Synthesis and biological properties of novel brefeldin A analogues

Article abstract of DOI:10.1021/jm400615g

New brefeldin A (1) analogues were obtained by introducing a variety of substituents at C15. Most of the analogues exhibited significant biological activity. (15R)-Trifluoromethyl-nor-brefeldin A (3), (15R)-vinyl-nor-brefeldin A (5), their epimers 4 and 6 as well as (15S)-ethyl-nor-brefeldin A (2) were prepared from the key building blocks 12 or 24 by Julia-Kocienski olefination with tetrazolyl sulfones and subsequent macrolactonization. The vinyl derivative 5 allowed analogues to be synthesized by hydroboration and Suzuki-Miyaura coupling. The following biological properties were assessed: (a) inhibition of cell growth of human cancer cells (NCI), (b) induction of morphological changes of the Golgi apparatus of plant and mammalian cells, and (c) influence on the replication of the enterovirus CVB3. Furthermore, conformational aspects were studied by X-ray crystal structure analysis and molecular mechanics calculations, including docking of the analogues into the brefeldin A binding site of an Arf1/Sec7-complex.

Full text of DOI:10.1021/jm400615g

Article
pubs.acs.org/jmc
Synthesis and Biological Properties of Novel Brefeldin A Analogues
Kai Seehafer,† Frank Rominger,† Gunter Helmchen,*,† Markus Langhans,‡ David G. Robinson,‡
̈
§
§
⊥
⊥
̈
Basa
̧
k Ozata, Britta Brugger, Jeroen R. P. M. Strating, Frank J. M. van Kuppeveld,
̈
and Christian D. Klein#
†Institute of Organic Chemistry, University of Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany
‡Department of Plant Cell Biology, Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, 69120
Heidelberg, Germany
§Heidelberg University Biochemistry Center, University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
⊥Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan
1, 3584 CL Utrecht, The Netherlands
#Institute of Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Im Neuenheimer Feld 364, 69120
Heidelberg, Germany
S
* Supporting Information
ABSTRACT: New brefeldin A (1) analogues were obtained by introducing a variety of substituents at C15. Most of the
analogues exhibited significant biological activity. (15R)-Trifluoromethyl-nor-brefeldin A (3), (15R)-vinyl-nor-brefeldin A (5),
their epimers 4 and 6 as well as (15S)-ethyl-nor-brefeldin A (2) were prepared from the key building blocks 12 or 24 by Julia−
Kocienski olefination with tetrazolyl sulfones and subsequent macrolactonization. The vinyl derivative 5 allowed analogues to be
synthesized by hydroboration and Suzuki−Miyaura coupling. The following biological properties were assessed: (a) inhibition of
cell growth of human cancer cells (NCI), (b) induction of morphological changes of the Golgi apparatus of plant and mammalian
cells, and (c) influence on the replication of the enterovirus CVB3. Furthermore, conformational aspects were studied by X-ray
crystal structure analysis and molecular mechanics calculations, including docking of the analogues into the brefeldin A binding
site of an Arf1/Sec7-complex.
accompanied by its biogenetic precursor brefeldin C and the
oxidation product 7-dehydrobrefeldin A.3 Brefeldin A displays a
variety of biological properties, including antifungal,4 antiviral,5
antinematodal,6 and antimitotic7 activities. Most important,
cytostatic activity through apoptosis against numerous human
cancer cell lines was found and prompted 1 to be a target for
biomedical chemistry.8,9
INTRODUCTION
■
Brefeldin A (1) (Figure 1)1 is a secondary metabolite of
Ascomycetes species. It was first isolated in 1958 by Singleton et
al.2 from Penicillium decumbens. Brefeldin A is usually
Another aspect of 1 has led to its use as a reagent of
considerable importance for biomedical research. Treatment of
eukaryotic cells with 1 induces a reversible morphological
disruption, in which the cis Golgi apparatus redistributes into
the endoplasmic reticulum (ER), an effect that is caused by the
breakdown of vesicle-mediated protein transport between both
organelles.10 The underlying event involves binding of 1 to a
protein complex that is responsible for vesicle budding. This
consists of the small G-protein Arf1 (ADP ribosylation factor
1) and a guanidine exchange factor (GEF).11,12 The GDP/GTP
Received: April 25, 2013
Published: June 27, 2013
Figure 1. Natural brefeldins.
© 2013 American Chemical Society
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exchange in Arf1 is catalyzed by Sec7 domain of the GEF.
Brefeldin A acts as a noncompetitive inhibitor by binding
reversibly at the interface of both proteins. In 2003, Cherfils et
al.13 and Goldberg et al.14 published X-ray crystal structures of
the Arf1/brefeldin A/Sec7-complex and furnished insights into
the binding mode of 1.
Although 1 exhibits strong cytostatic effects paired with low
toxicity in mice (LD50 > 200 mg/kg, intraperitoneal),4 it could
not be established as an anticancer drug because of its low
bioavailability and poor pharmacokinetics.15 Subsequent to our
previous synthesis of 1 (see below), we decided to synthesize
new analogues of 1 that could provide better in vivo stability
and solubility, perhaps even possessing improved cytostatic
activity when compared with 1. The new analogues were
submitted to the National Cancer Institute (NCI), USA, for
screening in their Development Therapeutics Program.16
Furthermore, the effect on the Golgi apparatus in plant and
mammalian cells and replication inhibition of the enterovirus
CVB3 were investigated. The analogues were computationally
assessed by molecular modeling and docking into the X-ray
structure of the Arf1/brefeldin A/Sec7-complex.
A number of analogues of 1 have been reported in
conjunction with anticancer investigations. The most successful
approach is due to Cushman et al. who developed several
classes of prodrugs, which in the end yield 1 itself as active
principle.17b−e A second approach was alteration of the
structure of 1 itself. Most of the compounds generated in this
way showed no or reduced biological activity compared with
1.17 There is only one exception, 15-nor-brefeldin A, which was
synthesized by our group;17h this compound showed significant
activity in tests at the NCI.8b This prompted us to further
explore structural variations at C15. It was decided to begin
with the new analogues listed in Figure 2.1
Figure 3. Retrosynthetic analysis (Σ: protecting group).
converted into the enantiomerically enriched alcohol 8 by
asymmetric addition19 of diethylzinc using (−)-DBNE20 as
chiral ligand. At a reaction temperature of 0 °C, 8 was obtained
in 88% yield with 87% ee. TBS-deprotection followed by
Mitsunobu reaction with 1-phenyl-1H-tetrazole-5-thiol and
protection of the secondary hydroxyl group gave the sulfide
10, which furnished the tetrazolyl sulfone 11 by Mo(VI)-
catalyzed oxidation.21
The Julia−Kocienski olefination of tetrazolyl sulfone 11 and
the from 1 derived aldehyde 1217g,22 proceeded with essentially
complete stereocontrol to give the E-olefin 13 in 73% yield
(Scheme 2).23 Selective deprotection gave the hydroxy ester 14,
which was saponified to the corresponding hydroxy acid, which
was then subjected to macrolactonization under Yamaguchi
conditions, yielding 15 in excellent yield of 88%.24 MEM-
deprotection with concentrated HBr17h,25 and purification by
recrystallization furnished 2 in 75% yield.
Synthesis of (15R)- and (15S)-Trifluoromethyl-nor-
brefeldin A (3, 4). Considering that selective substitution of
hydrogen by fluorine can affect both pharmacokinetics and
pharmacodynamics of a bioactive compound dramatically,26 we
synthesized an analogue of 1 with the 15-CH3 group exchanged
for a CF3 group (Scheme 3). Using our general strategy, we
first prepared the tetrazolyl sulfone 21, starting with the
commercially available diol 16. A Mitsunobu reaction followed
by a Swern oxidation yielded the aldehyde 18. Reaction of 18
with the Ruppert−Prakash reagent and a catalytic amount of
TBAF furnished the racemic alcohol 19.27 Subsequent
oxidation of the sulfide 19 and TES-protection of the secondary
alcohol 20 provided the racemic building block 21.
