Structures, reactivities, and antibiotic properties of the marinopyrroles A-F

Article abstract of DOI:10.1021/jo1002054

Cultivation of actinomycete strain CNQ-418, retrieved from a deep ocean sediment sample off the coast of La Jolla, CA, has provided marinopyrroles A-F. Sharing just 98% 16S rRNA gene sequence identity with S. sannurensis, the strain likely represents a new Streptomyces species. The metabolites contain an unusual 1,3′-bipyrrole core decorated with several chlorine and bromine substituents and possess marked antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The congested N,C-biaryl bond establishes an axis of chirality that, for marinopyrroles A-E, is configurationally stable at room temperature. Moreover, the natural products are fashioned strictly in the M-configuration. The Paal-Knorr condensation was adapted for the synthesis of the 1,3′-bipyrrole core. Halogenation of this material with N-bromosuccinimide cleanly furnished the 4,4′,5,5′- tetrahalogenated core that characterizes this class of marine-derived metabolites.

Full text of DOI:10.1021/jo1002054

pubs.acs.org/joc
Structures, Reactivities, and Antibiotic Properties of the
Marinopyrroles A-F
Chambers C. Hughes, Christopher A. Kauffman, Paul R. Jensen, and William Fenical*
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography,
University of California, San Diego, La Jolla, California 92093-0204
wfenical@ucsd.edu
Received February 9, 2010
Cultivation of actinomycete strain CNQ-418, retrieved from a deep ocean sediment sample off the
coast of La Jolla, CA, has provided marinopyrroles A-F. Sharing just 98% 16S rRNA gene sequence
identity with S. sannurensis, the strain likely represents a new Streptomyces species. The metabolites
contain an unusual 1,30-bipyrrole core decorated with several chlorine and bromine substituents and
possess marked antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).
The congested N,C-biaryl bond establishes an axis of chirality that, for marinopyrroles A-E, is
configurationally stable at room temperature. Moreover, the natural products are fashioned strictly
in the M-configuration. The Paal-Knorr condensation was adapted for the synthesis of the 1,30-
bipyrrole core. Halogenation of this material with N-bromosuccinimide cleanly furnished the
4,40,5,50-tetrahalogenated core that characterizes this class of marine-derived metabolites.
Introduction
produce antibiotics, although since the 1940s a plethora of
antibiotics have been isolated from actinomycetes collected
from the soil. Initially, the extent of evolutionary divergence
between terrestrial and marine actinomycete strains was
underestimated. Since then, comparisons of 16S rRNA
sequence data have shown that deep-ocean sediments harbor
new actinomycete taxa, some of which are fully restricted to
life in the sea.3 Cultivation of these strains in the laboratory
has yielded many structurally unique metabolites of consid-
erable biological interest.4
The capacity of pathogenic bacteria, such as methicillin-
resistant Staphylococcus aureus (MRSA), to develop resis-
tance to antimicrobial compounds requires the “persistent
discovery” of novel structural pharmacophores to combat
infection.1 Bacterial resistance has developed against even
the glycopeptide antibiotics (vancomycin and teicoplanin),
which are widely considered the drugs of “last resort”. The
first fully vancomycin-resistant S. aureus (VRSA) strain was
isolated from the clinic in 2002.2
As described in a previous communication,5 cultivation
of the marine Streptomyces strain CNQ-418 in a seawater-
based medium provided (-)-marinopyrroles A (1) and B (2),
Marine bacteria of the order Actinomycetales (actino-
mycetes) have received little attention for their ability to
(1) (a) von Nussbaum, F.; Brands, M.; Hinzen, B.; Weigand, S.; Haebich,
D. Angew. Chem., Int. Ed. 2006, 45, 5072–5129. (b) Walsh, C. Antibiotics:
Actions, Origins, Resistance; ASM Press: Washington, DC, 2003. (c) Schito,
G. C. Clin. Microbiol. Infect. 2006, 12, 3–8. (d) Palavecino, E. In Methicillin-
resistant Staphylococcus Aureus (MRSA) Protocols; Ji, Y., Ed.; Humana Press:
Totowa, 2007; pp 1-19.
(2) Smith, T. L.; Pearson, M. L.; Wilcox, K. R.; Cruz, C.; Lancaster,
M. V.; Robinson-Dunn, B.; Tenover, F. C.; Zervos, M. J.; Band, J. D.;
White, E.; Jarvis, W. R. N. Engl. J. Med. 1999, 340, 493–501.
(3) Maldonado, L. A; Fenical, W.; Jensen, P. R.; Kauffman, C. A.;
Mincer, T. J.; Ward, A. C.; Bull, A. T.; Goodfellow, M. Int. J. Syst. Evol.
Microbiol. 2005, 55, 1759–1766.
(4) (a) Fenical, W.; Jensen, P. R. Nat. Chem. Biol. 2006, 2, 666–673. (b)
^
Hughes, C. C.; MacMillan, J. B.; Gaudencio, S. P.; Jensen, P. R.; Fenical, W.
Angew. Chem., Int. Ed. 2009, 48, 725–727.
(5) Hughes, C. C.; Prieto-Davo, A.; Jensen, P. R.; Fenical, W. Org. Lett.
2008, 10, 629–631.
3240 J. Org. Chem. 2010, 75, 3240–3250
Published on Web 04/20/2010
DOI: 10.1021/jo1002054
r
2010 American Chemical Society

Hughes et al.
JOCArticle
CHART 1. Marinopyrroles A-F (1-6)
in nature.7 As such, the marinopyrroles represent the first
naturally occurring 1,30-bipyrroles. Perhaps for this reason,
reports that describe the synthesis of 1,30-bipyrroles are few.8
Results and Discussion
Sequence comparison of 16S rRNA, a 1542 nucleotide
constituent of the prokaryotic 30S ribosomal subunit, has
become a standard method for phylogenetic analysis of new
bacterial isolates.9 Strain CNQ-418 shares 98.1% 16S rRNA
gene sequence identity with its nearest neighbor, Streptomyces
sannurensis (GenBank accession no. bankit EU116310). Given
that the ribosomal machinery is ancient and highly conserved,
small deviations in this sequence often reflect considerable
genetic divergence. Though there is disagreement over the
exact percentage of 16S rRNA variance that constitutes a new
species, less than 97-99% sequence identity has been pro-
posed as a benchmark. As such, CNQ-418 likely represents a
new Streptomyces species.
Initial observations using proton NMR and UV/vis spec-
troscopy revealed the largely aromatic character of marino-
pyrrole A (1). The presence of halogen atoms was concluded
upon examining the LR-ESI mass spectrum [m/z (M þ H)þ
509, 511, 513; m/z (M þ Na)þ 531, 533, 535], which showed an
isotope composition indicative of multiple chlorine substitu-
ents. Analysis of high-resolution mass spectral data indicated
the molecular formula C22H12Cl4N2O4 [m/z (M - H)- calcd
for C22H1135Cl4N2O4 506.9473, found 506.9447]. Analogues
2-6 displayed a similar UV profile. Marinopyrroles B (2)
and E (5) possess an additional bromine atom (C22H11-
BrCl4N2O4), while marinopyrroles C (3) and D (4) have an
additional chlorine atom (C22H11Cl5N2O4). Marinopyrrole F
(6) contains an extra degree of unsaturation and analyzed for
the molecular formula C22H11Cl3N2O4.
Planar Structure. Elucidation of the planar structure of
marinopyrrole A (1) via NMR methods proved difficult.
Only two salicyloyl substituents could be confidently defined
by interpretation of COSY, HSQC, and HMBC NMR
experiments (Figure 1). The presence of the o-hydroxyl func-
tionality on each of these rings was concluded from the
chemical shifts of the O-bearing aromatic carbons (δ 161.1
and 162.5) and from its reactivity with methylating and acylat-
ing reagents. Two sharp singlets at δ 11.18 and 10.42 were
attributed to the peri-hydroxyl protons, and a broad 1H signal
at δ 9.73 was assigned to an acidic N-H proton (Table 1).
A single aromatic singlet at δ 6.72, which showed a strong
correlation to one of the carbonyl carbons (δ 186.8), as well as
to carbons at δ 128.8 and 124.1, remained unassigned. With
only two proton resonances available to map 2D-heteronuclear
correlations, the structure of the central nitrogen-containing
CHART 2. Axially Chiral Bipyrroles 7-9
axially chiral, densely halogenated natural products com-
posed of two salicyloyl substituents on a 1,30-bipyrrole core
(Chart 1). The requirement of seawater for growth of this
strain indicates a significant degree of adaptation to the
marine environment. Under optimized culturing conditions,
described herein, (-)-marinopyrroles C (3), (-)-D (4), (-)-E
(5), and (()-F (6) were also isolated from the culture broth of
CNQ-418. The most abundant metabolite, marinopyrrole A
(1), was obtained in a 50 mg L-1 yield after HPLC purifica-
tion. Compared to 1, (-)-marinopyrroles C-E (3-5) pos-
sess an additional halogen atom attached to either the
bipyrrole nucleus or one of the two salicyloyl moieties. The
structure of (()-marinopyrrole F (6) includes a rare eight-
membered ether ring. Each metabolite, except (()-6, was
isolated as a single atropo-enantiomer exhibiting configura-
tional stability at room temperature.
