Characterization and Synthesis of Eudistidine C, a Bioactive Marine Alkaloid with an Intriguing Molecular Scaffold

Article abstract of DOI:10.1021/acs.joc.6b02380

An extract of Eudistoma sp. provided eudistidine C (1), a heterocyclic alkaloid with a novel molecular framework. Eudistidine C (1) is a racemic natural product composed of a tetracyclic core structure further elaborated with a p-methoxyphenyl group and a phenol-substituted aminoimidazole moiety. This compound presented significant structure elucidation challenges due to the large number of heteroatoms and fully substituted carbons. These issues were mitigated by application of a new NMR pulse sequence (LR-HSQMBC) optimized to detect four- and five-bond heteronuclear correlations and the use of computer-assisted structure elucidation software. Synthesis of eudistidine C (1) was accomplished in high yield by treating eudistidine A (2) with 4(2-amino-1H-imidazol-5-yl)phenol (4) in DMSO. Synthesis of eudistidine C (1) confirmed the proposed structure and provided material for further biological characterization. Treatment of 2 with various nitrogen heterocycles and electron-rich arenes provided a series of analogues (5-10) of eudistidine C. Chiral-phase HPLC resolution of epimeric eudistidine C provided (+)-(R)-eudistidine C (1a) and (-)-(S)-eudistidine C (1b). The absolute configuration of these enantiomers was assigned by ECD analysis. (-)-(S)-Eudistidine C (1b) modestly inhibited interaction between the protein binding domains of HIF-1α and p300. Compounds 1, 2, and 6-10 exhibited significant antimalarial activity against Plasmodium falciparum.

Full text of DOI:10.1021/acs.joc.6b02380

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pubs.acs.org/joc
Characterization and Synthesis of Eudistidine C, a Bioactive Marine
Alkaloid with an Intriguing Molecular Scaffold
Susanna T. S. Chan,† Roger R. Nani,‡ Evan A. Schauer,‡,& Gary E. Martin,§ R. Thomas Williamson,§
Josep Saurí,§ Alexei V. Buevich,§ Wes A. Schafer,§ Leo A. Joyce,§ Andrew K. L. Goey,∥ William D. Figg,∥
Tanya T. Ransom,† Curtis J. Henrich,†,⊥ Tawnya C. McKee,†,$ Arvin Moser,Δ Scott A. MacDonald,Δ
Shabana Khan,⊗ James B. McMahon,† Martin J. Schnermann,*,‡ and Kirk R. Gustafson*,†
‡
†Molecular Targets Laboratory, Center for Cancer Research and Chemical Biology Laboratory, Center for Cancer Research,
National Cancer Institute, Frederick, Maryland 21702-1201, United States
§NMR Structure Elucidation, Process Research and Development, Merck & Co., Inc., Rahway, New Jersey 07065, United States
∥Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, United States
⊥Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland
21702-1201, United States
ΔAdvanced Chemistry Development, Inc. (ACD/Laboratories), Toronto Department, 8 King Street East Suite 107, Toronto, Ontario
M5C 1B5, Canada
⊗National Center for Natural Products Research, School of Pharmacy, University of Mississippi, Mississippi 38677, United States
S
* Supporting Information
ABSTRACT: An extract of Eudistoma sp. provided eudisti-
dine C (1), a heterocyclic alkaloid with a novel molecular
framework. Eudistidine C (1) is a racemic natural product
composed of a tetracyclic core structure further elaborated
with a p-methoxyphenyl group and a phenol-substituted
aminoimidazole moiety. This compound presented significant
structure elucidation challenges due to the large number of
heteroatoms and fully substituted carbons. These issues were
mitigated by application of a new NMR pulse sequence (LR-
HSQMBC) optimized to detect four- and five-bond
heteronuclear correlations and the use of computer-assisted
structure elucidation software. Synthesis of eudistidine C (1)
was accomplished in high yield by treating eudistidine A (2)
with 4(2-amino-1H-imidazol-5-yl)phenol (4) in DMSO. Synthesis of eudistidine C (1) confirmed the proposed structure and
provided material for further biological characterization. Treatment of 2 with various nitrogen heterocycles and electron-rich
arenes provided a series of analogues (5−10) of eudistidine C. Chiral-phase HPLC resolution of epimeric eudistidine C provided
(+)-(R)-eudistidine C (1a) and (−)-(S)-eudistidine C (1b). The absolute configuration of these enantiomers was assigned by
ECD analysis. (−)-(S)-Eudistidine C (1b) modestly inhibited interaction between the protein binding domains of HIF-1α and
p300. Compounds 1, 2, and 6−10 exhibited significant antimalarial activity against Plasmodium falciparum.
β-lactam monamphilectine A,7 and odoamide, a cytotoxic
INTRODUCTION
■
cyclicpeptide from a cyanobacterium.8 There has been
Natural products benefit from “privileged” structures that
evolved to interact with essential biopolymers such as proteins
significant progress with new genomic tools that enable
sequencing-based recognition and annotation of natural
and nucleic acids. These secondary metabolites encompass
products with known biosynthetic machineries, and the
extensive diversity in terms of both their molecular architecture
structures of encoded secondary metabolites can often be
and biological activity and remain a key source of lead
accurately predicted.9−11 However, the identification of
completely new molecular scaffolds such as complex polycyclic
alkaloids cannot typically be accomplished using genomic
methods alone. Such discoveries often depend on approaches
compounds and new therapeutic agents.1 Nitrogen-rich natural
products represent a broad class of bioactive compounds that
interact with important cellular targets, particularly in the
context of anticancer and anti-infective agents.2−4 Some recent
examples of nitrogenous metabolites include the myxobacterial
cytotoxin haprolid,5 the synoxazolidinones, antimicrobial
guanidine alkaloids from a marine ascidian,6 the antimalarial
Received: September 29, 2016
Published: November 8, 2016
© XXXX American Chemical Society
A
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utilizing activity-guided fractionation and de novo structural
elucidation. In this area, NMR-based structural studies of
severely mass-limited samples on the nanomole scale have been
facilitated by a new generation of miniaturized cryogenically
cooled NMR probes12−14 and the development of sensitivity
enhanced NMR pulse sequences.15−18 There has also been
dramatic progress in the development of computational
approaches that provide predictive structure determination
derived from experimental data.19−22 The work described
herein illustrates a range of contemporary structural elucidation
strategies and techniques, including total synthesis. These were
applied in a concerted fashion in the context of the novel
heterocyclic alkaloid, eudistidine C (1).
