Santacruzamate A, a potent and selective histone deacetylase inhibitor from the panamanian marine cyanobacterium cf. symploca sp.

Article abstract of DOI:10.1021/np400198r

A dark brown tuft-forming cyanobacterium, morphologically resembling the genus Symploca, was collected during an expedition to the Coiba National Park, a UNESCO World Heritage Site on the Pacific coast of Panama. Phylogenetic analysis of its 16S rRNA gene sequence indicated that it is 4.5% divergent from the type strain for Symploca and thus is likely a new genus. Fractionation of the crude extract led to the isolation of a new cytotoxin, designated santacruzamate A (1), which has several structural features in common with suberoylanilide hydroxamic acid [(2), SAHA, trade name Vorinostat], a clinically approved histone deacetylase (HDAC) inhibitor used to treat refractory cutaneous T-cell lymphoma. Recognition of the structural similarly of 1 and SAHA led to the characterization of santacruzamate A as a picomolar level selective inhibitor of HDAC2, a Class I HDAC, with relatively little inhibition of HDAC4 or HDAC6, both Class II HDACs. As a result, chemical syntheses of santacruzamate A as well as a structurally intriguing hybrid molecule, which blends aspects of both agents (1 and 2), were achieved and evaluated for their HDAC activity and specificity.

Full text of DOI:10.1021/np400198r

Article
pubs.acs.org/jnp
Santacruzamate A, a Potent and Selective Histone Deacetylase
Inhibitor from the Panamanian Marine Cyanobacterium cf. Symploca
sp.
Christopher M. Pavlik,† Christina Y. B. Wong,‡ Sophia Ononye,† Dioxelis D. Lopez,‡ Niclas Engene,§
Kerry L. McPhail,⊥ William H. Gerwick,§,∥ and Marcy J. Balunas*,†,‡,§,∥
†Division of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269,
United States
‡Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT), Ciudad del Saber, Clayton, Apartado Postal
0816-02852, Panama City, Panama
́
§Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography and Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, United States
⊥College of Pharmacy, Oregon State University, Corvallis, Oregon 97331, United States
∥Smithsonian Tropical Research Institute (STRI), Ancon
, Apartado Postal 0843-03092, Panama City, Panama
́ ́
S
* Supporting Information
ABSTRACT: A dark brown tuft-forming cyanobacterium,
morphologically resembling the genus Symploca, was collected
during an expedition to the Coiba National Park, a UNESCO
World Heritage Site on the Pacific coast of Panama.
Phylogenetic analysis of its 16S rRNA gene sequence indicated
that it is 4.5% divergent from the type strain for Symploca and
thus is likely a new genus. Fractionation of the crude extract
led to the isolation of a new cytotoxin, designated santacruzamate A (1), which has several structural features in common with
suberoylanilide hydroxamic acid [(2), SAHA, trade name Vorinostat], a clinically approved histone deacetylase (HDAC)
inhibitor used to treat refractory cutaneous T-cell lymphoma. Recognition of the structural similarly of 1 and SAHA led to the
characterization of santacruzamate A as a picomolar level selective inhibitor of HDAC2, a Class I HDAC, with relatively little
inhibition of HDAC4 or HDAC6, both Class II HDACs. As a result, chemical syntheses of santacruzamate A as well as a
structurally intriguing hybrid molecule, which blends aspects of both agents (1 and 2), were achieved and evaluated for their
HDAC activity and specificity.
he packaging of DNA into nucleosomes is an essential
process for gene transcription in eukaryotic cells, one step
such as cancer,6,7 HIV/AIDS,8 stroke and other neurological
disorders,9,10 and parasitic diseases.11 The first clinically useful
HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA, 2, or
Vorinostat), was approved for clinical use in 2006 in patients
with refractory cutaneous T-cell lymphoma.12 In 2009, another
HDAC inhibitor, romidepsin (Istodax), was also approved for
cutaneous T-cell lymphoma.13
There are several classes of HDAC enzymes of human
relevance, including Class I (HDAC 1−3, 8), Class IIa (HDAC
4, 5, 7, 9), Class IIb (HDAC 6, 10), Class III (sirtuins 1−7),
and Class IV (HDAC 11).14 The first HDAC inhibitor
approved for clinical use, SAHA (2), is considered a pan-
HDAC inhibitor with activity across several classes of HDAC
enzyme. Romidepsin, the only other HDAC inhibitor currently
on the market, has selectivity for Class I HDAC isozymes but is
not selective within the class, exhibiting activity against HDACs
1, 2, 3, and 8.15,16 Another marine cyanobacterial metabolite,
T
of which involves the winding of DNA around histone proteins
to form chromatin.1,2 Histone deacetylases (HDACs) and
histone acetyltransferases (HATs) are key enzymes that
regulate this process, and thus they have substantial impact
on gene transcription. HDAC enzymes remove acetyl groups
from lysine residues in histones, thus increasing their overall
positive charge and the resulting binding affinity between
histones and negatively charged DNA; this process leads to
repression of gene transcription. In contrast, HATs acetylate
histones, thus reducing their positive charge and allowing
unwinding of the DNA that must precede gene transcription or
replication.3 HDAC inhibition prevents the removal of acetyl
groups from histones, causing their accumulation in cell nuclei
and resulting in several downstream effects including apoptosis,
differentiation, and reduced proliferation of cancer cells.1,3,4 In
addition, HDACs have recently been shown to have a
multitude of non-histone protein substrates, which may also
contribute to these downstream cellular effects.5 Such effects
are of potential relevance to the treatment of several diseases
Received: March 8, 2013
Published: October 28, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Molecular-phylogenetic inference of the SCA-producing strain PAC-19-FEB-10-1 (GenBank acc. no. JX458089.1, highlighted with an
arrow). The closest related group is the genus Symploca (reference strain: PCC 8002T, GenBank acc. no. AB039021) with a p-distance of 4.5% based
on the SSU rRNA gene sequence divergence (gray box). The “tropical marine Symploca” clades are highlighted with orange boxes. The phylogram is
based on SSU (16S) rRNA gene sequences using the bayesian (MrBayes) and maximum likelihood (PhyML) methods, and support values are
indicated as posterior probability and bootstrap support at nodes. Specimens designated with (R) represent reference-strains obtained from Bergey’s
Manual. The scale bar is indicated at 0.03 expected nucleotide substitution per site, corrected using the general time reversal (GTR) model.
