Isolation, biological activity evaluation, structure elucidation, and total synthesis of eliamid: A novel complex i inhibitor

Article abstract of DOI:10.1002/chem.201201879

Eliamid is a secondary metabolite isolated from two bacterial strains. This molecule features a linear polyketide backbone terminated by a tetramic acid amide moiety. Among other biological activities, eliamid shows a high and specific cytostatic action on human lymphoma and cervix carcinoma cell lines. The 2,4-anti relative configuration of the C-2,C-4-dimethyl substituted amide fragment was assigned by means of Breit's rule. The absolute configuration of all stereocenters was determined by a combination of degradation methods, structural similarity analysis and total synthesis. The stereogenic centers were introduced by vinylogous Mukaiyama aldol reaction and two consecutive Myers alkylations. The use of pentafluorophenyl ester as acylation agent allowed the efficient formation of tetramic acid amide. The longest linear sequence in the synthesis consist of 13 steps and proceeds with 12 % overall yield. Differential spectroscopy experiments with beef heart submitochondrial particles established that eliamid is a potent inhibitor of the NADH-ubiquinone oxidoreductase complex. Additionally, biosynthesis of eliamid was investigated by feeding experiments with ¹³C-labeled precursors. How valuable is Breit's rule? Isolation, biological activity profiling, structure elucidation and total synthesis of polyketide natural product eliamid are reported. This molecule is characterized by a tetramic acid amide fragment, which is rather rare among secondary metabolites isolated from myxobacteria. Copyright

Full text of DOI:10.1002/chem.201201879

FULL PAPER
DOI: 10.1002/chem.201201879
Isolation, Biological Activity Evaluation, Structure Elucidation, and Total
Synthesis of Eliamid: A Novel Complex I Inhibitor
Gerhard Hçfle, Klaus Gerth, Hans Reichenbach, Brigitte Kunze, Florenz Sasse,
Edgar Forche, and Evgeny V. Prusov*[a]
Abstract: Eliamid is a secondary me-
tabolite isolated from two bacterial
strains. This molecule features a linear
polyketide backbone terminated by a
tetramic acid amide moiety. Among
other biological activities, eliamid
shows a high and specific cytostatic
action on human lymphoma and cervix
carcinoma cell lines. The 2,4-anti rela-
tive configuration of the C-2,C-4-di-
methyl substituted amide fragment was
assigned by means of Breitꢁs rule. The
absolute configuration of all stereocen-
ters was determined by a combination
of degradation methods, structural sim-
ilarity analysis and total synthesis. The
stereogenic centers were introduced by
vinylogous Mukaiyama aldol reaction
and two consecutive Myers alkylations.
The use of pentafluorophenyl ester as
acylation agent allowed the efficient
formation of tetramic acid amide. The
longest linear sequence in the synthesis
consist of 13 steps and proceeds with
12% overall yield. Differential spectro-
scopy experiments with beef heart sub-
mitochondrial particles established that
eliamid is a potent inhibitor of the
NADH–ubiquinone
oxidoreductase
complex. Additionally, biosynthesis of
eliamid was investigated by feeding ex-
periments with 13C-labeled precursors.
Keywords: complex I inhibitors
·
natural products · structure elucida-
tion · tetramic acids · total synthesis
Introduction
Activity-guided isolation of secondary metabolites from mi-
croorganisms, plants and fungi has evolved as a powerful
technology for the discovery of biologically relevant natural
products and their cellular targets. At the Helmholtz-Zen-
trum fꢀr Infektionsforschung (HZI), more than 100 structur-
ally novel compounds produced by myxobacteria have been
discovered, characterized, and, in part, developed, for exam-
ple, as anticancer drugs.[1] Later, the focus shifted to other
groups of gliding bacteria, and molecular biology of the pro-
ducing organisms.[2] Here, we describe the isolation, biologi-
cal activity evaluation, structure elucidation, total synthesis
and studies on the biosynthesis of eliamid, a potent com-
plex I inhibitor with some other interesting activities.
In a screening of myxobacteria for secondary metabolites
with antifungal activity, a substance with a conspicuous UV
adsorption was discovered in the HPLC traces of extracts
from two bacterial strains: Soce241,[3] which also produces
soraphen,[4] and Soce439, which produces ambruticins,[5]
both antifungal compounds. The new isolate was named eli-
amid (1, Figure 1) acknowledging the place (Arismeliya,
Egypt) where the Soce241 strain was collected. The carbon
Figure 1. Structures of eliamid (2D) and mirabimide E.
skeleton was identified to be a linear polyketide with a ter-
minal tetramic amide moiety.
Results and Discussion
Isolation: The producing organisms, Sorangium cellulosum
Soce439 (DSMZ 11529) and Soce241 were isolated from
the soil samples taken at Pfeffingen, Switzerland and Aris-
meliya, Egypt, respectively. The fermentation was run in a
100 L bioreactor with 70 L of medium in the presence of
XAD-16 adsorbing resin (2% v/v, 1.4 kg) for 11 days. After
separation of the resin by filtration, it was eluted with a 1:1
[a] Prof. Dr. G. Hçfle, Dr. K. Gerth, Prof. Dr. H. Reichenbach,
Dr. B. Kunze, Dr. F. Sasse, Dr. E. Forche, Dr. E. V. Prusov
Helmholtz-Zentrum fꢀr Infektionsforschung (HZI)
Inhoffenstr. 7, Braunschweig (Germany)
Fax : (+49)531-6181-9499
E-mail: evgeny.prusov@helmholtz-hzi.de
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201879.
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mixture of methanol/acetone followed by pure methanol.
The eluates were concentrated independently until separa-
tion of the water phase, and were extracted with ethyl ace-
tate. The combined organic phases were evaporated and the
residue purified by two consecutive chromatographic sepa-
rations on normal silica gel to give eliamid (460 mg) as a vis-
cous oil.
Biological activity evaluation: The biological evaluation of
eliamid (1) with a panel of transformed cell lines showed a
specific cytostatic action on human lymphoma and cervix
carcinoma cell cultures (Table 1). Additionally, a significant
Table 1. Cytostatic activity of eliamid on various transformed cells lines.
Cell line
Organism/source
IC50 [ngmLÀ1
]
Figure 2. Inhibitory effect of eliamid on NADH oxidation in beef heart
submitochondrial particles (SMP). The samples contained 66.3 mgmLÀ1
beef heart protein. The rate of NADH oxidation without inhibitor was
L-929
PtK2
mouse fibroblast
0.5
30.0
1.0
15.0
0.5
3.0
20.0
15.0
rat kangaroo kidney
human cervix carcinoma
human leukemia
0.51
N
.