In addition to the marinopyrroles, there are only three
known examples of axially chiral bipyrroles;1,10-bipyrrole
7,6a 2,20-bipyrrole 8,6b and most recently, 3,30-bipyrrole 96c
(Chart 2). Compounds 7-9 are synthetic in origin and
require a resolution step, either fractional recrystallization
of a brucine salt or chiral HPLC purification, to attain
atropo-enantiomeric purity. Furthermore, of the three pos-
sible bipyrroles that involve bonding through nitrogen only
achiral 1,20-bipyrroles have previously been shown to occur
(7) Wu, J.; Vetter, W.; Gribble, G. W.; Schneekloth, J. S., Jr.; Blank, D.
H.; Gorls, H. Angew. Chem., Int. Ed. 2002, 41, 1740–1743.
(8) (a) Mingoia, F. Tetrahedron 2001, 57, 10147–10153. (b) Rochais, C.;
Lisowski, V.; Dallemagne, P.; Rault, S. Tetrahedron Lett. 2004, 45, 6353–
6355. (c) Santo, R. D.; Massa, S.; Costi, R.; Simonetti, G.; Retico, A.
Farmaco 1994, 49, 229–236.
(9) (a) Weisburg, W. G.; Barns, S. M.; Pelletier, D. A.; Lane, D. J. J.
Bacteriol. 1991, 173, 697–703. (b) Fox, G. E.; Wisotzkey, J. D.; Jurtshuk, P.,
Jr. Int. J. Syst. Bacteriol. 1992, 42, 166–170. (c) Stackebrandt, E.; Goebel, B.
M. Int. J. Syst. Bacteriol. 1994, 44, 846–849. (d) Drancourt, M.; Bollet, C.;
Carlioz, A.; Martelin, R.; Gayral, J.-P.; Raoult, D. J. Clin. Microbiol. 2000,
38, 3623–3630. (e) Stach, J. E. M.; Maldonado, L. A.; Masson, D. G.; Ward,
A. C.; Goodfellow, M.; Bull, A. T. Appl. Environ. Microbiol. 2003, 69, 6189–
6200.
(6) (a) Chang, C.; Adams, R. J. Am. Chem. Soc. 1931, 53, 2353–2357. (b)
Webb, J. L. A. J. Org. Chem. 1953, 18, 1423–1427. (c) Dey, S.; Pal, C.; Nandi,
D.; Giri, V. S.; Zaidlewicz, M.; Krzeminski, M.; Smentek, L.; Hess, B. A.;
Gawronski, J.; Kwit, M.; Babu, N. J.; Nangia, A.; Jaisankar, P. Org. Lett.
2008, 10, 1373–1376.
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Hughes et al.
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Marinopyrrole D (4) also analyzed for the molecular for-
mula C22H11Cl5N2O4. The carbon signal at δC 185.3 was
assigned to C-60 based on the HMBC correlation from H-30
(δ 6.69). The signal at δC 185.1 was then assigned to C-6. Here,
an HMBC correlation from the proton signal at δ 7.32 (H-120)
to C-60 allowed entry into the adjacent salicyloyl spin system.
In this isomer, COSY and HMBC correlations showed that
the chlorine substituent was attached to the same salicyloyl
ring at C-110 (δC 123.7). COSY correlations between protons
of the other salicyloyl ring (H-9 to H-12) were uninterrupted.
The NMR spectra for marinopyrrole E (5), C22H11-
BrCl4N2O4, were similar to the spectra for marinopyrrole
D (4). As before, the signals at δC 185.3 and δC 185.1 were
assigned to C-60 and C-6. COSY and HMBC correlations
showed that the halogen atom was attached to the lower
salicyloyl ring at C-110 (δC 120.0).
FIGURE 1. Partial NMR-based structure of marinopyrrole A (1).
portion of the molecule, consisting of 14 contiguous quaternary
carbon centers, could not be solved.10
Elucidation of the planar structure of marinopyrrole F (6),
1
C22H11Cl3N2O4, required more analysis. In the H NMR
The planar bipyrrole structure common to the marino-
pyrroles was finally defined by an X-ray crystal structure
determination of marinopyrrole B (2). Slow evaporation of a
pure solution of 2 in toluene at room temperature yielded
crystals that were amenable to X-ray analysis. The X-ray
structure showed that a polyhalogenated 1,30-bipyrrole com-
prised the core of the molecule, a motif that no natural
product to date has featured. Fortuitously, given the gamut
of fruitless attempts to obtain an X-ray quality crystal from
1, the bromine atom in 2 facilitated the X-ray assignment of
an M-configuration to the natural product (vide infra).
The bipyrrole skeleton having been revealed, the planar
structures for marinopyrroles A (1) and C-E (3-5) were
then readily determined by interpretation of 2D NMR and
mass spectral data. Since the additional singlet at δ 6.72 in
the proton spectrum of 1 showed a strong HMBC correlation
to the C-60 carbonyl group, it was positioned at C-30 (see
Figure 1). The HMBC correlation from H-30 to the C-60
ketone carbon was instrumental in uncovering the halogena-
tion pattern in pentahalogenated analogues 3-5. Combined
with the correlation from H-120 to C-60, the two salicyloyl
substituents could be distinguished from each other (see
Figure 1).
Marinopyrrole C (3) analyzed for the molecular formula
C22H11Cl5N2O4. The carbon signal at δC 186.7 was assigned
to C-60 based on the HMBC correlation from H-30 (δ 6.81).
The other carbonyl signal at δC 184.5 was then assigned to
C-6. An HMBC correlation from the proton doublet at δ
7.66 (H-120) to C-60 allowed entry into the adjacent salicyloyl
spin system. Uninterrupted COSY correlations between the
protons of this salicyloyl ring showed the absence of a
substituent. Instead, HMBC and COSY correlations indi-
cated that the chlorine atom was attached to the other
salicyloyl ring at C-11 (δC 123.8).11
spectrum of 6, a low-field peri-hydroxyl proton signal, one of
two, was conspicuously missing, and the chemical shift of the
C-6 carbonyl carbon was shifted 10 ppm upfield (δC 175.8)
compared to 1-5 (see Table 1). Formation of this trichloro
analogue, we thought, may arise from intramolecular dis-
placement of the chlorine atom at C-4 or C-50 in 1 by the
pendant C-8 or C-80 phenolic group. An HMBC NMR
correlation from H-30 to an aromatic carbon at δ 145.8
suggested attachment of C-50 to an electronegative oxygen
atom. Reaction of the C-8 phenolic group via intramole-
cular cyclization would also account for the upfield shift of
C-6, since the hydrogen bond between the peri-proton and
the C-6 carbonyl group would no longer exist. In addition,
this putative cyclization step is consistent with the observa-
tion that nucleophilic aromatic substitution readily occurs
at C-50 in 1 with several external nucleophiles (vide infra).
An X-ray structure verified our proposed structure of
marinopyrrole F (6) as an eight-membered ether derivative
of 1 (Figure 2).
Absolute Configuration. X-ray analysis of marinopyrrole B
(2) allowed for assignment of an M-configuration to this
metabolite. The circular dichroism spectrum of 2, when
compared to the spectra of the other metabolites, showed
that marinopyrroles A (1), C (3), D (4), and E (5) were also
M-configured. Each CD spectrum showed negative first and
positive second Cotton effects in the salicyloyl-absorbing region
(λ 300-400 nm) (see the Supporting Information). This pattern
suggested a negative exciton coupling between the salicyloyl
substituents of 1-5, agreeing with an M-configuration about
the biaryl bond. The enantiopurity of metabolites 1-5 was
confirmed using chiral HPLC (Chiralcel OD-H), which indi-
cated that biosynthetic formation of the N,C-biaryl bond
proceeds in a highly atropo-selective manner.