The synthesis of the second building block, 24, is described
in Scheme 4. We decided to use TBS rather than MEM as
protecting group in order to simplify workup and NMR spectra
of the synthetic intermediates. First, TBS-protection of the
hydroxyl groups of 1 to give 22 was carried out as described by
Cushman and co-workers.17c Subsequent saponification and
esterification with diazomethane gave the hydroxy ester 23.
This was subjected to selective oxidative cleavage with OsO4/
periodate to furnish the aldehyde 24 in excellent yield.
The building blocks 21 and 24 were combined by a Julia−
Kocienski olefination under Barbier-type conditions to provide
compound 25 (Scheme 5). Selective TES-deprotection in the
presence of the OTBS groups was accomplished with the
triethylamine trihydrofluoride complex to yield hydroxy ester
26 in 90% yield. Saponification and macrolactonization under
Figure 2. Brefeldin A analogues.
Our syntheses are based on a strategy, see Figure 3, which we
have recently reported.17g Key intermediate is the aldehyde A
that is derived from 1 by saponification and oxidative cleavage
of the lower side chain with OsO4/periodate. Aldehyde A can
be combined with various tetrazolyl sulfones B in a convergent
and stereoselective manner by Julia−Kocienski olefination. A
subsequent macrolactonization usually proceeds with good
yield. A particularly versatile early target was (15R)-vinyl-nor-
brefeldin A (5), from which further derivatives could be
efficiently generated in a late stage of their synthesis via
addition reactions and Suzuki coupling.
RESULTS AND DISCUSSION
■
Synthesis of (15S)-Ethyl-nor-brefeldin A (2). The
synthesis of the requisite side chain precursor, the tetrazolyl
sulfone 11, is described in Scheme 1. The aldehyde 7, accessible
in two steps from commercially available 1,5-pentanediol,18 was
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a
Scheme 1
a
Reagents and conditions: (a) Et2Zn, (−)-DBNE, toluene, 0 °C, 24 h, 87% ee; (b) HCl, THF, rt, 20 min; (c) (i) PPh3, DEAD, 1-phenyl-1H-
tetrazole-5-thiol, THF, 0 °C to rt, 16 h, (ii) TBS-Cl, imidazole, DMAP, CH2Cl2, 0 °C to rt, 3 h; (d) (NH4)6Mo7O24·4H2O, H2O2, EtOH, rt, 16 h.
a
Scheme 2
a
Reagents and conditions: (a) compound 11, KHMDS, 1,2-dimethoxyethane, −78 °C to rt, 18 h; (b) HCl, THF, rt, 2 h; (c) (i) LiOH, THF/H2O,
rt, 2 h, (ii) 2,4,6-trichlorobenzoylchloride, NEt3, THF, rt, 1.5 h, (iii) DMAP, toluene, reflux, 5 h; (d) (i) conc HBr, THF, rt, 1.5 h, (ii)
recrystallization.
a
Scheme 3
a
Reagents and conditions: (a) PPh3, DIAD, 1-phenyl-1H-tetrazole-5-thiol, THF, 0 °C to rt, 16 h; (b) (COCl)2, DMSO, NEt3, CH2Cl2, −78 °C, 45
min; (c) CF3−SiMe3, TBAF, THF, 0 °C to rt, 1 h; (d) (NH4)6Mo7O24·4H2O, H2O2, EtOH, rt, 16 h; (e) TES-Cl, imidazole, DMAP, CH2Cl2, rt, 20
min.
a
Scheme 4
a
Reagents and conditions: (a) TBS-Cl, imidazole, DMAP, CH2Cl2, 40 °C, 20 h; (b) (i) KOH, MeOH, 40 °C, 30 min, (ii) CH2N2, EtOAc, rt; (c) (i)
K2OsO4·2H2O, NMO, acetone/H2O, rt, 3 h, (ii) NaIO4, THF/H2O, rt, 2 h.
Yamaguchi conditions24 furnished the epimers 27 and 28,
which could be separated by HPLC. Standard deprotection
gave the target compounds 3 and 4, respectively. The relative
configuration of the epimers at C15 could be assigned by a X-
ray crystal structure analysis of 3 (Figure 4). Structural
assignment of C15-epimers is also generally possible by H
NMR because for all cyclic compounds there are characteristic
differences in chemical shifts (see below).
Synthesis of (15R)- and (15S)-Vinyl-nor-brefeldin A (5,
6). (15S)-Ethyl-nor-brefeldin A (2) displayed promising
anticancer activity in tests at NCI (see below). Thus,
exploration of the corresponding vinyl derivative, (15R)-vinyl-
nor-brefeldin A (4), was of interest; this compound was also
useful as relay for late stage syntheses of further analogues of 1.
The synthesis of 4 relied on a new procedure for
enantioselective synthesis of branched allyl alcohols, an
iridium-catalyzed allylic hydroxylation, developed by our
1
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a
Scheme 5
a
Reagents and conditions: (a) KHMDS, 1,2-dimethoxyethane, −78 °C to rt, 16 h; (b) NEt3·3HF, CH3CN, rt, 30 min; (c) (i) NaOH, THF/MeOH,
60 °C, 1 h, (ii) 2,4,6-trichlorobenzoylchloride, NEt3, THF, rt, 2 h, (iii) DMAP, toluene, reflux to rt, 16 h, (iv) separation of 27 and 28 by HPLC; (d)
NEt3·3HF, CH3CN, 60 °C, 18 h.
15-(2-Arylethyl)-nor-brefeldin A Derivatives 39 via
Suzuki-Miyaura Coupling. Both (15S)-ethyl-nor-brefeldin A
(2) and (15R)-vinyl-nor-brefeldin A (5) showed good
cytostatic activity in the NCI assessment (see below), it was
therefore of interest to further vary the substituent at C15.
First, aryl groups were introduced by Suzuki−Miyaura coupling
between di-TBS-protected (15R)-vinyl-nor-brefeldin A (36)
and aryl halides. The target compounds and yields are listed in
Table 1. The crucial step of these syntheses was the preparation
of a suitable borane by hydroboration of 36, selectively at the
vinyl group rather than double bond Δ10,11. This problem was
Figure 4. X-ray crystal structure of compound 3.
solved by reaction of 36 with the bulky borane 9-BBN at 70 °C
group.28 We decided to apply this procedure in a catalyst-
controlled diastereoselective manner to the brefeldin A-derived
for 5 min; the subsequent Suzuki-Miyaura coupling gave 38a−f
in moderate to good overall yield. Deprotection under our
intermediate 33 (Scheme 7). The side-chain precursor of this
standard conditions gave the target compounds 39a−f.
compound was prepared as follows (Scheme 6). Aldehyde 18
was subjected to a HWE olefination to give the enoate 29,
which was reduced to the allylic alcohol 30. Subsequent
carbonate formation and oxidation of the sulfide 31 provided
nor-Brefeldin A Derivatives with Polar Substituents at
C15. The di-TBS-protected (15R)-vinyl-nor-brefeldin A (36)
allowed a broad spectrum of compounds considerably more
polar than the previous ones to be prepared. The synthesis of
the tetrazolyl sulfone 32 in excellent overall yield.
the diastereomeric diols 42 and 43 was carried out first
(Scheme 8) in order to obtain compounds more soluble in
water than 1. Asymmetric dihydroxylation (AD) reaction29
using an osmium complex of a bulky cinchona alkaloid was
expected to favor reaction at the vinyl group. With Sharpless’
commercially available AD-mix-β (containing (DHQD)2PHAL
as chiral ligand),30 diastereoisomers 40 and 41 were obtained in
overall yield of 44% in a ratio of ca. 3:1, respectively. The
diastereoisomers were separated by HPLC. Deprotection and
recrystallization gave pure diols 42 and 43.
Julia-Kocienski olefination of 24 with 32 under Barbier-type
conditions yielded the methyl carbonate 33 E-selectively in 84%
yield. Next the aforementioned asymmetric iridium-catalyzed
allylic hydroxylation was applied, using (π-allyl)Ir-complexes
C3 (Figure 5) or ent-C3 as catalysts;28 alcohols 34 and 35 were
generated in high yield and diastereoselectivity of 95:5 and
5:95, respectively. Saponification followed by macrolactoniza-
tion gave the diastereoisomers 36 and 37, which were subjected
to the standard deprotection already described to give the
diastereomeric target compounds 5 and its epimer 6,
respectively.