largazole, isolated from another Symploca sp., has distinctive
structural similarity to the active moiety of romidepsin and also
selectively inhibits Class I HDACs, although does not show
selectivity within the class.16 This general lack of selectivity is
believed to result in many of the undesirable side effects of
these HDAC-targeting agents,14 and recent research has
focused on the search for isozyme-selective HDAC inhibitors.17
It is hoped that agents with potent activity against one HDAC
isozyme, with little or no effect on other isozymes, will result in
increased clinical utility and a decreased incidence of side
effects.
The Panama-based International Cooperative Biodiversity
Group (ICBG) has focused on natural product drug discovery
from Panamanian plants and microorganisms with a particular
emphasis on the discovery of antiparasitic and anticancer lead
molecules.18−20 In this regard, our work with marine
cyanobacteria has been especially productive, and a number
of anticancer and antiparasitic agents have been isolated from
these organisms.21−23 In the current work, we report on the
isolation of a selective HDAC inhibitor with extraordinary
potency against HDAC2, a Class I HDAC enzyme. Due to the
collection of the source organism from near Santa Cruz Island
in Panama’s Coiba National Park, a UNESCO World Heritage
Site, this carbamate derivative has been named santacruzamate
A (1).
RESULTS AND DISCUSSION
■
A dark brown cyanobacterium, morphologically resembling the
genus Symploca (strain PAC-19-FEB-10-1, Figure S1), was
collected from a coral and rock reef near Santa Cruz Island
during an expedition to Coiba National Park on the Pacific
coast of Panama. Microscopically, the specimen is composed of
fine (9−10 μm wide) filaments with isodiametric cells covered
with a barely visible sheath (Figure S1, Supporting
Information).
The SSU (16S) rRNA gene sequence was obtained from the
strain PAC-19-FEB-10-1 (GenBank acc. no. JX458089.1, Figure
1) and used to infer the evolution of this specimen in relation
to other groups of cyanobacteria. This phylogenetic inference
revealed that the closest related reference strain was Symploca
atlantica PCC 8002R (GenBank acc. no. AB039021). However,
the uncorrected gene sequence divergence between this clade
and the original type-strain was 4.5% over 1162 base pairs in
the 16S rRNA gene. This high evolutionary divergence, in
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combination with distinct biogeographic and ecological
divergences, suggested that strain PAC-19-FEB-10-1 composes
an independent group, distinct from the genus Symploca.
Phylogenetically closely related to PAC-19-FEB-10-1 was the
hoiamide C-producing strain PNG05-8 (GenBank acc. no.
HM072003) from Papua New Guinea (p-distance = 0.1% gene
sequence divergence).24 It should be noted that there is a
second lineage of “tropical marine Symploca” (p-distance =
5.8% gene sequence divergence from the “tropical marine
Symploca” clade containing PAC-19-FEB-10-1 and PNG05-8),
which includes the dolastatin 10-producing strain VP642b
(AY032933) and the symplostatin 1-producing strain VP377
(AF306497).25 Thus, it becomes clear that not only should
“tropical marine Symploca” be separated from the current genus
Symploca but also there are at least two different groups within
the “tropical marine Symploca” that may be distinct from one
another at the genus level.
A polar fraction from the initial normal-phase flash
chromatography of the crude extract, fraction H (eluted with
3:1 ethyl acetate−methanol), was tested at 10 μg/mL and
found to have potent activity against the malaria parasite
(99.9% inhibition of parasite growth) and MCF-7 cancer cells
(50% cell death indicated by negative growth, perhaps
indicative of overt cytotoxicity), with no activity in
leishmaniasis or Chagas’ disease assays (7.9% and 9.2%
inhibition, respectively). Isolation efforts continued using
reversed-phase solid-phase extraction (RP-SPE) and RP high-
performance liquid chromatography (HPLC) and yielded a
single molecule, santacruzamate A (1).
Table 1. NMR Data for Santacruzamate A (1) in CDCl3
a
b
position δH, mult. (J in Hz)
δC, mult.