Although metabolites 1-5 are fashioned by nature in
enantiopure form, the molecules could be racemized at
elevated temperatures.12 Heating marinopyrrole A (1) and
B (2) in toluene at 120 °C over 20 h was sufficient for
complete racemization. Interestingly, racemization of mar-
inopyrroles 3-5 either required prolonged heating in 1,2-
dichlorobenzene at 150 °C (as in 3) or could not be noticeably
accomplished at all (as in 4 and 5), even at 180 °C. It appears
that the steric influence of the halogen substituents at C-11
(10) In general, compounds that have few protons have proven to be
resistant to confident structure assignment by NMR methods. When H/C
ratios reach 0.5 and below, and the number of heteroatoms increases, the use
of 2D NMR experiments such as COSY, TOCSY, HMQC, and HMBC are
rendered ineffective. Marinopyrrole A has a H/C ratio (nonexchangeable H)
of 0.41. This limitation was first described by Professor Phillip Crews, UC
Santa Cruz, seventh US-Japan Conference on Marine Natural Products
Chemistry (June 2007).
(11) The placement of the halogen atom in 3-5, para to the hydroxyl
group, agrees with a biosynthetic mechanism involving an electrophilic
ꢀ
halogenating species. See: van Pee, K. H. Annu. Rev. Microbiol. 1996, 50,
375–399.
(12) Hall, D. M.; Harris, M. M. J. Chem. Soc. 1960, 490–494.
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and C-110 significantly raises the energetic barrier for rota-
tion about the biaryl bond.
In contrast, marinopyrrole F (6) was isolated in racemic
form. The material produced no measurable optical rotation
and lacked the CD Cotton effects apparent in 1-5. Although
formation of the oxacycle in 6 may proceed without stereo-
selection, another possibility is that this biaryl bond itself is
not configurationally stable at room temperature. To ascer-
tain this, a sample of enantioenriched 6 was collected using
chiral HPLC. Analysis of this material, stored in methanol
for 18 h at room temperature, showed complete racemiza-
tion. Apparently, the fused ether ring lowers the barrier for
atropisomerism in 6. The optical purity degrades at room
temperature, despite the sterically hindered environment still
present about the biaryl bond.
Reactivity. Monodeoxypyoluteorin (10)13 was also isolated
from cultures of actinomycete strain CNQ-418, providing
direct evidence for the biosynthetic origin of marinopyrrole A
(1). The structure and antibacterial activity of pyoluteorin14a-c
and the related pyrrolomycins14d-fare well-documented. We
inferred, however, that the 1,30-bipyrrole structure of the
marinopyrroles would exhibit reactivity that is distinct from
its simpler pyrrole congeners. A distinct mode of reactivity
might point toward a unique target or mechanism-of-action. In
order to probe the inherent reactivity of the marinopyrrole
structure and define structure-activity relationships, a semi-
synthetic study of marinopyrrole A (1) was undertaken.
FIGURE 2. ORTEP drawing of marinopyrrole F (6).
dimethylamine (1 to 16) via an aromatic substitution reac-
tion.15 The displacement of chloride by these nucleophiles
occurred exclusively at C-50, as illustrated by analysis of 2D
HMBC experiments (Table 2). The mutual HMBC correla-
tion from H-30 and H-140 in 14-16 to C-50 established the site
of substitution. As expected, the carbon resonance of C-50
was greatly influenced by the presence of the newly attached
heteroatom, shifting C-50 to δC 146.7 in 14, to δC 131.6 in 15,
and to δC 143.8 in 16. The C-50 chemical shift in methyl ether
14 (δC 146.7) was in close agreement with the shift in phenyl
ether 6 (δ 145.8).
The tendency for aromatic substitution at C-50 was further
demonstrated when, after prolonged heating at 145 °C in
N,N-dimethylacetamide (DMA), marinopyrrole A (1) was
converted to marinopyrrole F (6) (see Scheme 2). Subsequent
treatment of 6 with dimethylamine effected, in quantitative
yield, conversion to 16. Thus, in addition to 1, ether 6 can also
function as an effective arylating agent. Monodeoxypyoluteor-
in and its derivatives, notably, do not participate in nucleophilic
aromatic substitution under similar conditions.
Lastly, when subjected to elevated temperatures in pyr-
idine, marinopyrrole A (1) reacted with the ε-amine of N-R-
benzyloxycarbonyllysine methyl ester hydrochloride to give
imine 17 (Scheme 3). The adduct was isolated as a mixture of
two imine conformers after purification by reversed-phase
HPLC.16,17 HMBC correlations from H-14 [δ 3.32 (1H), 3.17
(2H), and 3.02 (1H)] to carbonyl signals at δC 161.9 and 161.5
showed the presence of an imine. Several other HMBC
correlations revealed that imine formation occurred exclu-
sively at C-6. First, HMBC correlations from H-12 (δ 7.09
and 7.00) to C-6 were evident.18 In addition, the H-30 singlets
at δ 6.66 and 6.53, one from each imine conformer, and the
H-120 doublets at δ 7.75 and 7.40 showed HMBC correla-
tions to the intact C-60 ketone (δC 185.9 and 187.1). The
mechanism by which the molecule targets actin and elicits
cytotoxicity in eukaryotic cells, detailed in a previous pub-
lication by our group,19 may indeed depend upon imine
Manipulation of the polar functionality on the molecule’s
periphery was first examined (Scheme 1). Both phenolic
groups of marinopyrrole A (1) were cleanly acetylated to
provide 11 via treatment with acetic anhydride. The natural
product was per-methylated using dimethylsulfate to afford
12. Finally, as a testament to the strong hydrogen-bonding
interaction between each carbonyl group and its peri-proton,
the pyrrole nitrogen could be selectively methylated in a cold
ethereal solution of diazomethane to give compound 13.
Yields of 13 were much lower when the reaction was carried
out in methanol, a solvent that likely disrupts the peri-
hydrogen bonding.
The highly halogenated bipyrrole structure of marinopyr-
role A (1) and its analogues suggested that the molecules may
act as electrophiles. Accordingly, 1 was treated with oxygen-,
sulfur-, and nitrogen-containing nucleophiles (Scheme 2).
Under basic conditions and elevated temperatures, 1 reacted
with methanol (1 to 14), N-acetylcysteamine (1 to 15), and
(13) (a) Durham, D. G.; Hughes, C. G.; Rees, A. H. Can. J. Chem. 1972,
50, 3223–3228. (b) Petruso, S.; Bonanno, S.; Caronna, S.; Ciofalo, M.;
Maggio, B.; Schillaci, D. J. Heterocycl. Chem. 1994, 31, 941–945.
(14) (a) Takeda, R. J. J. Am. Chem. Soc. 1958, 80, 4749–4750. (b) Takeda,
R. J. Bull. Agr. Chem. Soc. Jpn. 1959, 23, 126–130. (c) Birch, A. J.; Hodge, P.;
Rickards, R. W.; Takeda, R.; Watson, T. R. J. Chem. Soc. 1964, 2641–2644.
(d) Kaneda, M.; Nakamura, S.; Ezaki, N.; Iitaka, Y. J. Antibiot. 1981, 34,
1366–1368. (e) Ezaki, N.; Koyama, M.; Shamura, T.; Tsuruoka, T.; Inouye,
S. J. Antibiot. 1983, 36, 1263–1267. (f) Ezaki, N.; Koyama, M.; Kodama, Y.;
Shomura, T.; Tashiro, K.; Tsuruoka, T.; Inouye, S. J. Antibiot. 1983, 36,
1431–1438.
(16) The imine was resistant to in situ reduction with sodium cyanobor-
ohydride.
(17) The atropo purity of 17 degraded slightly, though not enough to
account for the 1:1 mixture of diastereomers. The natural product, liberated
during prolonged storage of the imine, was isolated in 74% ee.
(18) Both H-12 resonances were not clearly resolved from the other
aromatic signals.
(19) Hughes, C. C.; Yang, Y.-L.; Liu, W.-T.; Dorrestein, P. C.; La Clair,
J. J.; Fenical, W. J. Am. Chem. Soc. 2009, 131, 12094–12096.
(15) Vlasov, V. M. Russ. Chem. Rev. 2003, 72, 681–703.
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TABLE 2. Selected 1H and 13C NMR Spectral Data for 14-16
(CDCl3)
SCHEME 1. “Capping” the Polar Functionality in 1a
14 (X = O)
15 (X = S)
16 (X = N)
position
δC
δH, multb
δC
δH, multb
δH, multc
a
a
a
δC
30
122.4
146.7
186.3
60.9
6.66, s
119.9
131.6
187.6
35.4
6.82, s
122.0
143.8
186.6
41.5
6.69, s
50
aReagents and conditions: (a) Ac2O, DMAP, pyr, rt, 62%; (b) Me2SO4,
K2CO3, Me2CO, rt, quant; (c) CH2N2, ether, 0 °C, 86%. DMAP =
4-(dimethylamino)pyridine.
60
140
3.79, s
2.37, m
2.67, m
2.30, s
a75 MHz. b500 MHz. c600 MHz.
a
SCHEME 2. Nucleophilic Aromatic Substitution at C-50
SCHEME 3. Imine Formation at C-6
the products were obtained either in diminished enantiopurity
or in racemic form, as in 14 (0% ee), 15 (76% ee), and 16
(0% ee).