The diol 40 was used to access (15R)-(hydroxymethyl)-nor-
brefeldin A (46) (Scheme 9). The aldehyde 44 is prone to
a
Scheme 6
a
Reagents and conditions: (a) NaH, (EtO)2P(O)CH2O2Et, THF, −78 °C to rt, 14 h; (b) DIBAL-H, THF, −78 °C, 2.5 h; (c) ClCO2Me, pyridine,
CH2Cl2, 0 °C to rt, 1 h; (d) (NH4)6Mo7O24·4H2O, H2O2, EtOH, 0 °C to rt, 16 h.
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a
Scheme 7
a
Reagents and conditions: (a) KHMDS, 1,2-dimethoxyethane, −78 °C to rt, 18 h; (b) C3 (Figure 5) or ent-C3, KHCO3, DMF/H2O, rt, 3 h; (c) (i)
NaOH, THF/MeOH, 60 °C, (ii) 2,4,6-trichlorobenzoylchloride, NEt3, THF, rt, 2.5 h, (iii) DMAP, toluene, reflux; (d) NEt3·3HF, CH3CN, 60 °C,
15 h.
Table 1. Preparation of Aryl Derivatives 38a−f and 39a−f via
a
Suzuki−Miyaura-Type Coupling and Deprotection
Figure 5. (π-Allyl)Ir-complex C3.
epimerization. This could be suppressed by carrying out
periodate cleavage with 40 in a phosphate buffer system. The
aldehyde was reduced with NaBH4 to give 45 cleanly after 2
min and in 88% yield. A short reaction time is necessary in
order to prevent intramolecular transesterifiaction (see below).
Finally, standard TBS-deprotection and recrystallization gave
triol 46 in excellent yield. The homologous alcohol 48 was
prepared from the vinyl derivative 36 by hydroboration with 9-
BBN and oxidation, followed by standard desilylation (Scheme
10).
With alcohol 47 in hand, it appeared logical to generate
ethers such as 51 and 52 (Scheme 11). This turned out to be
more difficult than anticipated because of facile intramolecular
transesterification (see below). After some experimentation, the
following procedures gave good results. The methyl ether 49
was obtained by treating a solution of 47 in methyl iodide with
silver(I)-oxide, according to a procedure described by Greene
et al.31 for a total synthesis of 1. The benzyl ether 50 was
similarly prepared in good yield by treating 47 in BnBr as
solvent with a catalytic amounts of TBAI. Standard
deprotection gave brefeldin A ether analogues 51 and 52 in
high yield.
a
Reagents and conditions: (a) (i) 9-BBN, THF, 70 °C, 5 min, (ii) Ar-
X, Pd(dppf)Cl2, Ph3As, Cs2CO3, DMF/H2O, rt or 50 °C; (b) (i)
NEt3·3HF, CH3CN, 60 °C, 15 h, (ii) recrystallization. Reagents and
b
The intramolecular transesterification mentioned above
occurred upon attempted etherification of alcohol 47 with
NaH and BnBr, which gave only a very low yield of the benzyl
ether but mainly gave rise to the ring expanded secondary
alcohol 53, which was obtained in almost quantitative yield
after optimization of the reaction conditions (Scheme 12).
Standard deprotection gave the triol 54, which was
characterized by X-ray crystal structure analysis (Figure 10C).
Given di-TBS-protected compound 36, it was straightfor-
ward to synthesize 15-acetyl-nor-brefeldin A (56) via a Wacker
conditions: (i) HCl, MeOH, 40 °C, 2 h, (ii) recrystallization.
oxidation32 (Scheme 13), anticipating that reaction at the
secondary atom of the vinyl group would be favored. The
Wacker oxidation was carried out with palladium(II)-chloride,
copper(I)-chloride and oxygen to give methyl ketone 55 in 41%
yield after 23 h at rt. In addition, the corresponding aldehyde
was isolated in 17% yield. Deprotection of 55 with triethyl-
amine trihydrofluoride gave the methyl ketone 56 in 87% yield.
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a
a
Scheme 8
Scheme 11
a
a
Reagents and conditions: (a) (i) AD-Mix-β, MeSO2NH2, t-BuOH/
Reagents and conditions: (a) Ag2O, MeI, rt, 7.5 h or Ag2O, TBAI,
BnBr, rt, 7 h; (b) (i) NEt3·3HF, CH3CN, 60 °C, 15 h, (ii)
recrystallization.
H2O, rt, 16 h, (ii) separation of 40 and 41 by HPLC; (b) with 40, (i)
NEt3·3HF, CH3CN, 60 °C, 17 h, (ii) recrystallization; with 41, (i)
HCl, THF, 40 °C, 2 h, (ii) recrystallization.
a
Scheme 12
a
Scheme 9
a
Reagents and conditions: (a) NaIO4, THF/0.25 M NaH2PO4/
a
Na2HPO4 (pH 7), rt, 1 h; (b) NaBH4, THF/H2O, rt, 2 min; (c) (i)
NEt3·3HF, CH3CN, 60 °C, 15 h, (ii) recrystallization.
Reagents and conditions: (a) NaH, THF, 0 °C, 30 min; (b) (i)
NEt3·3HF, CH3CN, 60 °C, 15 h, (ii) recrystallization.
a
a
Scheme 10
Scheme 13
a
Reagents and conditions: (a) (i) 9-BBN, THF, 70 °C, 5 min, (ii)
a
Reagents and conditions: (a) PdCl2, CuCl, O2, N,N-dimethylaceta-
mide/H2O, rt, 23 h; (b) (i) NEt3·3HF, CH3CN, 60 °C, 15 h, (ii)
recrystallization.
NaOH, H2O2, 0 °C, 20 min; (b) (i) NEt3·3HF, CH3CN, 60 °C, 15 h,
(ii) recrystallization.
a
The analogues derived by Suzuki−Miyaura coupling showed
high biological activity. It appeared of interest to add an amide
group to the aryl moiety in order to increase polarity and
hydrogen bonding capacity (Scheme 14). Accordingly the
amine 38d was acylated with acetyl chloride to furnish amide
57 after 5 min in 89% yield. Standard deprotection gave the
amide 58 in good yield.
Scheme 14 .
Biological Properties of the Brefeldin A Analogues.
Anticancer Activity Tested at NCI. Except for 39f, the new
analogues of 1 were submitted to the National Cancer Institute
(NCI, Maryland, USA) of the U.S. National Institutes of Health
for evaluation of their anticancer activity within the
Developmental Therapeutics Program (DTP). The substances
were first applied to 60 human cancer cell lines including lung,
colon, CNS, ovarian, renal, prostate, and breast cancer cell lines
a
Reagents and conditions: (a) MeCOCl, NEt3, THF, 0 °C, 5 min; (b)
(i) NEt3·3HF, CH3CN/MeOH, 60 °C, 15 h, (ii) recrystallization.
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Table 2. Cytostatic/Cytotoxic Activity of the Active Analogues and 1 against Human Cancer Cell Lines
a
b
c
Compounds that were only assessed by a 1-dose test are not included. Internal ID number of the NCI. Mean values of two different 5-dose tests.
Values of one 5-dose test.
d
as well as leukemia and melanoma cells at a high concentration
of 10 μM (1-dose test). Compounds displaying significant
activity were then tested at five different concentrations on the
same cells (5-dose test), establishing the parameters GI50
(growth inhibition 50%; concentration of drug at which cell
proliferation is inhibited by 50%), TGI (total growth inhibition;
concentration of drug at which cell proliferation is inhibited by
100%), and LC50 (lethal dose 50%; concentration of drug at
which 50% of cell population is killed).16 Table 2 provides the
results of the five-dose tests as averages of all cell lines. Detailed
information, for instance the activities for each cell line, will also
soon be published in the database of the NCI.8b As an example,
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the five-dose test results for the highly active analogue 5 are
given in the Supporting Information.
30 min results in a fusion of the Golgi with the ER with the
Golgi marker then becoming distributed throughout the ER
network and nuclear envelope (Figure 6B). The same
phenotype was observed after application of the analogues
39a, 39c, 58 and 39e (Figure 6C−F). Similarly, the analogues 2
and 5 when applied to Arabidopsis roots elicited the same
phenotype as 1 itself, namely the formation of a “brefeldin A-
compartment“ with large central core of TGN elements and
surrounding Golgi stacks (compare A with B−D in Figure 7).