KB 3.1
HL-60
U-937
A-431
A-498
A-549
human lymphoma
human epidermis carcinoma
human kidney carcinoma
human lung carcinoma
the characteristic absorption maxima for the different cyto-
chromes (Figure 3). Treatment of SMP with eliamid almost
completely inhibited reduction of cytochrome a+a3 (a band
at 605 nm), cytochrome b (a band at 563 nm) and the cyto-
chromes c+c1 (a band at 553 nm) by NADH; this indicates
that the inhibitory effect of eliamid is on the substrate side
of cytochrome b. Cytochrome b of complex III can be re-
duced by NADH by complex I (NADH–ubiquinone oxidor-
eductase) or by succinate by complex II (succinate–ubiqui-
none oxidoreductase). Reduction kinetics of cytochrome b
with either NADH or succinate as substrate showed that eli-
amid inhibited the reduction of cytochrome b only when
NADH was the electron donor. This indicates that the in-
hibitory effect of eliamid, like that of piericidine, myxala-
mide and actinopyron, is mediated by interaction with the
complex I (NADH–ubiquinone oxidoreductase) of the eu-
karyotic respiratory chain.
reduction of motility, partially one-side only, was observed
within 20–30 min after application of 0.2–0.3 mgmLÀ1 solu-
tions of eliamid to a colony of brine shrimps, Artemia salina.
However, death occurred rather gradually so that even after
24 h some shrimps remained alive, while the damage effect
was found to be persistent. Treatment of soil nematodes
(Panagrellus spec.) with 5 mgmLÀ1 eliamid was lethal to all
animals. Additionally, eliamid showed moderate inhibitory
activity on fungi and yeast (Table 2).
Table 2. Antifungal activity of eliamid at 20 mg per test plate.
Test organism
Diameter of inhibition zone [mm]
Mucor hiemalis
Alternaria solani
Fusarium oxysporum
Sclerotina sclerotorium
Hansenula anomala
Sporidiobolus ruineniae
22
15
19
16
20
16
To determine the mode of action of eliamid, its effect on
the mitochondrial respiratory chain was examined. Eliamid
(1) strongly inhibited NADH oxidation in beef heart submi-
tochondrial particles (SMP) with an IC50 of 8 ngmLÀ1
(20 nm; Figure 2). The site of inhibition of eliamid within
the respiratory chain was investigated by means of differen-
tial spectroscopy by using a DW 2000 UV/Vis SLM double
beam spectrophotometer. Upon reduction with physiological
substrates, for example, NADH, fully oxidized cytochromes
in front of the block became reduced, whereas those behind
it remained oxidized. The differential spectrum of NADH-
reduced minus air-oxidized SMP without inhibitor showed
Figure 3. The effect of eliamid on the reduction of cytochromes by
NADH. Top line: differential spectrum (reduced minus oxidized) of SMP
reduced with NADH without inhibitor; bottom line: the same, but in the
presence of 15 mgmLÀ1 eliamid.
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Eliamid: A Novel Complex I Inhibitor
FULL PAPER
Structural elucidation and total synthesis: The IR spectrum
of eliamid shows three strong bands at 1632, 1680 and
1719 cmÀ1, whereas the UV spectrum shows two broad
maxima at 223 and 234 nm. High resolution mass spectrom-
etry and elemental analysis established the m/z value of elia-
mid as 389.2558 and the elemental composition as
C23H35NO4, which corresponds to seven double bond equiva-
1
lents (DBEs). Examination of the H and 13C NMR spectro-
scopy revealed the presence of an a,b-unsaturated keton, a
three substituted double bound and two carboxyl group
equivalents (Table 3). The positions of the unsaturated
Table 3. 1H and 13C NMR spectroscopy data of eliamid in [D4]MeOH
(400 MHz).
Scheme 1. Determination of the absolute configuration of the tetramic
acid.
Atom
13C d [ppm]
1H d [ppm]
Multiplet
JHH [Hz]
1
2
178.5
37.7
42.2
42.2
29.8
48.9
48.9
136.0
128.6
40.7
205.8
138.3
139.0
14.8
16.5
19.7
16.2
18.0
11.4
182.7
93.5
171.5
56.9
17.3
59.6
–
–
m
ddd
ddd
m
dd
dd
–
–
–
3.98
1.30
1.52
1.69
1.84
2.01
–
5.02
4.21
–
strategy of unraveling the unknown configurations by degra-
dation was changed.
Instead, the structure was compared with similar natural
products for which the corresponding configurations were
known, such as myxalamide D (7),[7] piericidin A1 (6)[8] and
actinopyrone A (8)[9] (Figure 4). This strategy takes advant-
3a
3b
4
5a
5b
6
7
8
9
10
11
12
13
14
15
16
17
1’
2’
3’
4’
5’
13.6, 7.5, 5.5
13.6, 8.8, 6.2
–
13.2, 8.1
13.2, 6.2
–
9.5
9.5, 6.7
–
–
7.0, 1.0
7.0, 1.0
7.0
6.2
–
6.6
–
–
–
–
d
dq
–
–
–
6.91
1.92
1.11
0.89
1.70
1.13
1.77
–
5.21
–
4.63
1.46
3.95
qd
dd
d
d
s
d
s
–
s
–
q
d
s
6.6
6.6
–
6’
ketone, double bond and methyl substituents along the
1
carbon skeleton were established by direct H,1H COSY and
1H,13C HMQC NMR correlation spectroscopies, together
1
with the long range H,13C HMBC correlations. The tetramic
acid fragment, which accounts for the last two DBEs, was
deduced from the chemical shift and long range HMBC cor-
relations of CH-2ꢁ (dH =5.21 ppm, dC =93.5 ppm) to qC-1ꢁ
(d=182.7 ppm) and qC-3’ (d=171.5 ppm) and by compari-
son with the spectroscopic data for mirabimide E (2).[6]
The absolute configuration of the stereocenter in the tet-
ramic acid fragment was determined by oxidative and hy-
drolytic degradation of natural product according to the mir-
abimide E (2) precedent (Scheme 1).[6] Analysis of the reac-
tion mixtures by chiral gas chromatography unambiguously
established that the tetramic acid moiety has the absolute
configuration of l-alanine. Further attempts to elucidate the
absolute configurations of remaining stereocenters by ozo-
nolysis or other degradation methods eventually led to the
consumption of all available material, at which point the
Figure 4. Structural similarities between eliamid and other complex I in-
hibitors.
age of Celmerꢁs rule[10] and is based on the fact that similar
structural motifs might arise from shared polyketide gene
clusters.
Also, of particular importance here were the resonances
of the 3-Ha and 3-Hb methylene protons (Figure 5), which
were recorded at 1.27 and 1.50 ppm, respectively. It was al-
ready observed earlier that deoxygenated polypropionate
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E. V. Prusov et al.
From here, several routes were reasonable for the intro-
duction of the tetramic acid amide moiety.[15] We opted to
employ the method of Tønder,[16] which relies on perfluoro-
phenyl esters as acylation agents (Scheme 3). Thus, the car-
boxylic acid 19 obtained by lithium peroxide hydrolysis of
amide 18 was converted to the activated ester 20 according
to the modified Steglich protocol.[17] Ester 20 was then al-
lowed to react with a slight excess of potassium salt of
pyAla-OMe (5)[18] to provide the desired product in 38%
yield (62% b.o.r.s.m.). The TBS protecting group was
cleaved with Et3N·3HF, and subsequent oxidation of the al-
lylic alcohol 22 with the Dess–Martin periodinane provided
the putative eliamid.
