Isolation and structural elucidation of armeniaspirols A-C: Potent antibiotics against gram-positive pathogens

Article abstract of DOI:10.1002/chem.201201635

In an antibiotic lead discovery program, the known strain Streptomyces armeniacus DSM19369 has been found to produce three new natural products when cultivated on a malt-containing medium. The challenging structural elucidation of the isolated compounds was achieved by using three independent methods, that is, chemical degradation followed by NMR spectroscopy, a computer-assisted structure prediction algorithm, and X-ray crystallography. The compounds, named armeniaspirol A-C (2-4), exhibit a compact, hitherto unprecedented chlorinated spiro[4.4]non-8-ene scaffold. Labeling experiments with [1-¹³C] acetate, [1,2-¹³C2] acetate, and [U-¹³C] proline suggest a biosynthesis through a rare two-chain mechanism. Armeniaspirols displayed moderate to high in vitro activities against Gram-positive pathogens such as methicillin-resistant S. aureus (MRSA) or vancomycin resistant E. faecium (VRE). As analogue 2 was active in vivo in an MRSA sepsis model, and showed no development of resistance in a serial passaging experiment, it represents a new antibiotic lead structure.

Full text of DOI:10.1002/chem.201201635

FULL PAPER
DOI: 10.1002/chem.201201635
Isolation and Structural Elucidation of Armeniaspirols A–C: Potent
Antibiotics against Gram-Positive Pathogens
Cosima Dufour,[a] Joachim Wink,[a] Michael Kurz,[a] Herbert Kogler,[a] Helene Olivan,[b]
Serge Sablꢀ,[c] Winfried Heyse,[a] Martin Gerlitz,[a] Luigi Toti,[a] Antje Nußer,[a]
Astrid Rey,[d] Cedric Couturier,[d] Armin Bauer,[a] and Mark Brçnstrup*[a]
Dedicated to Professor Reinhard W. Hoffmann
Abstract: In an antibiotic lead discov-
ery program, the known strain Strepto-
myces armeniacus DSM19369 has been
found to produce three new natural
products when cultivated on a malt-
containing medium. The challenging
structural elucidation of the isolated
compounds was achieved by using
three independent methods, that is,
chemical degradation followed by
NMR spectroscopy, a computer-assist-
ed structure prediction algorithm, and
X-ray crystallography. The compounds,
named armeniaspirol A–C (2–4), exhib-
it a compact, hitherto unprecedented
proline suggest a biosynthesis through
a rare two-chain mechanism. Arme-
niaspirols displayed moderate to high
in vitro activities against Gram-positive
pathogens such as methicillin-resistant
S. aureus (MRSA) or vancomycin re-
sistant E. faecium (VRE). As analogue
2 was active in vivo in an MRSA sepsis
model, and showed no development of
resistance in a serial passaging experi-
ment, it represents a new antibiotic
lead structure.
chlorinated spiroACTHNUTRGNE[UNG 4.4]non-8-ene scaf-
fold. Labeling experiments with [1-13C]
acetate, [1,2-13C2] acetate, and [U-13C]
Keywords: antibiotics · biosynthe-
sis · drug discovery · natural prod-
ucts · spiro compounds · structure
elucidation
Introduction
isms, because this source provided the majority of antibiotics
on the market today.[4] The discovery strategy was based on
“chemical screening” of microbial fermentation broths, in
which all natural compounds that passed criteria of chemical
novelty were isolated on a preparative scale; the biological
activity profiles of the pure compounds were characterized
in subsequent steps.[5-7] The strategy led to the identification
of armeniaspirols, a new class of natural products. The struc-
ture of the armeniaspirols and a part of the data have been
disclosed before in a patent application.[8] In a recent com-
munication, semisynthetic modifications of the natural prod-
ucts and the total synthesis of a 5-chloro analogue of arme-
niaspirols have been reported.[9] In this paper, we describe
the discovery, structure elucidation, first experiments on the
biosynthesis, and the in vitro and in vivo antibacterial prop-
erties of armeniaspirols A–C.