Synthetic Studies. Although the preparation of N-phenyl-
azoles via Ullman coupling is well documented, direct
construction of an N,C-linked bipyrrole in this way has
not been previously demonstrated.20,21 A total synthesis of
marinopyrrole A (1) that featured Ullman coupling of two
pyrroles would, therefore, be novel (Scheme 4). In this
reaction, a copper(I) salt catalyzes carbon-nitrogen bond
formation between monodeoxypyoluteorin (10) and C-3-
iodo- or -bromomonodeoxypyoluteorin (19, 20). Chela-
tion of both diamine ligands (e.g., N,N-dimethylethane-
1,2-diamine) and the pendant carbonyl functionality in 10
should promote oxidative addition of organocopper(I)
species 18 to the copper(III) intermediate.
A short sequence consisting of Friedel-Crafts acylation
of pyrrole with 2-methoxybenzoyl chloride, demethylation,
acetylation, and selective chlorination of the pyrrole pro-
vided O-acetyl monodeoxypyoluteorin (21) (Scheme 5).13b
The phenol functionality was liberated in aqueous acid to
give 10, which agreed in all respects with the natural material
obtained from the culture of strain CNQ-418. To prepare a
aReagents and conditions: (a) K2CO3, MeOH, 65 °C, 42% (63% b.o.
rsm); (b) N-acetylcysteamine, K2CO3, DMF, 80 °C, 55%; (c) HNMe2,
THF, 65 °C, 42% (62% b.o.rsm); (d) DMA, 145 °C, 62%; (e) HNMe2,
THF, 55 °C, quant DMF = N,N-dimethylformamide, DMA = N,N-
dimethylacetamide. b.o.rsm = based on recovered starting material.
formation between 1 and a key amine-containing residue of
the target.
Marinopyrrole A (1) is constitutionally and configura-
tionally stable to a variety of both strongly acidic conditions
(e.g., trifluoroacetic acid in dichloromethane) and strongly
basic conditions (e.g., 6 N aqueous sodium hydroxide in
methanol) but can be racemized at temperatures above
100 °C. Although the conversion of 1 to compounds 11-16
was accomplished under less rigorous conditions, the enantio-
purity of many of these derivatives eroded. The optical purity of
the material was maintained when reaction occurred distal
to the biaryl bond, as in the formation of 13. When the reac-
tion proceeded at a site ortho to the biaryl bond, however,
(20) (a) Kunz, K.; Scholz, U.; Ganzer, D. Synlett 2003, 2428–2439. (b)
Antilla, J, C.; Baskin, J. M.; Barder, T. E.; Buchwald, S. L. J. Org. Chem.
2004, 69, 5578–5587. (c) Cristau, H.-J.; Cellier, P. P.; Spindler, J.-F.;
Taillefer, M. Chem.;Eur. J. 2004, 10, 5607–5622. (d) Cai, Q.; Zhu, W.;
Zhang, H.; Zhang, Y.; Ma, D. Synthesis 2005, 496–499. (e) Taillefer, M.; Xia,
N.; Ouali, A. Angew. Chem., Int. Ed. 2007, 46, 934–936. (f) Correa, A.; Bolm,
C. Angew. Chem., Int. Ed. 2007, 46, 8862–8865. (g) Mino, T.; Harada, Y.;
Shindo, H.; Sakamoto, M.; Fujita, T. Synlett 2008, 614–620.
(21) For a review of the synthesis of axially chiral biaryl compounds, see:
Bringmann, G.; Mortimer, A. J. P.; Keller, P. A.; Gresser, M. J.; Garner, J.;
Breuning, M. Angew. Chem., Int. Ed. 2005, 44, 5384–5427.
J. Org. Chem. Vol. 75, No. 10, 2010 3245

Hughes et al.
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SCHEME 4. Proposed Ullman Coupling for the Synthesis of
(()-1
SCHEME 5. Synthesis of Monodeoxypyoluteorin (10) and 3-
Halo Analoguesa
coupling partner for the Ullman reaction, 21 was converted
to 22 in 57% yield using 1 equiv of N-bromosuccinimide
(NBS). Acetate 22 afforded 19, the structure of which
was verified with an X-ray crystal structure determination
(see the Supporting Information). Similarly, 21 reacted with
N-iodosuccinimide (NIS) in 65% yield to give 23, which after
deprotection yielded 20.22 N-Bromination to form 24 oc-
curred when excess NBS was employed.23
Attempts to couple 3-halopyrroles 19, 20, 22, and 23 to
either 10 or 21, and effect formation of the key biaryl bond,
proved fruitless. Several modern methods were employed.20
Presumably, the reaction was not attainable due to unfavor-
able steric interactions between the substituents ortho to the
site of bond formation. Indeed, the efficiency of transition-
metal-catalyzed Ullman coupling reactions described in the
literature is largely reduced by the presence of one or more
ortho substituents. Even di-ortho-substituted biaryls are
often inaccessible or require forceful conditions (infinite
concentrations and high catalyst loading).20a Consequently,
the goal to prepare a tetra-ortho-substituted biaryl com-
pound, such as marinopyrrole A, in this way may be too
lofty.
aReagents and conditions: (a) aq HCl, MeOH, rt, 83%; (b) NBS (1
equiv), DCE, 90 °C, 57%; (c) NIS, DCE, 90 °C, 65%; (d) NBS (2 equiv),
DCE, 90 °C, 82%; (e) aq HCl, MeOH, rt, 19 (94%), 20 (94%). NBS =
N-bromosuccinimide, NIS = N-iodosuccinimide, DCE = 1,2-dichlor-
oethane.
SCHEME 6. 1H-Chromeno[3,2-b]pyrrol-9-one 25 via Intramo-
lecular Ullman Reaction
Intermolecular Ullman coupling between monodeoxy-
pyoluteorin (10) and 3-halo substrates was often preceded
by intramolecular reaction of the organocopper species with
the pendant phenol. The acetate functionality in 22 and 23
was unstable to the reaction conditions, and the phenol
groups, once liberated, engaged in intramolecular coupling
to furnish a 1H-chromeno[3,2-b]pyrrol-9-one. Under opti-
mized conditions for the production of chromene 25, bro-
mide 19 was subjected to copper(I) iodide and potassium
carbonate at 110 °C (Scheme 6). A 41% yield of 25 was thus
obtained after HPLC purification. The preparation of 1H-
chromeno[3,2-b]pyrrol-9-ones has been described only once
before, employing a reductive cyclization of a nitro-coumarin
with triethylphosphite.24 Unlike monodeoxypyoluteorin
(10) and its 3-halo analogues, chromene 25 displayed little
activity against methicillin-resistant S. aureus and human
colon cancer cells (HCT-116).
core. 1,30-Bipyrroles have been previously synthesized ac-
cording to one of two methods: (1) condensation of a 1,4-
diketo compound onto a 3-aminopyrrole (Paal-Knorr
reaction) and (2) reaction of a β-pyrroyl-R,β-unsaturated
ester with tosylmethyl isocyanide (TosMIC).8 Adopting a
strategy based on the former method, Grignard reagent 26
was treated with oxalate 27 at low temperature to give the
product resulting from monoaddition, as described in the
literature (Scheme 7).25 Ozonolysis then provided aldehyde
28. This material was condensed with 3-aminopyrrole 2926 to
furnish bipyrrole 30, the structure of which was confirmed
through a series of 2D NMR experiments (see the Support-
ing Information). Brominated bipyrrole 31 was obtained via
treatment with NBS, although tetrachlorination of 30 using
excess NCS could not be accomplished. The structure of 31
was confirmed by X-ray analysis. Tetrabromide 31 should
Although we did not succeed in coupling two salicyloyl-
substituted pyrrole monomers to form marinopyrrole A, we
remained interested in the synthesis of the 1,30-bipyrrole
(22) Hasegawa, H.; Shiori, N.; Mogi, K.; Asaoka, T.; Ono, J.; Kuraishi,
T.; Katori, T. Jpn. Kokai Tokkyo Koho, Patent no. JP 63183562, 1988.
(23) For other examples of pyrrole N-halogenation, see: (a) De Rosa, M.
J. Org. Chem. 1982, 47, 1008–1010. (b) De Leon, C. Y.; Ganem, B.
Tetrahedron 1997, 53, 7731–7752.
(25) Macritchie, J. A.; Silcock, A.; Willis, C. L. Tetrahedron: Asymmetry
1997, 8, 3895–3902.
(26) Furneaux, R. H.; Tyler, P. C. J. Org. Chem. 1999, 64, 8411–8412.