Figure 5. Proposed 3D structures of eliamid.
compounds with a syn-2,4-dimethyl substitution pattern dis-
play significantly larger differences in the chemical shifts of
the diastereotopic methylene protons than the correspond-
ing anti-isomers. An excellent theoretical explanation for
this empirical rule and a meta-analysis of more than 60 com-
pounds of various classes containing 2,4-dimethyl substituted
chains were given by Breit and Schmidt in their recent
paper.[11] It follows that for syn-2,4-dimethyl amides the
chemical shift difference of methylene protons is usually
found to be in the 0.7–0.8 ppm region, whereas for anti-2,4-
diastereomers it is between 0.2 and 0.3 ppm. The observed
value of Dd=0.23 ppm would be consistent with an anti con-
figuration C-13 and C-14 methyl groups.
To our disappointment, the proton NMR spectrum of
compound 23 did not match that of the natural product.
Nevertheless, a good agreement was found for the resonan-
ces that lie in the down-field. The biggest deviation between
the spectra was observed for the signals of the CH3-5’, CH3-
13 and CH3-14 protons in the 0.7–1.1 ppm region (Figure 6).
It was, therefore, assumed that the second possible dia-
stereomer should be the natural product. Facing the low
yields using the Evans alkylation we switched to Myers
Based on the structural similarities to myxalamide D (7),
piericidin A1 (6) and actinopyrone A (8) the absolute con-
figuration at C-8 was proposed to be R. Additional reassur-
ance for the structural similarities was drawn from the fact
that all four compounds are inhibitors of the NADH–ubiq-
uinone oxidoreductase complex. Therefore, we decided to
take on the syntheses of both anti isomers. We started with
diastereomer A, which contains the same absolute configu-
ration of 2,4-dimethyl amide as 2,4-dimethyl ester moiety of
myxovirescin.[12]
The synthesis commenced with Kobayashiꢁs vinylogous
Mukaiyama aldol reaction between tiglic aldehyde and N,O-
silyl keten acetal (10) to provide 63% yield of aldol adduct
when the reaction was allowed
Figure 6. Comparison of the 1H NMR spectra of 23 (top) and eliamid
(bottom).
to
proceed
for
5 days
(Scheme 2).[13] Reductive cleav-
age of the chiral auxiliary pro-
vided alcohol 12, which was
converted to the allylic iodide
13. The subsequent Evans alky-
lation[14] produced the desired
amide 15, albeit in a moderate
yield. A second reductive cleav-
age and reaction with triflic an-
hydride furnished 16, which was
subjected to a second Evans al-
kylation. Despite the rather low
yields in this step, we were able
to obtain sufficient quantity of
material to proceed further
with the synthesis.
Scheme 2. Installation of stereocenters through vinylogous Mukaiyama aldol reaction and Evans alkylations.
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Eliamid: A Novel Complex I Inhibitor
FULL PAPER
quired to drive the reaction to completion. Then, a pro-
longed (one week) exposure to NaOH solution at 608C was
used to avoid concomitant cleavage of the TBS-ether during
hydrolysis of the pseudoephedrine amide. Formation of the
tetramic acid amide, TBS group cleavage and oxidation to
the ketone were performed as already described
(Scheme 5).
Scheme 3. Formation of the tetramic acid amide and completion of the
synthesis.
Scheme 5. Completion of the total synthesis of eliamid.
This time, the synthetic material was spectroscopically
identical to the authentic natural product (1H and 13C NMR
spectroscopy; Figure 7, see also the Supporting Information
for a detailed comparison). The optical rotation was found
to be acceptably close to that of the natural product: [a]D =
À658 (c=0.8 in MeOH) for the synthetic material versus
[a]D =À69.88 (c=0.5 in MeOH) for the authentic sample.
Additionally, synthetic eliamid was found to be equally
potent to the natural product in transformed cell line assays.
method[19] for the alkylation steps (Scheme 4). Gratifyingly,
transformation of allylic iodide 13 with the enolate derived
from N-propionyl pseudoephedrine (24) provided the de-
sired product in 90% yield. The auxiliary was removed with
lithium aminoborohydride and the resulting alcohol convert-
ed into alkyliodide 26. For the second alkylation step an
eight- to tenfold excess of pseudoephedrine enolate was re-
Figure 7. Spectral comparison for synthetic (top) and natural (bottom) el-
iamid.
Scheme 4. Myers alkylation approach to the acid 29.
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E. V. Prusov et al.
It is also worth noting that, the absolute configuration of
the C-4 and C-8 stereocenters in eliamid matches the corre-
sponding structural motif (C-24 and C-28) in the structurally
similar side chain of spirangien[20] (33; Figure 8). Together
semi-empirical rule was used to assign the relative stereo-
chemistry of stereocenters in natural products.
We also accomplished the total synthesis of eliamid in
thirteen chemical steps and 12% overall yield. The key
steps are a vinylogous Mukaiyama aldol reaction, Myers
enantioselective alkylations and tetramic acid amide forma-
tion from pentafluorophenyl ester. Further studies on the
chemistry and biology of eliamid and its modified analogues
are currently underway and will be reported in due course.
Experimental Section
General remarks: Optical rotations were determined on a Perkin–Elmer
241 instrument. IR and UV adsorption spectra were measured on Nicolet
20DXB and Shimadzu 2450 spectrometers, respectively. NMR spectra
were recorded in CD3OD and CDCl3 on a Bruker AM 300, AM 400 and
DMX-600 spectrometers. ESI mass spectra (reactant gas ammonia) were
obtained on a Finnigan MAT 95 spectrometer, high resolution data were
acquired by using peak matching (M/DM=10000). Analytical TLC
(TLC aluminium sheets silica gel Si 60 F254 (Merck), solvent: mixtures of
ethylacetate/petroleum ether, detection: UV absorption at 254 nm, dark
blue spots on staining with cerium(IV) sulfate phosphomolybdic acid in
sulfuric acid followed by charring. Unless otherwise stated, all reactions
were performed under inert gas blanket. All solvents used were commer-
cial absolute solvents over molecular sieves with water content less than
50 ppm (e.g., from Acros Organics). Sodium [1-13C, 99%]acetate, sodium
Figure 8. Comparison of eliamid (1) and spirangien (33).
with earlier described similarities to piericidin A1 (6), myxa-
lamide D (7) and actinopyron A (8) this implies that a
highly conserved polyketide synthase cluster exists in vari-
ous bacterial strains.
Biosynthesis: Several feeding experiments with 13C-labeled
precursors were performed on the “shaking flask” scale to
investigate the biosynthesis of eliamid. Though, no conclu-
sive results were obtained from experiments with
[1-13C]acetate and [1-13C]alanine, the 13C NMR spectrum of
[1-13C]propionate derived eliamid revealed a 15–18% 13C-
enrichment for all except one odd carbon atom, indicating a
contiguous incorporation of five propionate units (Figure 9).
[2-13C,
99%]acetate,
sodium
[1,2-13C2,
99%]acetate,
sodium
[1-13C]propionate and l-[methyl-13C, 98%]methionine were obtained
from Cambridge Isotope Laboratories.