The analogues 2, 5, 39a, and 39d are highly active, reducing
cell growth of colon cancer cells, renal cancer cells and
melanoma cells at a comparable concentration as 1 does.
Moreover, these derivatives display a stronger anticancer effect
than 1 on breast cancer cell lines. Slightly lower activities were
determined for the naphthyl-derivative 39b, 51, and 52. (15R)-
Trifluoromethyl-nor-brefeldin A (3), 39c, 39e, 48, and 58 are
less active than 1, requiring a 10-fold higher concentration to
achieve the same growth inhibition as 1. Compounds 6, 46, 56,
and nor-brefeldin A,17h previously synthesized in our group,
cause inhibition only at higher concentrations. Almost inactive
are the compound 4, the tetrols 42 and 43, and the ring
expanded analogue 54 that were not selected for the five-dose
screen and are not listed in the table. The same is also true for
the (6R)-hydroxybrefeldin A, (7S)-aminobrefeldin C, and the
lactam analogue of 1 previously synthesized in our
laboratory.17g,33
Effects of Brefeldin A Analogues on Plant Cells. As
previously carried out,34 we have screened the analogues for
brefeldin A activity on tobacco leaf protoplasts transiently
expressing the fluorescent cis Golgi marker protein Man1-RFP,
and on root cells of an Arabidopsis line stably expressing the
trans Golgi marker ST-YFP and the trans Golgi network
(TGN) marker VHAa1-RFP. As seen in Figure 6, under control
conditions the Golgi marker in protoplasts assumes a punctate
appearance corresponding to the hundreds of Golgi stacks
normally present in a plant cell (Figure 6A). Addition of 1 for
Figure 7. Effects of 1 (B) and brefedlin A analogues 2 (C) and 5 (D)
on Arabidopsis root cells stably expressing the Golgi marker ST-YFP
and the TGN marker VHAa1-RFP. Roots were treated with 90 μM of
1 (B) and analogues of 1 (C,D) for 30 min before observing in the
CLSM. The control (A) shows the normal Golgi and TGN
distribution in plant cells. Magnification bars (A−D) = 5 μm.
Golgi Disruption in Mammalian Cells. To study Golgi
disassembly, HeLa cells were incubated for either 30 or 60 min
in the presence of the various drugs. Cells were fixed and
processed as described in the Experimental Section. Visual-
ization of cis Golgi structures was performed using an anti-
GM130 antibody. Reassembly studies were performed after 60
min incubation in the presence of 1 or the indicated analogues,
followed by an additional 2 h incubation in medium in the
absence of drug. Immunolabeled cells were analyzed by
confocal microscopy (Figure 8). While all drugs tested induced
Golgi disassembly, only after removal of substance 39e was a
Golgi reassembly comparable to 1 observed. Substance 39a and
39b allowed partial recovery of Golgi structure, whereas the
analogue 5 induced an irreversible Golgi disruption.
Replication Inhibition of the Enterovirus CVB3. Brefeldin A
is a well-known inhibitor of the replication of enteroviruses.35,36
Enteroviruses are a large group (>250 members) of viruses,
which includes among others poliovirus (the causative agent of
poliomyelitis), the rhinoviruses (common cold), and the
coxsackieviruses (the main cause of viral myocarditis,
pancreatitis, and meningitis). Enteroviruses have a single-
stranded RNA genome of positive-orientation, which is
replicated on reorganized intracellular membranes by assem-
blies of viral proteins and hijacked cellular proteins.37,38 These
host proteins include GBF1,39−41 which is inhibited by 1.42,43
To test whether our analogues could also inhibit enterovirus
replication, we infected cells with a recombinant coxsackievirus
B3 (CVB3) that carried a Renilla luciferase gene41 and
Figure 6. Effects of 1 (B) and analogues 39a, 39c, 58, and 39e (C−F)
on tobacco mesophyll protoplasts transiently expressing the cis Golgi
marker Man1-RFP. Protoplasts were treated with 90 μM of 1 and
analogues of 1 for 30 min before observing in the CLSM. The control
(A) shows the normal Golgi distribution in plant cells. Magnification
bars (A−F) = 5 μm.
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As shown in Figure 9, we found that at 5 and 50 μM all
tested compounds inhibited replication of CVB3 to about the
same extent as 1. Only at 0.5 μM, several analogues inhibited
replication to about one log value less than 1 did, except for the
analogue 5, which was about one log more potent than 1, and
39e, which inhibited virus replication clearly less potent than 1
at both 5 and 0.5 μM. This effect on replication was specific and
not due to toxicity of the compounds, since at the tested
concentrations none of the compounds cause a notable
reduction of cell viability (see Supporting Information). Thus,
all analogues of 1 tested have retained the capability to inhibit
the replication of enteroviruses. The vinyl derivative 5 also
shows particularly strong inhibition of cancer cell growth,
whereas 39e displayed a less strong inhibition of cancer cell
growth.
Crystal Structures and NMR Spectra of the Brefeldin A
Analogues. X-ray crystal structures of the analogues 2, 3, 5, 6,
39a, 42, 48, and 54 were determined in order to compare the
conformations of the new analogues with that of 1. Super-
impositions of the structures of a representative set of
analogues with the structure of 1 are displayed in Figure 10.
For derivative 5, a very close fit is apparent (Figure 10A), with a
root-mean-square deviation (rmsd) of only 0.03 Å (based on
the core atoms). This is also true for the analogues 2, 3, 39a,
42, and 48, which only differ from 5 in the substituent at C15.
However, by changing the configuration at C15, as in analogue
6, both the conformation of the five- and the 13-membered ring
are strongly affected, leading to a much increased rmsd of 0.57
Å (Figure 10B). The major change is seen for the upper, polar
side chain of the 13-membered ring. While these derivatives
show the enoate moiety (OC−CC) in the natural
Figure 8. HeLa cells were treated with 1 and the analogues 5 and 39e.
Control cells were incubated in medium. After 60 min incubation of
cells in the presence of drug, wash-out experiments were performed
after removal of the drug by incubating cells for additional 2 h in
medium without drug. Cells were immunolabeled with an anti-GM130
antibody and visualized by confocal microscopy.
subsequently incubated the cells for 7 h in the presence or
absence of compound to allow the replication of the viral
genome. Upon viral genome replication, luciferase activity
increases exponentially and can be readily assessed as a measure
of viral genome replication. At a concentration of ∼5 μM, 1
normally causes a nearly complete inhibition of replication. To
assess the potency of the compounds, we tested this
concentration and a 10-fold higher and lower concentration.
Figure 9. HeLa R19 cells were infected with RLuc-CVB3 virus as described in the Experimental Section procedures and treated with the analogues, 1
or a corresponding dilution of DMSO as a negative control for 7 h. Cells were lysed and the Renilla luciferase activity as a measure of virus replication
was measured. Luciferase activity is expressed relative to the control with the same DMSO dilution and shown on a logarithmic scale. Triplicates
measurements are shown as means SEM. Like 1, at the concentrations used, the analogues do not considerably affect the viability of the cells
within the time frame of the experiment (see Supporting Information).
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Arf1/brefeldin A/Sec7-complex from the PDB48 (ID code:
1RE014) was prepared for docking using the additional
programs of the GOLD package. Thus, the ligand 1 was
removed from the binding pocket, a new binding pocket was
defined (CZ of TYR81, radius 10 Å), and different rotamers
were allowed for four amino acids (ARG19, TRP66, TYR81,
VAL270), which are thought to be important with regard to the
interactions with the substituents at C15 of the analogues.
Furthermore, water molecules in this region and in the vicinity
of the binding site were defined as flexible. Otherwise, the
default settings of the GOLD package were used.
The analogues 2 and 5 scored similar docking scores as 1. All
the other analogues with the natural configuration at C15
achieved much higher docking scores. These analogues were
docked in a position and orientation (pose) that resembles the
pose of 1 in the X-ray structure. As example, docking results for
(15R)(2-phenylethyl)-nor-brefeldin A (39a) are described in
Figure 11 (left); the first three docked poses are superposed.
