Marinomycins A−D, Antitumor-Antibiotics of a New Structure Class from a Marine Actinomycete of the Recently Discovered Genus “ Marinispora ”

Abstract

Four antitumor-antibiotics of a new structure class, the marinomycins A-D (1-4), were isolated from the saline culture of a new group of marine actinomycetes, for which we have proposed the name "Marinispora". The structures of the marinomycins, which are unusual macrodiolides composed of dimeric 2-hydroxy-6-alkenyl-benzoic acid lactones with conjugated tetraene-pentahydroxy polyketide chains, were assigned by combined spectral and chemical methods. In room light, marinomycin A slowly isomerizes to its geometrical isomers marinomycins B and C. Marinomycins A-D show significant antimicrobial activities against drug resistant bacterial pathogens and demonstrate impressive and selective cancer cell cytotoxicities against six of the eight melanoma cell lines in the National Cancer Institute's 60 cell line panel. The discovery of these new compounds from a new, chemically rich genus further documents that marine actinomycetes are a significant resource for drug discovery.

Full text

Marinomycins A-D, Antitumor-Antibiotics of a New Structure
Class from a Marine Actinomycete of the Recently Discovered
Genus “
Marinispora
”
Hak Cheol Kwon, Christopher A. Kauffman, Paul R. Jensen, and William Fenical*
Contribution from the Center for Marine Biotechnology and Biomedicine, Scripps Institution of
Oceanography, UniVersity of California at San Diego, La Jolla, California 92093-0204
Received September 8, 2005; E-mail: wfenical@ucsd.edu
Abstract:
Four antitumor-antibiotics of a new structure class, the marinomycins A-D(1-4), were isolated
from the saline culture of a new group of marine actinomycetes, for which we have proposed the name
“
Marinispora
”. The structures of the marinomycins, which are unusual macrodiolides composed of dimeric
2-hydroxy-6-alkenyl-benzoic acid lactones with conjugated tetraene-pentahydroxy polyketide chains, were
assigned by combined spectral and chemical methods. In room light, marinomycin A slowly isomerizes to
its geometrical isomers marinomycins B and C. Marinomycins A-D show significant antimicrobial activities
against drug resistant bacterial pathogens and demonstrate impressive and selective cancer cell cytotoxicities
against six of the eight melanoma cell lines in the National Cancer Institute’s 60 cell line panel. The discovery
of these new compounds from a new, chemically rich genus further documents that marine actinomycetes
are a significant resource for drug discovery.
Introduction
The actinomycetes are Gram-positive bacteria, which for more
than 50 years provided a significant source for bioactive
secondary metabolites. These mainly soil-derived microorgan-
isms have yielded more than 10 000 bioactive compounds
including more than 70% of the natural antibiotics discovered.1
Unfortunately, beginning in the late 1980s, the rate of discovery
of new drug candidates from terrestrial actinomycetes began to
decrease, which rendered continued exploration of this source
inefficient. Ultimately, this led many of the major international
pharmaceutical industries to abandon terrestrial actinomycetes
in favor of alternate sources of chemical diversity such as
directed and combinatorial synthesis. With the dramatic increase
in the emergence of drug-resistant infectious diseases, the need
to discover and develop new antibiotics has never been greater.
Unfortunately, the current sources for chemical diversity do not
seem to be generating new antibiotic drugs in response to this
emerging challenge.
Because actinomycete bacteria are very common in soils, they
are introduced into the oceans in large numbers, by runoff and
river flows, leading to the hypothesis that the vast majority of
strains isolated from marine sources may be of terrestrial origin.2
With this in mind, and considering the difficulty in exploring
marine ecosystems, it is no wonder that the oceans have been
largely overlooked as a source for these chemically prolific
bacteria. Quite recently, it has been recognized that actino-
mycetes adapted for life in the sea do indeed occur.3Using
various sampling tools, including methods that provide access
to deep ocean sediments, we have cultivated multiple new
groups of actinomycetes from marine samples. These studies,
which combine new sampling and culture methods with
phylogenetic evaluation of the strains observed, have demon-
strated that numerous new actinomycete taxa are present in
marine habitats. This approach led to the discovery of the
chemically rich genus Salinispora4,5 and, more recently, a new
marine actinomycete genus, originally designated as MAR2,6
for which we now suggest the name “Marinispora”.
The first Marinispora strain to be subjected to chemical study,
strain CNQ-140, was isolated from a sediment sample collected
at a depth of 56 m offshore of La Jolla, CA. Strain CNQ-140
was cultivated in a seawater-based medium and then extracted
with the adsorbent resin XAD-7. The resin was eluted with
acetone, the solvent was removed under reduced pressure and
the residue was partitioned between ethyl acetate and water.
Removal of the ethyl acetate provided an extract that demon-
strated in vitro cytotoxicity against HCT-116 human colon
carcinoma (IC50 )1.2 µg/mL). Activity guided fractionation
of the ethyl acetate extract by a diversity of chromatographic
methods led to the isolation of four macrodiolides, marinomycins
A-D(1-4). These interesting compounds were subsequently
purified, and their structures were assigned by combined spectral
and chemical methods. The marinomycins possess signifi-
cant antibiotic activities, with MIC values of 0.1-0.6 µM,
(1) Berdy, J. J. Antibiot. 2005,58,1-26.
(2) Goodfellow, M.; Haynes, J. A. Biological, Biochemical, and Biomedical
Aspects of Actinomycetes; Academic Press: New York, 1984; pp 453-
472.
(3) Mincer, T. J.; Jensen, P. R.; Kauffman, C. A.; Fenical, W. Appl. EnViron.
Microbiol. 2002,68, 5005-5011.
(4) 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. Appl. Microbiol.
2005,55, 1759-1766.
(5) Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C. A.; Jensen, P.
R.; Fenical, W. Angew. Chem., Int. Ed. 2003,42, 355-357.
(6) Jensen, P. R.; Mincer, T. J.; Williams, P. G.; Fenical, W. Antonie Van
Leeuwenhoek 2005,87,43-48.
Published on Web 01/13/2006
1622 9J. AM. CHEM. SOC. 2006,
128
, 1622-1632 10.1021/ja0558948 CCC: $33.50 © 2006 American Chemical Society

against methicillin-resistant Staphylococcus aureus (MRSA) and
vancomycin-resistant Enterococcus faceium (VREF). In addi-
tion, the marinomycins inhibit cancer cell proliferation with
average LC50 values of 0.2-2.7 µM against the NCI’s 60 cancer
cell line panel. The results of that testing further revealed that
the marinomycins had potent and selective cytotoxicities against
six of the eight melanoma cell lines.
Results and Discussion
Marinomycin A (1) was obtained as a yellow powder that
analyzed for the molecular formula C58H76O14 by interpretation
of HR-MALDI-FTMS ([M +Na]+m/z1019.5190) and NMR
data. The IR spectrum of 1displayed absorption bands at 3375
and 1713 cm-1, indicating the presence of hydroxyl and ester
functionalities, while a UV absorption band at 359 nm was
suggestive of a highly conjugated polyene moiety.7Proton NMR
spectral data, including correlations from COSY and J-resolved
experiments, illustrated signals attributable to a conjugated
tetraene [(δ6.35 (1H, dd, J)11.0, 2.5 Hz), 6.49 (1H, dd, J)
14.0, 10.2 Hz), 6.52 (1H, dd, J)14.6, 10.5 Hz), 6.63 (1H, dd,
J)14.0, 10.5 Hz), 6.74 (1H, dd, J)14.6, 11.0 Hz), 7.11
(1H, dd, J)15.5, 10.2 Hz), and 7.24 (1H, d, J)15.5 Hz)]
(Table 1). Two-dimensional NMR analysis, using HMBC and
HMQC experiments, showed that these proton signals correlated
with 13C NMR bands at δ128.7, 129.5, 131.2, 132.1, 132.4,
132.8, 135.7, and 138.3. Other characteristic features of the 1H
NMR spectrum of 1were the presence of five oxymethine
protons [δ4.29 (1H, br m), 4.30 (1H, br m), 4.64 (1H, br m),
4.78 (1H, br m), and 6.55 (1H, m)], five methylene protons [δ
1.76-2.50], one secondary methyl group at δ1.37 (3H, d, J)
6.3 Hz), and two additional nonconjugated trans olefinic protons
[δ5.94 (1H, dd, J)15.0, 6.5 Hz) and 6.22 (1H, dt, J)15.0,
7.6 Hz)]. The relative lack of branching demonstrated in these
1H NMR signals suggested marinomycin A was composed of a
linear, polyketide-type chain. Overall analysis of the NMR data
indicated that marinomycin A possessed the molecular formula
C29H38O7, exactly one-half of the molecular formula, C58H76O14,
determined by HRMS. Thus, it became clear that marinomycin
A was a symmetrical macrodiolide dimer composed of two
identical C29 units. This was further indicated by acetylation,
which yielded the deca-acetate 1a, illustrating that marinomycin
A possesses at least 10 hydroxyl groups.
(7) Bruno, T. J.; Svoronos, P. D. N. Handbook of basic tables for chemical
analysis; CRC press: Florida, 2000; p 222.
Table 1.