COSY HMBC (H→C)
1
7.23, m
7.30, m
7.22, m
126.5, CH
128.8, CH
128.6, CH
138.9, qC
128.6, CH
128.8, CH
35.7, CH2
40.6, CH2
2, 6
1, 3
2
3, 5
2
4, 6
3
1, 5, 7
4
5
7.22, m
6
1, 3, 7
2, 4
6
7.30, m
1, 5
8
7
2.83, t (6.8)
3.53, q (6.8)
5.92, br s
3, 4, 5, 8
4, 7, 10
8
7, 9
8
9
10
11
12
13
14
15
16
17
172.5, qC
33.7, CH2
26.1, CH2
40.2, CH2
2.17, t (6.9)
1.80, pentet (6.8)
3.18, q (5.9)
4.92, br s
12
10, 12, 13
10, 11, 13
11, 12, 15
11, 13
12, 14
13
157.1, qC
60.8, CH2
14.7, CH3
4.10, q (6.9)
17
16
15, 17
16
1.23, t (7.3)
a
b
Measured at 400 MHz. Measured at 100 MHz.
H-8, H-11, and H-12 to an amide carbonyl at δC 172.5 (C-10)
established the connectivity of partial structures 1b and 1c and
further identified partial structure 1b as a γ-aminobutyric acid
residue. Finally, HMBC correlations from both H-13 and H-16
to an unusually shielded carbonyl at δC 157.1 (C-15) were used
to connect partial structures 1a and 1b. The distinctive
chemical shift of this C-15 carbonyl, along with the remaining
atoms required by the molecular formula, was consistent with a
carbamate functionality spanning these two spin systems. The
final, achiral molecular structure of santacruzamate A (1) was
thus established.
By HRESIMS, a [M + H]+ peak consistent with a molecular
formula of C15H22N2O3 was obtained, indicative of six degrees
of unsaturation. 1H NMR spectroscopic analysis of the purified
metabolite (1) revealed a relatively uncomplicated NMR
spectrum with two amide protons (δH 5.92 and 4.92), five
phenyl protons (δH 7.22−7.30), six sets of methylene protons
(δH 4.10, 3.53, 3.18, 2.83, 2.17, and 1.80), and one methyl
group (δH 1.23). The dispersions in chemical shift and coupling
patterns helped to determine the respective linkage of these
protons in four distinct spin systems (1a−1d). The most
deshielded methylene signal, a quartet at δH 4.10, was
consistent with protons attached to a carbon bearing an
oxygen atom (by HSQC, δC 60.8) and proximal to a methyl
Figure 2. Partial structures of santcruzamate 1a-1d, and their
connectivity by HMBC (arrows).
Santacruzamate A (1) has several structural features in
common with SAHA (2), a clinically approved histone
deacetylase inhibitor used to treat refractory cutaneous T-cell
lymphoma.12 The target of SAHA includes all isozyme subtypes
of histone deacetylases, the consequence of which is to
upregulate the transcription of cell cycle regulators, nuclear
transcription factors, and pro-apoptotic genes, thus bringing
about an overall antineoplastic effect. Some structural similarity
to 1 can also be seen with two other marine natural products:
psammaplin H (4), a moderate HDAC inhibitor isolated from a
marine sponge with which santacruzamate shares the ethyl
carbamate functionality,26 and grenadamide (5) from a marine
cyanobacterium, with which santacruzamate shares the
phenethylamine moiety and which has shown activity in a
central nervous system (CNS) assay.27
SAHA (2) binds to HDAC enzymes such that the phenyl cap
sits above the enzyme pocket into which the aliphatic chain
inserts, positioning the hydroxamic acid adjacent to the
enzymatic zinc at the distal end of the pocket.2 Given this
binding mechanism and the structural similarity between 1 and
SAHA, it was hypothesized that santacruzamate A might also be
an HDAC inhibitor. Consequently, the chemical syntheses of
1
group (for which the resonance was located by H−1H COSY
at δH 1.23), thus defining partial structure 1a. Two methylene
groups in different spin systems had 1H and 13C NMR chemical
shifts indicative of proximity to nitrogen atoms (δH 3.53 and
3.18), both of which resonated as quartets. Delineation of a
second spin system (1b) began with the higher field of these
quartets (δH 3.18; δC 40.2), which was coupled to both an
1
amide H singlet at δH 5.92 and a methylene resonance at δH
1.80 (CH2-12, δC 26.1). The pentet splitting pattern of this
latter signal suggested a third contiguous methylene group, and
this was confirmed via COSY correlations between the H2-12
and H2-11 (δH 2.17; δC 33.7) signals. The more downfield
quartet for a methylene adjacent to nitrogen (CH2-8, δH 3.53
and δC 40.6) was proximate to another methylene (CH2-7, δH
2.83, δC 35.7), thereby defining a −CH2−CH2− spin system
(1c). The fourth spin system (1d) was represented by three
overlapping 1H multiplets in the aromatic region that integrated
to five protons, as is typical of a monosubstituted phenyl group
(Table 1). HMBC correlations from H-7 (δH 2.83) and H-8
(δH 3.53) to C-4 (δC 138.9) linked partial structures 1c and 1d
to define a phenethylamine moiety. HMBC correlations from
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and 3 at 1 μM selectively inhibited HDAC2 with relatively little
inhibition of HDAC4 (data not shown). In contrast, at 1 μM
SAHA completely inhibited both Class I and II HDAC
enzymes. On the basis of these promising results, IC50 values
for the natural product 1, synthetic 1, the 1−SAHA hybrid, and
authentic SAHA were determined using HDAC2, HDAC4, and
HDAC6, a Class IIb HDAC. Against HDAC2, SAHA (2)
showed an IC50 of 85.8 nM, whereas natural and synthetic 1
yielded values of 119 and 112 pM, respectively. Thus, 1 is over
700-fold more potent for HDAC2 than the clinically useful
drug SAHA. The 1−SAHA hybrid (3) was of diminished
activity compared to 1, with an IC50 value of 3.5 nM against
HDAC2. Intriguingly, both samples of 1 and the 1−SAHA (3)
hybrid were found to have IC50 values of more than 1 μM
against HDAC4, indicating a strong selectivity for Class I
HDAC inhibition. Santacruzamate A (1) and 1−SAHA (3)
were further tested against HDAC6 and found to have IC50
values of 433.5 and 385.8 nM, respectively, while SAHA (2)
showed limited selectivity, with an IC50 of 38.9 nM (Table 2).