Figure 10. Superpositions of the X-ray crystal structures of different
brefeldin A analogues (purple) and 1 (gray). (A) Analogue 5; (B)
analogue 6; (C) analogue 54.
occurring s-trans conformation, conformation with respect to
the bonds C3−C4 and C4−C5 differ considerably. The ring
expanded compound 54 (Figure 10C) is conformationally
similar to 1 around the five-membered ring, while the
conformation of the macrocycle differs considerably.
Figure 11. Close-up view of the X-ray crystal complex (PDB ID code:
1RE0) consisting of 1 (purple) in the binding pocket between Arf1
(green ribbon) and Sec7 (yellow ribbon). Left, overlay of the first
three docked poses of 39a (turquoise); right, overlay of the docked
pose of 6 (turquoise).
The differences in the conformational situation of the C15-
1
epimers 3 and 4 as well as 5 and 6 are reflected by their H
NMR spectra (see Supporting Information). Thus, for the
analogues 3 and 5, possessing the same configuration as 1, one
of the diastereotopic protons at C-13 resonates at <1 ppm and
the olefinic proton at C-3 at 7.5 ppm, while for the epimers 4
and 6 the signal of the 13-H is found at 1.4−1.5 ppm and that
of the 3-H at 6.8 ppm.
Molecular Modeling and Protein−Ligand Docking
with the Brefeldin A Analogues. We also investigated the
structures of all new analogues by molecular modeling, using
the MacroModel44 software and a procedure developed by Still
et al. that is based on the mmffs force field and a Monte Carlo
Multiple Minimum (MCMM) search; they applied this
procedure to 7-dehydro-brefeldin A for validation.45
Three conformers with different orientation of the aryl group
are accepted, implicating that bulky residues at C15 are well
tolerated.
The compounds with the non-natural configuration at C15,
4, 6, and the ring expanded 54 gave much lower docking scores
than 1. These compounds show an anomalous pose, as
illustrated by analogue 6 (Figure 11, right).
Relationship of Structure and Cytostatic Activity.
Several classes of the analogues of 1 can be distinguished (cf.
Table 2).
The analogues 2, 5, 39a, and 39b, carrying a nonpolar
substituent at C15, are highly active compounds, provide high
docking scores, and also show a conformation and docked pose
similar to 1. Even bulky residues at C15, i.e., the 1-naphthyl
moiety in 39b, are well accepted.
The compounds 4 and 6, with the non-natural configuration at
C15, and the ring expanded compound 54 are nearly inactive.
The calculated low energy conformers of these compounds
differ considerably from that of 1, and they possess a low
docking score and abnormal docking pose.
The alcohols 42 and 43 with the highly polar 1,2-
dihydroxyethyl substituent at C15 display extremely low
activity. Very low activity was also obtained for 46, carrying a
hydroxymethyl group at C15, and low activity was found for 48,
and 56, with a 2-hydroxyethyl- and an acetyl group,
respectively, at C15. All these analogues provide high docking
scores and conformational similarity to 1. Accordingly,
conformational similarity to 1 appears as necessary but not as
sufficient condition for cytostatic activity.
As observed in our previous investigation,17g the calculated
minimal energy structures and the X-ray crystal structures of
the analogues are superposable for the macrocyclic moiety,
while conformers differ for the cyclopentane ring. For this, the
conformer with an equatorial 7-OH group is generally preferred
in the calculations, while in all crystal structures determined by
us the conformer with an axial 7-OH group pertains. However,
the energy difference between these conformers is small,
calculated as only 0.79 kJ/mol for 1. Inspection of the crystal
structure of 1 indicates that hydrogen bonding in the crystal is
responsible for the preference of the axial conformer (cf.17g).
The conformer with an equatorial 7-OH group was found in
the crystal structure of the Arf1/brefeldin A/Sec7-complex.
Docking experiments were carried out with the calculated
conformers of lowest energy, using GOLD 5.146 with
ChemPLP as scoring function and the UCSF Chimera
package47 for visualization. The X-ray crystal structure of the
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with 4% paraformaldehyde in phosphate buffered saline (PBS) for 20
min at rt. Following a 20 min incubation in 50 mM ammonium
chloride at rt, cells were permeabilized for 5 min at rt with 0.5%
Triton-X-100 in PBS. Blocking was performed for 15 min at rt in 5%
BSA in PBS. Cells were then incubated with an anti-GM130 antibody
(BD Transduction Laboratories) for 30 min at rt. As secondary
antibody Alexa Flour 546-coupled-goat-antimouse (Invitrogen, cat. no.
A11030) was used. After each step, cells were washes twice for 5 min
in PBS. Cells were mounted in Prolong Gold antifade mounting
reagent (Invitrogen) and stored at 4 °C. Immunolabeled cells were
protected against light. Immunofluorescence was then analyzed with a
Zeiss LSM510 confocal microscope. A Plan-Apochromat 63×/1.4 Oil
DIC objective was used to visualize the cells. The images were
processed by FIJI ImageJ 1.46j.
Replication Inhibition of the Enterovirus CVB3. Subconfluent
monolayers of HeLa R19 cells were infected for 30 min at 37 °C with a
Renilla luciferase-expressing Coxsackievirus B3 (RLuc-CVB3)41 at a
multiplicity of infection (MOI) of 0.1 CCID50 (50% cell culture
infectious dose). After infection, the virus dilution was replaced by
medium with 0.5, 5, or 50 μM of compound (from a 5 mM stock in
DMSO) or the same amount of DMSO only as a negative control.
After 7 h, the cells were processed either to measure the amount of
luciferase produced as a measure for replication using a Renilla
luciferase assay system (Promega, Madison (WI), USA), or for a
viability assay using a CellTiter 96 AQueous Non-Radioactive Cell
Proliferation Assay (Promega, Madison (WI), USA). Triplicate
measurements were normalized to the average of the negative control
wells with the same DMSO dilution.
Compounds with a large apolar substituent containing a small
polar moiety, 39c with a 7-quinolyl and 39d with a 4-
aminophenyl group, command interest. While the former is
almost inactive, the letter is highly active. However, the N-
acetyl derivative 58, derived from 39d, is inactive.
CONCLUSIONS
■
We have synthesized (15R)-trifluoromethyl-nor-brefeldin A
(3), (15R)-vinyl-nor-brefeldin A (5), their C15-epimers 4 and
6, as well as (15S)-ethyl-nor-brefeldin A (2). The vinyl
derivative 5 allowed further analogues to be generated
efficiently by hydroboration/Suzuki−Miyaura coupling and
other addition reactions. Conformational analyses were carried
out by X-ray crystal structure determinations and molecular
mechanics calculations. For all brefeldin A analogues, computa-
tional docking into the brefeldin A (1) binding site of the Arf1/
Sec7 complex was conducted.
The analogues were tested for their cytostatic effects in
human cancer cells. Activity was found for nearly all
compounds. High activity was obtained for 2 and 5 as well as
39a and 39b, bearing a phenyl and a 4-aminophenyl group at
C15, respectively. In addition, the ability of inducing
morphological changes in the Golgi apparatus in plant and
mammalian cells as well as inhibition of the replication of the
enterovirus CVB3 in HeLa cells was investigated. In most cases,
effects similar to those of 1 were found; however, the analogue
5 displayed 10-fold higher activity than 1 against the
enterovirus CVB3.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and analytical data for all compounds,
determination of enantio- or diastereoselectivities for com-
pounds 8, 34, and 35, copies of 1H NMR and 13C NMR spectra
of all compounds, NCI 5-dose test results for compound 5, X-
ray crystal data for compounds 2, 3, 5, 6, 39a, 42, 48, and 54,
cell viability during the replication inhibition of CVB3. This
material is available free of charge via the Internet at http://
pubs.acs.org.
EXPERIMENTAL SECTION
Details on the synthesis of all compounds are given in the Supporting
Information. The purity of all tested compounds was determined by
analytical HPLC to be >95%.