Fermentation: The producing organism, Sorangium cellulosum Soce439
(DSMZ 11529) was isolated in 1989 from soil samples taken at Pfeffingen
(Switzerland). A 100 L bioreactor (Giovanola, Monthey, Switzerland)
equipped with a flat-blade turbine stirrer and containing 70 L production
medium (10 gLÀ1 glucose, 5 gLÀ1 soy peptone (Marcor), 2 gLÀ1 yeast ex-
tract, 1 gLÀ1 MgSO4·7H2O, 1 g LÀ1 CaCl2·2H2O, ethylenediaminetetraace-
tic acid, 0.008 gLÀ1 iron
ACTHNUGRTENUNG(III) sodium salt, pH adjusted to 7.2) and 2% (v/
Additionally,
in
a
feeding
experiment
with
v) XAD16 adsorbing resin (Rohm and Haas), was inoculated with 5 L of
a 4 day old shake culture grown in the same medium. The fermentation
was run for 11 days at 328C with an aeration rate of 0.1 VVÀ1 minÀ1 and
a stirrer velocity of 250 rpm. The pH was maintained at 7.2 with 10% (g/
v) KOH. The foam formation was blocked by addition of the silicon anti-
foam Tegosipon (Goldschmidt AG, Essen). Eliamid is absorbed quantita-
tively on the XAD adsorbing resin, which is separated by filtration
through a process filter.
[13CH3]methionine, a 5% 13C-enrichment in the methoxy
group of tetramic acid was observed.
Isolation: The XAD-16 adsorbing resin (1.4 kg) was eluted with 1:1 mix-
ture of methanol/acetone (4 L) followed by 6 L of pure methanol. The
extracts were independently concentrated until separation of the water
phase, and extracted with ethyl acetate (2ꢃ3 L). The combined organic
phases were evaporated, in vacuo, to give 15.8 g of crude extract, which
was dissolved in petroleum ether (with minimal addition of methanol)
and purified by chromatography over silica gel (400 g, 10 cm column di-
ameter) with a stepwise gradient elution (petroleum ether to petroleum
ether/tert-butylmethylether 1:1) with a total of 7 L of eluent. The eliamid
containing fractions were identified by TLC and concentrated to give
2.26 g of crude product. Final purification by chromatography over silica
gel (20–45 mm, 5ꢃ55 cm column, elution with petroleum ether/tert-butyl-
methylether/methanol 80:19:1, UV detection at 227 nm) provided elia-
mid (460 mg) as a viscous oil.
Figure 9. Biosynthesis of eliamid.
Conclusion
In conclusion, we report the isolation, biological activity
evaluation, structure elucidation and total synthesis of the
new natural product eliamid. The compound exhibits a
broad spectrum of biological activities and could serve as a
lead structure for the development of novel anticancer
drugs. The relative configuration of the 2,4-dimethyl desoxy-
propionate amide was correctly predicted by Breitꢁs rule. To
the best of our knowledge, this is the first time that this
Analytical data: TLC Rf =0.37 (petroleum ether/Et2O 1:1, UV detec-
tion); HRMS EI: 389.2558 (calcd for C23H35NO4 389.2566); elemental
analysis (%) calcd: C 70.92, H 9.06, N 3.60; found: C 70.94, H 8.78, N
3.67; IR (CHCl3): n˜ =1719, 1680, 1632 cmÀ1; UV/Vis (CH3OH): lmax (e)=
223 (19200), 234 nm (19300 molÀ1 dm3 cmÀ1). For the 1H and 13C NMR
spectroscopy results see Table 3.
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Eliamid: A Novel Complex I Inhibitor
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Degradation studies
3H), À0.07 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=À5.1, À4.7,
10.8, 12.9, 14.0, 17.4, 18.1, 25.6, 36.7, 69.3, 83.4, 121.2, 130.7, 134.1,
137.1 ppm; HRMS: calcd for C17H34O2SiNa 321.2226, found 321.2226.
Oxidative degradation: A solution of eliamid (5 mg) in acetone (1 mL)
was oxidized with sodium periodate (20 mg in 0.3 mL H2O) and potassi-
um permanganate (3 mg) according to the procedure described for mira-
bimid E.[6] After 2 h of stirring at room temperature the reaction was
quenched with methanol (2 mL) and evaporated. The residue was esteri-
fied by treatment with HCl in (1m) iPrOH (0.5 mL) at 1008C for 30 min,
evaporated, trifluoroacetylated with (CF3CO)2O (100 mL in 0.5 mL
CH2Cl2, 10 min at 1008C) and analyzed by gas chromatography on the
Chirasil L-val column. The chromatogram revealed the presence of N-tri-
fluoroacetyl-l-alanine isopropyl ester.
tert-Butyl((2E,4R,5R,6E)-8-iodo-3,5,7-trimethylocta-2,6-dien-4-yloxy)di-
methylsilane (13): Methyltriphenoxyphosphonium iodide (1.34 g, 3 mmol,
1.25 equiv) in DMF (3 mL) was added dropwise to a stirred (at 08C) sol-
ution of alcohol 12 (720 mg, 2.42 mmol, 1 equiv) in DMF (3 mL). The re-
action mixture was allowed to warm to room temperature and stirred for
30 min. The reaction mixture was diluted with hexane (35 mL) and the
organic layer was extracted with cold aqueous 1N NaOH (2ꢃ10 mL) and
water (2ꢃ10 mL). The organic layer was dried over Na2SO4, filtered, and
concentrated in vacuo to afford iodide 13 (910 mg, quantitative) as a
yellow oil. The crude product was immediately used in the subsequent re-
action without any further purification.
Hydrolytic degradation: NaOH (0.5N, 70 mL) was added to a solution of
eliamid (5 mg) in ethanol (0.1 mL). After 50 min the reaction mixture
was evaporated in vacuo and portioned between AcOEt and water at
pH 3. From the organic phase, 3.5 mg of acid 4 were isolated after purifi-
cation. The water phase was subjected to butanol extraction (3ꢃ1 mL),
the combined extracts were evaporated to give 0.1 mg of the tetramic
acid 5 (H-py-Ala-OMe).
Compound 4: 1H NMR (CDCl3): d=0.79 (d, J=7 Hz, 3H), 1.09 (d, J=
7 Hz, 3H), 1.10 (d, J=7 Hz, 3H), 1.09 (d, J=7 Hz, 3H), 1.28 (m, 1H),
1.45 (m, 1H), 1.62 (d, J=1 Hz, 3H), 1.65 (m, 1H), 1.75 (m, 1H), 1.75 (s,
3H), 1.83 (dd, J=7, 1 Hz, 3H), 1.98 (m, 1H), 2.50 (m, 1H), 4.01 (dq, J=
10, 7 Hz, 1H), 5.08 (d, J=10 Hz, 1H), 6.77 ppm (q,d, J=7, 1 Hz, 1H);
DCI-MS (isobutane) after treatment with diazomethane: m/z 295 [M+
H+].
(2S,4E,6R,7R,8E)-7-(tert-Butyldimethylsilyloxy)-N-((1S,2S)-1-hydroxy-1-
phenylpropan-2-yl)-N,2,4,6,8-pentamethyldeca-4,8-dienamide (25): nBuLi
(2.5m in hexanes, 2.1 mL, 5.5 mmol) was added to a suspension of LiCl
(720 mg, 18 mmol, dried for 18 h at 1308C under high vacuum) and diiso-
propylamine (0.8 mL, 5.5 mmol) in THF (5 mL) at À788C. The resulting
suspension was warmed to 08C for 10 min. An ice-cooled solution of
(R,R)-pseudoephedrine propionamide (0.6 g, 2.71 mmol) in THF (3 mL)
was added at À788C and the mixture was stirred for 1 h at À788C,
15 min at 08C, and 5 min at room temperature. Iodide 13 (0.35 g,
0.79 mmol) was added to this solution at 08C, and the reaction mixture
was stirred at room temperature for 14 h. The yellow reaction mixture
was treated with half-saturated aqueous NH4Cl (100 mL) and extracted
with EtOAc (4ꢃ100 mL). The combined organic layers were dried over
Na2SO4 and concentrated, in vacuo. Flash chromatography (petroleum
ether/Et2O 2:1) yielded amide 25 (330 mg, 90% yield) as a yellow oil.