(24) Paparao, C.; Rao, K. V.; Sundaramurthy, V. Synthesis 1981, 234–
236.
3246 J. Org. Chem. Vol. 75, No. 10, 2010

Hughes et al.
JOCArticle
SCHEME 7. Synthesis of the Tetrahalogenated 1,30-Bipyrrole
none of the semisynthetic derivatives were more active
against MRSA than the natural products. Advanced inter-
mediates 30 and 31 showed little antimicrobial activity and
cytotoxicity, pointing to the importance of the salicyloyl
substituents.
Core of the Marinopyrrolesa
Many of the MRSA-active compounds also displayed
significant cytotoxicity toward the human colon cancer line,
HCT-116, with IC50’s predominantly between 1 and 5 μg
mL-1 (see Table 3). The therapeutic window for the mar-
inopyrroles, therefore, may be too narrow for the treatment
of bacterial infection in humans. That the marinopyrroles,
given their inherent cytotoxicity, may function as effective
cancer chemotherapy agents remains an issue to be explored.
Conclusion
Herein, we report the full composition and final structure
assignments and reactivity of a family of densely haloge-
nated, axially chiral metabolites from a marine actinomycete
strain identified as a member of the genus Streptomyces
(strain CNQ-418). The marinopyrroles are antibiotics of an
unprecedented structure class, containing the first naturally
occurring 1,30-bipyrrole. Except for (()-marinopyrrole F
(6), the metabolites are M-configured and configurationally
stable at room temperature. Given the difficulty associated
with the transition metal-catalyzed formation of tetra-ortho-
substituted biaryls in chemical synthesis, the biosynthetic
mechanism by which this coupling occurs for the marino-
pyrroles is of considerable interest. That the N,C-biaryl bond
is formed in a completely atropo-selective manner is, like-
wise, an appealing biosynthetic concept. As such, elucidation
of the biosynthetic gene cluster responsible for marinopyr-
role production may set the stage for a biomimetic total
synthesis and even elicit the development of novel biaryl
coupling reactions with larger scope.
aReagents and conditions: (a) 1. 27, THF, -78 °C, 90%; 2. O3, -78 °C,
then Me2S; (b) 29, THF, PTSA, 23% (two steps); (c) NBS, DCE, 90 °C,
86%. PTSA = p-toluenesulfonic acid, NBS = N-bromosuccinimide,
DCE = 1,2-dichloroethane
TABLE 3. Antimicrobial and Cytotoxic Properties of Selected
Marinopyrroles and Derivatives (μg mL-1 27,28
)
compd
MRSAa (MIC90
)
HCT-116b (IC50
)
(-)-1
(-)-2
(-)-3
(()-6
11
12
13
14
15
0.31
0.63
0.16
3.1
4.5
5.3
0.21
2.9
0.42
NSA
NSA
1.1
7.2
4.4
1.6
NSA
NSA
0.78
6.3
16
1.6
aMRSA = methicillin-resistant Staphylococcus aureus. Positive con-
trol: vancomycin (MIC90 = 0.20-0.39 μg mL-1) and penicillin G
(MIC90 = 6.3-12 μg mL-1). bHCT-116 is a human colon cancer cell
line. Positive control: etoposide (IC50 = 0.29-2.9 μg mL-1). NSA = no
significant activity (>8 μg mL-1).
Experimental Section
Cultivation of CNQ-418. The strain was cultured in 2.8 L
Fernbach flasks (20 ꢀ 1 L) in a seawater-based medium (per 1 L
seawater: 10 g of starch, 4 g of yeast extract, 2 g of peptone, 1 g of
exist as a racemic mixture of two configurationally stable
atropo-enantiomers at room temperature, but this could not
be strictly confirmed by chiral chromatography.
CaCO3, 40 mg of Fe2(SO4)3 4H2O, 100 mg of KBr) and shaken
3
Antibiotic Properties. The marinopyrroles A-C (1-3),
obtained in sufficient quantity and purity for testing, dis-
played significant activity against methicillin-resistant S.
aureus (MRSA) with MIC90 values of less than 1 μg mL-1
(Table 3). Marinopyrrole F (6) and derivatives 11-16,
encompassing modifications of 1 either at a polar functional
group on the periphery or at core halogen substituents, were
much less active against MRSA. The loss in activity of 11-13
reflects the importance of the hydrogen-bonding capacity of
the salicyloyl hydroxyl groups and the N-H functionality.
Methylation of the latter functionality proved particularly
detrimental to activity. The requirement of a free N-H group
for activity against MRSA may point toward a crucial
hydrogen-bonding interaction between the molecule and its
bacterial target. The loss in antimicrobial activity of 6, 15,
and 16 may reflect the central role that the C-50 chlorine
substituent plays in the mechanism of antibiotic action.
Ether 14 retained much of the activity characteristic of
metabolites 1-3, presumably, since the C-50 methoxy sub-
stituent can also function as an effective leaving group in
nucleophilic aromatic substitution reactions. Remarkably,
at 230 rpm at 27 °C.
Isolation and Purification of the Marinopyrroles. After 17 days
of cultivation, sterilized XAD-18 resin (20 g L-1) was added,
and the resin was collected 24 h later by filtration, washed with
deionized water, and eluted with acetone. The acetone was
removed under reduced pressure, and the resulting aqueous
layer was extracted with ethyl acetate (3 ꢀ 600 mL). The
combined extracts were dried, filtered, and concentrated. The
crude extract was loaded onto silica gel (20 g) and fractionated
on a silica column (80 ꢀ 60 mm, h ꢀ w) using a stepwise gradient
(0.5% EtOAc in hexanes, 30% EtOAc in hexanes, EtOAc, 10%
MeOH in EtOAc, MeOH). The fractions were concentrated to
give F1 (81.6 mg), F2 (1.34 g), F3 (199.8 mg), F4 (144.8 mg), and
F5 (422.4 mg). A portion of F2 (134 mg) was dissolved in MeOH
(3.0 mL), and the marinopyrroles were purified by reversed-
phase HPLC (80% MeOH in H2O, 0.2% TFA). Pure marino-
pyrrole A (1) (92.1 mg) was thus obtained. Further purification
by reversed-phase HPLC (65-68% CH3CN in H2O, 0.2%
TFA) provided, in order of elution, marinopyrrole F (6) (2.7
mg), C (3) (1.2 mg), B (2) (2.0 mg), D (4) (1.8 mg), E (5) (0.5 mg).
Marinopyrrole A (1): UV/vis (CH3CN/H2O) λmax = 270, 324,
348 nm; [R]D -69 (c 0.39, MeOH); IR (film) ν~ = 1623, 1595 cm-1
;
1H NMR see Table 1; 13C NMR 186.8, 185.9, 162.5, 161.1,
J. Org. Chem. Vol. 75, No. 10, 2010 3247

Hughes et al.
JOCArticle
136.3, 136.2, 131.9, 130.3, 128.8, 124.1, 123.8, 123.1, 120.4,
120.3, 119.1, 118.9, 118.9, 118.7, 118.4, 117.8, 112.7, 110.7;
HRMS (ESI-TOF) m/z (M - H)- calcd for C22H1135Cl4N2O4
1.1 mmol) dropwise via syringe. The mixture was heated at 40 °C
overnight, allowed to cool, filtered through Celite with acetone
washings, and concentrated. The product was purified by
reversed-phase C-18 HPLC (73% CH3CN in H2O, 0.2%
TFA, tR = 23 min) to furnish 27.3 mg (100%) of 12 as a white
506.9473, found 506.9447; m/z (M þ Na)þ calcd for C22H12
-
35Cl4N2O4Na 530.9449, found 530.9439.
Marinopyrrole B (2): UV/vis (CH3CN/H2O) λmax = 274, 322,
solid: [R]D -92 (c 0.42, MeOH); IR (film) ν~ = 1647, 1597 cm-1
;
348 nm; [R]D -72 (c 0.20, MeOH); IR (film) ν~ = 1622, 1599 cm-1
;
1H NMR δ 7.40 (t, J = 7.8 Hz, 1H), 7.24-7.18 (m, 3H), 6.98-
6.93 (m, 2H), 6.75 (d, J = 8.2 Hz, 1H), 6.67 (t, J = 7.8 Hz, 1H),
6.25 (s, 1H), 4.05 (s, 3H), 3.80 (s, 3H), 3.75 (s, 3H); 13C NMR δ
184.2, 182.9, 157.3, 157.0, 131.9, 131.9, 130.7, 129.4, 129.1,
127.5, 127.4, 126.4, 125.6, 125.0, 123.6, 121.2, 119.8, 119.6,
111.7, 111.5, 110.5, 109.6, 55.6, 55.5, 34.7; HRMS (ESI-TOF)
m/z (M þ H)þ calcd for C25H1935Cl4N2O4 551.0099, found
551.0100.