■
The Effects of Brefeldin A Analogues on Plant Cells. Plants of
Arabidopsis thaliana stably transformed with VHAa1-RFP/N-ST-
YFP49 were grown from surface-sterilized seeds in half-strength
Murashige and Skoog (MS) medium50 with 1% (w/v) sucrose in a
controlled climate room at 22 °C with a 16 h day length. Transient
expression in protoplasts of Nicotiana tabacum cv. Petit Havana was
done exactly as described in Langhans et al. in 2011.34 Drug
Treatment: Control experiments were performed with a standard
concentration of 90 μM 1 (stock solution 5 mg/mL 1 in DMSO) in
half MS media for 30 min or without 1 in half MS media and then
observed under the microscope. In following experiments plants were
treated 30 min with 90 μM of different brefeldin A analogues as
described before. Analogues were dissolved in DMSO. Confocal
microscopy: 80 μL protoplast solution of N. tabacum and A. thaliana
roots were transferred to slides as previously described in Langhans et
al. in 2011.34 Cells or plant material were observed under a Zeiss
Axiovert LSM510 Meta microscope using a CApochromat 63/1.2 W
corr water immersion objective for protoplasts and roots. Special
settings were designed for observing single or double expression with
different XFPconstructs. Fluorescence was detected by the Meta-
detector using main beam splitters HFT 488/543. The following
fluorophores (excited and emitted by frame switching in the single or
multitracking mode) were used: YFP (488 nm/529−550 nm) and
RFP (543 nm/593−625 nm). Pinholes were adjusted to 1 Airy Unit
for each wavelength. Postacquisition image processing was performed
using the Zeiss LSM 510 image Browser (4.2.0.121), CorelDrawX4
(14.0.0.567) and ImageJ (1.46o).
Accession Codes
The Cambridge Crystallographic Data Center (CCDC)
contains the supplementary crystallographic data for this
paper in the Cambridge Structural Database (CSD). CCDC
numbers: 925984 for compound 2, 925985 for compound 3,
925986 for compound 5, 925987 for compound 6, 925988 for
compound 39a, 925989 for compound 42, 925990 for
compound 48, and 925991 for compound 54.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49-6221-548421. Fax: +49-6221-544205. E-mail: g.
helmchen@oci.uni-heidelberg.de.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Deutsche Forschungsgemein-
schaft (Graduiertenkolleg 850) and the Dr. Rainer Wild-
Stiftung and grants from The Netherlands Organisation for
Scientific Research (NWO) (an NWO-ALW open program
grant and a VICI grant to F.v.K. and a VENI grant to J.S.). The
authors thank Patrick Rieger for his kind assistance with
cultivation of Eupenicillium brefeldianum. We thank the National
Cancer Institute (NCI, U.S. National Institutes of Health,
Golgi Disruption in Mammalian Cells. As previously
described,51 HeLa cells were cultivated in Dulbecco’s Modified Eagle
Medium (DMEM) supplemented with 10% fetal bovine serum, 100
units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine.
1 and its analogues were added to cells at a final concentration of 5
μM. Cells were incubated at 37 °C for 30 or 60 min and then fixed
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onto ARF protein. Nature 1992, 360, 350−352. (b) Helms, J. B.;
Rothman, J. E. Inhibition by brefeldin A of a Golgi membrane enzyme
that catalyses exchange of guanine nucleotide bound to ARF. Nature
1992, 360, 350−352.
Maryland, USA) for performing the anticancer screening within
their Developmental Therapeutics Program (DTP).
ABBREVIATIONS USED
■
(12) For the mechanism of vesicle budding and the function of G-
proteins in this context see: (a) Rothman, J. E.; Wieland, F. T. Protein
Sorting by Transport Vesicles. Science 1996, 272, 227−234.
9-BBN, 9-borabicyclo[3.3.1]nonane; (−)-DBNE, (1S,2R)-
(−)-2-(dibutylamino)-1-phenyl-1-propanol; DEAD, diethyl
azodicarboxylate; DIAD, diisopropyl azodicarboxylate; dppf,
1,1′-bis(diphenylphosphino)ferrocene; KHMDS, potassium
bis(trimethylsilyl)amide; MEM, methoxyethoxymethyl; NMO,
4-methylmorpholine N-oxide; TBAI, tetrabutylammonium
iodide; TES, triisopropylsilyl; TBS, tert-butyldimethylsilyl
(b) Brugger, B.; Wieland, F. T. Mechanismen der COPI-
̈
Vesikelbiogenese. Biospektrum 2004, 1, 30−33. (c) Bonifacino, J. S.;
Lippincott-Schwartz, J. Coat proteins: shaping membrane transport.
Nature Rev. Mol. Cell Biol. 2003, 4, 409−414.
(13) Renault, L.; Guibert, B.; Cherfils, J. Structural snapshots of the
mechanism and inhibition of a guanine nucleotide exchange factor.
Nature 2003, 426, 525−530.
(14) Mossessova, E.; Corpina, R. A.; Goldberg, J. Crystal Structure of
ARF1·Sec7 Complexed with Brefeldin A and Its Implications for the
Guanine Nucleotide Exchange Mechanism. Mol. Cell 2003, 12, 1403−
1411.
(15) Phillips, L. R.; Supko, J. G.; Malspeis, L. Analysis of Brefeldin A
in Plasma by Gas Chromatography with Electron Capture Detection.
Anal. Biochem. 1993, 211, 16−22.
(16) For more information see: Developmental Therapeutics Program;
Division of Cancer Treatment and Diagnostics, NCI/NIH; http://dtp.
nci.nih.gov.
REFERENCES
■
(1) In text, the common numbering for 1 is used (cf. Figures 1 and
2). This begins at the carbonyl group and continues anticlockwise
around the molecule. In the Supporting Information, for identifying
compound names and spectral data, IUPAC numbering is used.
(2) Singleton, V. L.; Bohonos, N.; Ullstrup, A. J. Decumbin. A new
compound from a species of Penicillium. Nature 1958, 181, 1072−
1073.
(3) (a) Sunagawa, M.; Ohta, T.; Nozoe, S. Biosynthesis of Brefeldin
A. Introduction of Oxygen at the C-7 Position. J. Antibiot. 1983, 36,
25−29. (b) Yamamoto, Y.; Hori, A.; Hutchinson, C. R. Biosynthesis of
Macrolide Antibiotics. 6. Late Steps in Brefeldin A Biosynthesis. J. Am.
Chem. Soc. 1985, 107, 2471−2474.
(17) For brefeldin analogues derived from 1 see: (a) Zhu, J.-W.;
Hori, H.; Nojiri, H.; Tsukuda, T.; Taira, Z. Synthesis And Activity Of
Brefeldin A Analogs As Inducers Of Cancer Cell Differentiation And
Apoptosis. Bioorg. Med. Chem. Lett. 1997, 7, 139−144. (b) Argade, A.
B.; Devraj, R.; Vroman, J. A.; Haugwitz, R. D.; Hollingshead, M.;
Cushman, M. Design and Synthesis of Brefeldin A Sulfide Derivatives
as Prodrug Candidates with Enhanced Aqueous Solubilities. J. Med.
Chem. 1998, 41, 3337−3346. (c) Argade, A. B.; Haugwitz, R. D.;
Devraj, R.; Kozlowski, J.; Fanwick, P. E.; Cushman, M. Highly Efficient
Diastereoselective Michael Addition of Various Thiols to (+)-Brefeldin
A. J. Org. Chem. 1998, 63, 273−278. (d) Fox, B. M.; Vroman, J. A.;
Fanwick, P. E.; Cushman, M. Preparation and Evaluation of Sulfide
Derivatives of the Antibiotic Brefeldin A as Potential Prodrug
Candidates with Enhanced Aqueous Solubilities. J. Med. Chem. 2001,
44, 3915−3924. (e) Anadu, N. O.; Davisson, V. J.; Cushman, M.
Synthesis and Anticancer Activity of Brefeldin A Ester Derivatives. J.
Med. Chem. 2006, 49, 3897−3905. (f) Ref 17g. For brefeldin
̈
(4) Harri, E.; Loeffler, W.; Sigg, H. P.; Stahelin, H.; Tamm, C. Uber
̈
̈
die Isolierung neuer Stoffwechselprodukte aus Penicillium brefeldia-
num DODGE. Helv. Chim. Acta 1963, 46, 1235−1243.
(5) Tamura, G.; Ando, K.; Suzuki, S.; Takatsuki, A.; Arima, K.
