Latrunculin A and its C-17-O-carbamates inhibit prostate tumor cell invasion and HIF-1 activation in breast tumor cells

Article abstract of DOI:10.1021/np070587w

The marine-derived macrolides latrunculins A (1) and B, from the Red Sea sponge Negombata magnifica, have been found to reversibly bind actin monomers, forming a 1:1 complex with G-actin and disrupting its polymerization. The microfilament protein actin is responsible for several essential functions within the cell such as cytokinesis and cell migration. One of the main binding pharmacophores of 1 to G-actin was identified as the C-17 lactol hydroxyl moiety that binds arginine 210 NH. Latrunculin A-17-O-carbamates 2-6 were prepared by reaction with the corresponding isocyanates. Latrunculin A (1) and carbamates 4-6 displayed potent anti-invasive activity against the human highly metastatic human prostate cancer PC-3M cells in a Matrigel assay at a concentration range of 50 nM to 1 μM. Latrunculin A (1, 500 nM) decreased the disaggregation and cell migration of PC-3M-CT+ spheroids by 3-fold. Carbamates 4 and 5 were 2.5- and 5-fold more active than 1, respectively, in this assay with less actin binding affinity. Latrunculin A (1, IC₅₀ 6.7 μM) and its 17-O-[N-(benzyl)carbamate (6, IC₅₀ 29 μM) suppress hypoxia-induced HIF-1 activation in T47D breast tumor cells.