Rf =0.32 (petroleum ether/Et2O, 2:1); [a]D =À57.5 (c=2.0 in CH2Cl2);
1H NMR (300 MHz, CDCl3) major rotamer: d=7.21–7.42 (m, 5H), 5.31
(q, J=6.6 Hz, 1H), 4.98 (dd, J=9.4, 0.9 Hz, 1H), 4.62 (d, J=7.5 Hz, 1H),
4.37–4.51 (m, 1H), 3.62 (d, J=7.9 Hz, 1H), 2.88 (s, 3H), 2.42–2.57 (m,
1H), 2.13 (dd, J=13.8, 4.5 Hz, 1H), 1.98 (dd, J=13.8, 9.4 Hz, 1H), 1.52–
1.61 (m, 9H), 1.14 (d, J=7.0 Hz, 3H), 1.03 (d, J=6.6 Hz, 3H), 0.84 (s,
9H), 0.72 (d, J=6.8 Hz, 3H), À0.03 (s, 3H), À0.07 ppm (s, 3H);
13C NMR (75 MHz, CDCl3): d=179.3, 142.5, 137.2, 131.9, 131.1, 128.3,
127.6, 126.3, 121.0, 83.4, 43.5, 37.1, 34.7, 25.8, 18.1, 17.7, 16.5, 16.0, 14.4,
12.9, 10.9, À4.8, À5.0 ppm; HRMS: calcd for C30H52NO3Si 502.3716,
found 502.3716.
Compound 5: 1H NMR (CDCl3): d=1.32 (d, J=7 Hz, 3H), 3.77 (s, 3H),
4.08 (q, J=7 Hz, 1H), 4.99 ppm (s, 1H); DCI-MS (isobutane): m/z (%):
128 (73) [M+H+], 255 (100) [2M+H+].
Total synthesis
(R)-3-((2E,4R,5R,6E)-5-(tert-Butyldimethylsilyloxy)-2,4,6-trimethylocta-
2,6-dienoyl)-4-isopropyloxazolidin-2-one (11): Tiglic aldehyde (9; 3 mL,
2.6 g, 30 mmol) was added to a stirred solution of TiCl4 (1.0m in CH2Cl2,
15 mL, 15 mmol) in CH2Cl2 (150 mL) at À788C. This was immediately
followed by addition of vinylketene silyl N,O-acetal 10 (5.08 g, 15 mmol)
in CH2Cl2 (10 mL). After being stirred for 5 days at À508C, the reaction
was quenched by the addition of a 1:1 mixture of saturated aqueous
NaHCO3 and saturated aqueous Rochelle salt (100 mL total volume).
The resulting mixture was stirred at room temperature until the white
slurry was dissolved. The organic layer was separated and concentrated
under reduced pressure. The residue was purified by column chromatog-
raphy over silica gel (petroleum ether/EtOAc, 3:1) to afford 2.9 g (63%)
of aldol adduct.
(2S,4E,6R,7R,8E)-7-(tert-Butyldimethylsilyloxy)-2,4,6,8-tetramethyldeca-
4,8-dien-1-ol:
A solution of n-butyllithium in hexanes (2.5m, 2 mL,
5 mmol, 10 equiv) was added to a solution of diisopropylamine (0.8 mL,
5.5 mmol, 11 equiv) in tetrahydrofuran (10 mL) at À788C. The resulting
solution was stirred at À788C for 10 min, then warmed to 08C, and held
at that temperature for 10 min. Borane–ammonia complex (151 mg,
5 mmol, 10 equiv) was added in one portion, and the suspension was stir-
red at 08C for 15 min and subsequently warmed to 238C. After 15 min,
the suspension was cooled to 08C. A solution of amide 25 (260 mg,
0.52 mmol, 1 equiv) in tetrahydrofuran (2 mL) was added via cannula
over 3 min. The reaction mixture was warmed to 238C, held at that tem-
perature for 2 h, and then cooled to 08C, at which point excess hydride
was quenched by the careful addition of 1N aqueous hydrochloric acid
solution (12 mL). The mixture was stirred for 30 min at 08C and then ex-
tracted with four 15 mL portions of Et2O. The combined organic extracts
were washed sequentially with 3N aqueous hydrochloric acid solution
(10 mL), 2N aqueous sodium hydroxide solution (10 mL), and brine
(10 mL). The ether extracts were dried over magnesium sulfate and con-
centrated. Purification of the residue by flash column chromatography
(petroleum ether/Et2O, 1:1) afforded the intermediate alcohol as a color-
less oil (160 mg, 94% yield). Rf =0.58 (petroleum ether/Et2O, 1:2); [a]D =
À4.6 (c=1 in CH2Cl2); 1H NMR (300 MHz, CDCl3): d=5.33 (qt, J=6.6,
1.1 Hz, 1H), 5.00 (dd, J=9.4, 1.0 Hz, 1H), 3.66 (d, J=7.2 Hz, 1H), 3.39–
3.53 (m, 2H), 2.44–2.58 (m, 1H), 2.09 (dd, J=12.1, 4.8 Hz, 1H), 1.69–
1.90 (m, 2H), 1.62 (d, J=1.1 Hz, 3H), 1.58 (d, J=6.6 Hz, 3H), 1.53–1.56
(m, 3H), 0.86 (s, 9H), 0.85 (d, J=6.4 Hz, 3H), 0.77 (d, J=7.0 Hz, 3H),
À0.02 (s, 3H), À0.06 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=137.3,
132.5, 130.6, 120.6, 83.0, 68.7, 44.3, 37.0, 33.6, 25.8, 18.2, 18.0, 16.3, 16.3,
2,6-Lutidine (2.3 mL, 2.14 g, 20 mmol) and TBSOTf (3 mL, 3.5 g,
13.3 mmol) were added to a solution of the above aldol adduct (2.9 g,
9.5 mmol) in CH2Cl2 (50 mL) at 08C. The mixture was stirred 15 min at
08C and allowed to warm to room temperature. The reaction was
quenched with sat. aq. NaHCO3, the organic layer was separated, dried
over Na2SO4 and concentrated, in vacuo. Purification by column chroma-
tography over silica gel (petroleum ether/EtOAc, 6:1) afforded 3.82 g
(95%) of imid 11. Analytical data were identical to that reported in
ref. [9].
(2E,4R,5R,6E)-5-(tert-Butyldimethylsilyloxy)-2,4,6-trimethylocta-2,6-dien-
1-ol (12): Lithium borohydride (88 mg, 4 mmol, 1.5 equiv) was added to
a cooled (08C, ice-bath) solution of imide 11 (1.12 g, 2.67 mmol) in Et2O/
MeOH (20 mL/0.16 mL) and allowed to stir at this temperature for 4 h.