The combination of picomolar potency and the greater than
3500-fold selectivity for Class I HDACs makes santacruzamate
A (1) an exciting lead compound.
Subsequently, the natural product 1 and synthetic com-
pounds were tested for cytotoxicity to HCT-116 colon
carcinoma cells, HuT-78 cutaneous T-cell lymphoma cells,
and human dermal fibroblast (hDF) cells. SAHA (2) was used
as the positive control and showed a GI50 value of 0.4 μM in
HCT-116 cells, 3.0 μM for HuT-78 cells, and 6.1 μM in hDF
cells (Table 2). 1−SAHA was slightly less potent in HCT-116
cells (GI50 of 2.3 μM) but of somewhat greater potency against
HuT-78 cells (GI50 0.7 μM), with no activity using hDF cells
(GI50 >100 μM). The natural and synthetic 1 were found to be
of only moderate potency in HCT-116 cells, with GI50 values of
29.4 and 28.3 μM, respectively. However, both potently
inhibited the growth of HuT-78 cells with GI50 values of 1.4
and 1.3 μM, respectively, and neither exhibited activity against
hDF cells (GI50 >100 μM). The 20-fold differential activity of 1
found against HCT-116 and HuT-78 cells poses several
interesting avenues for further research, including determining
if these cellular responses are due to HDAC inhibition or to
other cellular effects. Additional testing using the National
Cancer Institute (NCI) in vitro panel of 60 cancer cell lines is
ongoing, and studies to investigate the levels of cellular
acetylation are planned.
compound 1 and a structurally intriguing hybrid structure
blending aspects of both 1 and SAHA were undertaken (Figure
3). In brief, GABA was converted to the carbamate
intermediate 4-((ethoxycarbonyl)amino)butanoic acid via
reaction with K2CO3 and ethyl chloroformate in water (76%
yield).28 This intermediate was coupled to phenethylamine
using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydro-
chloride (EDC-HCl), triethylamine (TEA), and catalytic 4-
(dimethylamino)pyridine (DMAP) to yield santacruzamate A
(1, 92% yield, overall yield 70%). To create an 1−SAHA hybrid
structure (3, Figure 3B), phenethylamine was coupled to
monomethyl glutarate via the peptide coupling described above
(88% yield), and then this intermediate was converted to the
hydroxamic acid via standard literature procedures using
hydroxylamine hydrochloride and KOH in methanol to afford
compound 3 (90% yield, overall yield 79%).29
The synthetic products 1 and 3, as well as the original natural
product santacruzamate A (1), were all evaluated for isozyme-
selective inhibition of HDAC2, a Class I HDAC, and HDAC4,
a Class IIa HDAC. Initial results revealed that compounds 1
Given the remarkable potency and selective HDAC
inhibition of santacruzamate A (1), it is of interest to create
structural analogues so as to enhance cellular activity as well as
Figure 3. Synthetic schemes for preparation of (A) SCA (1) and (B) the SCA−SAHA hybrid (3).
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Table 2. Biological Activity of Santacruzamate (1) and Synthetic Compounds against Class I and Class II HDACs Using
HDAC2, HDAC4, and HDAC6 as Well as in Cellular Cytotoxicity Testing Using HCT-116 Colon Cancer, HuT-78 Cutaneous
T-Cell Lymphoma, and Human Dermal Fibroblast (hDF) Cells
HDAC2
HDAC4
HDAC6
HCT116
Hut-78
hDF
a
IC50 (nM)
IC50 (nM)
IC50 (nM)
SI
GI50 (μM)
GI50 (μM)
GI50 (μM)
1 (natural product)
1 (synthetic)
3
0.119
0.112
3.5
>1000
>1000
>1000
n.d.