We recently reported the isolation, structural elucidation, and
synthesis of eudistidines A (2) and B (3) from an extract of the
marine ascidian Eudistoma sp.23 The extract was chemically
investigated because it showed activity in a high-throughput
screen for inhibitors of the protein−protein interaction
between p300 and HIF-1α. We found that 2, but not 3,
inhibited this interaction. In the current study, we further
interrogated the same extract and identified a related
compound that exhibits increased chemical complexity. This
compound maintains the tetracyclic core of 2 but is further
elaborated at the C-10 position with an unusual aryl-substituted
aminoimidazole moiety. Structure elucidation of 1 was
complicated by the proton-deficient core of the molecule.
The assignment was accomplished using a combination of
NMR spectroscopic analyses, including a new pulse sequence
specifically optimized for long-range heteronuclear correlations
and structure predictive software. Synthetic studies confirmed
the structure of eudistidine C (1) and revealed that a variety of
electron-rich arenes can react with 2 under mild conditions to
provide a series of aryl-substituted analogues. Chiral resolution
of racemic 1 provided the two C-10 epimers, and only (−)-(S)-
eudistidine C (1b) had significant activity in the p300 and HIF-
1α assay. In total, these studies illustrate the utility of applying
synergistic technologies in concert to characterize the chemical
and biological diversity of new secondary metabolites.
Table 1. NMR Spectroscopic Data for Eudistidine C (1) in
CD3OD
a
b
position
δC
δN
δH (mult,J in Hz)
HMBC
1
245.8
2
167.5
94.3
8.45 (d, 5.0)
6.77 (d, 5.3)
1, 3, 3a, 3b, 8c
1, 2, 3a, 3b, 8b
3
3a
147.4
117.6
126.1
130.2
136.5
129.7
147.0
3b
4
8.06 (d, 8.0)
7.58 (t, 7.5)
7.81 (t, 7.5)
7.65 (d, 8.0)
3a, 6, 7a
3b, 7
5
6
4, 7a
7
3b, 5, 8
7a
8
256.0
169.6
8a
159.5
157.3
8b
8c
c
9
n.o.
10
73.7
130.9
129.8
115.2
161.7
55.8
11
12/16
13/15
14
7.68 (d, 8.9)
6.91 (d, 8.8)
10, 12/16, 14
11, 13/15, 14
RESULTS AND DISCUSSION
■
Our initial bioassay-guided fractionation of the Eudistoma sp.
extract provided eudistidines A (2) and B (3).23 Further
14-OMe
17
3.76 (s)
14
1
126.1
investigation of a side fraction from this effort revealed a H
c
18
n.o.
NMR signature indicative of a related eudistidine analogue.
Repeated chromatographic separations on LH-20 media
ultimately provided 10.7 mg of eudistidine C (1).
19
148.0
c
20
n.o.
21
127.5
120.0
131.9
115.7
159.3
Eudistidine C (1) was isolated as an optically inactive yellow
oil with a molecular formula of C28H21N7O2, as established by
HRESI mass spectrometry, which required 22 degrees of
unsaturation. The highly aromatic nature of 1 was confirmed by
26 sp2 carbons (δC 167.5, 161.7, 159.5, 159.3, 157.3, 148.0,
147.4, 147.0, 136.5, 131.9, 131.9, 130.9, 130.2, 129.8, 129.8,
129.7, 127.5, 126.1, 126.1, 120.0, 117.6, 115.7, 115.7, 115.2,
115.2, 94.3) and only two sp3 carbons (δC 73.7 and 55.8) in the
22
23/27
24/26
25
6.85 (d, 8.8)
6.30 (d, 8.5)
21, 23/27, 25
22, 24/26, 25
c
28
n.o.
a
15N assignments were based on 1H−15N HMBC correlations. The δN
values were not calibrated to an external standard but were referenced
b
to neat NH3 (δ 0.00) using the standard Bruker parameters. 1H−13C
1
13C NMR spectrum (Table 1). H NMR (CD3OD) revealed
(optimized for 8.3 Hz) and H−15N (optimized for 8 Hz) HMBC
1
c
signals attributable to one methoxy group (δH 3.76) and 14
aromatic protons, which constituted four protonated spin
systems based on COSY data: a pair of mutually coupled
heteroaromatic protons [δH 8.45 (d, J = 5.0 Hz), 6.77 (d, J =
5.3 Hz)], a 1,2-disubstituted benzene ring [δH 8.06 (d, J = 8.0
Hz), 7.81 (t, J = 7.5 Hz), 7.65 (d, J = 8.0 Hz), 7.58 (t, J = 7.5
Hz)], and two para-substituted benzene rings [δH 7.68 (2H d, J
= 8.9 Hz), 6.91 (2H d, J = 8.8 Hz) and 6.85 (2H d, J = 8.8 Hz),
correlations are listed. Not observed.
6.30 (2H d, J = 8.5 Hz)]. Direct comparison of the 1D and 2D
NMR data sets for eudistidine C (1) with those recorded for
eudistidines A (2) and B (3) supported the presence of the
same tetracylic eudistidine core structure (rings A−D),
composed of fused pyrimidine and imidazole rings with
B
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embedded guanidine and amidine functionalities (Figure 1 and
the Supporting Information). However, the chemical shift of C-
While computer-assisted structure elucidation (CASE)
provided a structure consistent with our proposal, we felt that
additional evidence was required to complete the assignment
due to the proton-deficient nature of 1. The LR-HSQMBC
experiment is a recently described NMR pulse sequence
optimized to detect very long-range heteronuclear correla-
tions.24 It can be used to detect
J
CH
and
J
NH
correlations,
4,5
4,5
and the utility of these long-range data was clearly
demonstrated in the NMR characterization of cervinomycin
A2.25 LR-HSQMBC is a high-sensitivity pulse sequence that
optimizes responses for very small nJCH/NH couplings (≤0.2 Hz
for nJCH), and the resulting data complements experiments such
as HMBC for defining structural elements that have few if any
detectable protons. The HMBC experiment provides access to
2,3J correlations and only occasionally 4J heteronuclear
correlations, while LR-HSQMBC data sets are populated with
numerous four- and five-bond correlations.26 These long-range
correlations can help define structural elements with multiple
quaternary carbons and heteroatoms, and link these moieties
with protonated portions of the molecule. Crucial 4JCH and 5JCH
correlations observed by LR-HSQMBC (Figure 2), including
Figure 1. Selected 1H−13C HMBC (black arrows), 1H−15N (red
arrows), and COSY (bold blue lines) correlations for eudistidine C
(1).