1H, 13C, and HMBC NMR Data for Marinomycin A (1)
position
δ
H mult (
J
,
g
Hz)
a
δ
C
b
HMBC
a
1, 1′169.7
2, 2′122.7
3, 3′156.7
4, 4′7.04 dd (8.2, 1.6) 115.6 1 (1′),3(3′),2(2′),6(6′),5(5′)
5, 5′7.27 t (8.2) 131.0 3 (3′),7(7′),6(6′)
6, 6′7.31 dd (8.2, 1.6) 116.2 7 (7′),2(2′),4(4′),8(8′),5(5′)
7, 7′137.0
8, 8′7.24 d (15.5) 129.5 7 (7′),9(9′), 10 (10′),6(6′)
9, 9′7.11 dd (15.5, 10.2) 132.4 7 (7′), 11 (11′), 10 (10′),8(8′)
10, 10′6.49 dd (14.0, 10.2)f132.8 11 (11′), 12 (12′),8(8′)
11, 11′6.63 dd (14.0, 10.5)f135.7 12 (12′), 13 (13′)
12, 12′6.52 dd (14.6, 10.5)f132.1 11 (11′), 10 (10′), 13 (13′), 14 (14′)
13, 13′6.74 dd (14.6, 11.0) 131.2 15 (15′), 11 (11′), 12 (12′), 14 (14′)
14, 14′6.35 dd (11.0, 2.5) 128.7 12 (12′), 13 (13′), 16 (16′), 29 (29′)
15, 15′138.3
16, 16′2.43c,f49.9 17 (17′), 18 (18′), 29 (29′), 15 (15′),
14 (14′)
2.32 br d (12.2)f
17, 17′4.29 br md,f70.2
18, 18′2.03 me45.6 19 (19′), 17 (17′), 20 (20′)
1.76 br d (14.2)
19, 19′4.78 br m 73.1
20, 20′5.94 dd (15.0, 6.5) 136.8 21 (21′), 19 (19′), 22 (22′)
21, 21′6.22 dt (15.0, 7.6) 128.3 20 (20′), 19 (19′), 22 (22′), 23 (23′)
22, 22′2.50 mc,f42.6 23 (23′), 20 (20′), 21 (21′)
2.38 mc,f
23, 23′4.30 br md,f67.8
24, 24′2.10 me,f44.5 23 (23′), 25 (25′), 26 (26′), 22 (22′)
25, 25′6.55 m 71.7 1 (1′)
26, 26′2.18 br ddd
(14.2, 6.5, 3.0) 46.1 25 (25′), 24 (24′), 27 (27′), 28 (28′)
2.00 me
27, 27′4.64 br m 64.0
28, 28′1.37 d (6.3) 24.9 27 (27′), 26 (26′)
29, 29′1.88 s 17.8 16 (16′), 15 (15′), 13 (13′), 14 (14′)
a300 MHz, pyridine-d5.b75 MHz, pyridine-d5.c-eOverlapping signals.
fChemical shifts were assigned using 1H-1H COSY and homo-J-resolved
1H NMR spectral data. gCoupling constants derived from analysis of homo-
J-resolved 1H NMR spectral data.
Marinomycins A
−
D, Antitumor-Antibiotics
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J. AM. CHEM. SOC. 9VOL. 128, NO. 5, 2006 1623

The 13C NMR and HMQC spectra of 1allowed all protons
to be assigned to their respective carbons. The oxymethine
protons correlated with oxygenated carbons carbons at δ64.0,
67.8, 70.2, 71.7, and 73.1, while the methylene protons
correlated with five methylene carbons at δ42.6, 44.5, 45.6,
46.1, and 49.9. The aliphatic methyl group protons correlated
with a carbon signal at δ24.9, and the two nonconjugated
olefinic protons to carbon signals at δ128.3 and 136.8. In
addition, the 1H NMR spectrum of 1showed additional signals
attributed to a phenyl group [δ7.04 (1H, dd, J)8.2, 1.6 Hz),
7.27 (1H, t, J)8.2 Hz), and 7.31 (1H, dd, J)8.2, 1.6 Hz)],
and a vinyl methyl group [δ1.88 (3H, s)]. The HMQC NMR
spectrum showed that the phenyl protons correlated to carbons
at δ115.6, 116.2, and 131.0, while the olefinic methyl group
was observed at δ17.8. As expected, the ester carbon signal
was observed at δ169.7.
Comprehensive collation of 2D NMR data from 1H-1H
COSY, TOCSY, ROESY, HMQC, and HMBC experiments led
to the construction of a 2-hydroxy-6-alkenyl-benzoic acid ester
with the tetraene chain in the 6 position. The chemical shifts of
the aromatic ring in this specific constellation were comparable
to those from distantly related compounds available in the
literature.8Key HMBC correlations (Table 1) allowed the vinyl
methyl group to be positioned at C-15, while the full carbon
chain from C-16 to C-28, including the presence of oxygen at
C-17, -19, -23, -25, and -27, was readily assigned by these
combined data. An HMBC correlation from the carbinol proton
at C-25 to the ester carbon (C-1) indicated the site of lacton-
ization was at C-25. The geometries of three of the double bonds
in the tetraene chain (C-8-C-13) were determined to be trans
(E) on the basis of their characteristic coupling constants (Jg
14.0 Hz) observed in the homo J-resolved 1H NMR spectrum.
The ∆14,15 trisubstituted olefin was assigned as Eon the basis
of a prominent ROESY NMR correlation between the vinyl
methyl protons (H3-29) and H-13.
Methanolysis of marinomycin A with NaOMe in MeOH
cleaved both lactone linkages to yield the monomer methyl ester
6(Scheme 1), which was fully characterized by LC-MS, and
1D and 2D NMR methods (see Experimental Section). This
experiment, and the complete NMR analysis described above,
defined marinomycin A as a dimeric macrodiolide possessing
an unprecedented 44-membered ring.
The relative stereochemistry of marinomycin A (1) was
assigned on the basis of spectral analysis and chemical
modification. The relative stereochemistries of the polyol
functionalities (C-17, C-19, C-23, C-25, and C-27) were initially
assigned by application of Kishi’s Universal NMR Database,9
and by conversion to two different acetone ketals. Kobayashi
et al. showed that the relative stereochemistry of 1-ene-3,5-
diols and 1,3,5-triols can be predicted by comparison of 13C
NMR chemical shifts at C-4 and C-5 of the stereoisomers with
the model compounds non-2-en-1,4,6-triol and decan-1,3,5,7-
tetraol, respectively.9Comparison of the 13C NMR data derived
from the methanolysis product 6with those from the above
model compounds (Figure 1) allowed the relative stereochem-
istries at several centers to be assigned. The 13C NMR chemical
shift of C-19 (δ69.4) in the spectrum of 6(DMSO-d6) was
very close to the C-4 value (δ69.0) of the syn-diol in non-2-
en-1,4,6-triol. Similarly, the 13C NMR chemical shift of C-25
(δ63.8) of 6was very close to that of the C-5 carbon (δ64.0)
of anti,anti-decan-1,3,5,7-tetraol.
Treatment of 1with 2,2-dimethoxypropane and pyridinium-
p-toluenesulfonate in methanol afforded the bis-acetone ketal
8, resulting from ketal formation at the C-17, C-19 and C-17′,
C-19′hydroxyl groups (Scheme 2). The chemical shifts of the
acetonide methyl groups were observed at δ19.6 and δ30.1
(HSQC spectral data), indicating the six-membered 1,3-dioxane
ring was in a chair conformation. In the NMR method described
by Rychnofsky,10 the 13C NMR chemical shifts of methyl groups
in a syn-acetonide (chair form) are commonly at δ20 and δ30
(axial and equatorial methyls), while the methyl signals of an
anti-acetonide (the skew form yields identical methyl groups)
are both observed at δ25. On the basis of the observed acetonide
methyl chemical shifts at δ19.6 and 30.1, the hydroxyl groups
at C-17, 19 (and C-17′,19′) were assigned syn configurations,
respectively.
(8) (a) Kim, J. W.; Shin-ya, K.; Furihata, K.; Hayakawa, Y.; Seto, H. J. Org.
Chem. 1999,64, 153-155. (b) Hedge, V. R.; Puar, M. S.; Dai, P.; Patel,
M.; Gullo, V. P.; Das, P. R.; Bond, R. W.; McPhail, A. T. Tetrahedron
Lett. 2000,41, 1351-1354.
(9) Kobayashi, Y.; Czechtizky, W.; Kishi, Y. Org. Lett. 2003,5,93-96.
(10) Rychnovsky, S. D.; Rogers, B. N.; Richardson, T. I. Acc. Chem. Res. 1998,
31,9-17.
Scheme 1.
Methanolysis of Marinomycin A (1) and Marinomycin B (2) To Yield Esters 6and 7, Respectively
Figure 1.
13C NMR chemical shift comparisons of the carbinol carbons in
methyl ester 6(A) with those of model compounds (B).
ARTICLES
Kwon et al.
1624 J. AM. CHEM. SOC. 9VOL. 128, NO. 5, 2006

In a similar fashion, treatment of the methanolysis product 6
with 2,2-dimethoxypropane and pyridinium-p-toluenesulfonate
in methanol afforded two diacetonides, 9and 10, which were
readily separated by HPLC methods (Scheme 2). NMR data
for both bis-ketals showed acetonide methyl group signals at δ
19.6, 24.8 (×2), and 30.1, consistent with the presence of both
syn and anti acetonides, and consistent with the assignment
predicted by Kishi’s Universal Database analysis. On the basis
of these NMR experiments, the relative stereochemistry of the
three 1,3-diols in 1were assigned as 17,19-syn (17′,19′-syn),
23,25-anti (23′,25′-anti), and 25,27-anti (25′,27′-anti).