The emergence of resistance against all known classes of an-
tibiotics represents a significant and increasing threat to
public health.[1] At the same time, anti-infective drug discov-
ery efforts in the past two decades have declined overall,
and were mostly focused on incremental chemical modifica-
tions of existing drugs.[2,3] To discover antibacterial leads
with new chemotypes, we investigated bacterial microorgan-
[a] Dr. C. Dufour, Dr. J. Wink, Dr. M. Kurz, Dr. H. Kogler, W. Heyse,
Dr. M. Gerlitz, Dr. L. Toti, A. Nußer, Dr. A. Bauer,
Dr. M. Brçnstrup
Sanofi R&D
Departments of Parallel Synthesis & Natural Products
Analytical Sciences, Innovation & External Networking
Industriepark Hoechst, H823, 65926 Frankfurt (Germany)
E-mail: mark.broenstrup@sanofi.com
[b] H. Olivan
Sanofi R&D, Department of Analytical Sciences
Bldg. 9C, 1 Av Pierre Brossolette
91385 Chilly Mazarin (France)
Results and Discussion
An investigation of the Streptomyces armeniacus type strain
DSM19369, cultivated on standard medium composed of
soymeal, glucose, cornsteep liquor, CaCO3, and NaCl at
pH 7.0 for 5 days, led to the identification of streptopyrrole
(1) in the methanolic extract. Compound 1 is an antibiotic
with an unusual benzopyranopyrrole scaffold and a known
metabolite of S. armeniacus and S. rimosus (Figure 1,
Figure 2 and the Supporting Information, Section 1).[10–12]
When the strain was cultivated on ISP-2, a medium com-
[c] S. Sablꢀ
Sanofi R&D, Department of Analytical Sciences
Friedel-Rotonde, 13 quai Jules Guesde
94403 Vitry sur Seine (France)
[d] Dr. A. Rey, Dr. C. Couturier
Sanofi R&D, TSU Anti-infectives
Bldg 15, 195, Route d’Espagne-BP
13669 Toulouse (France)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201635.
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16123

Figure 2. Chemical structures of streptopyrrol (1), armeniaspirols A–C
(2–4), the pyrrolomycin derivatives 5–7, and the hydrogenation product
8. The numbering of the carbon atoms corresponds to armeniaspirol B.
thine groups, as well as nine quaternary carbons and one hy-
droxyl group. The main chemical shift differences between
the three compounds were observed in the aliphatic region.
The COSY and HMBC data suggested the presence of dif-
ferently branched aliphatic chains (an n-hexyl chain for 2, a
4-methyl pentyl (isohexyl) chain for 3, and a 4-methyl hexyl
chain for 4) linked to an aromatic ring. The aromatic ring of
the three compounds was found to be tetrasubstituted, as
Figure 1. HPLC chromatograms of methanolic extracts of S. armeniacus
(DSM19369) cultivations using a) soja medium and b) ISP-2 medium The
UV traces at l=210 nm of the retention time window between 5 and
6 min are displayed. Major peaks correspond to 1 (5.38), 2 (5.34), 3
(5.25) and 4 (5.48 min). The chromatography conditions were as follows:
Column: Phenomenex Luna C18 (2), 3 mm, 30ꢂ2 mm with Phenomenex
pre-column (4 mm ꢂ 2 mm, C18 ODS Octadecyl); mobile phase A: water
with 1% formic acid; mobile phase B: acetonitrile with 1% formic acid;
elution gradient: 5 to 100% B in 5.5 min, 100% B over 1.5 min at a flow
rate of 1 mLminÀ1 at 408C.
1
the H spectrum showed only two doublets at d=6.60 and
7.41 ppm (3J=8.8 Hz). The CH2 in position 13 was found to
be in the para position relative to the aromatic proton at
d=7.41 ppm, as these protons have the same HMBC pat-
tern, namely two strong correlations at d=171.2 and
170.3 ppm. From their chemical shifts, it was likely that
these two last carbons were linked to a phenolic OH group
(very broad signal observed at d=11.70 ppm; broad IR
band at 3213 cmÀ1). The two remaining quaternary aromatic
carbons C6 and C4 were observed at d=112.2 and
107.9 ppm, respectively, both showing a strong coupling (3J)
to the proton in position 2. Since the signal at d=112.2 ppm
experienced also intensive couplings with the methylene
groups in position 13 (2J coupling) and 14 (3J coupling), this
carbon could be assigned to position 6 linked to the aliphat-
ic chain. Finally, the aromatic proton in position 3 was
found also coupled to a carbon at d=185.8 ppm, probably
corresponding to a a,b-unsaturated keto-like group.