10 in 1 (δC 73.7) differed significantly from the C-10
hemiaminal (δC 94.4) in eudistidine A (2) and amide (δC
172.0) in eudistidine B (3). HMBC correlations established
that ring E in 1 was a p-methoxybenzene ring that was
substituted at C-10 (Figure 1). The remaining para-substituted
benzene ring (G) had a resonance at δC 159.1 that was
indicative of a hydroxyl substituent.
With the tetracyclic eudistidine core and two para-
substituted phenyl groups firmly established, molecular formula
considerations required the final structure of eudistidine C (1)
incorporate an additional C3H3N3 moiety and 3 degrees of
unsaturation. The three remaining carbons were quaternary sp2
carbons with chemical shifts of δC 148.0, 127.5, 126.1. The only
HMBC correlation to these carbons was a correlation from H-
23/27 (δH 6.85) to the carbon at δC 127.5, which revealed that
ring G was connected to the C3 fragment at this position. No
NH protons were ever observed using a variety of different
NMR experiments (1H, 1H−15N HSQC, 1H−15N HMBC) and
deuterated solvents (CD3OH, DMSO-d6, DMF-d7). The
carbon chemical shifts, in particular the signal at δC 148.0,
suggested the remaining fragment was an amino-substituted
imidazole ring (ring F) connected via the carbon at δC 126.1 to
C-10 and to ring G through the carbon at δC 127.5. Thus, the
novel heptacyclic structure of eudistidine C (1) was proposed.
However, significant structural ambiguity remained due to the
large number of adjacent quaternary carbons and non-
protonated nitrogen atoms, along with the lack of observable
NH signals. The experimental 13C NMR data recorded with
eudistidine C (1) were compared with the DFT-calculated
theoretical carbon chemical shifts for structure 1. The
computational results were generally in good agreement with
the experimentally derived data (Supporting Information), with
the largest outliers being carbons, C-11 and C-19, which had
Δδ values of 3.8 and 4.5 ppm, respectively. The molecular
formula and complete set of NMR data (1H, 13C, COSY,
HSQC, HMBC) recorded for 1 were then analyzed using
ACD/Structure Elucidator Suite software, which allows for de
novo structure elucidation and generates a complete set of
structures that fit the correlations observed in the experimental
data.21,22 The program generated a total of 419 structural
isomers, the 13C NMR data sets for each structure were
calculated, and the structures ranked on the basis of cumulative
error between the calculated and experimental NMR values.
The structure we proposed for eudistidine C (1) was the
highest ranked structure identified by the Structure Elucidator
software (Supporting Information).
Figure 2. Key four-bond (blue arrows) and five-bond (red arrows)
1H−13C correlations measured with a 2 Hz optimized LR-HSQMBC
experiment digitized with 768 F1 increments.
those from H-12/16 (ring E) to C-8a (ring D) and C-17 (ring
G) as well as from H-23/27 and H-24/26 (ring F) to C-17,
helped confirm the composition and connectivity of the rings in
eudistidine C (1). When the LR-HSQMBC correlations were
added to the data set used with the ACD/Structure Elucidator,
the number of candidate structures was reduced from 419 to
335 and the total processing time was reduced from 247 to 35
s. As before, structure 1 was the highest ranked candidate.
Despite the valuable data obtained by the LR-HSQMBC
experiment, no long-range correlations were ever recorded for
C-19 or any of the nitrogen atoms associated with ring F. Thus,
a portion of eudistidine C (1) remained only partially
characterized, with inadequate spectroscopic data to fully
define the structure. The synthesis of eudistidine C (1) was
viewed as the best way to definitively prove its structure as well
as provide access to useful quantities of 1 and its analogues.
Inspection of 1 suggested it might arise from a dehydration-
driven union of eudistidine A (2) and 3-arylaminoimidazole 4.
We recently reported the preparation of 2 using a
condensation/cyclization reaction cascade between 4-(2-
aminophenyl)pyrimidin-2-amine and 4-(methoxyphenyl)-
glyoxal.23 While this synthetic route was concise, isolation of
the final product required a scale-limiting preparative HPLC
step. We subsequently found that synthetic 2 can be isolated
directly from the homogeneous reaction solution by simple
addition of 1:1 acetone/hexanes to provide a solid product.
This material is the HBr salt of 2 in sufficient purity to use
C
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Figure 3. Synthesis of eudistidine C (1).
1
Figure 4. H NMR spectral characteristics of eudistidine C (1) in CD3OD: (A) synthetic eudistidine C TFA salt, (B) synthetic eudistidine C free
base, (C) natural product eudistidine C free base form.
directly in the synthetic studies described below. Alternately,
the solid material can be converted to its free base form
through preparative HPLC in the presence of NH4OH to give
material with NMR spectral features identical to those of
natural eudistidine A (2). This four-step, scalable preparation of
eudistidine A (2) allowed us to investigate the coupling
reaction to form eudistidine C (1) and other analogues. The
aminoimidazole coupling partner needed to generate 1 was
prepared in one step from the commercially available methyl
ether through demethylation with BBr3 to give the desired
phenol 4 (Experimental Section). The identity and purity of 4
were verified by NMR analysis (Supporting Information). After
a screen of reaction conditions, we found the combination of
the HBr salt of 2 and 4 (1.5 equiv) in DMSO at room
temperature afforded racemic eudistidine C (1) in 64% yield
following preparative HPLC (Figure 3). Notably, the free base
of 2 does not undergo this reaction without additional acidic
promotor. The synthetic material had NMR spectral features
(1H and 13C) identical to those of the natural compound 1
when compared in their free base forms or when both spectra
D
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were obtained in CD3OD/0.1% TFA (Supporting Informa-
tion). A 1:1 mixture of natural and synthetic eudistidine C
Table 2. Reaction of Eudistidine A (2) with Heteroarene and
Arene Nucleophiles
1
provided a discrete set of H and 13C NMR resonances, which
confirmed the identical nature of these materials. Significant
spectral differences were observed between the free base and
salt forms of these alkaloids (Figure 4). Protonation state,
counterion, and sample concentration can all be essential for
accurate spectral comparison of natural and synthetic samples
of complex heterocycles, such as the eudistidines.
