Isolation of lomaiviticins C-E, transformation of lomaiviticin C to lomaiviticin A, complete structure elucidation of lomaiviticin A, and structure-activity analyses

Article abstract of DOI:10.1021/ja3074984

We describe the isolation of (-)-lomaiviticins C-E (6-8), elucidation of the complete absolute and relative stereochemistry of (-)-lomaiviticin A (1), the synthetic conversion of (-)-lomaiviticin C (6) to (-)-lomaiviticin A (1), and the first evidence that the dimeric diazofluorene of (-)-lomaiviticin A (1) plays a defining and critical role in antiproliferative activity.

Full text of DOI:10.1021/ja3074984

Communication
pubs.acs.org/JACS
Isolation of Lomaiviticins C−E, Transformation of Lomaiviticin C to
Lomaiviticin A, Complete Structure Elucidation of Lomaiviticin A, and
Structure−Activity Analyses
Christina M. Woo,† Nina E. Beizer,† Jeffrey E. Janso,‡ and Seth B. Herzon*,†
†Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
‡Natural Products − Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, 445 Eastern Point Road,
Groton, Connecticut 06340, United States
S
* Supporting Information
kinamycins; the absolute stereochemistry of the carbohydrates
was not defined.3
ABSTRACT: We describe the isolation of (−)-lomaiviti-
cins C−E (6−8), elucidation of the complete absolute and
relative stereochemistry of (−)-lomaiviticin A (1), the
synthetic conversion of (−)-lomaiviticin C (6) to
(−)-lomaiviticin A (1), and the first evidence that the
dimeric diazofluorene of (−)-lomaiviticin A (1) plays a
defining and critical role in antiproliferative activity.
Lomaiviticins A (1) and B (2) are powerful antibiotics, with
minimum inhibitory concentrations (MICs) of 6−25 ng/spot,
and 1 is an exceptionally potent anticancer agent, with half-
maximal inhibitory concentration (IC50) values in the 72 nM−
7.3 pM range against 24 cancer cell lines.1a,4 Evidence suggests
that 1 may operate by a novel mechanism of action, but studies
of 1 and 2 have been impossible to initiate because of the
absence of a synthetic route to either target and their low levels
of natural production (reported fermentation yields: 1 mg/L
for 1 and 0.17 mg/L for 2).1a
omaiviticins A (1) and B (2) are complex C2-symmetric
L
bacterial metabolites that contain a dimeric
diazotetrahydrobenzo[b]fluorene (diazofluorene) skeleton
bearing 2−4 dideoxyglycoside residues, oleandrose and N,N-
dimethylpyrrolosamine (Figure 1).1 They are structurally
As part of our synthetic investigations,3,5 we sought to
reisolate lomaiviticin A (1). Accordingly, “Salinispora pacifica”
strain DPJ-0019 was acquired from the USDA Agricultural
Research Service as NRRL 50168. Careful examination of the
concentrated fermentation extracts revealed the presence of 1
and three novel metabolites, which we have named
lomaiviticins C−E (6−8). Lomaiviticin B (2) was not detected
[ultra high-performance liquid chromatography (UPLC)/MS
analysis]. We describe herein the structure elucidation of
(−)-lomaiviticins C−E (6−8), the direct conversion of
(−)-lomaiviticin C (6) to (−)-lomaiviticin A (1), and the
first evidence implicating the dimeric diazofluorene structure of
(−)-lomaiviticin A (1) as underlying its potent antiproliferative
effects. We have also elucidated the complete relative and
absolute stereochemistry of 1, which has remained an
unresolved issue for over a decade. These latter data bear
particular significance to synthetic studies,3,5,6 since both D- and
L-oleandrose are found in nature7 and the absolute stereo-
chemistry of pyrrolosamine8 has never been determined.
“S. pacifica” DPJ-0019 was fermented in SPYESS medium in
the presence of Dianion HP-20 resin. The resin was collected
and extracted with methanol, and the concentrated extract was
purified by reversed-phase chromatography. Lomaiviticins C−E
(6−8) were isolated by mass-spectrometry-guided fractiona-
tion9 in yields of 37−62, 10−19, and 1.6−3.6 mg/L,
respectively, over several fermentations.
Figure 1. Structures of lomaiviticins A (1) and B (2) and kinamycins
A−C (3−5).
related to the monomeric diazofluorenes known as the
kinamycins (3−5), which were first isolated by Omura and
̅
co-workers.2 The constitution and relative stereochemistry of
the carbohydrate and aglycon substructures of 1 and 2 were
elucidated by extensive 1D and 2D NMR, IR, and high-
resolution mass spectrometry (HRMS) analyses. The absolute
stereochemistry of the aglycon residue was not rigorously
established but was assigned by analogy to that of the
Lomaiviticin C (6) was obtained as a burgundy amorphous
solid. HRMS analysis indicated a molecular formula of
C68H82N4O24 (m/z 670.2724 [M + 2H]2+, error = 1.2 ppm).