The absolute stereochemistry of marinomycin A (1) was
determined by application of the modified Mosher ester NMR
method using the acetonides 9and 10. Treatment of acetonide
9, in separate experiments, with (R)-(-)-R-methoxy-R-(tri-
fluoromethyl)phenylacetyl chloride (R-MTPA-Cl) and (S)-(+)-
MTPA-Cl, yielded the S-Mosher ester 11a and R-Mosher ester
11b, respectively. Analysis of 1H NMR chemical shift differ-
ences (∆δS-R) between 11a and 11b revealed that the absolute
stereochemistry of C-23 is S(Figure 2).11 Similarly, preparation
of the S-MTPA ester (12a) and R-MTPA ester (12b) from 10,
followed by NMR analysis, revealed the absolute stereochem-
istry at C-27 is R(Figure 2).
The absolute stereochemistry of the allylic alcohol at C-19
in 1was difficult to assign by the Mosher method because the
hydroxyl group tended to eliminate on attempted acylation under
a variety of mild reaction conditions. To approach establishing
the absolute stereochemistry at this center, a series of derivatives
were prepared involving hydrogenation of the olefinic bonds
in 1. Catalytic hydrogenation of 1(10% Pd/C) yielded three
HPLC-resolvable stereoisomers of perhydro-marinomycin A in
a 1/2/1 ratio. The isomer mixture was fractionated [C-18 reverse
phase, acetonitrile-water (9:1)] to yield 13, the major stereo-
isomer of unknown configuration at C-15 (Scheme 3). Succes-
sive acetonide formation, acetylation, and acetonide deprotection
(11) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc.
1991,113, 4092-4096.
Scheme 2.
(A) Formation of Acetonide 8Illustrating the 13C NMR Chemical Shifts of the Acetonide Methyl Carbons; (B) Acetonides 9and
10 Illustrating the 13C NMR Chemical Shifts of the Acetonide Methyl Carbons; (C) The Conformations of
syn
- and
anti
-1,3 Diol Acetonides
Illustrating the Predicted 13C NMR Chemical Shifts of the Acetonide Methyl Groups
Figure 2.
∆δS-Rvalues for the Mosher esters 11a/11b,12a/12b, and 17a/
17b.
Marinomycins A
−
D, Antitumor-Antibiotics
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J. AM. CHEM. SOC. 9VOL. 128, NO. 5, 2006 1625

of 13 yielded the 3,3′,23,23′,27,27′-hexa-acetate derivative 16
(Scheme 3). Treatment of 16 with R-MTPA-Cl and S-MTPA-
Cl, under standard acylation conditions, afforded tetra-S-MTPA-
ester (17a) and tetra-R-MTPA-ester (17b), respectively. Appli-
cation of both the classic Mosher ester 19F NMR method and
the modified Mosher ester analysis12 allowed assignment of the
absolute stereochemistry at C-17, C-17′, C-19, and C-19′. NMR
data, showing negative ∆S-Rvalues for the four fluorine signals
in the 19F NMR spectrum of 17a and 17b [17a δ5.38, 5.39,
5.46, and 5.55; 17b δ5.56 (×3), 5.68; TFA δ0.00], were
obtained (Figure 2).13 These results supported the assignment
of the absolute stereochemistry at C-17, C-17′, C-19, C-19′,
C-23, C-23′, C-25, and C-25′as S, while C-27 and C-27′were
assigned as R. While it is not always feasible to correctly
interpret the NMR shift data derived from multiple Mosher
esters, Riguera and co-workers have shown14 that the MTPA
esters of syn-1,3-diols can be confidently used to assign absolute
stereochemistry.
Marinomycin B (2) was isolated as a yellow powder that
analyzed for the molecular formula C58H76O14 by HRESI-MS
data ([obsd M +Na]+at m/z1019.5110), and by comprehensive
analysis of NMR data (Table 2). The UV absorption spectrum
of 2showed bands at 315 and 340 nm, which were suggestive
of the presence of the conjugated polyene as in 1. However,
the UV absorption spectrum of 2differed in fine structure,
indicating that 2is the geometric isomer of 1.7The configura-
tions of the double bonds in 2were assigned on the basis of
J-resolved and 1D proton-proton NMR coupling constant data
and upon analysis of ROESY NMR information. The geometries
of the ∆8,9 and ∆12,13 olefins were assigned as Zand E,
respectively, based upon proton coupling constants of JH-8,H-9
)11.0 Hz and JH-12,H-13 )14.6 Hz. ROESY NMR correlations
between the vinyl methyl protons (H3-29) and the proton at C-13
established the configuration of the ∆14,15 olefin as E. Unfor-
tunately, overlapping NMR signals for the protons at C-10 and
C-11 made assignment of the ∆10,11 olefin geometry by coupling
constant analysis impossible. However, ROESY NMR correla-
tions between H-9 (δ6.43) and H-12 (δ6.19) with the
overlapping H-10/H-11 overlapping pair (δ6.60), coupled with
a lack of correlation between H-9 and H-12 (1D ROE experi-
ment), indicated that the configuration of the ∆10,11 double bond
was E.
Comprehensive NMR analysis, utilizing data from COSY,
HMQC, and HMBC experiments, allowed the complete assign-
ment of the proton and carbon signals for 2, leading to the
(12) Seco, J. M.; Quinoa, E.; Riguera, R. Chem. ReV.2004,104,12-117.
(13) Rieser, M. J.; Hui, Y.-H.; Rupprecht, J. K.; Kozlowski, J. F.; Wood, K.
V.; McLaughlin, J. L.; Hanson, P. R.; Zhuang, Z.; Hoye, T. R. J. Am.
Chem. Soc. 1992,114, 10203-10213.
(14) Freire, F.; Seco, J. M.; Quin˜oa, E.; Riguera, R. J. Org. Chem. 2005,70,
3778-3790.
Scheme 3.
Preparation of Compound 16
a
aReagents and conditions: (a) (i) H2gas, 10% Pd/C, ethanol, (ii) C-18
RP HPLC using MeCN/H2O (9:1); (b) MeOH, 2,2-dimethoxypropane,
PPTS, 0 °C; (c) (i) acetic anhydride, pyridine, (ii) MeOH, PPTS, room
temperature, (iii) C-18 RP HPLC using MeCN/H2O (9:1).
Table 2.
1H and 13C NMR Data of Marinomycin B (2)in
Pyridine-
d
5and CDCl3
position
δ
H mult (
J
,Hz
n
)
300 MHz, pyridine-
d
5
δ
H mult (
J
,Hz
n
)
400 MHz, CDCl
3
δ
C
(pyridine-
d
5
)
δ
C
(CDCl
3
)
1, 1′170.5 171.6
2, 2′122.2 110.8
3, 3′160.0 163.4
4, 4′7.06 d (8.2) 6.91 d (8.2) 116.1 117.2
5, 5′7.32 t (8.2) 7.34 t (8.2) 132.0 134.5
6, 6′6.94 d (8.2) 6.70 d (8.2) 121.6 123.1
7, 7′139.2 140.8
8, 8′6.90 d (11.0) 6.66 d (11.0) 129.7 132.8
9, 9′6.43 m 6.13 mf130.2 129.8
10, 10′6.60 ma6.23 mg128.0 128.2l
11, 11′6.60 ma6.23 mg136.5 135.1
12, 12′6.19 m 6.13 mf128.3j128.4l
(14.6, 10.3)b,m
13, 13′6.71 dd (14.6, 11.0) 6.35 dd (14.6, 11.0) 130.8 131.8
14, 14′6.22dbrd
(11.0, 2.5)b,m5.86dbrd
(11.0, 2.5) 128.3j128.5l
15, 15′139.0 135.7
16, 16′2.42 mc,m1.81 mh,m49.1 49.1
17, 17′4.35 br md,m3.57 br tt (10.0, 4.0) 68.8 70.4
18, 18′2.0-2.2 µe1.34 br d (15.5) 44.6k44.2
1.83 br dt (15.5, 3.5) 1.08 m
19, 19′4.71 br m 3.91 br d (9.5) 71.6 71.9
20, 20′5.91 dd (15.2, 5.9) 5.29 br si136.5 135.1
21, 21′6.13 dt (15.2, 7.5)b5.29 br si127.4 128.6l
22, 22′2.50 mc,m1.90 m 42.0 41.1
2.53 mc,m
23, 23′4.17 br m 3.41 br tt (9.0, 4.5) 67.0 66.7
24, 24′2.0-2.2 µe1.71 mh,m44.6k43.0
25, 25′6.30 m 5.42 m 72.2 73.5
26, 26′2.0-2.2 µe1.66 mh,m44.6k46.1
1.79 mh,m
27, 27′4.38 br md,m3.99 m 64.0 65.0
28, 28′1.37 d (6.4) 1.17 d (6.4) 24.2 23.5
29, 29′1.92 s 1.68 s 17.8 17.3
OH 4.20 (4H, br s)
4.40 (2H, br s)
3-OH 11.68 s
a-kOverlapping signals. lInterchangeable signals. mChemical shifts were
assigned by interpretation of 1H-1H COSY NMR data. nCoupling constants
determined by analysis of homo-J-resolved 1H NMR spectral data.
ARTICLES
Kwon et al.