The mild nature of this coupling procedure suggested that
alternative nucleophiles might also add to the C-10 position of
2. We first pursued this possibility with a variety of nucleophilic
heteroarenes, including N-methylindole, 3-methylindole, and
N-methylpyrrole. In all three cases, the corresponding adducts
5−7 could be obtained in useful preparative yield, although the
reaction required extended heating at 40 °C in the case of 3-
methylindole (Table 2). The connectivity of synthetic adducts
was confirmed by 2D NMR, with heterocycle modification
occurring at the expected site (C-3 of N-methylindole, C-2 of
3-methylindole, and C-2 of N-methylpyrrole). We further
examined the reaction of 2 with phenol, resorcinol, and
phloroglucinol. Compounds 8−10 were formed in suitable
yields, albeit requiring extended heating in DMSO in the case
of phenol and resorcinol. Following these results, the reactivity
of 2 with various heteroatom nucleophiles was also evaluated.
We found that with several representative alcohols, amines, and
thiols, no new adducts could be isolated or observed by LCMS.
Thus, it appears the electrophilic reactivity of 2 is confined to
carbon nucleophiles. These initial studies generated a series of
structural analogues of 1, helped to characterize the reactivity of
eudistidine A (2), and will enable future efforts to generate
additional analogues of the eudistidine class of natural products.
Investigation of the UV absorption properties of eudistidine
C (1) in MeOH revealed an absorption maximum at 403 nm.
The UV/vis absorption profiles of 1 in aqueous solutions were
quite similar to those recorded for eudistidine A (2),23 as they
exhibited pronounced pH-dependent spectral shifts. Local
absorbance maxima for 1 were observed at 403 and 446 nm,
with a hypsochromic shift in more acidic medium and an
isosbestic point at approximately 420 nm (Supporting
Information). Natural eudistidine C (1) is racemic, as it has
no optical rotation [α]D or Cotton effect in its CD spectrum,
and the enantiomers are related by inversion at C-10.
Eudistidine A (2) natural product is also racemic; however, it
has a hemiaminal group at C-10, creating the likelihood that a
ring D-opened minor tautomeric form of 2 facilitates rapid
racemization at this position. The racemic nature of 1 and its
inherent reactivity with nitrogen heteroaromatic compounds
under mild conditions suggests that the final aminoimidazole
coupling step in the biogenesis of eudistidine C (1) mirrors our
synthetic strategy and may not necessarily occur via an
enzymatic process. Ring D in 1 cannot open in a manner
similar to 2; thus, the two epimers at C-10 likely form during
the nucleophilic coupling step and then are configurationally
stable. The two enantiomers of the eudistidine C natural
product racemate could be resolved by HPLC using a chiral-
phase Lux Cellulose-4 analytical column (4.6 × 50 mm). The
enantiomers in the natural product occurred as a 1:1 mixture
with identical UV spectra but opposite sign Cotton effects
when monitored at 240 nm (Supporting Information).
Semipreparative separation (10 × 250 mm Lux-4 column) of
synthetic eudistidine C provided the two enantiomers in pure
form, and they showed equal magnitude but opposite sign
Cotton effects in their ECD spectra (Figure 5). The early
eluting enantiomer was assigned as (+)-(R)-eudistidine C (1a)
by comparing its experimentally measured ECD spectrum with
E
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Figure 5. Chiral-phase HPLC separation of racemic eudistidine C provided (+)-(R)-eudistidine C (1a) and (−)-(S)-eudistidine C (1b). (A) HPLC
UV trace monitored at 210 nm of the racemate separated on a Lux Cellulose-4 column (chlorinated cellulose phenylcarbamate) provided two peaks
(MH+, m/z 488) of approximately equal area. (B) ECD spectra of the early-eluting HPLC peak [blue; (+)-(R)-eudistidine C (1a)] and late-eluting
peak [pink; (−)-(S)-eudistidine C (1b)] had equal magnitude but opposite sign Cotton effects.
the DFT-calculated spectrum (Figure 6). The late-eluting
enantiomer was assigned as (−)-(S)-eudistidine C (1b) in a
similar manner (Supporting Information). Eudistidine A (2)
could also be resolved by chiral-phase HPLC into its two
enantiomers. However, when the eluent of each peak was
subsequently dried and reinjected on the HPLC two peaks of
equal area were observed, indicating that ring opening and
epimerization at C-10 had occurred.
Racemic eudistidine C (1) was tested in a dose−response
version of the screening assay to assess its ability to block
binding between the CH1 domain of p300 and the C-TAD
domain of HIF-1α. It showed approximately 48% inhibition of
the protein−protein interaction at a high test concentration of
500 μM. Testing of the individual enantiomers revealed that
(−)-(S)-eudistidine C (1b) had an IC50 of 276 μM, while
(+)-(R)-eudistidine C (1a) gave only 27% inhibition at 500
μM. The eudistidine C analogues 5−10 were also evaluated in
the HIF-1α/p300 assay. The phloroglucinol adduct (10) was
slightly more potent (IC50 = 141 μM), while the N-
methylindole (5), 3-methylindole (6), and N-methylpyrrole
(7) derivatives had IC50’s of 314, 399, and 356 μM,
respectively. The other analogues provided less than 50%
inhibition at 500 μM. In an effort to evaluate other biological
properties, eudistidines A (2) and C (1) were tested in the
NCI-60 panel of human tumor cell lines, but at an initial
concentration of 40 μM they were generally noncytotoxic and
thus did not undergo further 60-cell testing.27 Since a variety of
different alkaloid structural families are known to have
antimalarial properties, (+)-(R)-eudistidine C (1a), (−)-(S)-
eudistidine C (1b), eudistidine A (2), and analogues 6−10
F
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The new structural motif of eudistidine C (1) can be
accessed in a single step by reaction of eudistidine A (2) with
appropriate nucleophiles. The unusual propensity of eudistidine
A to react irreversibly with aromatic carbon nucleophiles, but
not with nucleophilic heteroatoms such as alkyl alcohols,
amines, or thiols, provided a means to generate synthetic
eudistidine C and also a variety of heteroarene and phenolic
structural analogues (5−10). The effective synthesis of
eudistidine C provided proof of its proposed structure and a
means to supply an extremely mass-limited natural compound.
Preparation of a series of structural analogues of 1 helped
delineate the electrophilic reactivity of ring D and the
hemiaminal moiety in eudistidine A (2) and also provided
additional eudistidine type alkaloids for biological evaluation.
Resolution of racemic eudistidine C into its two enantiomers
allowed assignment and characterization of (+)-(R)-eudistidine
C (1a) and (−)-(S)-eudistidine C (1b).
Figure 6. Comparison of calculated (red) and experimentally
measured (black) ECD spectra for (+)-(R)-eudistidine C (1a).
Initial biological characterization of eudistidine C suggests
potential applications in the context of cancer and malaria.