1H NMR see Table 1; 13C NMR 188.5, 185.3, 162.5, 161.4, 137.3,
136.4, 134.2, 133.7, 130.0, 124.4, 121.9, 121.7, 120.0, 119.2, 118.8,
118.6, 118.5, 118.1, 117.9, 114.7, 104.7 (one quaternary carbon
signal was not detected); HRMS (ESI-TOF) m/z (M - H)- calcd
for C22H1079Br35Cl4N2O4 584.8578, found 584.8588; m/z
(M þ Na)þ calcd for C22H1179Br35Cl4N2O4Na 608.8554, found
608.8570.
Marinopyrrole C (3): UV/vis (CH3CN/H2O) λmax = 270, 324,
346 nm; [R]D -100 (c 0.040, MeOH); IR (film) ν~ = 1623, 1597
(4,40,5,50-Tetrachloro-10-methyl-10H-1,30-bipyrrole-2,20-diyl)-
bis((2-hydroxyphenyl)methanone) (13). To a solution of meta-
bolite 1 (8.7 mg, 0.017 mmol) in ether (2.0 mL) at 0 °C was added
an ethereal solution of diazomethane via pipet (torched to
remove sharp edges). After 1 min, several drops of acetic acid
were added (until gas evolution ceased), and the solution was
concentrated. The product was purified by reversed-phase
HPLC (72% CH3CN in H2O, 0.2% TFA, tR = 33 min) to
afford 7.7 mg (86%) of 13 as a pale yellow solid: [R]D -62 (c 0.35,
MeOH); IR (film) ν~ = 1624, 1596 cm-1; 1H NMR δ 11.17 (s,
1H), 10.94 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 7.8 Hz,
1H), 7.49 (t, J = 7.8 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.01 (d,
J = 8.2 Hz, 1H), 6.91-6.87 (m, 2H), 6.59 (s, 1H), 6.55 (t, J = 7.8
Hz, 1H), 3.89 (s, 3H); 13C NMR δ 187.9, 186.7, 162.4, 161.9,
136.4, 136.1, 131.9, 131.6, 129.0, 125.1, 124.6, 122.6, 122.5,
120.3, 119.7, 119.1, 118.9, 118.5, 118.3, 117.6, 112.3, 108.7,
34.3; HRMS (ESI-TOF) m/z (M þ H)þ calcd for C23H1535Cl4-
N2O4 522.9786, found 522.9778.
(4,40,50-Trichloro-5-methoxy-10H-1,30-bipyrrole-2,20-diyl)bis-
((2-hydroxyphenyl)methanone) (14). To metabolite 1 (10.0 mg,
0.0196 mmol) and potassium cabonate (100 mg, 0.72 mmol) was
added MeOH (2.0 mL). The mixture was heated at 65 °C for 2 d,
allowed to cool, diluted with a saturated NH4Cl solution (4 mL),
and extracted with EtOAc (2 ꢀ 2 mL). The combined extracts
were washed with brine (1 mL), dried, filtered, and concen-
trated. The product was purified by reversed-phase HPLC (65%
CH3CN in H2O, 0.2% TFA, tR(14) = 25 min, tR(1) = 30 min) to
give 2.1 mg (21%) of 1 and 4.2 mg (42%) of 14 as a yellow
1
cm-1; H NMR see Table 1; 13C NMR (HSQC and HMBC)
186.7, 184.5, 162.4, 159.4, 136.1, 135.5, 131.1, 129.6, 128.9,
123.8, 123.8, 120.9, 118.9, 118.9, 118.3; HRMS (ESI-TOF) m/z
(M þ H)þ calcd for C22H1235Cl5N2O4 542.9240, found 542.9229.
Marinopyrrole D (4): UV/vis (CH3CN/H2O) λmax = 270, 320,
352 nm; [R]D -55 (c 0.080, MeOH); IR (film) ν~ = 1624, 1593
1
cm-1; H NMR see Table 1; 13C NMR (HSQC and HMBC)
185.3, 185.1, 161.0, 160.7, 136.3, 136.3, 130.3, 129.9, 128.4,
124.9, 123.7, 120.4, 120.1, 118.8, 118.0; HRMS (ESI-TOF) m/z
(M þ H)þ calcd for C22H1235Cl5N2O4 542.9240, found 542.9217.
Marinopyrrole E (5): UV/vis (CH3CN/H2O) λmax = 270, 320,
350 nm; [R]D -100 (c 0.005, MeOH); IR (film) ν~ = 1623, 1593
1
cm-1; H NMR: see Table 1; 13C NMR (HSQC and HMBC)
185.3, 185.1, 161.2, 161.0, 139.0, 136.0, 133.4, 129.7, 128.4,
124.9, 120.6, 120.4, 120.0, 119.1, 118.4; HRMS (ESI-TOF) m/z
(M þ H)þ calcd for C22H1279Br35Cl4N2O4 586.8735, found
586.8740.
Marinopyrrole F (6): UV/vis (CH3CN/H2O) λmax = 264, 336
nm; [R]D 0 [c 0.057, MeOH/Me2CO (2:1)]; IR (film) ν~ = 1620,
1601 cm-1 1H NMR (DMSO-d6) see Table 1; 13C NMR
;
(DMSO-d6) 183.8, 175.8, 158.2, 157.0, 145.8, 135.9, 134.0,
132.7, 130.9, 129.0, 127.7, 125.6, 123.2, 123.1, 121.6, 121.5,
120.9, 120.4, 118.8, 117.0, 105.7, 99.2; HRMS (ESI-TOF) m/z
(M þ H)þ calcd for C22H1235Cl3N2O4 472.9863, found
472.9858. (()-Marinopyrrole F (6) was prepared from marino-
pyrrole A (1): A solution of metabolite 1 (4.5 mg, 0.0088 mmol)
in DMA (1.0 mL) was heated at 145 °C for 3 d. The mixture was
allowed to cool and concentrated under full vacuum. The
product was purified by reversed-phase HPLC (65% CH3CN
in H2O, 0.2% TFA, tR = 27 min) to give 2.6 mg (62%) of (()-6
as a white solid. All analytical and spectroscopic data agreed
with the natural product.
4,40,5,50-Tetrachloro-10H-1,30-bipyrrole-2,20-diyl)bis((2-acetoxy-
phenyl)methanone) (11). To a solution of metabolite 1 (18.0 mg,
0.0353 mmol) and DMAP (1 mg) in pyridine (1.0 mL) was added
acetic anhydride (0.20 mL, 2.7 mmol) dropwise via syringe. The
mixture was stirred at room temperature for 5 h and concentrated.
The product was purified twice by reversed-phase HPLC (70%
CH3CN in H2O, 0.2% TFA, tR = 18 min; then 80% MeOH in
H2O, 0.2% TFA, tR = 25 min) to yield 12.9 mg (62%) of 11 as a
white solid: [R]D þ20 (c 0.38, MeOH); IR (film): ν~ = 1767, 1640
cm-1; 1H NMR δ 9.66 (br s, 1H), 7.58-7.51 (m, 2H), 7.38 (t, J =
7.8 Hz, 1H), 7.35-30 (m, 2H), 7.18 (d, J = 7.8 Hz, 1H), 7.12-7.08
(m, 2H), 6.52 (s, 1H), 2.20 (s, 3H), 2.19 (s, 3H); 13C NMR (125
MHz) δ 181.4, 180.2, 169.5, 169.3, 148.8, 148.4, 132.4, 132.2, 130.8,
130.1, 129.9, 129.2, 128.8, 125.4, 125.3, 125.3, 124.6, 123.8, 123.4,
123.3, 121.6, 120.4, 112.2, 111.2, 20.9, 20.7; HRMS (ESI-TOF) m/z
(M þ H)þ calcd for C26H1735Cl4N2O6 592.9841, found 592.9840.
(4,40,5,50-Tetrachloro-10-methyl-10H-1,30-bipyrrole-2,20-diyl)-
bis((2-methoxyphenyl)methanone) (12). To a solution of meta-
bolite 1 (25.2 mg, 0.0494 mmol) and potassium carbonate (200
mg) in acetone (1.0 mL) was added dimethyl sulfate (0.10 mL,
residue: [R]D þ5 (c 0.22, MeOH); IR (film) ν~ = 1619, 1592 cm-1
;
1H NMR δ 11.30 (s, 1H), 10.61 (s, 1H), 9.58 (br s, 1H), 7.72 (d,
J = 7.8 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.8 Hz,
1H), 7.35 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H),
6.95-6.89 (m, 2H), 6.66 (s, 1H), 6.53 (t, J = 7.8 Hz, 1H),
3.79 (s, 3H); 13C NMR δ 186.3, 186.1, 162.1, 161.1, 146.7,
136.0, 135.5, 131.7, 130.6, 123.3, 122.6, 122.4, 121.4, 119.9,
119.3, 118.9, 118.8, 118.8, 118.2, 117.4, 110.2, 96.5, 60.9;
HRMS (ESI-TOF) m/z (M þ H)þ calcd for C23H1635Cl3N2O5
505.0125, found 505.0118.