434
433
386
38.9
3647
3866
110
29.4
28.3
2.3
1.4
1.3
0.7
3.0
>100
>100
>100
6.1
2 (positive control)
85.8
0.33
0.4
a
SI = selectivity index calculated as HDAC6/HDAC2; n.d. = no data.
obtained. The extract was fractionated using flash Si gel column
chromatography (Aldrich, Si gel 60, 230−400 mesh, 40 × 180 mm)
using 300 mL each of 100% hexanes (A), 9:1 hexanes−EtOAc (B), 4:1
hexanes−EtOAc (C), 3:2 hexanes−EtOAc (D), 2:3 hexanes−EtOAc
(E), 1:4 hexanes−EtOAc (F), 100% EtOAc (G), 3:1 EtOAc−MeOH
(H), and 100% MeOH (I). Fraction H exhibited strong antimalarial
activity (99.9% inhibition of parasite growth at 10 μg/mL) and was
subjected to further fractionation using a Burdick & Jackson C18 RP-
SPE cartridge with a MeOH−H2O solvent gradient (1:1, 3:2, 7:3, 4:1
MeOH−EtOAc, 100% MeOH, 100% EtOAc). The fraction eluting
with 1:1 MeOH−H2O was subjected to RP-HPLC purification (55%
MeOH−45% H2O, 1.0 mL/min) to yield santacruzamate A (1, 4.0
mg, tR 10.9 min, 0.19% of extract).
to determine its mechanism of enzymatic inhibition. Due to its
small and linear peptide-type structure, 1 is amenable to
synthetic modifications to explore structure−activity relation-
ships, especially considering the structural similarities to known
HDAC-active compounds. Further modification of 1 could
result in the design of more potent analogues with the potential
for preclinical development and provide opportunities for facile
chemical scale-up. Indeed, the robust synthesis reported herein
has been employed to produce a focused library of additional
analogues, the biological properties of which are currently
under evaluation and will be reported in due course.
EXPERIMENTAL SECTION
Santacruzamate A (1): white, amorphous solid; mp 112−113 °C;
■
IR νmax (film) 3345, 3288, 1703, 1699, 1655, 1543, 1538, 1446, 1307,
General Experimental Procedures. Melting points were
collected on a Mel-Temp digital melting point apparatus. IR spectra
were recorded on a Shimadzu FT-IR 8400 spectrometer. NMR spectra
for the natural product SCA (1) were obtained on a JEOL Eclipse 400
MHz spectrometer and referenced to TMS. All other NMR spectra
1
1285, 1249, 1223, 1139, 1050, 1031 cm−1; H and 13C NMR data
(CDCl3, 400 and 100 MHz, respectively), see Table 1; ESIMS m/z
(%) 301.1 (8, [M + Na]+), 280.2 (25), 279.3 (100, [M + H]+);
HRESIMS [M + H]+ m/z 279.1721 (calcd for C15H23N2O3,
279.1709).
Morphological Characterization. Morphological characteriza-
tion was performed using an Olympus IX51 epifluorescent microscope
(1000×) equipped with an Olympus U-CMAD3 camera. Measure-
were recorded on a Bruker Avance 500 MHz spectrometer (500.13
̈
MHz 1H, 125.65 MHz 13C), with chemical shifts given in ppm
downfield from TMS. LC-MS data were collected on an Agilent ESI
single quadrupole mass spectrometer coupled to an Agilent HPLC
system with a G1311 quaternary pump, G1322 degasser, and a G1315
diode array detector using an Eclipse XDB-C18 (4.6 × 150 mm, 5 μm)
RP-HPLC column. High-resolution mass spectrometric data (HRMS)
were collected on a Micromass VB-QTOF tandem mass spectrometer.
HPLC purifications of natural product isolates were carried out on a
Merck Hitachi LaChrom HPLC system with an L-7100 pump, an L-
7614 degasser, and an L-7455 diode array detector using a Prontosil-
120 C18 (4.6 × 250 mm, 5 μM) RP-HPLC column, with the solvent
systems as indicated below. All chemicals were used as received from
Sigma-Aldrich or Acros without further purification. Hexanes,
tetrahydrofuran (THF), diethyl ether (Et2O), and dichloromethane
(CH2Cl2) were used directly from a Baker Cycle-tainer system. All
glassware was flame-dried under a vacuum, and all reactions were
performed under an argon atmosphere, unless otherwise noted. Thin-
layer chromatography was carried out on Fluka glass-backed TLC
plates with a fluorescent indicator and 0.2 mm silica gel layer thickness,
and p-anisaldehyde was used as a developing agent. Column
chromatography was done using 60 Å porosity, 32−63 μm silica gel.