Antiviral activity of Brefeldin A and Verrucarin A. J. Antibiot. 1968, 21,
160−161.
̌ ́
(6) Bacíkova, D.; Betina, V.; Nemec, P. Antinematodal activity of the
antibiotic cyanein. Naturwissenschaften 1964, 51, 445.
(7) Betina, V.; Montagnier, L. Action of cyanein on the synthesis of
nucleic acids and proteins in animal cells and bacterial protoplasts.
Bull. Soc. Chim. Biol. 1966, 48, 194−198.
́ ́ ́
(8) (a) Betina, V.; Horakova, K.; Barath, Z. Anti-HeLa cell effect of
cyanein. Naturwissenschaften 1962, 49, 241. (b) DTP Data; Division of
Cancer Treatment and Diagnostics, NCI/NIH; http://dtp.nci.nih.
gov/docs/dtp_search.html (NSC for 1, 56310; NSC for 15-nor-
brefeldin A, 746708).
analogues derived by total synthesis see: (g) Forster, S.; Persch, E.;
̈
Tverskoy, O.; Rominger, F.; Helmchen, G.; Klein, C.; Gonen, B.;
̈
Brugger, B. Syntheses and Biological Properties of Brefeldin
Analogues. Eur. J. Org. Chem. 2011, 878−891. (h) Hubscher, T.;
Helmchen, G. Enantioselective Formal Synthesis of Brefeldin A and
̈
(9) (a) Shao, R. G.; Shimizu, T.; Pommier, Y. Brefeldin A is a Potent
Inducer of Apoptosis in Human Cancer Cells Independently of p53.
Exp. Cell. Res. 1996, 227, 190−196. (b) Nojiri, H.; Hori, H.; Nojima, S.
Induction of Cell Differentiation and Apoptosis into Human
Malignant Tumor Cell Lines by Drugs Affecting Ganglioside
Biosynthesis. Glycoconjugate J. 1995, 12, 459. (c) Nojiri, H.; Manya,
H.; Isono, H.; Yamana, H.; Nojima, S. Induction of terminal
differentiation and apoptosis in human colonic carcinoma cells by
brefeldin A, a drug affecting ganglioside biosynthesis. FEBS Lett. 1999,
453, 140−144.
(10) Activity of 1 in mammalian cells: (a) Fujiwara, T.; Oda, K.;
Yokota, S.; Takatsuki, A.; Ikehara, Y. Brefeldin A Causes Disassembly
of the Golgi Complex and Accumulation of Secretory Proteins in the
Endoplasmic Reticulum. J. Biol. Chem. 1988, 263, 18545−18552.
(b) Lippincott-Schwartz, J.; Yuan, L. C.; Bonifacino, J. S.; Klausner, R.
D. Rapid Redistribution of Golgi Proteins into the ER in Cells Treated
with Brefeldin A: Evidence for Membrane Cycling from Golgi to ER.
̈
Analogues via Anionic Cyclization of an Alkenyl Epoxide. Synlett 2006,
1323−1326. (i) Forster, S.; Helmchen, G. Stereoselective Synthesis of
̈
a Lactam Analogue of Brefeldin C. Synlett 2008, 831−836. (j) Paek, S.-
M.; Seo, S.-Y.; Min, K.-H.; Shin, D. M.; Chung, Y. K.; Suh, Y.-G.
Synthesis of Lactam Analogue of 4-epi-Brefeldin A. Heterocycles 2007,
71, 1059−1066. (k) Gao, J.; Huang, Y.-X.; Wu, Y. Enantioselective
total synthesis of 13-O-brefeldin A. Tetrahedron 2008, 64, 11105−
11109. (l) Archambaud, S.; Aphecetche-Julienne, K.; Guingant, A. A
New Total Synthesis of (+)-Brefeldin C. Synlett 2005, 139−143.
(m) Archambaud, S.; Legrand, F.; Aphecetche-Julienne, K.; Collet, S.;
Guingant, A.; Evain, M. Total Synthesis of (+)-Brefeldin C, (+)-nor-
Me Brefeldin A and (+)-4-epi-nor-Me Brefeldin A. Eur. J. Org. Chem.
2010, 1364−1380. (n) Inai, M.; Nishii, T.; Mukoujima, S.; Esumi, T.;
Kaku, H.; Tominaga, K.; Abe, H.; Horikawa, M.; Tsunoda, T. Total
Synthesis of (+)-Brefeldin C Utilizing Aza-Claisen Rearrangement.
Synlett 2011, 1459−1461.
Cell 1989, 56, 801−813. Activity of 1 in plant cells: (c) Nebenfuhr, A.;
̈
Madison, S. L. Live-Cell Imaging of Dual-Labeled Golgi Stacks in
Tobacco BY-2 Cells Reveals Similar Behaviors for Different Cisternae
during Movement and Brefeldin A Treatment. Mol. Plant 2011, 4,
(18) Kozikowski, A. P.; Shum, P. W.; Basu, A.; Lazo, J. S. Synthesis of
structural analogues of lyngbyatoxin A and their evaluation as
activators of protein kinase C. J. Med. Chem. 1991, 34, 2420−2430.
(19) Reviews: (a) Soai, K.; Niwa, S. Enantioselective Addition of
Organozinc Reagents to Aldehydes. Chem. Rev. 1992, 92, 833−856.
(b) Pu, L.; Yu, H.-B. Catalytic Asymmetric Organozinc Additions to
Carbonyl Compounds. Chem. Rev. 2001, 101, 757−824.
896−908. (d) Nebenfuhr, A.; Ritzenthaler, C.; Robinson, D. G.
̈
Brefeldin A: Deciphering an Enigmatic Inhibitor of Secretion. Plant
Physiol. 2002, 130, 1102−1108 and literature cited therein.
(11) (a) Donaldson, J. G.; Finazzi, D.; Klausner, R. D. Brefeldin A
inhibits Golgi membrane-catalysed exchange of guanine nucleotide
5883
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Journal of Medicinal Chemistry
Article
(20) Soai, K.; Yokoyama, S.; Ebihara, K.; Hayasaka, T. A New Chiral
Catalyst for the Highly Enantioselective Addition of Dialkylzinc
Reagents to Aliphatic Aldehydes. J. Chem. Soc., Chem. Commun. 1987,
1690−1691.
(35) Irurzun, A.; Perez, L.; Carrasco, L. Involvement of membrane
traffic in the replication of poliovirus genomes: effects of brefeldin A.
Virology 1992, 191, 166−175.
(36) Maynell, L. A.; Kirkegaard, K.; Klymkowsky, M. W. Inhibition of
Poliovirus RNA Synthesis by Brefeldin A. J. Virol. 1992, 66, 1985−
1994.
(21) Blakemore, P. R.; Kocienski, P. J.; Marczak, S.; Wicha, J. The
Modified Julia Olefination in Vitamin D2 Synthesis. Synthesis 1999,
1209−1215.
(37) Hsu, N.-Y.; Ilnytska, O.; Belov, G.; Santiana, M.; Chen, Y.-H.;
Takvorian, P. M.; Pau, C.; van der Schaar, H.; Kaushik-Basu, N.; Balla,
T.; Cameron, C. E.; Ehrenfeld, E.; van Kuppeveld, F. J. M.; Altan-
Bonnet, N. Viral Reorganization of the Secretory Pathway Generates
Distinct Organelles for RNA Replication. Cell 2010, 141, 799−811.
(38) Nagy, P. D.; Pogany, J. The dependence of viral RNA
replication on co-opted host factors. Nature Rev. Microbiol. 2011, 10,
137−149.
(39) Wessels, E.; Duijsings, D.; Niu, T.-K.; Neumann, S.; Oorschot,
V. M.; de Lange, F.; Lanke, K. H. W.; Klumperman, J.; Henke, A.;
Jackson, C. L.; Melchers, W. J. G.; van Kuppeveld, F. J. M. A Viral
Protein that Blocks Arf1-Mediated COP-I Assembly by Inhibiting the
Guanine Nucleotide Exchange Factor GBF1. Dev. Cell 2006, 11, 191−
201.
(40) Belov, G. A.; Altan-Bonnet, N.; Kovtunovych, G.; Jackson, C. L.;
Lippincott-Schwartz, J.; Ehrenfeld, E. Hijacking Components of the
Cellular Secretory Pathway for Replication of Poliovirus RNA. J. Virol.