posed of glucose, malt extract, and yeast extract,[13] three
new peaks were observed in the HPLC chromatogram
(Figure 1). The exact masses associated with the peaks
measured by negative ion ESI-MS were m/z=382.0633,
382.0623, and 396.0767, respectively, corresponding to the
formula C18H19Cl2NO4, C18H19Cl2NO4, and C19H21Cl2NO4 for
the neutral compounds 2, 3, and 4, respectively. As the ana-
lytical data could not be matched to known natural prod-
ucts, the compounds were isolated in pure form by prepara-
tive reversed phase chromatography. From a shake flask fer-
mentation at the 30 L scale, 2, 3, and 4 were isolated in
amounts of 54, 45, and 67 mg, respectively. In addition, the
pyrrolomycin[14] derivatives 5–7 could be isolated as minor
side products and characterized (the Supporting Informa-
tion; Table 4 and Figure 5).
The 1H and 13C data collected for 2–4 are similar (the
Supporting Information; Table 2), suggesting closely related
structures. Only the assignment of 3 will be described here-
The sharp 1H singlet at d=2.66 ppm (3H, d (13C)=
À
25.4 ppm), which was assigned as a N CH3 group, presented
two strong correlations (3J coupling) with quaternary car-
bons at d=96.5 and 162.0 ppm, respectively. An additional
weaker correlation was observed to the carbon at d=
139.3 ppm (long-range coupling). The last quaternary
1
after. The analysis of the H- and 13C NMR data, along with
the multiplicity edited HSQC experiment, indicated the
presence of three methyl, three methylene and three me-
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Chem. Eur. J. 2012, 18, 16123 – 16128

Armeniaspirols A–C
FULL PAPER
carbon observed at d=127.2 ppm in the 1D 13C NMR spec-
trum did not exhibit any coupling in the HMBC spectrum.
Due to the lack of protons in the spiro-ring system, giving
rise to very few correlations in the HMBC spectrum, it was
not possible to make an unequivocal structural assignment
by NMR experiments. Therefore, a computer-assisted struc-
ture elucidation method (CASE) was applied (the Support-
cultivations, INADEQUATE spectroscopy was applied to
determine the carbon atoms that stem from a common ace-
tate precursor.[18] All carbons from the pyrrolinone ring as
well as the keto group of the adjacent benzofuranone origi-
nate from a proline precursor (Figure 3). The incorporation
1
ing Information; Section 1).[15] In brief, H and 13C chemical
shifts, multiplicities, carbon hybridization state, as well as
the COSY, HSQC and HMBC connectivities were used to
build a molecular connectivity diagram (MCD), which listed
1-, 2- and 3-bond correlations. A total of 19834 molecules
were generated by using the MCD as an input, from which 9
molecules passed all filtering criteria (check for chemical
feasibility, ring rules, removal of duplicates, etc.). In further
steps, 13C-spectra were predicted by increment methods and
neural network and hierarchical organization of spherical
environments (HOSE) approaches.[16] The structure with the
smallest deviations between predicted and experimental
chemical shifts corresponds to 3 (Figure 2).
The structure predicted from the CASE model was inde-
pendently validated through the Pd/C-catalyzed hydrogena-
tion of 2. The obtained derivatives 8 and 9 were amenable
to a full structure elucidation by NMR spectroscopy, as car-
bons 9 and 10 of the five-membered ring and the keto-func-
tion C7 have been completely reduced to methylene moiet-
ies (Figure 2 and the Supporting Information; Figure 4,
Tables 5 and 6). The quaternary carbon of the spiro-center
in 8 (C8 at d=103.90 ppm) experienced heteronuclear cou-
plings in the HMBC spectrum to all three methylene
groups, confirming the carbon skeleton proposed for struc-
tures 2–4.