Structural integrity and purity of the test compounds were determined
ments were provided as means
standard deviation. The filament
means were the average of three filament measurements, and cell
measurements the average of 10 adjacent cells in each of three
filaments. Morphological comparison and putative taxonomic
identification of the cyanobacterial specimen were performed in
accordance with modern classification systems.30,31
Gene Sequencing. Cyanobacterial specimens were preserved for
genetic analysis both as live material and in 10 mL RNAlater
(Ambion). Algal biomass (∼50 mg) was partly cleaned under an
Olympus VMZ dissecting microscope. Genomic DNA was extracted
using the Wizard Genomic DNA purification kit (Promega) following
the manufacturer’s specifications. DNA concentration and purity were
measured on a DU 800 spectrophotometer (Beckman Coulter). The
16S rRNA genes were PCR-amplified from isolated DNA using the
general primer set 106F and 1509R,32 while subsequent PCR reactions
were performed using the modified lineage-specific primers. The PCR
reaction volumes were 25 μL containing 0.5 μL (∼50 ng) of DNA, 2.5
μL of 10 × PfuUltra IV reaction buffer, 0.5 μL (25 mM) of dNTP mix,
0.5 μL of each primer (10 μM), 0.5 μL of PfuUltra IV fusion HS DNA
polymerase, and 20.5 μL of dH2O. The PCR reactions were performed
with an Eppendorf Mastercycler gradient as follows: initial
denaturation for 2 min at 95 °C, 25 cycles of amplification, followed
by 20 s at 95 °C, 20 s at 55 °C, and 1.5 min at 72 °C, and final
elongation for 3 min at 72 °C. PCR products were analyzed on agarose
gel (1%) in SB buffer and visualized by EtBr staining. The PCR
products were purified using a MinElute PCR purification kit (Qiagen)
before subcloning using the Zero Blunt TOPO PCR cloning kit
(Invitrogen), following the manufacturer’s specifications. Plasmid
DNA was isolated using the QIAprep Spin Miniprep kit (Qiagen)
and sequenced with M13 primers. The 16S rRNA gene sequence is
available in the DDBJ/EMBL/GenBank databases under accession
number JX458089-1.
1
by the composite of H and 13C NMR spectroscopy, melting point
range, LC-MS, and HRMS, and compounds were found to be >95%
pure.
Collection, Extraction, and Isolation. A cyanobacterium
morphologically resembling the genus Symploca (Figure S1) was
collected in March 2007 by hand using SCUBA at depths of 30−45
feet. The collection site was a coral and rock reef (Figure 1A) in Coiba
National Park (7°37.980 N, 81°47.091 W) in Veraguas, Panama. After
straining through a mesh bag to remove excess seawater, the sample
was stored in 1:1 EtOH−seawater at −20 °C. The voucher specimen,
number PAC-03/03/2007-1, is deposited at Scripps Institution of
Oceanography, UCSD (San Diego, CA, USA). The sample (221.5 g
dry weight) was thawed and extracted exhaustively with 2:1 CH2Cl2−
MeOH. After solvent evaporation, 2.1 g of a crude organic extract was
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Phylogenetic Inference. The 16S rRNA gene sequence of PAC-
19-FEB-10-1 was aligned with evolutionarily informative cyanobacteria
using the L-INS-I algorithm in MAFFT 6.71733 and refined using the
SSU secondary structure model for Escherichia coli J0169534 without
data exclusion. The best-fitting nucleotide substitution model
optimized by maximum likelihood (ML) was selected using corrected
Akaike/Bayesian information criterion (AICC/BIC) in jModeltest
0.1.1.35 The evolutionary histories of the cyanobacterial genes were
inferred using ML and Bayesian inference (BI) algorithms. The ML
inference was performed using PhyML36 for the GTR+I+G model
assuming heterogeneous substitution rates and gamma substitution of
variable sites (proportion of invariable sites (pINV) = 0.495, shape
parameter (α) = 0.452, number of rate categories = 4) with 1000
bootstrap-replicates. The Bayesian inference was conducted using
MrBayes 3.137 with four Metropolis-coupled MCMC chains (one cold
and three heated) run for 1 000 000 generations. The first 25% were
discarded as burn-in, and the following data set was sampled with a
frequency of every 100 generations.
Methyl 4-(Phenethylcarbamoyl)butanoate. Monomethyl glutarate
(1.00 g, 6.84 mmol) was taken up in CH2Cl2 (12 mL) and cooled to 0
°C. Phenethylamine (0.95 mL, 7.53 mmol) and triethylamine (1.91
mL, 13.68 mmol) were added to the solution followed by 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (1.44 g, 7.53
mmol) in one portion. 4-Dimethylaminopyridine (cat.) was added,
and the solution was stirred overnight at rt. The resulting mixture was
diluted with additional CH2Cl2 (20 mL) and washed sequentially with
20 mL of each of the following: 1.0 M HCl, saturated NaHCO3, H2O,
brine. The organic phase was then passed through Celite, dried over
Na2SO4, and concentrated to yield 4-(phenethylcarbamoyl)butanoate
(1.50 g, 6.02 mmol, 88% yield) as a clear crystalline solid: mp 58.3−
1
59.8 °C; H NMR (500 MHz, CDCl3) δ 7.30−7.36 (2H, m), 7.19−
7.28 (3H, m), 5.57 (1H, br s), 3.68 (3H, s), 3.52−3.57 (2H, m), 2.84
(2H, t, J = 6.94 Hz), 2.37 (2H, t, J = 7.25 Hz), 2.18−2.23 (2H, m),
1.95 (2H, pentet, J = 7.25 Hz); 13C NMR (126 MHz, CDCl3) δ 173.6,
172.0, 138.8, 128.6, 128.7, 126.5, 51.6, 40.5, 35.7, 35.5, 33.0, 20.8;
HRESIMS [M + H]+ m/z 250.1431 (calcd for C14H20NO3, 250.1443).
Synthesis of Santacruzamate A (1) and 1−SAHA Hybrid (3).
Full experiemental details are provided below for synthetic
intermediates and products of 1 and the 1−SAHA hybrid (3).