1626 J. AM. CHEM. SOC. 9VOL. 128, NO. 5, 2006

assignment of a planar structure for this compound. As in 1,
methanolysis of marinomycin B (2) with NaOMe in MeOH
yielded only one product, the methyl ester 7, which confirmed
that 2was also a symmetrical dimeric macrodiolide (Scheme
1).Marinomycin C (3) was also obtained as yellow powder that
analyzed for the formula C58H76O14 by HRESI-TOF MS data
([obsd M +Na]+at m/z1019.5110) and comprehensive NMR
data. As in marinomycins A and B, UV absorption bands at
319, 345, and 358 nm confirmed that 3also possessed the
conjugated phenyl-tetraene functionality. The 1H and 13C NMR
spectra of 3showed features similar to those of 1and 2, but in
this case the NMR data were more complex, indicating that 3
was an unsymmetrical dimer (Table 3). Methanolysis of 3with
NaOMe in MeOH yielded 6and 7in an approximately 1/1 ratio
by NMR and LC-MS analyses. Given this information, mari-
nomycin C (3) was assigned as an unsymmetrical dimer
possessing the all Etetraene functionality in one half and the
∆8,9 )Zolefin in the second half.
The assignment of the absolute stereochemistries of the polyol
carbons in marinomycins B and C (2and 3) were not approached
using the classic methods applied to marinomycin A. This
proved to be unnecessary, because it was observed that
marinomycin A could be photochemically converted to a
mixture of B and C under ordinary room light (T1/2 )ca. 1 h).
The NMR data, HPLC retention times, and optical rotations
derived from marinomycins B and C, derived by photoconver-
sion from A, were identical to those data derived from B and C
originally isolated from the fermentation broth.
Marinomycin D (4) was isolated as a yellow powder that
analyzed for the molecular formula C59H78O14, by HR ESI-TOF
MS (m/z1033.5267 [M +Na]+) and NMR methods. This
formula showed the addition of CH2to the molecular formulas
of the other marinomycins, suggesting an additional methylene
or methyl group had been added to the polyketide-like chain.
The UV absorption spectrum of 4was in good agreement with
that of marinomycin B. The 1H and 13C NMR spectra for 4
were almost identical to those of the Z,E,E,E-tetraene in 2(Table
4.). The major difference between the spectra for 2and 4was
the presence of a new triplet methyl group (C-29′,δ0.93).
HMBC NMR spectral data showed a strong correlation between
the methyl triplet (H-29′), and C-28′, and C-27′, thus indicating
that the linear chain in one half of marinomycin D had been
extended by one carbon. Because one half of marinomycin D
possessed a different carbon skeleton, we were unable to
chemically correlate the stereochemistry of 4to the fully defined
stereochemistry of marinomycin A. However, based upon the
optical properties of 4, and the excellent comparison of NMR
data to those from 2, we reasonably assume that the stereo-
chemistry is identical to 2at comparable centers. Having
rigorously defined the absolute stereochemistries of marinomy-
cins A-C at all oxymethine centers, we were surprised to find
that marinomycin A showed [R]D)+180°, while marinomycins
B-C showed negative [R]Dvalues of -245°and -151°,
respectively. The rotation of marinomycin D was [R]D)-233°,
a value similar to marinomycin B, its methylene lower homo-
logue. Because it seemed unreasonable that these simple olefin
geometrical isomers would show opposite rotations, we exam-
ined the CD spectra of 1-4(Figure 3). Unusual and complex
results were obtained that indicated that marinomycin A (1) was
more optically complex than B-D. The CD spectrum of
marinomycin A (1) showed a positive peak at 380 nm and a
negative peak at 360 nm, as well as a broad negative band
between 325 and 365 nm (Figure 3). Because the UV spectrum
of 1showed λmax at 325 (sh), 345 (sh), 359, and 378 nm, the
peaks at 380 and 360 nm seem to be due to a positive Cotton
effect by exciton coupling between the polyene chromophores
of the monomeric units. While the CD spectra of 2and 3did
not indicate an exciton coupling pattern, they did show a broad
negative band between 320 and 410 nm, which corresponds to
the UV absorption bands of 2and 3. In the end, we concluded
that these data were too complex to interpret, but that they
suggested differences in ring conformations, and perhaps that
the chirality was influenced by, if not derived from, interaction
of the polyene chains in marinomycin A.
To explore a possible conformational explanation, ROESY
NMR experiments were conducted with marinomycins A and
B. For marinomycin A, six strong NOE correlations, which
included H-6(6′) to H-9(9′), H-8(8′) to H-25(25′), H-12(12′)to
H-19(19′), H-14(14′) to H-17(17′), and H-29(29′) to H-13(13′),
were observed (Figure 4). The strong transannular NOEs
observed between H-12(12′) across the ring to H-19(19′), and
from H-14(14′) to H-17(17′), provided strong evidence that the
side chains in 1were parallel and in very close proximity. In
contrast, marinomycin B showed only two NOE correlations,
H-8(8′) to H-23(23′) and H-10(10′) to H-6(6′), which demon-
strated that 2existed in an entirely different ring conformation.
Simple molecular models of marinomycins A and B (see
Supporting Information), which were constructed based upon
Table 3.
1H and 13C NMR Data for Marinomycin C (3)
position
δ
H mult (
J
, Hz)
a
δ
C
b
position
δ
H mult (
J
, Hz)
a
δ
C
b
1 170.7 1′171.7
2 116.3 2′110.4
3 158.7 3′163.2
4 6.81 d (8.2) 116.3 4′6.92 d (8.1) 117.1
5 7.24 t (8.2) 132.8 5′7.36 t (8.1) 134.3
6 7.01 d (8.2) 118.0 6′6.71 d (8.1) 123.0
7 138.7 7′140.3
8 6.89 d (14.7) 130.8 8′6.69 d (10.6) 132.4l
9 6.66 dd (14.7, 10.0) 132.5l9′6.16 mi129.8
10 6.30-6.50 mc129.6 10′6.30-6.50 µc128.2p
11 6.30-6.50 mc134.6m11′6.30-6.50 µc134.7m
12 6.31 mc,q131.4 12′6.16 mi128.4p
13 6.40 mc,q131.8n13′6.40 mc,q131.6n
14 5.97 d (10.4) 127.9 14′5.88 d (11.0) 128.5p
15 136.3o15′136.1o
16 2.00-2.10 md49.6 16′1.80-2.10 µd48.3
17 3.80 me,q69.4 17′3.55 br mn,q69.3
18 1.50-1.70 mf43.7 18′1.40 br d (15.0) 44.4
0.90 mq
19 4.25 br mg73.5 19′4.02 br mj,q72.2
20 5.51 br sh135.6 20′5.35 br sk135.1
21 5.51 br sh127.9 21′5.35 br sk128.6p
22 2.15 md,q41.4 22′1.95 md,q41.0
23 3.75 br me,q66.7 23′3.46 br mn,q66.7
24 1.65-1.85 mf42.5 24′1.60-1.75 µf42.8
25 5.65 br m 71.2 25′5.55 br m 72.2
26 1.65-1.85 µf44.5 26′1.60-1.75 µf45.7
27 3.85 br me,q64.1 27′3.95 br mj,q64.3
28 1.18 d (6.5) 23.2 28′1.16 d (6.5) 23.2
29 1.72 s 17.7 29′1.68 s 16.4
OH 4.25 br sgOH 11.55 s/9.82 br s
4.61 br s, 4.49 br s
a300 MHz, CDCl3.b75 MHz, CDCl3.c-kOverlapping signals. l-pInter-
changeable signals. qChemical shifts were assigned by analysis of 1H-1H
COSY NMR data.
Marinomycins A
−
D, Antitumor-Antibiotics
ARTICLES
J. AM. CHEM. SOC. 9VOL. 128, NO. 5, 2006 1627

these NOE data and the ∆8,9 )Eand Zolefin geometries,
demonstrated that in marinomycin considerable transannular
interaction was indeed feasible, while in marinomycin B the
ring must expand to accommodate the Z-olefin. In marinomycin
A, the polyene-polyol chains adopt an opposing, coplanar, but
adjacent array that places the polyene chains within very close
proximity. In this configuration, the polyene chromophores are
in proximity and, conceivably, able to interact.
Interactions of polyolefinic systems are well known to
generate chirality in the absence of chiral carbons. In the polyene
carotenoid pigments, which lack chiral carbons, dimeric struc-
tures are produced by aggregation in polar solvents. This results
in defined interactions of their polyolefinic backbones creating
tortionally defined exciton coupling, which generate Cotton
effects in their CD spectra.15 An intramolecular exciton coupling
within marinomycin A (1), generated by conformational interac-
tions of the polyenes, rather than molecular aggregation, could
explain the differences in the rotation and CD data for 1and
2-4.
To explore this further, we recorded the CD spectrum of the
monomeric polyene 6under identical conditions. The spectrum
was essentially featureless with very poor to no absorptions in
the 350-400 nm range. Hence, we hypothesize that in marino-
mycin A (1), the all-Egeometry of the tetraene functionality
leads to transannular hydrogen bonding, thus creating a con-
figuration which may facilitate olefinic exciton coupling. In
contrast, in marinomycins B-D, NOE evidence suggests that
these rings are expanded to a circular configuration by virtue
of their ∆8,9 )Zolefinic bonds, resulting in few transannular
interactions.
It seem reasonable that the interactions described above could
explain the positive and negative [R]Dvalues for 1and for 2-4.
Although perhaps not the best models, reversals in the sign of
[R]Dvalues have been observed in the olefin geometrical isomers
of the secondary metabolites clathrynamide and hanliangicin.15
Bioactivity of the Marinomycins. The marinomycins showed
significant antibacterial and in vitro cancer cell cytotoxicities.