The mixture was quenched dropwise with
a saturated solution of
NaHCO3 (20 mL). When gas evolution ceased, the organic layer was sep-
arated, the aqueous layer was extracted with Et2O (20 mL), and the com-
bined organic layers were washed with a saturated solution of NaHCO3
(20 mL), dried over anhydrous Na2SO4, filtered, concentrated, in vacuo,
and purified by column chromatography over silica gel with petroleum
ether/EtOAc (6:1) as eluent to provide 720 mg (91%) of the allylic alco-
hol 12 as a colorless oil. Rf =0.51 (petroleum ether/EtOAc, 6:1); [a]D =
À1.1 (c=2.0 in CH2Cl2); 1H NMR (300 MHz, CDCl3): d=5.32 (q, J=
6.8 Hz, 1H), 5.22 (dd, J=9.8, 1.2 Hz, 1H), 3.99 (d, J=3.2 Hz, 2H), 3.63
(d, J=7.9 Hz, 1H), 2.53 (m, 1H), 1.67 (d, J=1.2 Hz, 3H), 1.57 (d, J=
6.6 Hz, 3H), 1.55 (s, 3H), 0.83 (s, 9H), 0.75 (d, J=6.8 Hz, 3H), À0.04 (s,
Chem. Eur. J. 2012, 00, 0 – 0
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www.chemeurj.org
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E. V. Prusov et al.
12.9, 11.3, À4.7, À5.0 ppm; HRMS: calcd for C20H40O2SiNa 363.2695,
(2R,4R,6E,8R,9R,10E)-Perfluorophenyl
2,4,6,8,10-pentamethyldodeca-6,10-dienoate
9-(tert-butyldimethylsilyloxy)-
found 363.2694
(30):
Pentafluorophenol
(92 mg 0.5 mmol) was added to a solution of DCC (103 mg, 0.5 mmol) in
EtOAc (2.5 mL) cooled to 08C. The solution was stirred for 30 min and
acid 29 (140 mg 0.35 mmol) was added as a solution in EtOAc (0.5 mL)
and the mixture was stirred for an additional 1.5 h. The mixture was
warmed to room temperature, stirred for 8 h, cooled to À108C and 2 mL
of hexane was added. The urea precipitate was collected by filtration
(450 mg), the solid was washed with cold hexane (1.5 mL), and the
mother liquor was evaporated. The residue was dissolved in CH2Cl2
(50 mL), washed with saturated aqueous NaHCO3 solution (2ꢃ40 mL),
saturated aqueous NaHCO3 (40 mL) containing dissolved sodium hy-
droxide (0.30 g), and water (40 mL), and then dried and evaporated. The
residue was chromatographed over silica gel (eluting with petroleum
ether/EtOAc, 10:1) to afford 200 mg (100%) of the activated ester 30.
Rf =0.8 (petroleum ether/EtOAc, 4:1); [a]D =À13.4 (c=3.4 in CH2Cl2);
1H NMR (600 MHz, CDCl3): d=5.32 (q, J=6.6 Hz, 1H), 4.97 (dd, J=
9.2 Hz, 1H), 3.64 (d, J=7.3 Hz, 1H), 3.18–3.24 (m, 1H), 2.88–2.95 (m,
1H), 2.47–2.54 (m, 1H), 2.08–2.14 (m, 1H), 1.90–1.96 (m, 2H), 1.54–1.60
(m, 9H), 1.26–1.37 (m, 4H), 0.85 (d, J=3.7 Hz, 3H), 0.84 (s, 9H), 0.76
(d, J=6.6 Hz, 3H), À0.03 (s, 3H), À0.07 ppm (s, 3H); 13C NMR
(75 MHz, CDCl3): d=173.0, 142.9, 141.0, 139.5, 137.4, 136.3, 132.1, 131.3,
120.7, 83.4, 55.7, 47.6, 41.2, 37.2, 37.1, 34.9, 28.6, 25.7, 25.5, 24.7, 18.8,
18.1, 17.9, 17.2, 16.2, 12.8, 11.0, À4.8, À5.1 ppm; HRMS: calcd for
C29H43F5O3SiNa 585.2799, found 585.2799.
tert-Butyl((2E,4R,5R,6E,9S)-10-iodo-3,5,7,9-tetramethyldeca-2,6-dien-4-
yloxy)dimethylsilane (26): Imidazole (61 mg, 0.9 mmol, 1.50 equiv) and
iodine (198 mg, 0.78 mmol, 1.35 equiv) were added sequentially to a solu-
tion of triphenylphosphine (190 mg, 0.72 mmol, 1.20 equiv) in dichloro-
methane (5 mL) at 238C. A solution of the above alcohol (200 mg,
0.6 mmol, 1 equiv) in dichloromethane (1 mL) was added to the resulting
fine suspension via cannula. After 2 h, silica (1 g) was added and di-
chloromethane was removed, in vacuo. The solid residue was loaded
onto a column of silica gel eluting with 5% ether petroleum ether to
afford iodide 26 as a colorless oil (220 mg, 83%). Rf =0.95 (petroleum
ether/Et2O, 10:1); 1H NMR (300 MHz, CDCl3): d=5.34 (qt, J=6.6,
1.1 Hz, 1H), 5.01 (dd, J=9.4, 1.0 Hz, 1H), 3.67 (d, J=7.0 Hz, 1H), 3.24
(dd, J=9.5, 4.5 Hz, 1H), 3.09 (dd, J=9.5, 6.5 Hz, 1H), 2.44–2.57 (m,
1H), 2.08 (dd, J=13.0, 6.0 Hz, 1H), 1.81 (dd, J=13.0, 8.0 Hz, 1H), 1.61–
1.73 (m, 1H), 1.59 (d, J=6.4 Hz, 3H), 1.57 (d, J=1.1 Hz, 3H), 1.53–1.56
(m, 3H), 0.94 (d, J=6.4 Hz, 3H), 0.86 (s, 9H), 0.78 (d, J=6.8 Hz, 3H),
À0.01 (s, 3H), À0.06 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=137.2,
131.6, 131.3, 120.6, 82.9, 47.1, 37.1, 33.1, 25.8, 20.3, 18.2, 18.0, 17.7, 16.4,
12.9, 11.4, À4.7, À5.0 ppm.
(2R,4R,6E,8R,9R,10E)-9-(tert-Butyldimethylsilyloxy)-N-((1R,2R)-1-hy-
droxy-1-phenylpropan-2-yl)-N,2,4,6,8,10-hexamethyldodeca-6,10-diena-
mide (28): nBuLi (2.5m in hexanes, 3.2 mL, 8 mmol) was added to a sus-
pension of LiCl (1 g, 25 mmol, dried for 18 h at 1308C under high
vacuum) and diisopropylamine (1.2 mL, 8.6 mmol) in THF (5.5 mL) at
À788C. The resulting suspension was warmed to 08C for 10 min. An ice-
cooled solution of (S,S)-pseudoephedrine propionamide (0.93 g,
4.2 mmol) in THF (3 mL) was added at À788C and the mixture was stir-
red for 1 h at À788C, 15 min at 08C, and 5 min at room temperature. A
solution of iodide 26 (220 mg, 0.5 mmol) in THF (1 mL) was then added
at 08C, and the reaction mixture was stirred at room temperature for
14 h. The yellow reaction mixture was treated with half-saturated aque-
ous NH4Cl (10 mL) and extracted with EtOAc (4ꢃ10 mL). The com-
bined organic layers were dried over Na2SO4 and concentrated, in vacuo.