(−)-(S)-Eudistidine C (1b) and analogues 5−7 and 10
inhibited interaction of the protein-binding domains of the
oncogenic transcription factor HIF-1a and its coactivator p300,
albeit only at relatively high concentrations. Small molecule
inhibitors of tight protein−protein interactions are notoriously
difficult to design or discover. Compounds based on the
eudistidine framework represent possible chemical probes into
the role of HIF-1α and p300 in the adaptation of cancer cells to
low oxygen environments and are leads for the development of
cancer therapeutics. Compounds 1a, 1b, 2, and 6−10 also
inhibited chloroquine-sensitive and chloroquine-resistant
strains of the malaria parasite P. falciparum at low micromolar
concentrations. Treatment and prevention of malaria, and
especially its drug-resistant variants, is a global health priority,
and current drug therapies are inadequate. In total, compound
1 and its readily accessible structural analogues provide a
potentially fruitful avenue for discovering novel antimalarial and
cancer therapeutics.
were also evaluated for inhibitory activity against chloroquine-
sensitive (D6) and chloroquine-resistant (W2) strains of the
Plasmodium falciparum parasite,28 and all showed significant
antimalarial effects (Supporting Information). The most potent
were eudistidine A (2), which had IC50 values of 1.4 and 1.1
μM, and the phloroglucinol analogue of eudistidine C (10),
which had IC50 values of 1.1 and 0.6 μM against the D6 and
W2 strains, respectively.
CONCLUSION
■
The novel heptacyclic structure of eudistidine C was assigned
using a combination of contemporary spectroscopic techniques
and CASE analyses. The recently described LR-HSQMBC
NMR pulse sequence with its ability to detect four- and five-
bond heteronuclear correlations is particularly effective for
assigning and verifying structural elements that are distant from
protonated portions of a molecule. This technique was most
applicable with eudistidine C (1), which has a severely proton-
deficient central core region. A hallmark of virtually all natural
products studies is the ability to define and verify the structure
of the compounds of interest. As such, the continuing
adaptation and application of new techniques such as NMR
experiments that extend the sensitivity/boundaries for data
acquisition are critically important. In addition, CASE programs
such as ACD/Structure Elucidator are becoming more powerful
and reliable, and they provide important tools that can be
effectively utilized in challenging structural elucidation
situations. The utility of CASE analysis was highlighted by
congruence of the structure obtained from conventional
interpretation of the spectroscopic data for eudistidine C with
the most favored structural candidate revealed by the software.
The new and often synergistic technologies now employed in
natural products discovery have confirmed the tremendous
untapped potential of this resource and sparked renewed
interest in the chemical and biological diversity of secondary
metabolites. In spite of recent advances, the architectural
complexity and unusual functional group arrays found in some
natural scaffolds still present structural elucidation challenges
for noncrystalline secondary metabolites. The current study
illustrates a range of contemporary structural elucidation
strategies and techniques, including total synthesis, that were
required to fully address structural questions associated with
eudistidine C (1).
EXPERIMENTAL SECTION
■
General Procedures. NMR spectra were obtained with an NMR
spectrometer equipped with a 3 mm cryogenic probe and operating at
1
1
600 MHz for H and 150 MHz for 13C. H−13C HMBC experiments
were optimized for nJ = 8.3 Hz, and 1H−15N HMBC experiments were
n
optimized for J = 8 Hz. (+)HRESIMS data for 1 were acquired on a
Q-TOF LC/MS. (+)HRESIMS data for 4−10 were conducted on a
LTQ-Orbitrap hybrid mass spectrometer system with an API
electrospray ion source in positive-ion mode. Preparative reversed-
phase HPLC was run on a system using a C18 column (10 μm, 110 Å,
75 × 30 mm) with 0.1% TFA in the aqueous phase or a C18 column (5
μm, 110 Å, 250 × 9.4 mm) with 0.1% formic acid. IR spectra were
acquired after MeOH solutions of the sample compounds were
applied to a NaCl plate and the solvent was removed under a stream of
N2, followed by high vacuum. The three-dimensional structure
depicted in the TOC graphic was generated by molecular mechanics
minimization using Spartan 14, and the representation was generated
in CYL view.29
Animal Material, Isolation, and Purification. Specimens of the
ascidian Eudistoma sp. were collected at a depth range of −3 to −10 m
on the Koror side of the collapsed Koror−Babeldaob Bridge in the
Koror/Airai Channel, Palau (07°21.64′ N, 134°30.17′ E), in
September 1998 by Dr. Patrick Colin, under contract through the
Coral Reef Research Foundation for the Natural Products Branch,
National Cancer Institute. Taxonomic identification of the ascidian
was done by Francoise Monniot, and a voucher specimen
(0CDN5649) was deposited at the Smithsonian Institute, Washington,
G
DOI: 10.1021/acs.joc.6b02380
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
DC.The ascidian specimen (668 g, wet weight) was stored frozen until
it was extracted according to the procedures detailed by McCloud30 to
give 7.2 g of organic solvent (CH2Cl2−MeOH, 1:1) extract. A portion
of the organic extract (812.9 mg) was fractionated on two diol SPE
cartridges (2 g) eluting with 9:1 hexane−CH2Cl2 (fraction A), 20:1
CH2Cl2−EtOAc (fraction B), 100% EtOAc (fraction C), 5:1 EtOAc-
MeOH (fraction D), and 100% MeOH (fraction E) in a stepwise
manner. Repeated size-exclusion LH-20 column chromatography of
fraction E (439.5 mg) using MeOH as the eluent yielded eudistidine C
(1, 10.7 mg, 1.32%).
(+)HRESIMS m/z 488.1821 [M + H]+ (calcd for C28H22N7O2
488.1829).
Synthesis of Eudistidine C N-Methylindole Analogue (5).