Marinomycin A (1) was the most potent antibacterial agent
showing in vitro minimum inhibitory concentrations (MIC90)
of 0.13 µM against methicillin-resistant Staphylococcus aureus
(MRSA) and vancomycin-resistant Streptococcus faecium (VREF)
(Table 5). Given the presence of a polyene chain, and recogniz-
ing the apparent mechanism of action of the polyene antifungal
agents,17 we assumed that the marinomycins would be potent
antifungal agents. This turned out to be incorrect, as only
marinomycin A showed very weak activity below 10 µM against
Candida albicans. We also evaluated the antibacterial activity
of compounds 6,7,8, the hexa-acetate derivative of 8(8a),
and the 3,3′,23,23′,27,27′-hexa-acetate of 1(8b) (Table 5).
(15) Simonyi, M.; Bakadi, Z.; Zsila, F.; Deli, J. Chirality 2003,15, 680-698.
(16) (a) Ojika, M.; Itou, Y.; Sakagami, Y. Biosci. Biotechnol. Biochem.2003,
67, 1568-1573. (b) Kundim, B. A.; Itou, Y.; Sakagami, Y.; Fudou, R.;
Iizuka, T.; Yamanaka, S.; Ojika, M. J. Antibiot. 2003,56, 630-638.
(17) (a) Bolard, J. Biochim. Biophys. Acta 1986,864, 257-304. (b) De Kruijff,
B.; Demel, R. A. Biochim. Biophys. Acta 1974,339,57-70. (c) Finkelstein,
A.; Holz, R. In Membranes 2: Lipid Bilayers and Antibiotics; Eisenman,
G., Ed.; Marcel Dekker: New York, 1973; Chapter 5. (d) Matsumori, N.;
Yamaji, N.; Oishi, T.; Murata, M. J. Am. Chem. Soc. 2002,124, 4180-
4181. (e) Matsuoka, S.; Murata, M. Biochim. Biophys. Acta 2002,1654,
429-434.
Table 4.
1H and 13C NMR Data for Marinomycin D (4)
position
δ
H mult (
J
, Hz)
a
δ
C
b
position
δ
H mult (
J
, Hz)
δ
C
1, 1′171.7 18, 18′1.34 br d (14.6)/1.11 m 44.2
2, 2′110.8 19, 19′3.90 br d (9.0) 71.8
3, 3′163.5 20, 20′5.28 br se135.1
4, 4′6.91 d (8.1) 117.2 21, 21′5.28 br se128.6f
5, 5′7.35 t (8.1) 134.5 22, 22′1.91 m 41.1
6, 6′6.70 d (8.1) 123.1 23, 23′3.40 br t (9.0) 66.8
7, 7′140.8 24, 24′1.72 m 43.0/43.9
8, 8′6.66 d (11.0) 132.8 25, 25′5.43 m 73.4/73.7
9, 9′6.13 mc129.9 26, 26′1.62 m/1.72 m 46.1
10, 10′6.26 md128.2f27, 27′3.98 br m/3.65 br m 64.9/70.0
11, 11′6.26 md135.1 28 1.17 d (6.6) 23.5
12, 12′6.13 mc128.3f28′1.46 m 30.4
13, 13′6.36 dd
(14.6, 11.0) 131.8 29, 30′1.68 s 17.3
14, 14′5.86 br d (11.0) 128.4f29′0.93 t (7.3) 10.5
15, 15′135.7
16, 16′1.82 m 49.1 OH 4.20 (4H, br m)/4.40 (2H, br s)
17, 17′3.57 br m 70.4 3- and 3′-OH 11.68 s/11.70 s
a300 MHz, CDCl3.b75 MHz, CDCl3.c-eOverlapping signals. fInterchangeable signals.
Figure 3.
CD spectra of marinomycins A-D(1-4) in methanol.
ARTICLES
Kwon et al.
1628 J. AM. CHEM. SOC. 9VOL. 128, NO. 5, 2006

Compound 8showed a roughly 10-fold decrease in MRSA
activity as compared to marinomycin A.
More impressive potency and selectivity was observed when
the marinomycins were examined in the NCI’s panel of 60
cancer cell lines. Marinomycin A (1) showed significant tissue
type selectivity, with the most sensitive cancer types being six
of the eight melanoma cell lines (LOX IMVI, M14, SK-MEL-
2, SK-MEL-5, UACC-257, and UACC-62). The most sensitive
strain was SK-MEL-5 melanoma (LC50 )5.0 nM), which was
approximately 500 times more sensitive than the average LC50
value of 2.7 µM. Marinomycins B (2)andC(3) also showed
potent activities with average LC50 values of 0.9 and 0.2 µM,
respectively. Significantly, the compounds were only weakly
active against all six leukemia cell lines in the panel with LC50
values of approximately 50 µM.
As mentioned earlier, throughout this study we were con-
fronted with the tendency of all of the marinomycins to
photoisomerize in room light. A photochemical ∆8,9 olefin
isomerization of 1was observed when it was exposed to ambient
light, in glass (methanol) at room temperature. The time course
of this interconversion, monitored by LC-MS, resulted in an
equilibrium mixture of marinomycins A (1),B(2), and C (3)
within1hofexposure. Knowing this in advance greatly
facilitated the isolation and purification of the isomeric marino-
mycins under low light conditions. Cultivation in the dark
resulted in the production of mainly marinomycin A (1) and
much smaller amounts of 2and 3. While these observations
suggest that marinomycin A (1) could be the true natural
product, we could not rigorously prove this assumption. In
addition, given that marinomycin D has one ∆8,9 )Zolefin,
and the propensity of the olefin at this position in 1to
photoisomerize, it is conceivable that an all-Eisomer of 4
(structure 5) could also be the true natural product. By careful
LC-MS examination, we did observe a trace component of the
fermentation mixture with the correct molecular weight for 5.
However, this compound was in very small amounts, was highly
photochemically reactive, and could not be fully purified.
The marinomycins are the first examples of this new
macrodiolide class; however, the dimeric 2-hydroxy-6-alkyl-
benzoic acid lactone functionality has been observed in the
microbial metabolite (+)-SCH 351448, an activator of low-
density lipoprotein receptor (LDL-R) promoter.8b The marino-
mycins possess unique polyene-polyol structures, and have
unique photoreactivities and chiroptical properties. Their potent
and selective anticancer properties in the NCI panel suggest
these metabolites may inhibit tumor proliferation by a specific,
but as yet unknown, mechanism of action. The fact that these
polyenes lack substantive antifungal activity suggests they are
not membrane active like the well-known polyene antibiotics.17
Last, the marinomycins were isolated from the fermentation
broth of a marine actinomycete belonging to a new genus, for
which we suggest the name “Marinispora”. Based upon
phylogenetic analyses using 16s rRNA gene sequence data, this
genus clusters within the family Streptomycetaceae, but clearly
lies outside of all known terrestrial genera including the genus
Streptomyces.6More than 20 “Marinispora” isolates have been
recovered from a diversity of marine habitats, suggesting that
this new genus is widespread in the marine environment.
Chemical analysis of other “Marinispora” isolates has shown
the production of a significant number of diverse secondary
metabolites that are under current investigation.
Experimental Section
Isolation of CNQ-140 Strain, Cultivation, Extraction. Marinispora
strain CNQ-140 was isolated on medium A1+C (10 g of starch,4gof
peptone,2gofyeast extract,1gofcalcium carbonate, 18 g of agar,
1 L of seawater) from a marine sediment collected at a depth of 56 m
1 mile Northwest of the Scripps Institution of Oceanography Pier (La
Jolla, CA.) The strain was cultured in 20 ×1 L volumes of medium
Figure 4.
Planar views of the 44-membered rings in marinomycins A (1)andB(2) based upon transannular nuclear Overhauser enhancements observed
in ROESY NMR experiments.
Table 5.
Antimicrobial Bioassay Results for Marinomycins A-D
(1-4) and Synthetic Derivatives
a
compound
Staphylococcus aureus
methicillin-resistant
(MRSA)
MIC
90
µ
M
b
Enterococcus faecium
vancomycin-resistant
MIC
90
µ
M
b
Candida albicans
wild type
MIC
90
µ
M
c
10.13 0.13 7.8
20.25 NSA NSA
30.25 NSA NSA
40.25 NSA NSA
6NSA NSA N/T
7NSA NSA N/T
82.3 NSA N/T
8a 1.8 NSA N/T
8b NSA NSA N/T
aNSA )not significantly active (MIC90 values above 10.0 µM), N/T )
not tested. 8a )the hexa-acetate derivative of 8;8b )3,3′,23,23′,27,27′-
hexa-acetate of 1.bThe optical density (OD) was measured at 600 nm using
a Molecular Devices Emax microplate reader, and the MIC90 was determined
by the analysis program SOFTmax PRO. The MIC90 of vancomycin is
0.195-0.391 µg/mL, and that of penicillin G is 6.25-12.5 µg/mL. cAlamar
Blue was used as an indicator to measure cell proliferation. The dye yields
a colorimetric change that enables the MIC to be confidently estimated by
visual means. The MIC of amphotericin B is 1.56-0.78 µg/mL.
Marinomycins A
−
D, Antitumor-Antibiotics
ARTICLES
J. AM. CHEM. SOC. 9VOL. 128, NO. 5, 2006 1629

TCG (3 g of tryptone,5gofcasitone,4gofglucose,1Lofseawater)
while shaking at 230 rpm for 7 days. At the end of the fermentation
period, 20 g/L XAD-7 adsorbent resin were added to each flask, and
they were allowed to shake at a reduced speed for 2 additional hours.