Full text of DOI:10.1021/np070587w

396
J. Nat. Prod. 2008, 71, 396–402
Latrunculin A and Its C-17-O-Carbamates Inhibit Prostate Tumor Cell Invasion and HIF-1
Activation in Breast Tumor Cells
Khalid A. El Sayed,*,† Mohammad A. Khanfar,† Hassan M. Shallal,† A. Muralidharan,† Bhushan Awate,† Diaa T. A. Youssef,‡
Yang Liu,§ Yu-Dong Zhou,§ Dale G. Nagle,§ and Girish Shah†
Department of Basic Pharmaceutical Sciences, College of Pharmacy, UniVersity of Louisiana at Monroe, 700 UniVersity AVenue,
Monroe, Louisiana 71209, Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal UniVersity, Ismailia 41522, Egypt,
and Department of Pharmacognosy, School of Pharmacy, UniVersity of Mississippi, UniVersity, Mississippi 38677-1848
ReceiVed October 21, 2007
The marine-derived macrolides latrunculins A (1) and B, from the Red Sea sponge Negombata magnifica, have been
found to reversibly bind actin monomers, forming a 1:1 complex with G-actin and disrupting its polymerization. The
microfilament protein actin is responsible for several essential functions within the cell such as cytokinesis and cell
migration. One of the main binding pharmacophores of 1 to G-actin was identified as the C-17 lactol hydroxyl moiety
that binds arginine 210 NH. Latrunculin A-17-O-carbamates 2-6 were prepared by reaction with the corresponding
isocyanates. Latrunculin A (1) and carbamates 4-6 displayed potent anti-invasive activity against the human highly
metastatic human prostate cancer PC-3M cells in a Matrigel assay at a concentration range of 50 nM to 1 µM. Latrunculin
A (1, 500 nM) decreased the disaggregation and cell migration of PC-3M-CT+ spheroids by 3-fold. Carbamates 4 and
5 were 2.5- and 5-fold more active than 1, respectively, in this assay with less actin binding affinity. Latrunculin A (1,
IC50 6.7 µM) and its 17-O-[N-(benzyl)carbamate (6, IC50 29 µM) suppress hypoxia-induced HIF-1 activation in T47D
breast tumor cells.
Latrunculins A (1) and B are macrolides reported by Kashman
and co-workers from the Red Sea sponge Negombata magnifica
Kelly Borges and Vacelet (Podospongiidae).1 Latrunculins are
reported to decrease intraocular pressure and increase outflow
facility without corneal effects in monkeys.2,3 Latrunculin B and
analogues showed antiangiogenic, antimetastatic, and antimicrobial
activities.4 The most important biological effects of latrunculins
are their abilities to disrupt microfilament organization and inhibit
microfilament-mediated processes without affecting the organization
of the microtubular system.5 The latrunculins bind reversibly to
the cytoskeleton actin monomers, forming 1:1 complexes with
G-actin and disrupting polymerization.5 Actin-active agents are
attracting more attention in the field of cancer chemotherapy because
microfilament and microtubule proteins form versatile dynamic
polymers that can define cell polarity, organize cytoplasmic
organelles, control cell shape and promote stable cell-cell and
cell-matrix adhesions, and generate protrusive forces required for
migration.6–8 These functions usually fail and become abnormal
in cancer cells.6–8
On the basis of X-ray crystallography, the binding site of 1 has
been located between subdomains II and IV, in the vicinity of the
ATP binding cleft of the protein target in the actin monomer.9,10
The binding pharmacophores of 1 to G-actin were identified as
follows: C-1 carbonyl oxygen through water to glutamate 214
carboxy, C-17 lactol hydroxyl to arginine 210 NH (major binding),
C-17 pyran oxygen to tyrosine 69 hydroxy, thiazolidinone NH to
aspartate 157 carboxy, and thiazolidinone C-20 carbonyl oxygen
to threonine 186 hydroxy.9,10 Only the thiazolidinone NH group
acts as a hydrogen-bonding donor, while the rest of the binding
functions act as hydrogen-bonding acceptors.9,10
This study reports the anti-invasive and HIF-1 inhibitory activities
of latrunculin A (1) and its semisynthetic analogues (2-6).
Results and Discussion
Reflux of latrunculin A (1) in toluene with chloroethyl, bromo-
ethyl, chloropropyl, benzyl, and phenyl isocyanates in the presence
of catalytic amounts of triethylamine afforded the C-17-O-carbam-
ates, 2-6, respectively.
The HRMS analysis of compound 2 revealed the molecular
formula, C25H35ClN2O6S, with [M]+ and [M + 2]+, 3:1, isotopic
clusters characteristic for a monochlorinated compound. The
downfield shift of the C-17 carbon and H-18 signals in 2 (+1.2
and 1.00 ppm, respectively) compared with that of the starting
material 1 suggested possible carbamoylation at C-17, Table 1.11
1
The H and 13C NMR data further supported this fact and were
Semisynthetic carbamoylation products of the C-17 lactol group
in 1 were produced to study the pharmacological effects of the
addition of hydrogen bond donors and acceptors at this key position.
closely comparable to those of 1 except in the signals of an
additional C-17-O-[N-(2-chloroethyl)carbamoyl] moiety (Tables 1
and 2).11 The carbonyl carbon at δC 152.5 was assigned to C-1′.
3
This was based on its J-HMBC correlation with the methylene
Dedicated to Dr. G. Robert Pettit of Arizona State University for his
pioneering work on bioactive natural products.
* To whom correspondence should be addressed. Tel: 318-342-1725.
Fax: 318-342-1737. E-mail: elsayed@ulm.edu.
† University of Louisiana at Monroe.
1
multiplet H2-2′ (δH 3.61), which, in turn, showed H-1H COSY
coupling with the H2-3′ multiplet (δH 3.63).
Interpretation of the HRMS data of 3 suggested the molecular
formula C25H35BrN2O6S, with characteristic isotopic clusters for a
monobrominated compound. The 1H and 13C NMR data were
‡ Suez Canal University.
§ University of Mississippi.
10.1021/np070587w CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/26/2008