Eudistidine A HBr salt (20 mg, 0.048 mmol) was dissolved in DMSO
(0.5 mL), and N-methylindole (10 mg, 0.072 mmol) was added. The
solution was stirred at 22 °C for 24 h and then purified by reversed-
phase preparative HPLC, employing a gradient of 5−95% MeCN−
H2O (0.1% TFA) over 10 min to yield 5 (16 mg, 61%): IR (thin film)
1
λmax 1664, 1638, 1511, 1256, 1172 cm−1; H NMR (CD3OD, 600
MHz) δ 9.02 (1H, d, J = 5.8 Hz, H-2), 8.73 (1H, d, J = 8.0 Hz, H-4),
8.20 (1H, t, J = 7.7 Hz, H-6), 8.15 (1H, d, J = 8.1 Hz, H-7), 8.06 (1H,
d, J = 5.6 Hz, H-3), 7.99 (1H, t, J = 7.7 Hz, H-5), 7.60 (2H, d, J = 8.5
Hz, H-12/16), 7.43 (1H, d, J = 8.3 Hz, H-21), 7.21 (2H, m, H-18, H-
22), 7.13 (1H, d, J = 7.6 Hz, H-24), 6.98 (3H, m, H-13/15, H-23),
3.81 (3H, s, H-14OCH3), 3.80 (3H, s, H−N19OCH3); 13C NMR
(CD3OD, 150 MHz) δ 167.4 (C-2), 161.9 (C-14), 154.5 (C-8c),
154.3 (C-8a), 148.3 (C-3a), 147.0 (C-7a), 139.6 (C-20), 138.7 (C-6),
131.9 (C-5), 131.2 (C-11), 130.9 (C-7), 130.7 (C-18), 129.8 (C-12/
16), 127.5 (C-4), 126.2 (C-25), 123.6 (C-22), 121.4 (C-24), 121.1 (C-
23), 117.8 (C-3b), 115.3 (C-13/15), 114.0 (C-17), 111.0 (C-21),
103.5 (C-3), 71.5 (C-10), 55.9 (C-14OCH3), 33.1 (C-N19OCH3);
(+)HRESIMS m/z 444.1806 [M + H]+ (calcd for C28H22N5O
444.1819).
Synthesis of Eudistidine C 3-Methylindole Analogue (6). To
a solution of eudistidine A HBr salt (5.2 mg, 0.013 mmol) in DMSO
(1.0 mL) was added 3-methylindole (17 mg, 0.13 mmol). The orange/
brown solution was stirred at 40 °C for 7 days, after which time the
reaction was purified directly by preparative reversed-phase HPLC
(5−95% MeCN/0.1% TFA/water). The product-containing fractions
were concentrated in vacuo to afford 6 (2.5 mg, 35% yield) as a brown
solid: IR (thin film) λmax 3179, 1664, 1638, 1512, 1305, 1176 cm−1; 1H
NMR (CD3OD, 600 MHz) δ 9.04 (1H, d, J = 5.9 Hz, H-2), 8.76 (1H,
d, J = 8.1 Hz, H-4), 8.23 (1H, m, H-6), 8.21 (1H, m, H-7), 8.10 (1H,
d, J = 5.8 Hz, H-3), 8.02 (1H, m, H-5), 7.70 (2H, d, J = 8.7 Hz, H-12/
16), 7.51 (1H, d, J = 8.1 Hz, H-23), 7.25 (1H, d, J = 8.1 Hz, H-20),
7.13 (1H, t, J = 7.6 Hz, H-21), 7.07 (2H, d, J = 8.7 Hz, H-13/15), 7.04
(1H, t, J = 7.6 Hz, H-22), 3.84 (3H, s, 14-OCH3), 1.91 (3H, s, 25-
CH3); 13C NMR (CD3OD, 150 MHz) δ 167.5 (C-2), 162.2 (C-14),
154.4 (C-8c), 153.7 (C-8a), 148.3 (C-3a), 147.0 (C-7a), 138.8 (C-6),
137.5 (C-19), 132.2 (C-5), 131.1 (C-7), 130.6 (C-17), 130.5 (C-11),
130.5 (C-24), 129.7 (C-12/16), 127.5 (C-4), 124.2 (C-21), 120.5 (C-
22), 119.8 (C-23), 117.9 (C-3b), 115.6 (C-13/15), 112.7 (C-25),
112.2 (C-20), 103.7 (C-3), 71.6 (C-10), 55.9 (14-OCH3), 9.5 (25-
CH3); (+)HRESIMS m/z 444.1808 [M + H]+ (calcd for C28H22N5O
444.1819).
Eudistidine C (1): yellow oil; UV (MeOH) λmax (log ε) 208 (4.36),
219 (4.30), 278 (4.32), 348 (3.40), 439 (3.45) nm; IR λmax 1646, 1626,
1
1504, 1351, 1246, 1167 cm−1; H and 13C NMR data, see Table 1;
(+)HRESIMS m/z 488.1848 [M + H]+ (calcd for C28H21N7O2
488.1829).
Synthesis of Eudistidine A (2) HBr Salt. The reaction to form
eudistidine A (2) was run similarly to that reported previously, but the
procedure was carried on a larger scale and the isolation procedure was
significantly different.23 A solution of 2-bromo-4′-methoxyacetophe-
none (620 mg, 2.7 mmol) in DMSO (15 mL) and water (1.5 mL) was
heated at 65 °C for 3 h to generate 2-(4-methoxyphenyl)-2-
oxoacetaldehyde. The solution was cooled to 4 °C before a solution
of 4-(2-aminophenyl)pyrimidin-2-amine (300 mg, 1.8 mmol) in
DMSO (2 mL) was added slowly. The resulting solution was heated
at 60 °C for 1 h and then cooled back to 4 °C. I2 (450 mg, 1.8 mmol)
in DMSO (2 mL) was added slowly, and the brown solution was
heated for an additional 1 h at 60 °C. The resulting solution was
cooled to room temperature and then added to a stirring solution of
1:1 acetone−hexanes (250 mL). The red solid was filtered, washed
(1:1 acetone−hexanes), and then collected and dried (480 mg, 65%).
The compound could be subjected to reversed-phase HPLC
employing a gradient of 20−60% MeCN/H2O (1% NH4OH) over
10 min to yield the free base form of eudistidine A (2) that by
spectroscopic analysis was identical to the natural product.23 The HBr
salt of 2 was used in all the synthetic studies described below.
Eudistidine A (2) HBr salt: 1H NMR (DMSO-d6, 600 MHz) δ 9.21
(1H, d, J = 5.8 Hz, H-2), 8.99 (1H, d, J = 8.0 Hz, H-4), 8.40 (1H, d, J
= 5.7 Hz, H-3), 8.28 (1H, t, J = 7.8 Hz, H-6), 8.23 (1H, d, J = 8.1 Hz,
H-7), 8.17 (1H, br s, 10-OH), 8.10 (1H, t, J = 7.8 Hz, H-5), 7.71(2H,
d, J = 8.8 Hz, H-12/16), 7.05 (2H, d, J = 8.8 Hz, H-13/15), 3.79 (3H,
s, 14-OCH3); 13C NMR (DMSO-d6, 150 MHz) δ 165.8 (C-2), 160.3
(C-14), 152.2 (C-8c), 151.8 (C-8a), 145.3 (C-3a), 144.9 (C-7a), 137.8
(C-6), 131.3 (C-5), 129.7 (C-7), 128.9 (C-11), 128.3 (C-12/16),
126.9 (C-4), 116.5 (C-3b), 113.8 (C-13/15), 103.2 (C-3), 88.7 (C-
10), 55.4 (14-OCH3). Anal. Calcd: C, 55.49; H, 3.68; Br, 19.43;
N,13.62. Found: C, 49.39; H, 6.24; Br, 16.45; N. 14.35.31
Synthesis of 4-(2-Amino-1H-imidazol-5-yl)phenol (4). 5-(4-
Methoxyphenyl)-1H-imidazol-2-amine (100 mg, 0.53 mmol) was
dissolved in CH2Cl2 (5 mL), and the solution was cooled to 0 °C.