2007, 81, 558−567.
(41) Lanke, K. H. W.; van der Schaar, H. M.; Belov, G. A.; Feng, Q.;
Duijsings, D.; Jackson, C. L.; Ehrenfeld, E.; van Kuppeveld, F. J. M.
GBF1, a Guanine Nucleotide Exchange Factor for Arf, Is Crucial for
Coxsackievirus B3 RNA Replication. J. Virol. 2009, 83, 11940−11949.
(42) Mansour, S. J.; Skaug, J.; Zhao, X.-H.; Giordano, J.; Scherer, S.
(22) For syntheses of 24 not starting from 1 see: Haynes, R. K.; Lam,
W. W.-L.; Yeung, L.-L.; Williams, I. D.; Ridley, A. C.; Starling, S. M.;
Vonwiller, S. C.; Hambley, T. W.; Lelandais, P. Highly diaster-
eoselective conjugate addition of lithiated γ-crotonolactone (but-2-en-
4-olide) to cyclic enones to give syn-adducts: application to a brefeldin
synthesis. J. Org. Chem. 1997, 62, 4552−4553.
(23) Trost, B. M.; Crawley, M. L. A “chiral aldehyde” equivalent as a
building block towards biologically active targets. Chem.Eur. J. 2004,
10, 2237−2252. This article also contains an extensive bibliography
on previous total syntheses of 1.
(24) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. A
rapid esterification by means of mixed anhydride and its application to
large-ring lactonization. Bull. Chem. Soc. Jpn. 1979, 52, 1989−1993.
(25) Williams, D. R.; Jass, P. A.; Tse, H.-L. A.; Gaston, R. D. Total
Synthesis of (+)-breynolide. J. Am. Chem. Soc. 1990, 112, 4552.
(26) Reviews: (a) Muller, K.; Faeh, C.; Diederich, F. Fluorine in
̈
pharamceuticals: looking beyond intuition. Science 2007, 317, 1881−
1886. (b) Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V.
Fluorine in medicinal chemistry. Chem. Soc. Rev. 2008, 37, 320−330.
(c) O’Hagan, D. Understanding organofluorine chemistry. An
introduction to the C−F bond. Chem. Soc. Rev. 2008, 37, 308−319.
(27) (a) Ruppert, I.; Schlich, K.; Volbach, W. Die ersten CF3-
substituierten Organyl(chlor)silane. Tetrahedron Lett. 1984, 25, 2195−
2198. (b) Prakash, G. K. S.; Krishnamurti, R.; Olah, G. A. Fluoride-
induced trifluoromethylation of carbonyl compounds with trifluorme-
thyltrimethylsilane (TMS-CF3). A trifluormethide equivalent. J. Am.
Chem. Soc. 1989, 111, 393−395.
W.; Melanco̧ n, P. p200 ARF-GEP1: a Golgi-localized guanine
nucleotide exchange protein whose Sec7 domain is targeted by the
drug brefeldin A. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 7968−7973.
(43) Peyroche, A.; Antonny, B.; Robineau, S.; Acker, J.; Cherfils, J.;
Jackson, C. L. Brefeldin A Acts to Stabilize an Abortive ARF-GDP-
Sec7 Domain Protein Complex: Involvement of Specific Residues of
the Sec7 Domain. Mol. Cell 1999, 3, 275−285.
(28) (a) Gartner, M.; Mader, S.; Seehafer, K.; Helmchen, G. Enantio-
̈
and regioselective iridium-catalyzed allylic hydroxylation. J. Am. Chem.
Soc. 2011, 133, 2072−2075. (b) Spiess, S.; Welter, C.; Franck, G.;
Taquet, J.-P.; Helmchen, G. Iridium-catalyzed asymmetric allylic
substitutionsvery high regioselectivity and air stability with a catalyst
derived from dibenzo[a,e]cyclooctatetraene and a phosphoramidite.
Angew. Chem., Int. Ed. 2008, 47, 7652−7655. (c) Raskatov, J. A.;
(44) MacroModel is a product of Schrodinger, LLC; for detailed
̈
information see: MacroModel; Schrodinger, LLC: New York, 2013;
̈
http://www.schrodinger.com/products/14/11.
(45) (a) Chang, G.; Guida, W. C.; Still, W. C. An Internal Coordinate
Monte Carlo Method for Searching Conformational Space. J. Am.
Chem. Soc. 1989, 111, 4379−4386. (b) Method details: mmffs, energy
minimization by a conjugate gradient method (PRCG), Monte Carlo
Multiple Minimum (MCMM) conformational search using the
implemented automatic setup, H2O was used as solvent model.
(46) (a) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and Validation of a Genetic Algorithm for Flexible
Docking. J. Mol. Biol. 1997, 267, 727−748. (b) For more information
see: GOLD 5.1; Cambridge Crystallographic Data Centre: Cambridge,
UK; http://www.ccdc.cam.ac.uk/products/life_sciences/gold.
(47) (a) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G.
S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF ChimeraA
Visualization System for Exploratory Research and Analysis. J. Comput.
Chem. 2004, 25, 1605−1612. (b) For more information see: UCSF
Chimera, An Extensible Molecular Modeling System; University of
California, San Francisco: San Francisco, CA; http://www.cgl.ucsf.
edu/chimera.
Spiess, S.; Gnamm, C.; Brodner, K.; Rominger, F.; Helmchen, G. Ir-
̈
catalysed asymmetric allylic substitutions with cyclometalated
(phosphoramidite)Ir complexesresting states, catalytically active
(π-allyl)Ir complexes and computational exploration. Chem.Eur. J.
2010, 6601−6615.
(29) Reviews: (a) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K.
B. Catalytic asymmetric dihydroxylation. Chem. Rev. 1994, 94, 2483−
2547. (b) Lohray, B. B. Recent advances in the asymmetric
dihydroxylation of alkenes. Tetrahedron: Asymmetry 1992, 3, 1317−
1349.
(30) Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G. A.;
Hartung, J.; Jeong, K.-S.; Kwong, H.-L.; Morikawa, K.; Wang, Z.-M.;
Xu, D.; Zhang, X.-L. The osmium-catalyzed asymmetric dihydrox-
ylation: a new ligand class and a process improvement. J. Org. Chem.
1992, 57, 2768−2771.
(48) Biological Macromolecular Resource; RCSB Protein Data Bank;
http://www.rcsb.org/pdb/home/home.do.
́
(31) Greene, A. E.; Le Drian, C.; Crabbe, P. An efficient total
synthesis of ( )-Brefeldin-A. J. Am. Chem. Soc. 1980, 102, 7583−7584.
(32) Review: Tsuji, J. Synthetic applications of the palladium-
catalyzed oxidation of olefins to ketones. Synthesis 1984, 5, 369−384.
(33) Internal ID numbers of the NCI (NSC) for compounds not
listed in Table 2: 4 (NSC763496), 42 (NSC761153), 43
(NSC761155), 54 (NSC757950), (6R)-hydroxybrefeldin A
(NSC751150), (7S)-aminobrefeldin C (NSC671437), lactam ana-
logue of 1 (NSC652682).
(49) Dettmer, J.; Hong-Hermesdorf, A.; Stierhof, Y.-D.; Schumacher,
K. Vacuolar H+-ATPase Activity Is Required for Endocytic and
Secretory Trafficking in Arabidopsis. Plant Cell 2006, 18, 715−730.
(50) Murashige, T.; Skoog, F. A. A Revised Medium for Rapid
Growth and Bio Assays with Tobacco Tissue Cultures. Physiol. Plant.
1962, 15, 473−497.
(51) Rutz, C.; Satoh, A.; Ronchi, P.; Brugger, B.; Warren, G.;
̈
Wieland, F. T. Following the Fate In Vivo of COPI Vesicles Generated
(34) Langhans, M.; Forster, S.; Helmchen, G.; Robinson, D. G.
̈
In Vitro. Traffic 2009, 10, 994−1005.
Differential effects of the brefeldin A analogue (6R)-hydroxy-brefeldin
A in tobacco and Arabidopsis. J. Exp. Bot. 2011, 62, 2949−2957.
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