Flash chromatography (petroleum ether/Et2O, 2:1) yielded amide 28
(190 mg, 70% yield) as a yellow oil. Rf =0.33 (petroleum ether/Et2O,
(S)-1-((2R,4R,6E,8R,9R,10E)-9-(tert-Butyldimethylsilyloxy)-2,4,6,8,10-
pentamethyldodeca-6,10-dienoyl)-4-methoxy-5-methyl-1H-pyrrol-2(5H)-
one (31): nBuLi (2.5m, 0.05 mL, 0.125 mmol) was added to a solution of
py-Aly-OMe[18] (14 mg, 0.11 mmol) in THF (1 mL) at À508C, and the
mixture was stirred for 10 min. A solution of activated ester 30 (56 mg,
0.1 mmol) in THF (0.25 mL) was added dropwise over 15 min. The mix-
ture was allowed to stir for an additional 10 min, quenched with AcOH
(0.02 mL) and evaporated on silica. Column chromatography over silica
gel (petroleum ether/EtOAc, 4:1) provided the pure tetramic acid amide
31 (38 mg, 76%). Rf =0.3 (petroleum ether/EtOAc, 4:1); [a]D =12.55 (c=
2.9 in CH2Cl2); 1H NMR (600 MHz, CDCl3): d=5.30 (q, J=6.6 Hz, 1H),
5.03 (s, 1H), 4.91 (d, J=9.5 Hz, 1H), 4.60 (q, J=6.6 Hz, 1H), 3.89–3.95
(m, 1H), 3.86 (s, 3H), 3.62 (d, J=7.7 Hz, 1H), 2.48 (ddd, J=9.2, 7.2,
7.0 Hz, 1H), 2.01–2.07 (m, 1H), 1.59–1.69 (m, 3H), 1.57 (d, J=6.6 Hz,
3H), 1.54 (s, 3H), 1.53 (d, J=1.1 Hz, 3H), 1.46 (d, J=6.6 Hz, 3H), 1.30–
1.36 (m, 1H), 1.12 (d, J=6.6 Hz, 3H), 0.83 (s, 9H), 0.81 (d, J=5.9 Hz,
3H), 0.73 (d, J=7.0 Hz, 3H), À0.04 (s, 3H), À0.08 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=180.4, 177.4, 169.6, 137.4, 132.5, 130.7,
120.7, 93.0, 83.4, 58.6, 55.7, 48.1, 41.8, 37.1, 36.9, 28.6, 25.8, 18.4, 18.1,
17.8, 17.0, 16.4, 16.3, 12.9, 11.0, À4.8, À5.1 ppm; HRMS: calcd for
C29H51NO4SiNa 528.3475, found 528.3485.
1
2:1); [a]D = +35.2 (c=1.4 in CH2Cl2); H NMR (300 MHz, CDCl3) major
rotamer: d=7.23–7.42 (m, 5H), 5.31 (q, J=6.6 Hz, 1H), 4.91 (dd, J=
9.2 Hz, 1H), 4.45–4.65 (m, 2H), 3.61 (d, J=7.7 Hz, 1H), 2.90 (s, 3H),
2.70–2.80 (m, 1H), 2.42–2.57 (m, 1H), 2.00 (d, J=10.0 Hz, 1H), 1.49–1.73
(m, 13H), 1.10 (d, J=6.6 Hz, 3H), 1.03 (d, J=6.6 Hz, 3H), 0.84 (s, 9H),
0.75 (d, J=6.8 Hz, 3H), 0.72 (d, J=6.8 Hz, 3H), À0.03 (s, 3H),
À0.07 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=179.6, 142.5, 137.4,
132.3, 131.1, 128.3, 127.6, 126.4, 120.8, 83.5, 76.7, 58.1, 47.9, 41.9, 37.1,
34.3, 32.5, 28.5, 25.8, 18.9, 18.1, 17.8, 17.2, 16.4, 14.5, 12.9, 10.9, À4.7,
À5.1 ppm.
(2R,4R,6E,8R,9R,10E)-9-(tert-Butyldimethylsilyloxy)-2,4,6,8,10-pentame-
thyldodeca-6,10-dienoic acid (29): A round-bottomed flask (10 mL) was
charged with amide 28 (200 mg, 0.368 mmol, 1 equiv), tert-butyl alcohol
(2 mL), methanol (2 mL), and 2N aqueous sodium hydroxide solution
(1 mL, 2 mmol, 6 equiv). The mixture was stirred at 608C for 7 days and
then cooled to room temperature. The mixture was poured into a stirred
mixture of 1m aqueous HCl (20 mL) and dichloromethane (20 mL). The
aqueous layer was separated and extracted with dichloromethane (2ꢃ
10 mL). The combined organic extracts were washed with brine, dried
over sodium sulfate and concentrated, in vacuo. Flash chromatography
with petroleum ether/EtOAc (3:1) on silica afforded acid 29 (140 mg,
89%) as a colorless oil. Rf =0.3 (petroleum ether/EtOAc, 4:1); [a]D =
À8.33 (c=1.8 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d=5.31 (q, J=
6.6 Hz, 1H), 4.94 (dd, J=9.7 Hz, 1H), 3.63 (d, J=7.6 Hz, 1H), 2.44–2.62
(m, 2H), 1.99–2.11 (m, 1H), 1.62–1.73 (m, 1H), 1.52–1.62 (m, 9H), 1.32–
1.41 (m, 1H), 1.17 (d, J=7.1 Hz, 3H), 0.84 (s, 9H), 0.79 (d, J=6.6 Hz,
3H), 0.74 (d, J=7.1 Hz, 3H), À0.03 (s, 3H), À0.07 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=182.8, 137.4, 132.3, 131.0, 120.7, 83.4,
47.8, 41.1, 37.1, 31.2, 28.5, 25.8, 18.7, 18.1, 17.9, 16.7, 16.2, 12.9, 11.1, À4.7,
À5.0 ppm; HRMS: calcd for C23H44O3SiNa 419.2955, found 419.2957.
(S)-1-((2R,4R,6E,8R,9R,10E)-9-Hydroxy-2,4,6,8,10-pentamethyldodeca-
6,10-dienoyl)-4-methoxy-5-methyl-1H-pyrrol-2(5H)-one (32): Et3NÀ3HF
(0.15 mL) was added to a solution of imid 31 (24 mg, 47 mmol) in THF
(0.5 mL) in a plastic vessel under N2 atmosphere at 08C and the reaction
mixture was allowed to warm to room temperature and stirred for 7 days.