Single-crystal X-ray structure analysis of 2 and 4 enabled
the final proof of the structure and the determination of the
absolute configuration. Suitable crystals were obtained by
crystallization in an ethanol/heptane mixture. According to
the refinement of the fractional contribution, the Flack pa-
rameter[17] could be refined to 0.01 for structure 2 and to
0.15 for structure 4 with standard deviations less than 0.06.
Reference values are 0 for correct and +1 for inverted ab-
solute structure. Therefore, the absolute configurations of
the spiro-centers could be determined as R; the chiral
center in the aliphatic chain of 4 is S-configured.
Figure 3. Labelling studies on 2–4. The positions that show incorporation
of 13C-atoms after [U-13C] proline feeding are indicated by bold bonds;
acetate incorporation is indicated by arrows pointing from C2 to C1
based on feeding experiments with [1-13C] acetate and [1,2-13C2] acetate.
of acetate units within the pyrrolinone ring can be traced
back to the proline biosynthesis from a-ketoglutarate.[12,19]
Acetate is the source for all carbon atoms in the trisubstitut-
ed phenol ring and for all carbon atoms in the side chain of
2. A 13C label could not be detected in the N-methyl group
of the pyrrolinone ring and the terminal isobutyl group of 3,
and only to a small extent (relative intensity (Irel)=9% or
less, compared with Irel =48% for the adjacent C13/C14 ace-
tate) in the 2-methyl-butyryl group of 4. The specific posi-
tioning of acetate units relative to the annelated heterocycle
in the trisubstituted phenol ring is identical to that found in
1 and in pyralomycins in previous labeling studies.[12,19] It
clearly excludes a symmetric alkyl-resorcin intermediate
that reacts in a bimolecular manner with a proline derivative
(Figure 4).
The following hypothesis for the biosynthesis of 2–4 de-
rived from the data: The isolated pyrrolomycin derivatives
5–7 are probably central intermediates in the biosynthesis of
1–3. We propose that 5–7 are formed through a bimolecular
condensation of two polyketide chains to account for the
specific positioning of acetate units in the 6-membered aro-
matic ring. Bimolecular condensation reactions in natural
product biosynthesis are rare,[20] but have been suggested for
the biosynthesis of 1,[12] corallopyronin,[21] or bacterial stil-
benes.[22] We speculate that the CoA esters of acetic acid,
isobutyric acid and 2-methyl-butyric acids are all accepted
as a starter unit for the synthesis of 10–12. The biosynthesis
of the second chain starts with proline, which can be con-
verted to the dichloropyrrolyl carrier protein 13 by FADH2
halogenases, according to detailed studies on the biosynthe-
sis of pyrrolomycins and pyoluteorin.[14,23] After extension of
13 by a single C2 unit, condensation with 10–12 is proposed
to occur. A reduction/dehydration at the keto group in the
Considering their intriguing und unprecedented spiro-
ACHTUNGTRENNUNG[4.4]non-8-ene scaffold and the armenian origin of the pro-
ducer, we named the compounds armeniaspirol A (2), B (3),
and C (4). Remarkably, a spiro-structure that bears a simi-
larity to armeniaspirols has been postulated (but neither iso-
lated nor characterized) as an unstable intermediate in a
proposal for the biosynthesis of 1.[12]
To gain information on the biosynthetic precursors of ar-
meniaspirols, cultures of S. armeniacus were supplemented
with [1-13C] acetate, [1,2-13C2] acetate, or [U-13C] proline in
separate experiments (see the Supporting Information; Sec-
tion 2). After isolating the partially labeled analogues of 2–
4, the compounds were characterized by 13C NMR spectro-
scopy. For experiments based on [1,2-13C2] acetate-enriched
Chem. Eur. J. 2012, 18, 16123 – 16128
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16125

M. Brçnstrup et al.
data give a first sketch of the biosynthesis of 2–4 and its re-
lation to that of pyralomycins, pyrrolomycin, or streptopyr-
rols, a more detailed insight must be based on the future
analysis of the encoding gene cluster.
