)-13-Amino-1-hydroxy-6-methyl-1,6,7,8,9,
11a,12,13,14,14a-decahydro-4H-cyclopenta[f]oxacyclotridecin-4-one
(7): Ti(OEt)4 (140 mg, 0.614 mmol) was added to a solution of 7-
dehydro-BFA (3, 57 mg, 0.205 mmol) and (S)-2-methylpropane-2-
sulfinamide (37 mg, 0.305 mmol) in dry THF (2.1 mL), and the
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–15 °C and stirred at this temperature for 2 h [TLC monitoring;
ethyl acetate; Rf(28a) = 0.33, Rf(28b) = 0.22]. Subsequently, water
(5 mL) and THF (2 mL) were added, and the mixture was allowed
to warm to room temperature. After the mixture had been stirred
for 5 min, ethyl acetate (10 mL) was added, and the aqueous layer
was separated and extracted with ethyl acetate (3ϫ10 mL). The
combined organic layers were dried with Na2SO4 and filtered, and
the solvent was removed in vacuo. The crude product was subjected
to column chromatography on silica gel [ethyl acetate/methanol
(20:1); Rf(28a) = 0.44, Rf(28b) = 0.36]. The nonpolar fractions con-
tained two components, which could not be separated. The polar
fractions contained 28b accompanied by a trace of (S)-2-methyl-
propane-2-sulfinamide (36 mg). A solution of this mixture in meth-
anolic HCl (1 m, 1.88 mL) was stirred at room temperature for
45 min and then filtered through a short column of the strongly
basic ion exchange resin DOWEX 550A MONOSPHERE (OH),
which was rinsed with methanol (20 mL). The solvent was removed
under reduced pressure to yield 7 (23 mg, 40% from 3) as small
colorless plates (decomposition at 157 °C). [α]2D0 = 52.1 (c = 0.81,
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1
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13-H1), 1.20–1.34 (m, 1 H, 8-H1), 1.24 (d, J = 6.3 Hz, 3 H, CH3),
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1.50–1.95 (m, 6 H, 5-H, 6-H1, 13-H1, 14-H2, 12-H1), 1.95–2.13 (m,
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www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 878–891

Syntheses and Biological Properties of Brefeldin Analogues
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The NCI’s internal ID numbers (NSC) of the brefeldin ana-
logues described by us: NSC 652682 (BFA lactam analogue),
NSC 751150 [(6R)-hydroxy-BFA], NSC 671437 (7S)-amino-
BFC. The latter compound had already been submitted to the
NCI anonymously. Brefeldin analogues presented in earlier
publications: NSC 746705 (4-acetoxy-4-deoxy-BFC lactam an-
alogue), NSC 746706 (BFC lactam analogue), NSC 746707
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Received: September 16, 2010
Published Online: December 30, 2010
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