Received: August 2, 2012
Published: September 10, 2012
© 2012 American Chemical Society
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correlations between H-4 and C-1A, H-1A and C-4, H-4′ and
C-1A′, and H-1A′ and C-4′ in the HMBC spectrum of 6,
secured the location of the pyrrolosamine residues. ROE
contacts between H-1B and H-12 and between H-1B′ and H-
12′ established the location of the oleandrose residues. Distinct
signals in the 1H and 13C NMR spectra of 6 [δH = 6.72 ppm (s,
1H); δC = 120.9 ppm] that were conspicuously absent in the
spectra of 1 were also observed. A heteronuclear multiple-
quantum coherence (HMQC) experiment established the one-
bond connectivity of these atoms. These 1H and 13C resonances
correspond well to those of hydroxyfulvenes such as 9 (Figure
2) that have been previously synthesized in this laboratory (δH‑5
= 6.74 ppm, δC‑5 = 120.0 ppm, chloroform-d).5a The presence
of a hydroxyfulvene rather than two diazofluorenes is consistent
with the HRMS data for 6 and accommodates the C1 symmetry
of the metabolite. Carbon C-5 of 6, which bears the single
remaining diazo group, was observed at 78.3 ppm (78.8 ppm in
1). A strong IR absorbance at 2141 cm−1 also supported the
presence of a diazo group.
Figure 2. Structures of (−)-lomaiviticin C (6) and the synthetic
hydroxyfulvene 9.
a
Table 1. Selected NMR data for (−)-Lomaiviticin C (6)
position
δH (ppm)
δC (ppm)
HMBC (H → C)
1
2
198.9
47.8
3.88, d, J = 3.2 Hz
C-1, -3, -2′, -11b
Lomaiviticins D (7) and E (8) were isolated as burgundy
amorphous solids. HRMS of 7 and 8 suggested molecular
formulas of C69H84N4O24 (m/z 677.2818 [M + 2H]2+, error =
6.3 ppm) and C70H86N4O24 (m/z 684.2886 [M + 2H]2+, error
= 1.1 ppm), respectively, corresponding to addition of one or
two methylene units to 6. The NMR spectroscopic data for 7
and 8 revealed many similarities to 6, with the following
exceptions. First, in the 1H NMR spectrum of 7, two additional
singlet resonances at 3.57 and 3.56 ppm were detected, each
apparently integrating for 1.5H, and doubling of many other
3
83.6
4
5.38, s
67.4
C-2, -3, -4a, -11b, -1A
4a
131.9
5
78.3
5a/11a
6/11
11b
1A
134.6/128.9
185.1/185.0
137.0
4.47, d, J = 9.2 Hz
95.8
C-4, -2A
1′
2′
200.9
b
3.80
47.1
C-2, -4′, -3′, -11b′, -1′
C-2′, -3′, -4a′, -11b′, -1A′
1
resonances was observed. In the H NMR spectrum of 8, two
3′
4′
4a′
5′
83.6
additional singlets resonating at 3.57 and 3.56 ppm and
integrating for 3H each were observed. Analysis of the MS/MS
data for 7 revealed mass fragments with differences of m/z 144,
158, and 302 units from the parent ion. MS/MS analysis of 8
revealed mass fragments with differences of m/z 158 and 316
from the parent ion. Collectively, these data suggest that 7 and
8 are derivatives of 6 bearing an additional O-methyl
substituent on one (for 7) or both (for 8) oleandrose residues.
(−)-Lomaiviticin D (7) is an inseparable mixture of isomers in
which the methylation is proximal or distal to the diazofluorene
(Figure 3).
5.27, s
6.72, s
68.6
129.8
120.9
5a′/11a′
6′/11′
11b′
1A′
122.0/127.6
185.5/182.6
135.3
4.55, d, J = 9.2 Hz
95.6
C-4′, -2A′
a
NMR spectra (500 MHz) were recorded in methanol-d4 at 24 °C.
Positions 1−1A correspond to the half of 6 containing the
diazofluorene. Positions 1′−1A′ correspond to the half of 6 containing
the hydroxyfulvene. Full spectroscopic data are presented in the
b
Supporting Information. Obscured by an overlapping resonance.
The structure of 6 (Figure 2) was elucidated by 1D and 2D
NMR analysis and IR spectroscopy. Selected NMR data are
shown in Table 1.10 These data revealed that 6 shows some
homology to 1, including oleandrose and N,N-dimethylpyrro-
losamine residues, but more surprisingly that 6 is C1-symmetric.