Armeniaspirols exhibited moderate–high in vitro activities
against Gram-positive bacteria. The growth of pathogens
such as Methicillin-resistant or -sensitive S. aureus (MRSAs
ATCC33592 and ATCC33592, and MSSA ATCC13709),
penicillin-resistant S. pneumoniae (PRSP DSM11865), or
vancomycin resistant E. faecium (VRE DSM17050) was in-
hibited at low mgmLÀ1 concentrations in a BacTiter glow
assay (Table 1). In contrast, the compounds are inactive
against representative Gram-negative bacteria such as E.
coli and P. aeruginosa, or the fungus C. albicans. The com-
pounds displayed no cytotoxicity against the human hepato-
cyte HepG2 cell line up to 30 mm. Time-kill studies demon-
strated that 2 was bacteriostatic with a time-dependent
effect (Supporting Information; Section 3.2). In a serial pas-
saging experiment with S. aureus, development of resistance
against 2 was not observed within 30 passages (Supporting
Information; Section 3.3).
Figure 4. Mechanistic proposal for the biosynthesis of 2–4 through a two
chain condensation. The link between starting units and alkyl chain sub-
stitution patterns is as follows: the CoA-ester of acetic acid would lead to
10 (R1 =H, R2 =Me), that of isobutyric acid to 11 (R1 =Me, R2 =H), and
that of 2-methyl-butyric acid to 12 (R1, R2 =Me). Enz=Enzyme.
To investigate the antibacterial effect in vivo, compound 2
was studied in
a
MRSA-induced septicemia model
(Table 2). After intravenous administration, compound 2 im-
proved the survival rate and decreased blood bacteremia in
MRSA-infected mice in a dose-dependent manner. Howev-
er, the infection could not be completely cured at a dose of
15 mgkgÀ1 with 4 out of 6 animals surviving; at higher doses,
adverse cardiac side-effects were observed.
para position to the alkyl chain sequence would lead to the
stable intermediates 5–7. The conversion of 5–7 to the arme-
niaspirols 2–4 could proceed in a similar manner as suggest-
ed by Raggatt et al.[12] Initial model studies indicated that
simplified synthetic analogues of 5–7 could be successfully
transformed to the correctly substituted, chlorinated spiro-
Table 2. In vivo activities of 2 in a MRSA-induced septicemia model.[a]
2
Vancomycin
ACHTUNGTRENNUNG[4.4]non-8-ene scaffold under oxidative chlorination condi-
iv dose [mgkgÀ1 dÀ1
surviving animals [%]
log[CFU reduction]
]
3
0
0.05
10
33.3
0.9[b]
15
67
20
tions (the Supporting Information, Section 2.2).[9] The meth-
ylation of the nitrogen atom probably occurs from a methyl
source such as S-adenosyl methionine; because the feeding
of 13C-methionin suppressed the production of armeniaspir-
ol, the latter hypothesis could not be verified experimentally
(Supporting Information, Section 2). Whereas the current
100[b]
4.2[b]
2.8[b]
[a] Infection with MRSA-type strain ATCC33592 (6 animals/group for
survival, 5 animals/group for bacteremia). CFU (colony-forming units)
reductions are given as mean values; [b] P<0.001 versus vehicle-treated
group (vehicle: ethanol 5%/solutol 5%/Tris-NaCl buffer pH 9).
Table 1. In vitro activities of 2–4.
Strain
2
3
4
Ciprofloxacin
MIC
Nystatin
MIC
IC80
[mgmLÀ1
MIC
[mgmLÀ1
IC80
[mgmLÀ1
MIC
[mgmLÀ1
IC80
[mgmLÀ1
MIC
[mgmLÀ1
G
]
E
]
G
]
E
]
G
]
N
]
A
]
ACHTUNGTRENNUNG ]
[mgmLÀ1
S. aureus (ATCC13709)[b]
S. aureus (ATCC29213)[b]
S. aureus (ATCC33592)[b]
S. pneumonia (DSM11865)[a]
S. pyogenes (ATCC12344)[b]
0.3
0.35
<0.125
2.30
6.20
8.20
25.20
0.30
>64
>64
>64
1
1
0.7
0.85
<0.125
1.80
5.80
5.80
17.80
0.60
>64
>64
>64
1
2
0.5
0.75
<0.125
1.80
4.30
5.90
24.70
0.50
>64
>64
>64
1
2
0.5
0.5
0.5
–
–
–
–
–
–
–
–
–
–
2
0.25
4
16
16
32
2
>64
>64
>64
0.25
4
32
8
32
8
>64
>64
>64
0.125
4
64
8
64
4
>64
>64
>64
0.125
0.125
0.5
0.25
2
0.125
0.004
–
2
E. faecium (DSM17050)
E. faecalis (ATCC29212)[b]
M. smegmatis (ATCC607)[a]
P. aeruginosa (ATCC27853)[a]
E. coli (ATCC25922)[b]
C. albicans (FH2173)[b]
[a] Luminescence; [b] Absorption at 590 nm. MIC=minimal inhibitory concentration; IC=inhibitory concentration.