N-(2-(3,40,50-Trichloro-20,5-bis(2-hydroxybenzoyl)-10H-1,30-
bipyrrol-2-ylthio)ethyl)acetamide (15). To a solution of metabo-
lite 1 (7.0 mg, 0.014 mmol) and potassium carbonate (100 mg,
0.72 mmol) in DMF (1.0 mL) was added N-acetylcysteamine
(20 μL, 0.19 mmol) via syringe. The solution was heated at 80 °C
for 20 h, allowed to cool, diluted with a saturated NH4Cl
solution (4 mL), and extracted with EtOAc (2 ꢀ 2 mL). The
combined extracts were washed with brine (1 mL), dried,
filtered, and concentrated. The product was purified by
reversed-phase HPLC (50% CH3CN in H2O, 0.2% TFA, tR
=
24 min) to give 4.5 mg (55%) of 15 as a yellow residue: [R]D þ29
(c 0.27, MeOH); IR (film) ν~=1623, 1594 cm-1; 1H NMR δ 11.35
(s, 1H), 10.58 (br s, 1H), 10.19 (br s, 1H), 7.88 (d, J = 7.8 Hz,
1H), 7.57-7.53 (m, 2H), 7.30 (t, J = 7.8 Hz, 1H), 7.06 (d,
J = 8.2 Hz, 1H), 6.97 (t, J = 7.8 Hz, 1H), 6.93 (d, J = 8.2 Hz,
1H), 6.82 (s, 1H), 6.43 (t, J = 7.8 Hz, 1H), 5.70 (br s, 1H), 3.14
3248 J. Org. Chem. Vol. 75, No. 10, 2010

Hughes et al.
JOCArticle
(m, 2H), 2.67 (m, 1H), 2.37 (m, 1H), 1.95 (s, 3H); 13C NMR δ
187.6, 186.1, 170.5, 162.8, 161.1, 136.5, 136.0, 132.2, 131.6,
130.6, 128.5, 124.4, 124.0, 122.2, 119.9, 119.6, 119.2, 119.0,
119.0, 118.7, 118.5, 117.6, 109.8, 38.2, 35.4, 23.1; HRMS (ESI-
TOF) m/z (M þ H)þ calcd for C26H2135Cl3N3O5S 592.0267,
found 592.0274.
(4,40,50-Trichloro-5-(dimethylamino)-10H-1,30-bipyrrole-2,20-
diyl)bis((2-hydroxyphenyl)methanone) (16). To metabolite 1 (3.5
mg, 0.0069 mmol) was added a solution of dimethylamine in
THF (1.0 mL, 2.0 M) via syringe. The mixture was heated at
65 °C for 4 d, allowed to cool, and concentrated. The product
was purified by reversed-phase HPLC (60% CH3CN in H2O for
4 h at room temperature, the mixture was concentrated. The
product was purified by reversed-phase HPLC (50% CH3CN in
H2O, 0.2% TFA) to furnish 8.2 mg (94%) of 19 as a yellowish
solid: IR (film) ν~ = 1620, 1585 cm-1; 1H NMR δ 10.83 (s, 1H),
9.33 (br s, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H),
7.05 (d, J = 8.0 Hz, 1H), 6.95 (t, J = 7.8 Hz, 1H); 13C NMR δ
186.9, 161.8, 136.7, 133.3, 126.7, 119.7, 119.0, 118.3, 118.1, 115.0,
104.8; HRMS (ESI-TOF) m/z (M
C11H779Br35Cl2NO2 333.9037, found 333.9020.
þ
H)þ calcd for
(4,5-Dichloro-3-iodo-1H-pyrrol-2-yl)(2-hydroxyphenyl)metha-
none (20). To acetate 21 (24.0 mg, 0.0805 mmol) and N-iodosuc-
cinimide (26.0 mg, 0.116 mmol) was added DCE (2.0 mL). The
mixture was heated at 90 °C for 6 h, allowed to cool, poured into
EtOAc (50 mL), and washed with a saturated NaHCO3 solution
(10 mL) and brine (10 mL). The organic layer was dried, filtered,
and concentrated. The product was purified by column chroma-
tography (20% EtOAc in hexanes) to afford 22.3 mg (65%) of
acetylated iodide 23 as a foamy oil: IR (film) ν~ = 1765, 1745,
1623 cm-1; 1H NMR δ 7.57 (t, J = 7.8 Hz, 1H), 7.48 (d, J = 7.8
Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 2.16
(s, 3H); 13C NMR δ 182.5, 169.3, 148.2, 132.4, 130.1, 129.9,
129.8, 126.0, 123.2, 120.7, 119.3, 76.2, 20.8; HRMS (ESI-TOF)
m/z (M þ Na)þ calcd for C13H835Cl2INO3Na 445.8824, found
445.8818. To a solution of 23 (16.8 mg, 0.0396 mmol) in MeOH
(2.0 mL) was added 5 drops of concd HCl. After 18 h at room
temperature, the mixture was concentrated. The product was
purified by reversed-phase C-18 HPLC (55% CH3CN in H2O,
0.2% TFA) to furnish 14.2 mg (94%) of 20 as a yellowish solid:
IR (film) ν~ = 1621, 1588 cm-1; 1H NMR δ 10.82 (s, 1H), 9.28 (br
s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.06 (d,
J = 8.0 Hz, 1H), 6.96 (t, J = 7.8 Hz, 1H); 13C NMR δ 187.5,
161.7, 136.7, 133.6, 129.6, 119.3, 119.2, 119.0, 118.3, 118.1, 75.2;
HRMS (ESI-TOF) m/z (M þ H)þ calcd for C11H735Cl2INO2
381.8899, found 381.8897.
25 min, 60-100% CH3CN in H2O over 15 min, 0.2% TFA, tR(1)
=
35 min, tR(16) = 38 min) to yield 0.7 mg (20%) of 1 and 1.5 mg
(42%) of 16 as a yellow residue. Alternatively, to compound 6 (4.1
mg, 0.0087 mmol) was added a solution of dimethylamine in THF
(1.0 mL, 2.0 M). The mixture was heated at 55 °Cfor2d, allowedto
cool, and concentrated. The product was purified by reversed-
phase HPLC (70% CH3CN in H2O, 0.2% TFA, tR = 27 min) to
yield 4.9 mg (100%) of 16 as a yellow residue: [R]D þ3 (c 0.10,
MeOH); IR (film) ν~ = 1619, 1591 cm-1; 1H NMR (600 MHz) δ
11.41 (br s, 1H), 11.01 (br s, 1H), 10.28 (br s, 1H), 7.92 (d, J = 7.8,
1H), 7.63 (d, J = 7.8 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.31 (t, J =
7.8 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 6.96 (t, J = 7.8 Hz, 1H), 6.90
(d, J = 7.8 Hz, 1H), 6.69 (s, 1H), 6.48 (t, J = 7.8 Hz, 1H), 2.30 (s,
6H); 13C NMR δ 186.6, 186.2, 162.3, 161.6, 143.8, 136.0, 135.5,
132.0, 131.2, 124.0, 123.2, 122.7, 122.0, 119.5, 119.2, 118.9, 118.8,
118.8, 118.2, 117.3, 109.9, 105.8, 41.5; HRMS (ESI-TOF) m/z
(M þ H)þ calcd for C24H1935Cl3N3O4 518.0441, found 518.0431.