Then, the mixture was poured into sat. NaHCO3 solution (3 mL), the
aqueous layer was separated and extracted with CH2Cl2 (4ꢃ1.5 mL). The
combined extracts were dried (Na2SO4) and concentrated. The residue
was purified by flash chromatography on SiO2 (hexanes/EtOAc 75:25) to
give alcohol 32 (15 mg, 38 mmol, 81%) as a colorless oil. Rf =0.3 (petrole-
um ether/EtOAc, 4:1); [a]D = +38.3 (c=1.5 in CH2Cl2); 1H NMR
(600 MHz, CDCl3): d=5.47 (qd, J=6.9, 1.5 Hz, 1H), 5.02 (s, 1H), 4.96
(d, J=9.5 Hz, 1H), 4.59 (q, J=6.6 Hz, 1H), 3.89–3.96 (m, 1H), 3.86 (s,
3H), 3.56 (d, J=8.8 Hz, 1H), 2.56 (tq, J=9.6, 6.8 Hz, 1H), 2.10 (dd, J=
12.8, 5.1 Hz, 1H), 1.79 (dd, J=13.2, 8.8 Hz, 1H), 1.59–1.75 (m, 11H),
1.45 (d, J=6.6 Hz, 3H), 1.33 (ddd, J=13.0, 7.5, 5.5 Hz, 1H), 1.12 (d, J=
7.0 Hz, 3H), 0.84 (d, J=6.2 Hz, 3H), 0.77 ppm (d, J=6.6 Hz, 3H);
13C NMR (100 MHz, CDCl3): d=180.4, 177.2, 169.4, 137.1, 135.6, 129.1,
123.2, 93.0, 82.7, 58.6, 55.7, 48.1, 41.4, 36.8, 36.7, 28.6, 18.9, 17.5, 17.1,
16.7, 16.5, 13.1, 10.5 ppm; HRMS: calcd for C23H37NO4Na 414.2621,
found 414.2620.
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Eliamid: A Novel Complex I Inhibitor
FULL PAPER
(2R,4R,6E,8R,10E)-1-((S)-3-Methoxy-2-methyl-5-oxo-2,5-dihydro-1H-
pyrrol-1-yl)-2,4,6,8,10-pentamethyldodeca-6,10-diene-1,9-dione (1): Dess–
Martin periodinane (26 mg, 0.062 mmol, 2 equiv) was added to a stirring
solution of alcohol 32 (12 mg, 0.031 mmol, 1 equiv) in CH2Cl2 (1 mL) at
room temperature. After 1 h, the mixture was quenched by the simulta-
neous addition of equal volumes (~2 mL total) of aq. NaHCO3 and aq.
Na2S2O3. Once the layers cleared, the aqueous portion was extracted
twice with CH2Cl2 (1 mL) and dried over Na2SO4. The organic extracts
were filtered, concentrated under reduced pressure, and purified by
column chromatography on silica gel (petroleum ether/tBuOMe/MeOH,
80:19:1) to give 10 mg (83%) of eliamid as a colorless oil. Rf =0.37 (pe-
troleum ether/Et2O, 1:1), [a]D =À65 (c=0.8 in MeOH); 1H NMR
(600 MHz, CDCl3): d=6.71 (qd, J=6.9, 1.0 Hz, 1H), 5.07 (d, J=9.5 Hz,
1H), 5.01 (s, 1H), 4.58 (q, J=6.6 Hz, 1H), 4.01 (dq, J=9.5, 6.7 Hz, 1H),
3.86–3.92 (m, 1H), 3.86 (s, 3H), 2.00 (dd, J=13.2, 6.2 Hz, 1H), 1.85 (dd,
J=7.0, 0.9 Hz, 3H), 1.78 (dd, J=13.2, 8.1 Hz, 1H), 1.76 (s, 3H), 1.62–
1.70 (m, 1H), 1.63 (s, 3H), 1.51 (ddd, J=13.6, 8.8, 6.2 Hz, 1H), 1.43 (d,
J=6.6 Hz, 3H), 1.25 (ddd, J=13.0, 7.5, 5.5 Hz, 1H), 1.10 (d, J=7.0 Hz,
3H), 1.08 (d, J=7.0 Hz, 3H), 0.84 ppm (d, J=6.6 Hz, 3H), 13C NMR
(75 MHz, CDCl3): d=203.7, 180.3, 177.2, 169.4, 137.5, 136.5, 134.3, 127.5,
93.0, 58.6, 55.6, 47.8, 40.9, 39.8, 36.8, 28.7, 18.9, 17.7, 17.0, 16.2, 16.0, 14.8,
11.4 ppm; 1H NMR (600 MHz, [D4]MeOH): d=6.91 (qd, J=7.0, 1.1 Hz,
1H), 5.23 (s, 1H), 5.05 (d, J=9.5 Hz, 1H), 4.65 (q, J=6.6 Hz, 1H), 4.21
(dq, J=9.5, 6.6 Hz, 1H), 3.95–4.02 (m, 1H), 3.97 (s, 3H), 2.03 (dd, J=
13.2, 6.6 Hz, 1H), 1.94 (dd, J=7.0, 0.7 Hz, 3H), 1.87 (dd, J=13.2, 7.7 Hz,
1H), 1.80 (s, 3H), 1.68–1.75 (m, 1H), 1.72 (s, 3H), 1.52 (ddd, J=13.6, 7.8,
6.6 Hz, 1H), 1.46 (d, J=6.6 Hz, 3H), 1.30 (ddd, J=13.6, 7.8, 5.5 Hz, 1H),
1.13 (d, J=7.0 Hz, 3H), 1.11 (d, J=6.6 Hz, 3H), 0.91 ppm (d, J=6.2 Hz,
3H), 13C NMR (75 MHz, [D4]MeOH): d=205.8, 182.6, 178.5, 171.5,
139.0, 138.3, 135.9, 128.6, 93.5, 59.6, 56.8, 49.8 (overlapped with
[D4]MeOH), 42.0, 40.7, 37.7, 29.8, 19.6, 17.9, 17.3, 16.4, 16.2, 14.7,
11.4 ppm; HRMS: calcd for C23H36NO4 390.2634, found 390.2644.
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Acknowledgements
The authors acknowledge the experimental contribution of S. Reinecke.
We also indebted to Prof. Dr. M. Kalesse for his valuable inputs and P.
Okanya for help with manuscript preparation. We thank A. Raja for the
fluorescent microscopy pictures of eliamid treated cells. Financial support
from the DFG (grant PR 1328/1-1, to EVP) is gratefully acknowledged.
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Received: May 29, 2012
Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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ÞÞ
These are not the final page numbers!

E. V. Prusov et al.
How valuable is Breitꢀs rule? Isolation,
biological activity profiling, structure
elucidation and total synthesis of poly-
ketide natural product eliamid are
reported. This molecule is character-
ized by a tetramic acid amide frag-
ment, which is rather rare among sec-
ondary metabolites isolated from myx-
obacteria.
Natural Products
G. Hçfle, K. Gerth, H. Reichenbach,
B. Kunze, F. Sasse, E. Forche,
E. V. Prusov* ................. &&&& — &&&&
Isolation, Biological Activity Evalua-
tion, Structure Elucidation, and Total
Synthesis of Eliamid: A Novel
Complex I Inhibitor
Secondary metabolites……isolated from various
strains of myxobacterium Sorangium cellulosum
(photo in the background, taken by K. Gerth) often
possess interesting biological activities. Eliamid
(yellow) was found to inhibit the reduction of ubiq-
uinone through complex I (the red and blue curves)
at nanomolar concentrations. The difference in the
appearance of cell mitochondria can be clearly seen
on the small picture inserts.
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10
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www.chemeurj.org
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!