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Armeniaspirols A–C
FULL PAPER
rial); mobile phase A: water with NH4OAc (50 gLÀ1) at pH 7.0; mobile
phase B: isopropanol; elution gradient: 40 to 100% B over 30 min,
100% B over 15 min at a flow rate of 240 mLminÀ1; UV detection l:
222 nm; fraction volume: 1 L. Fractions 6 to 8 containing the armenia-
AHCTUNGERTGsNNUN pirol derivatives were combined, reduced to a volume of 1.5 L under
vacuum and lyophilized.
Conclusion
The cultivation of the well-characterized S. armeniacus
under new conditions led to the discovery of armeniaspirols.
This demonstrates that even well-known microorganisms
can serve as a source for new, active natural products. The
activation of silent, natural product-coding gene clusters by
rational means is therefore an important theme of current
natural product research.[24] The structures of armeniaspirols
could be elucidated through hydrogenation, computer-assist-
ed 13C NMR spectra prediction, and X-ray analysis. Due to
their unique scaffold, combined with moderate-high in vitro
activities against a panel of Gram-positive pathogens, and
their efficacy in an MRSA sepsis model, armeniaspirols rep-
resent new antibiotic lead structures. The first synthetic
route to armeniaspirols has facilitated the access to structur-
al analogues with improved antibiotic efficacy compared
with the profiles of 2–4.[9] In addition, sequence information
on the gene cluster(s) encoding for 1 and 2–4 should further
clarify their biosynthesis and the regulation of production.
For the purification of 2–4 by RP-18 chromatography, half (800 mg) of
the lyophilizate was purified by HPLC coupled with DAD-UV detection.
The column conditions were as followed: Pre-column: Waters - XTerra
(19 mmꢂ10 mm, 10 m C18); Column: Varian
- Dynamax Pursuit
(40 mmꢂ100 mm, 10 m C18); mobile phase A: water with NH4OAc
(50 gLÀ1) at pH 7.0; mobile phase B: acetonitrile; elution gradient: 10 to
95% B over 30 min, 95% B over 5 min at a flow rate of 170 mLminÀ1
;
UV detection l: 222 nm; fraction volume: 34 mL. Analysis by HPLC
showed that the fractions 32–33 contained armeniaspirol A, fractions 28–
30 contained armeniaspirol B and fraction 36 contained armeniaspirol C.
The second half (800 mg) of the lyophilizate was purified using an opti-
mized elution gradient: 20 to 95% B over 30 min, 95% B over 5 min at a
flow rate of 120 mLminÀ1; UV detection l: 222 nm; fraction volume:
24 mL. Analysis by HPLC showed that fraction 29 contained armenias-
pirol A, fractions 19–26 contained armeniaspirol B and fractions 33–35
contained armeniaspirol C.
After lyophilization, fractions 32+33 (column 1) added to fraction 29
(column 2) yielded 54.3 mg of armeniaspirol A (purity>95%), fractions
28–30 (column 1) added to fractions 19–26 (column 2) yielded 44.9 mg of
armeniaspirol B (purity>95%) and fraction 36 (column 1) added to frac-
tions 33–35 (column 2) yielded 67.3 mg of armeniaspirol C (purity>
95%).
Experimental Section
Analytical characterization of 2–4: A detailed physical characterization
and a full structural assignment by NMR spectroscopic analyses of 2–4 is
given in the Supporting Information.