4-[(Ethoxycarbonyl)amino]butanoic Acid. γ-Aminobutyric acid
(0.50 g, 4.85 mmol) was dissolved in H2O (7 mL). Once dissolved,
K2CO3 (1.74 g, 12.6 mmol) was added, and the resulting solution was
cooled to 0 °C. Ethyl chloroformate (0.63 mL, 6.31 mmol) was added
dropwise, and the solution was stirred at 0 °C for 2 h and then stirred
overnight at rt. The reaction mixture was then diluted with H2O (20
mL) and extracted with EtOAc (3 × 10 mL). The aqueous phase was
acidified to pH 2 with cold concentrated HCl and extracted with
EtOAc (3 × 20 mL). The EtOAc partitions were dried over Na2SO4
and concentrated to reveal a white solid, which was recrystallized from
cold hexanes to yield long white crystals (0.645 g, 3.68 mmol, 76%
N1-Hydroxy-N5-phenethylglutaramide (1−SAHA hybrid, 3). Hy-
droxylamine hydrochloride (5.55 g, 79.8 mmol) in methanol (150 mL)
was mixed with KOH (4.48 g, 79.8 mmol) at 40 °C in methanol (22
mL), cooled to 0 °C, and filtered. The butyric acid methyl ester (1.08
g, 4.43 mmol) was then added to the filtrate followed by addition
(over 30 min) of KOH (0.36 g, 6.49 mmol). The mixture was stirred
at rt overnight. The resulting solution was concentrated and
resuspended in 50 mL of ice cold H2O, and the pH was adjusted to
7.0 using acetic acid. The solution was cooled overnight at 0 °C to
yield 3 (0.98 g, 4.13 mmol, 90%) as a white, crystalline solid: mp 106−
107 °C; IR νmax (film) 3302, 3177, 2920, 2360, 2340, 1623, 1617,
1565, 1458, 1419, 1195, 1033, 940 cm−1; 1H NMR (500 MHz,
methanol-d4) δ 7.30−7.37 (2H, m), 5.52 (1H, br s), 3.51−3.58 (2H,
m), 2.84 (2H, t, J = 6.94 Hz), 2.37 (2H, t, J = 7.25 Hz), 2.21 (2H, t, J =
7.41 Hz), 1.96 (2H, pentet, J = 7.25 Hz); 13C NMR (126 MHz,
methanol-d4) δ 173.8, 170.8, 139.1, 128.4, 128.1, 125.9, 40.6, 35.1,
34.8, 31.6, 21.7; ESIMS m/z (%) 273.1 (8, [M + Na]+), 252.1 (15),
251.1 (100, [M + H]+), 218.2 (10); HRESIMS [M + H]+ m/z
251.1370 (calcd for C13H19N2O3, 251.1396).
1
yield): mp 44.1−45.0 °C; H NMR (500 MHz, methanol-d4) δ 4.12
(2H, q, J = 6.94 Hz), 2.85 (2H, t, J = 6.94 Hz), 2.20 (2H, t, J = 6.94
Hz), 1.82 (2H, pentet, J = 6.78 Hz), 1.25 (3H, t, J = 7.25 Hz); 13C
NMR (methanol-d4) δ 177.3, 157.1, 60.8, 40.6, 35.7, 26.1, 14.7.
Biological Assays. All assays were run using established protocols
or following kit directions. Brief experimental details are provided
below.
Ethyl 3-(Phenethylcarbamoyl)propylcarbamate (santacruza-
mate A, 1). 4-[(Ethoxycarbonyl)amino]butanoic acid (0.40 g, 2.28
mmol) was dissolved in CH2Cl2 (7 mL) and cooled to 0 °C.
Phenethylamine (0.327 mL, 2.60 mmol) and triethylamine (0.64 mL,
4.56 mmol) were added to the solution followed by 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (0.50 g, 2.60
mmol) in one portion. 4-Dimethylaminopyridine (cat.) was added,
and the solution was stirred at 0 °C for 60 min, then overnight at rt.
The resulting solution was diluted with additional CH2Cl2 (20 mL)
and washed sequentially with 10 mL of each of the following: 1.0 M
HCl, saturated NaHCO3, H2O, brine. The organic layer was dried over
Na2SO4 and concentrated to give a residue that was recrystallized by
trituration with hexanes. Upon cooling to 0 °C, the solution was
filtered to yield 1, which was obtained as a white solid (0.58 g, 92%
yield): mp 112−113 °C; IR νmax (film) 3345, 3288, 1703, 1699, 1655,
1543, 1538, 1446, 1307, 1285, 1249, 1223, 1139, 1050, 1031 cm−1; 1H
NMR (500 MHz, CDCl3) δ 7.30−7.36 (2H, m), 7.20−7.28 (3H, m),
5.96 (1H, br s), 4.96 (1H, br s), 4.12 (2H, q, J = 6.94 Hz), 3.55 (2H, q,
J = 6.73 Hz), 3.20 (2H, q, J = 5.88 Hz), 2.85 (2H, t, J = 6.94 Hz), 2.20
(2H, t, J = 6.94 Hz), 1.82 (2H, pentet, J = 6.78 Hz), 1.25 (3H, t, J =
7.25 Hz); 13C NMR (126 MHz, CDCl3) δ 172.5, 157.1, 138.9, 128.8,
128.6, 126.5, 60.8, 40.6, 40.2, 35.7, 33.7, 26.1, 14.7; ESIMS m/z (%)
301.1 (8, [M + Na]+), 280.2 (25) 279.3 (100, [M + H]+); HRESIMS
[M + H]+ m/z 279.1726 (calcd for C15H23N2O3, 279.1709).