(S,E)-Methyl 2-(benzyloxycarbonylamino)-6-((2-hydroxyphenyl)-
(4,40,5,50-tetrachloro-20-(2-hydroxybenzoyl)-10H-1,30-bipyrrol-2-
yl)methyleneamino)hexanoate (17). To metabolite 1 (8.6 mg,
0.017 mmol) and methyl 6-amino-2-(benzyloxycarbonylamino)-
hexanoate hydrochloride (Cbz-NH-Lys-OMe HCl, 40.6 mg,
3
2-(1,3-Dibromo-4,5-dichloro-1H-pyrrole-2-carbonyl)phenyl
Acetate (24). To a solution of acetate 21 (14.8 mg, 0.0763 mmol)
in DCE (1.0 mL) was added N-bromosuccinimide (30 mg, 0.17
mmol). The mixture was heated to 90 °C for 3 h, allowed to
cool, and concentrated. The product was purified by column
chromatography (10% EtOAc in hexanes) to give 22.0 mg
0.123 mmol) was added pyridine (0.5 mL). The mixture was
heated at 80 °C for 2 d, allowed to cool, diluted with a saturated
NaHCO3 solution (2 mL) and water (2 mL), and extracted with
EtOAc (2 ꢀ 3 mL). The combined extracts were washed with
brine (1 mL), dried, filtered, and concentrated. The product was
purified by reversed-phase HPLC (65% CH3CN in H2O, 0.2%
TFA, tR = 21 min) to give 4.9 mg (37%) of 17 as a 1:1 mixture of
diastereomers: 1H NMR (600 MHz, CD3CN) δ 7.75 (d, J = 7.8
Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.40
(d, J = 7.8 Hz, 1H), 7.38-7.26 (m, 11H), 7.16 (t, J = 7.8 Hz,
1H), 7.09 (d, J = 7.8 Hz, 1H), 7.01-6.89 (m, 6H), 6.79-6.73 (m,
2H), 6.66 (s, 1H), 6.53 (s, 1H), 6.47 (t, J = 7.8 Hz, 1H), 5.98 (m,
2H), 5.08-5.00 (m, 4H), 4.06 (m, 2H), 3.62 (s, 3H), 3.61 (s, 3H),
3.32 (m, 1H), 3.17 (m, 2H), 3.02 (m, 1H), 1.68-1.10 (m, 12H);
13C NMR (HSQC and HMBC) 187.1/185.9 (C-60), 173.5 (C-19),
161.9/161.5 (C-6), 156.9 (C-22), 52.4 (C-20); HRMS (ESI-TOF)
m/z (M þ H)þ calcd for C37H3335Cl4N4O7 785.1103, found
785.1098.
(82%) of 24 as a white solid: IR (film) ν~ = 1771, 1725 cm-1
1H NMR δ 7.69(t, J =7.8Hz, 1H), 7.60(d, J = 7.8Hz, 1H), 7.39
(t, J = 7.8 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 2.20 (s, 3H); 13
;
C
NMR δ 168.5, 162.7, 150.4, 135.6, 132.2, 126.5, 125.6, 124.2,
115.6, 114.6, 105.1, 101.3, 20.5; HRMS (ESI-TOF) m/z (M þ
Na)þ calcd for C13H779Br235Cl2NO3Na 475.8067, found
475.8078.
2,3-Dichlorochromeno[3,2-b]pyrrol-9(1H)-one (25). To bro-
mide 19 (22.2 mg, 0.0663 mmol), copper(I) iodide (8.0 mg,
0.042 mmol), and potassium carbonate (40 mg, 0.29 mmol)
was added DMF (2.0 mL). The mixture was heated at 110 °C for
2 d, allowed to cool, diluted with a saturated NH4Cl solution
(8 mL), and extracted with EtOAc (3 ꢀ 5 mL). The combined
extracts were washed with brine (4 mL), dried, filtered, and
concentrated. The product was purified by reversed-phase C-18
HPLC (70% MeOH in H2O, 0.2% TFA, tR = 25 min) to yield
6.9 mg (41%) of 25 as a white solid: IR (film) ν~ = 1646 cm-1; 1H
NMR (DMSO-d6) δ 13.82 (br s, 1H), 8.22 (d, J = 7.8 Hz, 1H),
7.81 (t, J = 7.8 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.50 (t,
J = 7.8 Hz, 1H); 13C NMR (DMSO-d6) δ 165.3, 154.9, 145.5,
133.4, 125.5, 124.3, 123.0, 122.8, 118.0, 115.9, 95.0; HRMS
(ESI-TOF) m/z (M þ H)þ calcd for C11H635Cl2NO2 253.9776,
found 253.9770.
(3-Bromo-4,5-dichloro-1H-pyrrol-2-yl)(2-hydroxyphenyl)metha-
none (19). To acetate 21 (115 mg, 0.386 mmol) and N-bromosuc-
cinimide (77.0 mg, 0.433 mmol) was added DCE (8.0 mL). The
mixture was heated at 90 °C for 4.5 h, allowed to cool, poured into
EtOAc (100 mL), and washed with a saturated NaHCO3 solution
(20 mL) and brine (20 mL). The organic layer was dried, filtered,
and concentrated. The product was purified by column chroma-
tography (10% EtOAc then 15% EtOAc in hexanes) to afford 83.6
mg (57%) of acetylated bromide 22 as a foamy oil: IR (film) ν~ =
1769, 1745, 1624 cm-1; 1H NMR δ 9.77 (br s, 1H), 7.56 (t, J = 8.0
Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.24 (d,
J = 8.0 Hz, 1H), 2.18 (s, 3H); 13C NMR δ 181.8, 169.2, 148.1,
132.4, 130.1, 129.8, 126.9, 125.8, 123.1, 120.9, 114.9, 106.2, 20.8;
HRMS (ESI-TOF) m/z (M þ Na)þ calcd for C13H879Br35Cl2NO3-
Na 397.8962, found 397.8957. To a solution of 22 (9.8 mg, 0.026
mmol) in MeOH (2.0 mL) was added 5 drops of concd HCl. After
Diethyl 10H-1,30-Bipyrrole-2,20-dicarboxylate (30). To a solu-
tion of ethyl 3-amino-1H-pyrrole-2-carboxylate (29) (160 mg,
1.04 mmol) and crude ethyl 2,5-dioxopentanoate (247 mg, 1.56
mmol) in dry THF (5.0 mL) was added p-toluenesulfonic acid
(PTSA, 2 mg). The mixture was heated at 60 °C overnight,
J. Org. Chem. Vol. 75, No. 10, 2010 3249

Hughes et al.
JOCArticle
allowed to cool, and concentrated. The product was purified by
column chromatography (30% EtOAc in hexanes) to furnish
66.7 mg (23%) of bipyrrole 30 as a colorless oil. Analytically
pure 30 was obtained using reversed-phase HPLC (52%
(q, J = 7.2 Hz, 2H), 4.13 (m, 2H), 1.26 (t, J = 7.2 Hz, 3H), 1.08
(t, J = 7.2 Hz, 3H); 13C NMR: δ 158.6, 158.2, 126.8, 125.9,
120.5, 120.0, 114.3, 106.0, 102.2, 100.5, 61.3, 60.7, 14.1, 13.8;
HRMS (ESI-TOF) m/z (M þ H)þ calcd for C14H1379Br4N2O4
588.7609, found 588.7607.
1
CH3CN in H2O, tR = 16 min): IR (film) ν~ = 1696 cm-1; H
NMR (600 MHz) δ 9.42 (br s, 1H), 7.06 (m, 1H), 6.89 (m, 1H),
6.88 (m, 1H), 6.29 (m, 1H), 6.25 (m, 1H), 4.16 (q, J = 7.2 Hz,
2H), 4.12 (q, J = 7.2 Hz, 2H), 1.22 (t, J = 7.2 Hz, 3H), 1.10 (t,
J = 7.2 Hz, 3H); 13C NMR δ 160.6, 160.0, 130.0, 129.7, 124.6,
120.4, 117.8, 117.5, 109.9, 108.5, 60.3, 59.6, 14.2, 13.9; HRMS
(ESI-TOF) m/z (M þ H)þ calcd for C14H17N2O4 277.1188,
found 277.1183.
Diethyl 4,40,5,50-Tetrabromo-10H-1,30-bipyrrole-2,20-dicar-
boxylate (31). To bipyrrole 30 (20.0 mg, 0.0724 mmol) and
N-bromosuccinimide (50 mg, 0.28 mmol) was added DCE (2.0
mL). The mixture was heated at 80 °C for 2 h, allowed to cool,
and concentrated. The product was purified by reversed-phase
C-18 HPLC (65% CH3CN in H2O, tR = 25 min) to furnish 36.7
mg (86%) of tetrabromide 31 as a white solid: IR (film) ν~ = 1699
cm-1; 1H NMR (600 MHz) δ 10.45 (br s, 1H), 7.17 (s, 1H), 4.20
Acknowledgment. This work was supported by the Na-
tional Institutes of Health, National Cancer Institute under
Grant Nos. CA44848 and CA052955. We thank Drs. Arnold
L. Rheingold and Antonio G. D’Pasquale (UCSD) for
providing X-ray diffraction data and Sara Kelly (SIO) for
performing the MRSA and HCT-116 bioassays.
Supporting Information Available: Selected 1H, 13C, and 2D
NMR spectra and HRMS data for compounds 1-6, 11-17, 19,
20, 22-25, 30, and 31. CD spectra for 1-6. Crystallographic
data for 6 (CCDC 764039), 19 (CCDC 764038), and 31 (CCDC
764040) (CIF). This material is available free of charge via the
Internet at http://pubs.acs.org.
3250 J. Org. Chem. Vol. 75, No. 10, 2010