BBr3 (1 M, 1.1 mL, 1.1 mmol) was added, and the reaction was
allowed to warm to room temperature. H2O (2 mL) was added slowly
to the resulting heterogeneous mixture. The aqueous layer was loaded
directly onto a C18 column and eluted with a gradient of 5−95%
MeCN−H2O (no modifier) to provide 4-(2-amino-1H-imidazol-5-
yl)phenol, as the HBr salt, as a white solid (100 mg, 70%):32 IR (thin
film) λmax 3271, 3142, 3077, 1698, 1674, 1602, 1511, 1352, 1199 cm−1;
1H NMR (DMSO-d6, 400 MHz) δ 12.1 (bs, 2H), 9.70 (s, 1H), 7.44
(d, 2H, J = 8.4 Hz), 7.31 (s, 2H), 7.15 (s, 1H), 6.81 (d, 2H, J = 8.4
Hz); 13C NMR (DMSO-d6, 100 MHz) δ 157.4, 147.2, 127.0, 125.9,
118.7, 115.6, 107.4; (+)HRESIMS m/z 176.0816 [M + H]+ (calc for
C9H10N3O 176.0818).
Synthesis of Eudistidine C N-Methylpyrrole Analogue (7).
Eudistidine A HBr salt (20 mg, 0.048 mmol) was dissolved in DMSO
(0.5 mL), and N-methylpyrrole (6 mg, 0.072 mmol) was added. The
solution was stirred at 22 °C for 24 h and then purified by reversed-
phase preparative HPLC, employing a gradient of 5−95% MeCN−
H2O (0.1% TFA) over 10 min to yield 7 (13 mg, 56%): IR (thin film)
1
λmax 1663, 1638, 1510, 1301, 1256, 1177 cm−1; H NMR (CD3OD,
600 MHz) δ 9.01 (1H, d, J = 5.3 Hz, H-2), 8.74 (1H, d, J = 7.7 Hz, H-
4), 8.23 (1H, t, J = 7.9 Hz, H-6), 8.20 (1H, d, J = 8.1 Hz, H-7), 8.06
(1H, d, J = 5.3 Hz, H-3), 8.01 (1H, t, J = 7.8 Hz, H-5), 7.42 (2H, d, J =
8.8 Hz, H-12/16), 7.04 (2H, d, J = 8.8 Hz, H-13/15), 6.82 (1H, br t, J
= 2.2 Hz, H-19), 6.26 (1H, m, H-21), 6.09 (1H, br t, J = 3.3 Hz, H-
20), 3.82 (3H, s, H-14OCH3), 3.45 (3H, s, H−N18OCH3); 13C NMR
(CD3OD, 150 MHz) δ 167.4 (C-2), 162.2 (C-14), 154.3 (C-8c),
153.2 (C-8a), 148.4 (C-3a), 146.7 (C-7a), 138.8 (C-6), 132.2 (C-5),
130.9 (C-7), 130.6 (C-11), 129.6 (C-12/16), 128.9 (C-17), 128.2 (C-
19), 127.6 (C-4), 117.8 (C-3b), 115.7 (C-13/15), 112.8 (C-21), 108.1
(C-20), 103.8 (C-3), 71.4 (C-10), 55.9 (C-14OCH3), 36.4 (C-
N18OCH3); (+)HRESIMS m/z 394.1656 [M + H]+ (calcd for
C24H20N5O 394.1662).
Synthesis of Eudistidine C (1). Eudistidine A HBr salt (20 mg,
0.048 mmol) was dissolved in DMSO (0.5 mL), and 4-(2-amino-1H-
imidazol-5-yl)phenol (4, 15 mg, 0.072 mmol) was added. The solution
was allowed to stand for 24 h. The solution was purified by reversed-
phase preparative HPLC, employing a gradient of 5−95% MeCN/
H2O (0.1% TFA) over 10 min to yield eudistidine C (1, 18 mg, 64%):
IR (thin film) λmax 3268, 3100, 1665, 1638, 1525, 1303, 1190 cm−1; 1H
NMR (CD3OD, 600 MHz) see the Supporting Information; 13C
NMR (CD3OD, 150 MHz) see the Supporting Information;
Synthesis of Eudistidine C p-Phenol Analogue (8). To a
solution of eudistidine A HBr salt (6.0 mg, 0.014 mmol) in DMSO
(0.3 mL) was added phenol (53 mg, 0.57 mmol). The orange/brown
solution was stirred at 40 °C for 6 days, after which time the reaction
was purified directly by preparative reversed-phase HPLC employing a
H
DOI: 10.1021/acs.joc.6b02380
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
gradient of 10−95% MeCN/H2O (0.1% formic acid) over 8 min. The
product-containing fractions were concentrated in vacuo to afford 8
(1.8 mg, 27% yield) as a yellow solid: IR (thin film) λmax 3100, 1663,
AUTHOR INFORMATION
Corresponding Authors
*E-mail: martin.schnermann@nih.gov.
*E-mail: gustafki@mail.nih.gov.
ORCID
■
1
1639, 1509, 1303, 1180 cm−1; H NMR (CD3OD, 600 MHz) δ 8.44
(1H, d, J = 5.0 Hz, H-2), 8.22 (1H, d, J = 7.7 Hz, H-4), 7.89 (1H, m,
H-6), 7.87 (1H, m, H-7), 7.66 (1H, m, H-5), 7.44 (2H, d, J = 9.4 Hz,
H-12/16), 7.34 (2H, d, J = 9.5 Hz, H-18/22), 6.90 (2H, d, J = 8.8 Hz,
H-13/15), 6.78 (1H, d, J = 5.1 Hz, H-3), 6.76 (2H, d, J = 8.5 Hz, H-
19/21), 3.77 (3H, s, 14-OCH3); 13C NMR (CD3OD, 150 MHz) δ
167.4 (C-2), 162.5 (C-8a), 160.7 (C-14), 158.3 (C-20), 156.3 (C-8c),
147.6 (C-7a), 147.5 (C-3a), 136.4 (C-6), 135.9 (C-11), 134.6 (C-17),
129.9 (C-7), 129.8 (C-5), 129.8 (C-12/16), 129.8 (C-18/22), 126.3
(C-4), 117.9 (C-3b), 116.1 (C-19/21), 114.7 (C-13/15), 93.7 (C-3),
79.6 (C-10), 55.7 (14-OCH3); (+)HRESIMS m/z 407.1497 [M + H]+
(calcd for C25H19N4O2 407.1503).