The carbon atoms C-2 and C-2′ were observed at 47.8 and 47.1
ppm, respectively, in good agreement with those of 1 (47.6
ppm). A rotating-frame nuclear Overhauser effect (ROE)
interaction between H-2 (3.88 ppm) and H-2′ (3.80 ppm), a 3J
coupling (3.2 Hz) between these same protons, and a
heteronuclear multiple-bond correlation (HMBC) confirmed
C-2/C-2′ as the conjoining bond between the monomeric
units. 2D nuclear Overhauser effect spectroscopy (NOESY)
analysis revealed contacts between H-2 and H-4 and between
H-2′ and H-4′, supporting a 1,3-cis stereorelationship and
consistent with the observation of W-plane couplings between
H-2 and H-4 and between H-2′ and H-4′ in the correlation
spectroscopy (COSY) spectrum of 6. ROE contacts between
H-1A and H-4 and between H-1A′ and H-4′, as well as
Figure 3. Structures of (−)-lomaiviticins D (7) and E (8).
Acidic digestion of (−)-lomaiviticin C (6) effected the
hydrolysis of both oleandrose residues and one pyrrolosamine
residue.11 Isolation and analysis by optical rotation revealed
that both carbohydrates are of the L form. The absolute
stereochemistry of the pyrrolosamine residue was used to
elucidate the stereochemistry of the aglycon substructure in 6:
observation of an NOE interaction between H-1A and H-4
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Communication
established the C-1A−H-1A and C4−O bonds as syn-
periplanar, and an additional NOE between H-2Aeq and H-12
necessitated that the ethyl substituent face the 2-position of the
aminosugar. These NOE interactions are uniquely accommo-
dated by the diastereomer shown.
Table 2. IC50 Values (in nM) of (−)-Lomaiviticins A (1) and
C−E (6−8) and (−)-Kinamycin C (5)
cell line
compound
K562
LNCaP
HCT-116
HeLa
Treatment of 6 with excess trifluoromethanesulfonyl azide
and trifluoroacetic acid formed semisynthetic (−)-lomaiviticin
A (1) (43%), which was indistinguishable (by 1H NMR,
HMQC, IR, HMRS, optical rotation, and HPLC retention
time) from natural material (Scheme 1).1a We previously
1
6
7
8
5
11
472
197
469
72
2
2
7
332
196
964
116
223
167
255
274
589
161
292
517
Scheme 1. Conversion of Natural (−)-Lomaiviticin C (6) to
(−)-Lomaiviticin A (1)
Cell viability studies revealed that lomaiviticins C−E (6−8)
are submicromolar antiproliferative agents (Table 2). The most
striking result of these assays is the large (up to 166-fold)
difference between the potencies observed for 1 and 6. In view
of the nearly identical structures of the two metabolites, these
data establish that the dimeric diazofluorene structure of 1 is
critical for potent activity. A large body of evidence suggests
that 1 is reductively activated,13 and we have observed that
dimeric diazofluorenes undergo reduction at rates several times
faster than related monomeric diazofluorenes.14 It is possible
that a similar rate enhancement is manifested in the reactivity of
1 and underlies its cytotoxicity.
We have previously presented evidence that the structure 6
(e.g., lomaiviticin C) forms from (−)-lomaiviticin A (1) under
reducing conditions,11 and it is possible that 6 is a product of
degradation of 1 during the fermentation process. However, if
this is the case, the transformation of 1 to 6 in the medium
must be rapid, as 1 does not accumulate in concentrations
detectable by UPLC/MS analysis prior to concentration.
Regardless, the synthetic transformation of (−)-lomaiviticin C
(6) to (−)-lomaiviticin A (1) provides a means to reestablish
cytotoxic activity and access quantities of 1 required for
chemical biological studies. The complete structure elucidation
of (−)-lomaiviticin A (1) will additionally facilitate its eventual
chemical synthesis.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and detailed characterization data for
all new compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
seth.herzon@yale.edu
Notes
The authors declare no competing financial interest.
reported diazo transfers to synthetic hydroxyfulvenes such as 9
using triflyl azide in the presence of base.5 Here, trifluoroacetic
acid significantly enhanced the yield, apparently by stabilizing
the product 1 without suppressing the diazo transfer. In our
hands, fermentation yields of (−)-lomaiviticin A (1) have never
exceeded 0.5 mg/L,12 while the yields of (−)-lomaiviticin C (6)
are as high as 62 mg/L. Thus, this sequence represents a >50-
fold increase in lomaiviticin A production. Perhaps more
importantly, this chemistry provides an unequivocal link
between lomaiviticins A (1) and C (6) and establishes the
complete structure of (−)-lomaiviticin A (1) for the first time.
ACKNOWLEDGMENTS
■
Financial support from the American Cancer Society, the
National Science Foundation (Graduate Research Fellowship
to C.M.W.), the Searle Scholars Program, and Yale University
(Dean’s Undergraduate Research Fellowship to N.E.B.) is
gratefully acknowledged. We thank Laura Abriola of the Yale
Small Molecule Discovery Center for conducting cell viability
assays. S.B.H. is a fellow of the David and Lucile Packard and
the Alfred P. Sloan Foundations, and is a Camille Dreyfus
Teacher−Scholar.
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(14) Woo, C. M.; Herzon, S. B. Unpublished results.
REFERENCES
■
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