The resin was then collected by filtration through cheesecloth, washed
with deionized water, and eluted twice with acetone. Evaporation of
the extraction solvent in vacuo left a wet residue that was taken up in
EtOAc, providing approximately 125 mg of dry extract per1Lof
culture after removal of solvent.
Isolation and Purification of Marinomycins A-D(1-4). The
combined EtOAc extract from two 20 ×1 L fermentations (5.0 g)
was subjected to silica gel column chromatography purification eluting
with solvent mixtures of n-hexanes-EtOAc (1:1), EtOAc, EtOAc-
MeOH (10:1), EtOAc-MeOH (5:1), EtOAc-MeOH (2:1), and 100%
MeOH, successively. The EtOAc-MeOH (10:1) eluting fraction
(Q140-3) showed the most potent HCT-116 cytotoxicity and by LC-
MS contained the marinomycins. These compounds were conspicuous
on silica gel and C-18 TLC because of their characteristic UV
absorbance (yellow fluorescence at 365 nm) and brown color develop-
ment when the plates were heated after spraying with a vanillin-H2SO4
reagent. Fraction Q140-3 was refractionated by C-18 reversed-phase
HPLC with MeCN-H2O (65:35) to obtain impure marinomycins A-D
as major components [retention times: Q140-3-A, 26.33-35.35 min;
Q140-3-B, 36.40-43.00 min; Q140-3-C, 43.50-51.65 min; Q140-
3-D, 51.65-58.00 min]. Final purification of marinomycins A-D(1-
4) was achieved by repeated C-18 RP HPLC with MeCN-H2O (65:
35). Typical recoveries of the marinomycins A-Dfroma1Lculture
were 2, 1.8, 2.4, and 0.5 mg, respectively.
Marinomycin A (1). Yellow powder, [R]D+180°(c0.11, EtOH).
UV (EtOH): 325 sh ()29 000), 345 sh ()41 600), 359 ()
52 300), 378 nm ()44 000). IR (neat): 3375, 2938, 2863, 1713,
1656, 1581, 1463, 1414, 1384, 1288, 1253, 1213, 1175, 1125, 1074,
1000, 969 cm-1. HR MALDI-FT MS: obsd m/z1019.5190 [M +Na]+,
C58H76O14Na requires 1019.5127. See Table 1 for NMR data.
Marinomycin B (2). Yellow powder, [R]D-245°(c0.15, EtOH).
UV (EtOH): 315 ()55 300), 340 ()50 300) nm. IR (neat): 3375,
2938, 1719, 1660, 1600, 1453, 1375, 1287, 1256, 1225, 1175, 1125,
1073, 1000, 978 cm-1. HR ESI-TOF MS: obsd m/z1019.5110
[M +Na]+,C
58H76O14Na requires m/z1019.5127. See Table 2 for NMR
data.
Marinomycin C (3). Yellow powder, [R]D-161°(c0.13, EtOH).
UV (EtOH): 319 sh ()40 800), 345 sh ()48 300), 358 ()
53 800), 375 ()41 400) nm. IR (neat): 3375, 2925, 1719, 1656,
1588, 1450, 1375, 1338, 1288, 1256, 1225, 1125, 1069, 1000, 975 cm-1.
HR ESI-TOF MS: obsd m/z1019.5110 [M +Na]+,C
58H76O14Na
requires 1019.5127. See Table 3 for NMR data.
Marinomycin D (4). Yellow powder, [R]D-233°(c0.03, EtOH).
UV (EtOH): 315 ()58 200), 340 ()50 600) nm. IR (neat): 3375,
2925, 1781, 1737, 1650, 1600, 1463, 1429, 1376, 1199, 1119, 1063,
1025, 978 cm-1. HR ESI-TOF MS: obsd m/z1033.5267 [M +Na]+,
C59H78O14Na requires 1033.5289. See Table 4 for NMR data.
Marinomycin A Deca-acetate (1a). Marinomycin A (1, 1 mg) was
treated with acetic anhydride and pyridine (500 µL each) for 12 h at
room temperature. Removal of the reactants under reduced pressure
provided the deca-acetate derivative 1a (1 mg). 1H NMR (500 MHz,
CDCl3): δ1.21-1.31 (br m), 1.78 (6H, br s), 1.80-1.90 (10H, m),
1.92-2.10 (30H, br m), 2.24-2.33 (10H, br m), 4.97 (6H, m), 5.19
(4H, m), 5.42 (2H, dd, J)15.0, 7.0 Hz), 5.59 (2H, dt, J)15.0, 7.5
Hz), 5.90 (2H, d, J)11.0 Hz), 6.26 (2H, dd, J)14.5, 11.0 Hz), 6.41
(2H, dd, J)15.0, 10.5 Hz), 6.44 (2H, dd, J)15.0, 10.5 Hz), 6.52
(2H, dd, J)14.5, 10.0 Hz), 6.71 (2H, d, J)15.0 Hz), 6.77 (2H, dd,
J)15.0, 10.0 Hz), 6.97 (2H, d, J)8.0 Hz), 7.32 (2H, t, J)8.0 Hz),
7.43 (2H, d, J)8.0 Hz). ESI-MS: m/z1439 [M +Na]+.
Marinomycin B Deca-acetate (2a). Marinomycin B (2, 1 mg) was
treated with acetic anhydride and pyridine at room temperature for 12
h. Removal of all reactants under reduced pressure provided marino-
mycin B deca-acetate (2a, 1 mg). 1H NMR (300 MHz, CDCl3): δ1.24
(6H, d, J)6.0 Hz), 1.62 (2H, m), 1.68 (6H, s), 1.75-1.90 (12H, m),
1.93 (6H, s), 1.97 (6H, s), 2.01 (12H, s), 2.14 (4H, m), 2.32 (3H, s),
2.35 (2H, m), 4.96 (6H, m), 5.17 (4H, m), 5.36 (2H, dd, J)15.5, 6.8
Hz), 5.54 (2H, dt, J)15.5, 6.8 Hz), 5.78 (2H, d, J)10.5 Hz), 6.14
(2H, dd, J)14.6, 9.5 Hz), 6.50 (8H, m), 6.79 (2H, dd, J)15.0, 10.0
Hz), 7.03 (2H, d, J)8.0 Hz), 7.37 (2H, t, J)8.0 Hz), 7.39 (2H, d,
J)8.0 Hz). LCMS: m/z1439 [M +Na]+.
Marinomycin C Deca-acetate (3a). Marinomycin C (3, 1 mg) was
acetylated by the same method as above to afford marinomycin C deca-
acetate (3a, 1 mg). 1H NMR (300 MHz, CDCl3): δ1.23 (3H, d, J)
6.0 Hz), 1.24 (3H, d, J)6.0 Hz), 1.69 (1H, m), 1.74 (3H, s), 1.76
(3H, s), 1.77-1.89 (12H, m), 1.92 (3H, s), 1.95 (9H, s), 1.99 (3H, s),
2.01 (9H, s), 2.15-2.25 (5H, m), 2.28 (3H, s), 2.30 (3H, s), 2.35 (2H,
m), 4.96 (6H, m), 5.17 (4H, m), 5.39 (2H, dd, J)15.5, 6.8 Hz), 5.55
(1H, dt, J)15.5, 6.8 Hz), 5.60 (1H, dt, J)15.5, 6.8 Hz), 5.84 (2H,
d, J)10.5 Hz), 6.14 (1H, dd, J)14.6, 10.0 Hz), 6.23 (1H, dd, J)
14.6, 10.5 Hz), 6.35-6.55 (8H, m), 6.69 (1H, d, J)15.5 Hz), 6.77
(1H, dd, J)15.5, 10.0 Hz), 6.96 (1H, d, J)8.0 Hz), 7.02 (1H, d, J
)8.0 Hz), 7.24 (1H, ovlp with solvent), 7.33 (1H, t, J)8.0 Hz), 7.37
(1H, t, J)8.0 Hz), 7.43 (1H, d, J)8.0 Hz). LCMS: m/z1439 [M
+Na]+.
Marinomycin D Deca-acetate (4a). Marinomycin D (4, 1 mg) was
acetylated using the same method used for marinomycin A to give the
deca-acetate 4a (1 mg) in pure form. 1H NMR (500 MHz, CDCl3): δ
0.92 (3H, t, J)7.5 Hz), 1.21-1.31 (br m, overlap with impurity),
1.68 (6H, s), 1.75-1.90 (12H, m), 1.93 (6H, s), 1.97 (6H, s), 2.01
(12H, s), 2.14 (4H, m), 2.28 (2H, br m), 2.31 and 2.32 (6H, each s),
4.86-5.0 (6H, m), 5.11-5.20 (4H, m), 5.36 (2H, dd, J)15.0, 7.0
Hz), 5.53 (2H, dt, J)15.0, 7.0 Hz), 5.77 (2H, d, J)11.0 Hz), 6.13
(2H, dd, J)14.6, 9.8 Hz), 6.37-6.60 (10H, m), 7.02 (2H, d, J)8.0
Hz), 7.24 (d, overlap with solvent peak), 7.37 (2H, t, J)8.0 Hz).
ESI-MS: m/z1453 [M +Na]+.