Alexei V. Buevich: 0000-0002-5968-9151
Present Addresses
$(T.C.M.) Diagnostic Biomarkers and Technology Branch,
Cancer Diagnosis Program, DCTD, National Cancer Institute,
Bethesda, MD 20850.
&(E.A.S.) Baylor College of Medicine, 1 Baylor Plaza, Houston,
TX 77030.
Notes
Synthesis of Eudistidine C Resorcinol Analogue (9). To a
solution of eudistidine A HBr salt (10 mg, 0.025 mmol) in DMSO (0.5
mL) was added resorcinol (31 mg, 0.29 mmol). The orange/brown
solution was stirred at 40 °C for 24 h, after which time the reaction
was purified directly by preparative reversed-phase HPLC employing a
gradient of 10−95% MeCN/H2O (0.1% formic acid) over 8 min. The
product-containing fractions were concentrated in vacuo to afford 9
(7.2 mg, 62% yield) as a yellow solid: IR (thin film) λmax 3100, 1662,
1640, 1527, 1303, 1258, 1185 cm−1; 1H NMR (DMSO-d6, 600 MHz)
δ 8.42 (1H, d, J = 4.7 Hz, H-2), 8.28 (1H, d, J = 7.8 Hz, H-4), 7.86
(1H, t, J = 7.5 Hz, H-6), 7.78 (1H, d, J = 8.1 Hz, H-7), 7.65 (2H, d, J =
8.8 Hz, H-12/16), 7.61 (1H, t, J = 7.5 Hz, H-5), 6.96 (2H, d, J = 9.1
Hz, H-13/15), 6.68 (1H, d, J = 8.8 Hz, H-18), 6.79 (1H, d, J = 4.9 Hz,
H-3), 6.16 (1H, d, J = 2.3 Hz, H-21), 6.14 (1H, dd, J = 8.7, 2.5 Hz, H-
19), 3.74 (3H, s, 14-OCH3); 13C NMR (DMSO-d6, 150 MHz) δ 165.9
(C-2), 162.9 (C-8a), 158.7 (C-14), 158.2 (C-20), 156.9 (C-22), 154.4
(C-8c), 145.8 (C-7a), 145.3 (C-3a), 134.9 (C-6), 133.0 (C-11), 128.8
(C-18), 128.7 (C-12/16), 128.3 (C-7), 128.0 (C-5), 125.3 (C-4),
119.7 (C-17), 116.6 (C-3b), 113.5 (C-13/15), 105.6 (C-19), 103.1
(C-21), 91.7 (C-3), 76.1 (C-10), 55.1 (14-OCH3); (+)HRESIMS m/z
423.1446 [M + H]+ (calcd for C25H19N4O3 423.1452).
Synthesis of Eudistidine C Phloroglucinol Analogue (10). To
a solution of eudistidine A HBr salt (5.6 mg, 0.014 mmol) in DMSO
(1.0 mL) was added phloroglucinol (16.4 mg, 0.130 mmol). The
orange/brown solution was stirred at 22 °C for 24 h, after which time
the reaction was purified directly by preparative reversed-phase HPLC
(5−95% MeCN/0.1% TFA/water). The product-containing fractions
were concentrated in vacuo to afford 10 (4.2 mg, 55% yield) as a
yellow solid: IR (thin film) λmax 3101, 1672, 1627, 1562, 1257, 1183
cm−1; 1H NMR (CD3OD, 600 MHz) δ 8.77 (1H, d, J = 5.2 Hz, H-2),
8.12 (1H, d, J = 8.1 Hz, H-4), 7.63 (1H, t, J = 7.4 Hz, H-6), 7.52 (1H,
d, J = 5.3 Hz, H-3), 7.41 (2H, d, J = 8.9 Hz, H-12/16), 7.19 (1H, t, J =
7.8 Hz, H-5), 7.12 (1H, d, J = 8.4 Hz, H-7), 7.01 (2H, d, J = 8.9 Hz, H-
13/15), 6.07 (1H, d, J = 1.9 Hz, H-21), 5.85 (1H, d, J = 1.9 Hz, H-19),
3.63 (3H, s, 14-OCH3); 13C NMR (CD3OD, 150 MHz) δ 168.3 (C-
2), 163.3 (C-20), 162.0 (C-14), 160.6 (C-18), 157.1 (C-22), 156.5 (C-
8c), 149.2 (C-3a), 144.6 (C-7a), 138.4 (C-6), 130.4 (C-12/16), 127.7
(C-4), 126.8 (C-11), 123.1 (C-5), 118.7 (C-7), 115.0 (C-13/15),
111.6 (C-3b), 104.4 (C-3), 103.3 (C-17), 98.5 (C-21), 90.6 (C-19),
78.6 (C-10), 55.8 (14-OCH3), not observed (C-8a); (+)HRESIMS m/
z 439.1392 [M + H]+ (calcd for C25H19N4O4 439.1401).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge D. Newman (NCI) for organizing
and documenting the collection, the Natural Products Support
Group at NCIFrederick for extraction, T. Bostaph (MTL)
and K. Goncharova (MTL) for screening data compilation and
analysis, J. Barchi (CBL) for NMR assistance, J. Kelly (CBL)
for mass spectrometry analysis, and S. Tarasov and M. Dyba
(Biophysics Resource Core, Structural Biophysics Laboratory,
CCR) and H. Bokesch (MTL) for assistance with high-
resolution mass spectrometry. This research was supported in
part by the Intramural Research Program of the NIH, National
Cancer Institute, Center for Cancer Research. This project was
also funded in part with Federal funds from the National
Cancer Institute, National Institutes of Health, under contract
HHSN261200800001E and by the USDA Agricultural
Research Service Specific Cooperative Agreement No. 58-
6408-1-603. The content of this publication does not
necessarily reflect the views or policies of the Department of
Health and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorsement by
the US Government.
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ASSOCIATED CONTENT
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Lett. 2010, 12, 4752−4755.
S
* Supporting Information
The Supporting Information is available free of charge on the
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