Plasmodium falciparum (Malaria) Assay. P. falciparum malaria
parasites were maintained and assayed in human erythrocytes, from a
chloroquine-resistant P. falciparum strain (Indochina W2).38
A
modified Trager and Jensen method was used to maintain cultures
in vitro in type O+ human erythrocytes. The bioassay involved the use
of synchronized ring form parasites that were incubated with extracts,
fractions, compounds, or controls (chloroquine was used as a positive
control) for 48 h within a humidified, airtight container, flushed with a
specialized gas mixture (5% CO2, 5% O2, and 90% N2). Parasite
percent growth (%G) was measured using an aliquot of culture
medium transferred to a new plate, permeabilized with Triton X, and
treated with PicoGreen. PicoGreen is a fluorescent nucleic acid stain
for quantitating double-stranded DNA. The bioassay measured
parasite %G by determining the quantity of PicoGreen intercalated
into intact parasitic DNA (erythrocytes are anucleate and so do not
absorb PicoGreen).
Leishmania donovani (Leishmaniasis) Assay. A WHO reference
strain of L. donovani (LD-1S/MHOM/SD/00-strain 1S) was
maintained in the promastigote stage using established protocols.39
The bioassay used the amastigote form of L. donovani, which is
induced using a change in pH, temperature, and medium. To avoid
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possible interference of the host cell, extracellular amastigotes were
used 96 h after conditions were changed. Extracts, fractions,
compounds, or controls (amphotericin B was used as positive control)
were combined with the axenic amastigotes for 72 h, after which time
parasite %G was measured using PicoGreen fluorescence.
Trypanosoma cruzi (Chagas’ Disease) Assay. Extracts, fractions,
compounds, or controls (nifurtimox was the positive control) were
tested using a transgenic, β-galactosidase-expressing, T. cruzi
(Tulahuen strain, clone C4).40 African green monkey kidney (Vero)
cells were plated with trypomastigotes added after 24 h. After invasion
of T. cruzi parasites into the Vero cells (48 h), the parasites changed to
the amastigote form, at which time extracts, fractions, compounds, or
controls were added. Inhibition of parasite growth (%IG) was
measured colorimetrically 5 days later, based on the cleavage of
chlorophenol red-β-D-galactopyranoside by the parasite-expressed β-
galactosidase.
ASSOCIATED CONTENT
■
S
* Supporting Information
Macroscopic and microscopic images of the santacruzamate A-
producing organism, NMR spectra for santacruzamate A (1)
including 1H, 13C, COSY, HSQC, and HMBC, and 1H and 13
C
NMR spectra for synthetic 1 and the 1−SAHA hybrid (3). This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +1-860-486-3051. Fax: +1-860-486-6857. E-mail: marcy.
balunas@uconn.edu.
Notes
HDAC Enzyme Assay. Three HDAC isozymes [HDAC2 (Class I),
HDAC4 (Class Ia), and HDAC6 (Class IIb)] were utilized to
determine percent inhibition and IC50 values of santacruzamate A (1)
and the 1−SAHA hybrid (3), using commercially available human
recombinant enzyme (BPS Bioscience) and fluorogenic HDAC assay
kits (HDAC2 kit from Active Motif; HDAC4 and HDAC6 kits from
BPS Bioscience). SAHA (2, Vorinostat; Sigma Aldrich, St. Louis, MO,
USA) served as a control for the enzyme inhibition assay. Assay data
were subjected to nonlinear regression analysis (GraphPad Software,
Inc., La Jolla, CA, USA). Enzyme inhibition assays were performed
with varying concentrations of 1, 2, or 3. Briefly, components were
added sequentially to a black, flat-bottom 96-well microtiter plate
(Sigma-Aldrich) as described by the manufacturer’s protocol, and the
reaction mixture was incubated for 30 min at 37 °C. The potent
HDAC inhibitor trichostatin A (included in the assay kit) was added
to the bifunctional HDAC assay developer at a final reaction
concentration of 1 μM to stop deacetylation and initiate the release
of the fluorophore. The reaction mixture was further incubated at
room temperature for 15 min. Fluorescence was measured on a
Spectra Max Gemini XPS (Molecular Devices, Sunnyvale, CA, USA)
using an excitation wavelength of 360 nm and a detection wavelength
of 460 nm. Inhibition assays were used to determine the half-maximal
inhibitory concentration, IC50, in HDAC2.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Funding for this research was provided by a Fogarty
International Center (FIC) International Cooperative Bio-
diversity Group (ICBG) grant based in Panama (U01
TW006634) and by an FIC International Research Scientist
Development Award (IRSDA) (K01 TW008002). We thank
M. K. Hadden (UConn) for access to mammalian cell culture
facilities, A. Anderson (UConn) for initial HDAC results, D.
Wright (UConn) for dry solvents, and T. Rasmussen (UConn)
for hDF cells. We are grateful to the Autoridad Nacional del
Ambiente (ANAM) for providing access to biological samples
in Panama and to C. Thornburg for assistance in collection of
the sample.
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