Methanolysis of 1 To Yield Ester 6. Marinomycin A (1,10mg)
was dissolved in 5% NaOMe in MeOH and stirred for 10 h at 40 °C.
The reaction mixture was neutralized with 1 N aqueous HCl, the
aqueous phase was extracted with EtOAc, and the residue after solvent
removal was purified by C18 HPLC using MeCN-H2O (4:6) to yield
compound 6(9 mg): [R]D-18.2°(c0.16, EtOH). 1H NMR (400 MHz,
DMSO-d6): δ1.03 (3H, d, J)5.9 Hz, H-28), 1.24 (2H, m, H-24),
1.30 (2H, m, H-26), 1.37 (1H, br ddd, J)14.0, 6.6, 3.7 Hz, H-18),
1.47 (1H, ddd, J)14.0, 8.1, 6.6 Hz, H-18), 1.75 (3H, s, H-29), 2.04
(2H, m, H-22), 2.13 (2H, br d, J)5.9 Hz, H-16), 3.66 (1H, m, H-23),
3.68 (1H, m, H-17), 3.79 (3H, s, OMe), 3.78 (1H, m, H-27), 3.84 (1H,
m, H-25), 4.07 (1H, m, H-19), 4.34 (3H, br s, OH), 4.50 (1H, br s,
OH), 4.69 (1H, br s, OH), 5.36 (1H, dd, J)15.0, 6.6 Hz, H-20), 5.54
(1H, dt, J)15.0, 7.3 Hz, H-21), 5.91 (1H, d, J)11.0 Hz, H-14),
6.22 (1H, dd, J)14.6, 11.0 Hz, H-12), 6.35 (1H, d, J)15.4 Hz,
H-8), 6.39 (1H, dd, J)14.6, 10.3 Hz, H-10), 6.50 (1H, dd, J)14.6,
11.0 Hz, H-11), 6.55 (1H, dd, J)14.6, 11.0 Hz, H-13), 6.77 (1H, d,
J)8.1 Hz, H-4), 6.88 (1H, dd, J)15.4, 10.3 Hz, H-9), 7.07 (1H, d,
J)8.1 Hz, H-6), 7.16 (1H, t, J)8.1 Hz, H-5), 8.50 (s, 3-OH). 13C
NMR (100 MHz, DMSO-d6)δ17.3 (C-29), 24.4 (C-28), 41.0 (C-22),
44.7 (C-26, C-18), 47.4 (C-24), 48.4 (C-16), 51.9 (OMe), 62.9 (C-27),
63.8 (C-25), 66.7 (C-17, C-23), 69.4 (C-19), 114.4 (C-6), 114.6 (C-4),
120.6 (C-2), 126.5 (C-21), 126.8 (C-14), 127.7 (C-8), 129.9 (C-5), 130.3
(C-13), 130.4 (C-12), 131.2 (C-9), 131.6 (C-10), 134.9 (C-7), 135.0
(C-11), 135.3 (C-20), 138.2 (C-15), 155.2 (C-3), 168.1 (C-1). ESI-LC-
MS: C-18 column, rt 12.2 min (10% MeCN/H2O-100% MeCN/H2O,
20 min). UV (DAD): 358, 374 (sh) nm. LRMS: [M +Na]+m/z553
amu.
Photoisomerization of Marinomycin A (1) To yield Marino-
mycins B and C (2, 3). A solution of marinomycin A (1, 7.0 mg) in
methanol (5 mL) was exposed to intense room light for 2 h. The solution
was analyzed by reversed-phase HPLC, which showed the presence of
three major compounds. The products were isolated by semipreparative
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Kwon et al.
1630 J. AM. CHEM. SOC. 9VOL. 128, NO. 5, 2006

C-18 RP HPLC using 65% MeCN/H2O to obtain 1(3.2 mg), 2(0.4
mg), and 3(1.8 mg). The 1H NMR spectrum of each isolated compound
(1-3), as well as their UV spectra, were identical to the compounds
isolated from the original fermentation extract. The optical rotation
values were 1,[R]D+168°(c0.16, EtOH); 2,[R]D-310°(c0.02,
EtOH); 3,[R]D-151°(c0.09, EtOH), which were similar to those of
the compounds isolated from the fermentation mixture.
Methanolysis of 2 To Yield Ester 7. Marinomycin B (2, 2 mg) in
5% NaOMe in methanol was stirred for5hat40°C, and the reaction
mixture was processed as above for 1. The crude product was analyzed
by LC-MS, which showed compound 7as the major product, rt 12.8
min (10% MeCN/H2O-100% MeCN/H2O, 20 min). [R]D-12°(c0.05,
EtOH). UV (DAD): 315, 340 nm. LR-ESI-MS: [M +Na]+m/z553.
1H NMR (500 MHz, DMSO-d6): δ1.03 (3H, d, J)6.0 Hz, H-28),
1.22-1.37 (5H, m), 1.46 (1H, ddd, J)14.0, 7.0, 6.5, H-18), 1.75
(3H, s, H-29), 2.05 (2H, m, H-22), 2.12 (2H, br d, J)6.0 Hz, H-16),
3.67 (2H, m, H-23, H-17), 3.74 (3H, s, OCH3), 3.77 (1H, m, H-27),
3.84 (1H, m, H-25), 4.06 (1H, m, H-19), 4.20 (1H, br d, J)4.5 Hz,
OH), 4.29 (2H, br s, OH), 4.45 (1H, br d, J)5.0 Hz, OH), 4.65 (1H,
br d, J)3.5 Hz, OH), 5.35 (1H, dd, J)15.5, 6.5 Hz, H-20), 5.53
(1H, dt, J)15.5, 7.0 Hz, H-21), 5.87 (1H, d, J)11.5 Hz, H-14),
6.20 (1H, m), 6.24 (1H, d, J)11.5 Hz), 6.31 (1H, m), 6.51 (2H, m,
H-10, H-11), 6.55 (1H, dd, J)14.5, 11.5 Hz, H-13), 6.80 (1H, d, J)
8.0 Hz, H-4), 6.82 (1H, d, J)8.0 Hz, H-6), 7.26 (1H, t, J)8.0 Hz,
H-5), 10.05 (1H, br s, OH).
Methanolysis of 3 To Yield Esters 6 and 7. Marinomycin C (3,2
mg) was also treated with 5% NaOMe in methanol for5hat40°C,
and the product mixture was analyzed by LC-MS as above. The LC-
MS chromatogram illustrated the presence of compounds 6, rt 12.2
min (m/z553 amu [M +Na]+), and 7, rt 12.8 min (m/z553 amu [M
+Na]+), as the major products.
Bisacetonide 8. Marinomycin A (1, 1.0 mg) was dissolved in 2,2-
dimethoxypropane (1 mL) and methanol (0.2 mL), and pyridinium-p-
toluenesulfonate (2 mg) was added. The reaction was allowed to stir
for6hat0°C. The reaction was then quenched with 5% aqueous
NaHCO3, and the aqueous phase was extracted thrice with CH2Cl2.
The CH2Cl2solution was dried (anhyd. MgSO4), the solvent was
removed under reduced pressure, and the residue was purified by C-18
RP HPLC (82:18: MeCN-H2O) to provided compound 8(0.6 mg).
1H NMR (500 MHz, pyridine-d5): δ1.27-1.33 (8H, m), 1.36 (6H, d,
J)6.0 Hz), 1.46-1.55 (8H, m), 1.56 (6H, s), 1.60 (6H, s), 1.85 (6H,
s), 2.00-2.20 (14H, m), 2.26 (2H, br d), 2.40-2.53 (8H, m), 4.22 (2H,
br m), 4.32 (2H, br m), 4.57 (4H, br m), 5.82 (2H, dd, J)15.5, 6.2
Hz), 6.20 (2H, dt, J)15.5, 7.3 Hz), 6.23 (2H, d, J)11.0 Hz), 6.36
(2H, dd, J)14.6, 10.2 Hz), 6.45 (2H, m), 6.49 (2H, dd, J)14.6,
10.2 Hz), 6.62 (2H, dd, J)15.0, 11.0 Hz), 6.72 (2H, dd, J)14.6,
11.0 Hz), 7.04 (2H, d, 8.0 Hz), 7.10 (2H, dd, 15.5, 10.2 Hz), 7.25
(2H, d, J)15.5 Hz), 7.28 (2H, t, J)8.0 Hz), 12.13 (2H, br s). ESI
LCMS (C-18, 80% MeCN/H2O): retention time 6.6 min, m/z1099
[M +Na]+.