Crystallographic data: CCDC-809579 (2) and CCDC-809580 (4) contain
the supplementary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
Armeniaspirol A (2): (2R)-3’,4’-dichloro-7-hexyl-6-hydroxy-1’-methyl-spi-
(1’H)-dione): [a]2D0 = +52.7 (c=
ro[benzofuran-2ACTHNUTRGENN(GU 3H),2’-[2H]pyrrole]-3,5’ACHTUTGNRENNUGN
0.75 in methanol); 1H NMR (600 MHz, 303 K in [D6]DMSO): d=6.72
(d); 7.48 (d); 2.67 (s); 2.57 (m); 1.51 (m); 1.30/1.25 (m); 1.25 (m); 1.24
(m); 0.82 (t) ppm; 13C NMR (150 MHz, 303 K in [D6]DMSO): d=168.3
(s); 113.6 (d); 124.3 (d); 109.2 (s); 171.4 (s); 112.6 (s); 187.0 (s); 96.4 (s);
138.8 (s); 127.5 (s); 162.0 (s); 25.4 (q); 21.7 (t); 27.9 (t); 28.3 (t); 31.0 (t);
21.9 (t); 13.8 (q) ppm; IR (ATR): n˜ =3211 (O-H); 2955, 2926, 2857 (C-
H); 1721, 1706 (C=O); 1619, 1600 (C=C) cmÀ1; UV/Vis (water/acetoni-
trile): lmax =210, 233, 301 nm; HR-ESI-MS: m/z calcd for C18H18Cl2NO4:
382.0612 [MÀH]À; found: 382.0633; m/z calcd for C18H19Cl2NO4:
383.06911 [M]; found: 383.07058; elemental analysis calcd (%) for
C18H19Cl2NO4: C 56.26; H 4.98; N 3.65; found: C 56.74; H 5.16; N 3.75.
Cultivation of Streptomyces sp. ST 108140 (DSM 19369): 100 mL of nu-
trient solution (soymeal (10 gLÀ1), glucose (10 gLÀ1), cornsteep liquor
(1.5 gLÀ1) CaCO3 (2.0 gLÀ1), and NaCl (1.5 gLÀ1) at pH 7.0) in a sterile
300 mL Erlenmeyer flask was inoculated with the strain Streptomyces sp.
ST 108140, and the culture was incubated for 7 days at 288C and 180 rpm
on a rotating shaker. To induce the production of 2–4, nutrient solution
(100 mL of 4.0 gLÀ1 glucose, 4.0 gLÀ1 yeast extract, 10.0 gLÀ1 malt ex-
tract, 2.0 gLÀ1 CaCO3, pH 7.0) in a sterile 300 mL Erlenmeyer flask was
inoculated with the strain Streptomyces sp. ST 108140 and the culture
was incubated for 7 days at 288C and 180 rpm on a rotating shaker.
10 mL of the preliminary cultures were then used to prepare the main
cultures.
Armeniaspirol B (3); (2R)-3’,4’-dichloro-6-hydroxy-1’-methyl-7-(4-meth-
ACHUTNGRENyNUG lACHUTGNENRNUGpentyl)-spiro[benzofuran-2ACHTUTGNREN(NUNG 3H),2’-[2H]pyrrole]-3,5’ACHTUNGTRENN(UGN 1’H)-dione):
For the liquid main culture, a sterile 300 mL Erlenmeyer flask containing
the nutrient solution (100 mL of 4.0 gLÀ1 yeast extract, 10.0 gLÀ1 malt ex-
tract, 4.0 gLÀ1 glucose, pH 7.0) was inoculated with 10 mL of a prelimi-
[a]2D0 = +69.2 (c=0.70 in methanol); 1H NMR (600 MHz, 303 K in
[D6]DMSO): d=6.60 (d); 7.41 (d); 2.66 (s); 2.57 (m); 1.52/1.47 (m); 1.17
(m); 1.49 (m); 0.81 (d); 0.81 (d); 11.70 (bs) ppm; 13C NMR (150 MHz,
303 K in [D6]DMSO) d=170.3 (s); 114.6 (d); 124.1 (s); 107.9 (s); 171.2
(s); 112.2 (s); 185.8 (s); 96.5 (s); 139.3 (s); 127.2 (s); 162.0 (s); 25.4 (q);
21.9 (t); 25.9 (t); 38.1 (t); 27.2 (d); 22.4 (q); 22.4 (q) ppm; IR (ATR): n˜ =
3213 (O-H); 2954, 2925, 2866 (C-H); 1720, 1704 (C=O); 1620, 1601 (C=
C) cmÀ1; UV/Vis: lmax =209, 232, 300 nm; HR-ESI-MS: m/z calcd for
C18H18Cl2NO4: 382.0612 [MÀH]À; found: 382.0623; m/z calcd for
C18H19Cl2NO4: 383.06911 [M]; found: 383.06958; elemental analysis calcd
(%) for C18H19Cl2NO4: C 56.26; H 4.98; N 3.65; found: C 56.52; H 5.15;
N 3.59.
ACHTUNGTRENNUNGnary culture and incubated at 288C and 180 rpm on a shaker. The maxi-
mum production of armeniaspirols was reached after 120–168 h. A 96–
120 hour-old submerged culture (inoculation quantity approx. 5–10%)
from the same nutrient solution as described above was sufficient for in-
oculating from 10 to 200 L fermenters. Ten liter fermenters were operat-
ed under the following conditions: Inoculum approximately 10%; nu-
trients as described above; incubation temperature of 288C; stirrer speed
of 112 rpm; aeration of 8 LminÀ1; pH regulation from pH 7.8 to 7.5, no
pO2 regulation. The pH was regulated using 10% KOH or 10% H2SO4,
respectively. Foam formation was suppressed by for example, repeatedly
adding Clerol FBA 265 (Cognis Deutschland GmbH). Maximum produc-
tion is reached after approximately 72 to 96 h.
Armeniaspirol C (4): (2R)-3’,4’-dichloro-6-hydroxy-1’-methyl-7-[(4S)-4-
methylhexyl]-spiro[benzofuran-2ACTHNUGTRENNUG(3H),2’-[2H]pyrrole]-3,5’ACHUTNGTREN(NUGN 1’H)-dione):
[a]2D0 = +66.3 (c=0.86 in Methanol); 1H NMR (600 MHz, 303 K in
[D6]DMSO) d=6.73 (d); 7.49 (d); 2.68 (s); 2.57 (m); 1.52 (m); 1.29/1.11
(m); 1.31 (m); 1.26/1.06 (m); 0.79 (t); 0.81 (d) ppm; 13C NMR (150 MHz,
303 K in [D6]DMSO) d=168.2 (s); 113.6 (d); 124.2 (s); 109.2 (s); 171.3
(s); 112.6 (s); 186.9 (s); 97.4 (s); 138.9 (s); 127.5 (s); 162.1 (s); 25.4 (q);
22.0 (t); 25.5 (t); 35.5 (t); 33.5 (d); 28.8 (t); 11.0 (q); 18.9 (q) ppm; IR
(ATR): n˜ =3212 (O-H); 2960, 2926, 2865 (C-H); 1720, 1704 (C=O); 1619,
1600 (C=C) cmÀ1; UV/Vis: lmax =208, 232, 300 nm; HR-ESI-MS: m/z
Preparative isolation of 2–4: After the Streptomyces sp. ST 108140 fer-
mentation had come to an end, the culture broth (30 L) was separated
from the biomass by centrifugation. The biomass was extracted with a
mixture of 90% methanol and 10% water (4ꢂ5 L). The methanol extract
was reduced to 5 L under vacuum and then loaded together with the cul-
ture broth (30 L) onto a preparative column. The HPLC conditions were
as followed: Column: Mitsubishi Chemical Corporation-MCI gel
(130 mmꢂ200 mm, packed with approx. 4 L of 75–150 m CHP-20P mate-
Chem. Eur. J. 2012, 18, 16123 – 16128
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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16127

M. Brçnstrup et al.
calcd for C19H20Cl2NO4: 396.0768 [MÀH]À; found: 396.0767; m/z calcd
for C19H21Cl2NO4: 397.08476 [M]; found: 397.08398.
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Received: May 9, 2012
Published online: November 9, 2012
16128
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Chem. Eur. J. 2012, 18, 16123 – 16128