Bisacetonides 9 and 10. Methyl ester 6(5.0 mg) was dissolved in
2,2-dimethoxypropane (3 mL), and methanol (1 mL) and pyridinium-
p-toluenesulfonate (5 mg) were added. The reaction was allowed to
proceed for 12 h at room temperature and then quenched with 5%
aqueous NaHCO3, extracted 3 times with CH2Cl2, the solvent extracts
were combined and dried (anhydrous MgSO4), and the solvent was
removed under reduced pressure. The residue obtained was fractionated
by silica gel HPLC (2:1, n-hexanes-ethyl acetate) to provide com-
pounds 9(1.0 mg) and 10 (1.3 mg). For acetonide 9,1H NMR (500
MHz, pyridine-d5): δ1.15 (3H, d, J)6.0 Hz, H-28), 1.34-1.40 (2H,
m, H-18), 1.41 (3H, s, acetonide CH3), 1.45 (3H, s, acetonide CH3),
1.49 (3H, s, acetonide CH3), 1.51 (3H, s, acetonide CH3), 1.51 (1H, m,
H-26), 1.65 (1H, m, H-26), 1.73 (1H, m, H-24), 1.82 (1H, m, H-24),
1.86 (3H, s, H-29), 2.23 (1H, dd, J)13.0, 5.5 Hz, H-16), 2.45 (3H,
m, H-16, H-22), 3.94 (3H, s, OCH3), 3.98 (1H, br m, H-27), 4.14 (1H,
br m, H-17), 4.23 (1H, br m, H-23), 4.45 (2H, br m, H-19, H-25), 5.74
(1H, dd, J)15.5, 6.0 Hz, H-20), 6.11 (2H, m, H-21, H-14), 6.38 (1H,
dd, J)14.5, 11.0 Hz, H-12), 6.50 (1H, dd, J)14.5, 10.0 Hz, H-10),
6.61 (1H, dd, J)14.5, 11.0 Hz, H-11), 6.70 (1H, dd, J)14.5, 11.0
Hz, H-13), 7.03 (1H, d, J)8.0 Hz, H-8), 7.03 (1H, d, J)8.0 Hz,
H-4), 7.06 (1H, dd, J)15.0, 10.0 Hz, H-9), 7.28 (2H, m, H-5, H-6).
LCMS (C-18 RP column, 75% MeCN/H2O), retention time 6.9 min
(UV DAD λmax 356, 374 (sh) nm), ESI MS m/z633 [M +Na]+. For
acetonide 10,1H NMR (500 MHz, pyridine-d5): δ1.34 (3H, d, J)
6.0 Hz, H-28), 1.40 (1H, m, H-18), 1.41 (3H, s, acetonide CH3), 1.46
(3H, s, acetonide CH3), 1.49 (3H, s, acetonide CH3), 1.52 (3H, s,
acetonide CH3), 1.54 (1H, m, H-18), 1.66 (2H, m, H-24, H-26), 1.73
(2H, m, H-24, H-26), 1.86 (3H, s, H-29), 2.26 (2H, m, H-16, H-22),
2.43 (1H, m, H-22), 2.44 (1H, m, H-16), 3.94 (3H, s, OCH3), 3.98
(1H, br m, H-23), 4.15 (1H, br m, H-17), 4.26 (1H, br m, H-27), 4.41
(1H, br m, H-25), 4.48 (1H, br m, H-19), 5.72 (1H, dd, J)15.5, 6.0
Hz, H-20), 5.92 (1H, dt, J)15.5, 7.0 Hz, H-21), 6.14 (1H, d, J)
11.0 Hz, H-14), 6.38 (1H, dd, J)14.5, 11.0 Hz, H-12), 6.49 (1H, dd,
J)14.5, 10.0 Hz, H-10), 6.60 (1H, dd, J)14.5, 11.0 Hz, H-11),
6.70 (1H, dd, J)14.5, 11.0 Hz, H-13), 7.04 (1H, d, J)15.0 Hz,
H-8), 7.04 (1H, d, J)8.0 Hz, H-4), 7.08 (1H, dd, J)15.0, 10.0 Hz,
H-9), 7.28 (2H, m, H-5, H-6). LCMS (C-18 RP column, 75% MeCN/
H2O), retention time 6.6 min (UV DAD λmax 356, 373 (sh) nm), ESI
MS m/z633 [M +Na]+.
Mosher MTPA Esters 11a/11b. Acetonide 9(1.0 mg) was divided
into two portions, and each was dissolved in 500 µL of pyridine-d5in
a 5 mm NMR tube. To each NMR tube were added 10 µLof(R)-
MTPACl and 10 µLof(S)-MTPACl, respectively. After 12 h, the
reaction was complete and 1D 1H NMR spectra for (S)-mosher ester
11a and (R)-mosher ester 11b were recorded (see Supporting Informa-
tion for NMR data).
Mosher MTPA Esters 12a/12b. Acetonide 10 (1.3 mg) was divided
into two portions, and each one was treated with pyridine-d5and (R)-
and (S)-MTPACl in separate 5 mm NMR tubes as above. The 1H NMR
spectra for the (S)-Mosher ester 12a and (R)-Mosher ester 12b were
recorded (see Supporting Information for NMR data).
Hydrogenation of Marinomycin A (1) To Yield 13a-c. To a
solution of marinomycin A (1, 4 mg) in ethanol (4 mL) was added
10% Pd/C (25 mg), and the mixture was stirred at room temperature,
under an atmosphere of H2for 3 h. The reaction mixture was filtered
and concentrated under reduced pressure. The residue obtained was
analyzed by C-18 LC-MS, which showed the three isomeric perhydro-
marinomycin A derivatives, that were isolated by C-18 RP HPLC (9:1
MeCN-H2O) to give compounds 13a,13b, and 13c, as viscous oils,
in a 1/2/1 ratio by LC-MS analysis. LC-MS data (C-18 RP column,
linear gradient elution: 10-100% MeCN/H2O in 25 min) retention
times: 13a 23.5 min, 13b 24.2 min, and 13c 24.9 min. UV spectra
(DAD): all peaks show λmax 210, 245 nm. LRMS (ESI): 13a,13b,
13c,m/z1017 [M +H]+,m/z1039 [M +Na]+.
Conversion of Perhydro-marinomycin A to Hexa-acetate 16. To
a solution of compound 13b (1.6 mg) in methanol (0.5 mL) were added
2,2-dimethoxypropane (3 mL) and pyridinium-p-toluenesulfonate (5
mg), and the solution was stirred for6hinanicebath. A 5% aqueous
NaHCO3solution was then added, and the mixture was extracted with
CH2Cl2as in the preparation of acetonide 8. The CH2Cl2layer was
dried (anhydrous MgSO4) and concentrated under reduced pressure to
obtain the bisacetonide 14 (1.5 mg). Acetylation of 14 with Ac2O/py
provided compound 15 (1.6 mg, not characterized), which was dissolved
in methanol, and pyridinium-p-toluenesulfonate was added and then
stirred for2hatroom temperature. The reaction mixture was then
quenched with 5% aqueous NaHCO3, the solution was partitioned
between CH2Cl2and H2O, and the organic layer was separated, dried
(anhydrous MgSO4), and concentrated under reduced pressure. The
residue was purified by C-18 RP HPLC (MeCN-H2O, 9:1), to provide
the hexa-acetate 16 (1 mg). 1H NMR (300 MHz, CDCl3): δ0.84 (3H,
dJ)5.9 Hz), 0.86 (3H, d, J)5.9 Hz), 1.24 (48H, br m), 1.43 (12H,
m), 1.55 (br s, overlap with H2O peak), 1.89 (8H, br s), 1.98 (12H, s),
Marinomycins A
−
D, Antitumor-Antibiotics
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J. AM. CHEM. SOC. 9VOL. 128, NO. 5, 2006 1631

2.28 (6H, s), 2.35 (2H, t, J)7.8 Hz), 2.64 (4H, br s), 3.81 (2H, br m),
3.91 (2H, br m), 4.90 (2H, br m), 4.96 (2H, br m), 5.11 (2H, br m),
6.95 (2H, d, J)7.8 Hz), 7.08 (2H, d, J)7.8 Hz), 7.32 (2H, t, J)
7.8 Hz). LCMS (C-18 RP column, 75% MeCN/H2O(0-5 min)-100%
MeCN (-10 min) rt for 16: 10.2 min. UV DAD spectrum: 210, 235
(sh) nm. LRMS (ESI): m/z1269 [M +H]+,m/z1291 [M +Na]+.
Tetra-MTPA Esters of 16 (17a/17b). To a solution of 16 (0.5 mg)
in CH2Cl2(2 mL) were added triethylamine (10 µL), (dimethylamino)-
pyridine (1 mg), and (R)-MTPACl (10 µL) in sequence. The reaction
mixture was stirred for 24 h at room temperature, and then N,N-
dimethyl-1,3-propanediamine (12 µL) was added and stirring was
continued for an additional 10 min. The solution was concentrated under
reduced pressure, and the residue was fractionated by Sephadex LH-
20 column chromatography using CH2Cl2-MeOH (1:1). The fractions
containing the MTPA ester were combined and purified by silica gel
column chromatography (n-hexanes-EtOAc; 2:1) to afford 0.5 mg of
the (S)-MTPA ester 17a. In a similar fashion, 16 was treated with (S)-
MTPACl (10 µL) and pyridine (100 µL) to afford, after fractionation
as above, 0.7 mg of the (R)-MTPA ester 17b (see Supporting
Information for NMR data).
Acknowledgment. This research is a result of generous
financial support from the National Institutes of Health, National
Cancer Institute, under grant R37 CA44848, and by the
University of California Industry-University Cooperative Re-
search Program (IUCRP, grant BioSTAR 10354). P.R.J. and
W.F. are scientific advisors to and stockholders in Nereus
Pharmaceuticals, the corporate sponsor of the IUCRP award.
The terms of this arrangement have been reviewed and approved
by the University of California, San Diego, in accordance with
its conflict of interest policies.
Supporting Information Available: Spectral data sets (1D
and 2D NMR, ESI-MS, UV-bis, HRMS, etc.) for 1-4,1H and
13C NMR spectra, ESI-MS data for 6-17, and molecular models
showing the predicted ring configurations of marinomycins A
andB(1,2). This material is available free of charge via the
Internet at http://pubs.acs.org.
JA0558948
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Kwon et al.
1632 J. AM. CHEM. SOC. 9VOL. 128, NO. 5, 2006