Latrunculin A Inhibits Prostate Tumor Cell InVasion
Table 1. 13C NMR Data of Compounds 2-6a
Journal of Natural Products, 2008, Vol. 71, No. 3 397
δC
position
2
3
4
5
6
1
2
3
4
5
6
7
8
165.8, qC
117.4, CH
158.1, qC
32.6, CH2
30.7, CH2
131.8, CH
126.1, CH
127.8, CH
136.9, CH
29.2, CH
31.8, CH2
31.5, CH2
62.4, CH
35.0, CH2
68.2, CH
35.2, CH2
98.1, qC
62.9, CH
25.4, CH2
176.2, qC
24.7, CH3
21.9, CH3
152.5, qC
42.2, CH2
43.2, CH2
165.5, qC
117.4, CH
158.2, qC
32.6, CH2
30.6, CH2
131.8, CH
126.0, CH
127.4, CH
136.4, CH
29.3, CH
31.8, CH2
31.2, CH2
62.3, CH
35.0, CH2
68.2, CH
35.2, CH2
98.1, qC
62.9, CH
25.5, CH2
176.9, qC
25.0, CH3
21.9, CH3
152.2, qC
48.0, CH2
42.1, CH2
165.6, qC
117.4, CH
158.3, qC
32.6, CH2
30.6, CH2
132.0, CH
126.0, CH
127.4, CH
136.4, CH
29.2, CH
32.2, CH2
31.5, CH2
62.4, CH
35.0, CH2
68.2, CH
35.2, CH2
98.1, qC
62.9, CH
25.4, CH2
177.0, qC
24.7, CH3
21.9, CH3
152.4, qC
37.7, CH2
31.8, CH2
42.3, CH2
165.5, qC
117.4, CH
158.2, qC
32.5, CH2
30.5, CH2
132.0, CH
125.9, CH
127.4, CH
136.4, CH
29.3, CH
31.8, CH2
31.4, CH2
62.4, CH
35.0, CH2
68.2, CH
35.3, CH2
98.1, qC
62.9, CH
25.4, CH2
178.6, qC
24.6, CH3
21.8, CH3
149.6, qC
137.1, qC
121.5, CH
129.1, CH
124.5, CH
129.1, CH
121.5, CH
165.5, qC
117.4, CH
158.1, qC
32.5, CH2
30.5, CH2
131.9, CH
126.0, CH
127.4, CH
136.5, CH
29.3, CH
32.6, CH2
31.5, CH2
62.4, CH
35.0, CH2
68.2, CH
35.2, CH2
98.1, qC
62.9, CH
25.4, CH2
177.2, qC
24.7, CH3
21.8, CH3
151.2, qC
41.5, CH2
141.5, qC
127.0, CH
129.1, CH
126.5, CH
129.1, CH
127.0, CH
9
10
11
12
13
14
15
16
17
18
19
20
21
22
1′
2′
3′
4′
5′
6′
7′
8′
a In CDCl3, 100 MHz. Carbon multiplicities were determined by DEPT135° or APT experiments. qC ) quaternary, CH ) methine, CH2
methylene, CH3 ) methyl carbons.
)
similar to those of 2 with the replacement of chlorine at C-3′ by
bromine (Tables 2). The methylene multiplet H2-3′ (δH 3.69), which
correlated with the methylene carbon at δC 42.1, was assigned on
its regulatory proteins are crucial for cell migration in most cells.15a,b
During cell migration, the actin cytoskeleton is dynamically
remodeled, and this reorganization produces the force necessary
for cell migration.15a,b Since latrunculin A (1) attenuated invasive-
ness of PC-3M cells, its effect was examined on the actin
cytoskeleton of PC-3M cells. PC-3M cells display an invasive
phenotype, and they usually attach to the surface, exhibiting
stretched actin fibers with long exon-type extensions (Figure 2A).
Upon stimulation with 50 nM CT, the cells displayed rapid
reorganization of the actin cytoskeleton, leading to visible changes
in cell morphology within 10 min. The CT-stimulated cells
demonstrated long actin-rich extensions resembling filopodia or
microspikes or web-like actin-containing fibers linking these
filopodium-like structures. However, the addition of latrunculin A
(1) resulted in rapid changes in cell morphology and actin
distribution as characterized by the remarkable shrinkage of actin
stress fibers, possibly caused by the depolymerization disruption
of actin (Figure 2B). Compound 1 also abolished CT-induced
changes in the actin cytoskeleton. These results are consistent with
the actions of latrunculin on PC-3M cell invasion (Figure 1) and
suggest that anti-invasive actions of 1 are mediated through the
disruption of actin cytoskeleton remodeling.
Spheroid disaggregation provides the measure of cell disaggre-
gation as well as cell migration, and this model simulates the process
of tissue disaggregation and invasion of cells in vivo.13b,15c This
assay is based on disaggregation of cancer cell spheroids and radial
migration of released cells on extracellular matrix (ECM).13a–c,14d
Current evidence has shown that tumor cells from primary tumors
in vivo are generally released in clumps, which attach to a favorable
ECM and are later released gradually to migrate in all
directions.13a–13c,14d Therefore, the spheroid disaggregation model
is closer to in situ tumor metastasis than the linear Matrigel invasion
assays. The results shown in Figure 3A demonstrate that latrunculin
A (1, 500 nM) decreased disaggregation and cell migration of PC-
3M-CT+ spheroids 3-fold. Figure 3B shows the antimetastatic
1
the basis of its H-1H COSY coupling with the H2-2′ multiplet
(δH 3.47, δC 48.0).
Analysis of the HRMS and 1H and 13C NMR data indicated 4 to
be nearly identical in structure to 2, except for the presence of an
additional methylene carbon in the carbamate side chain (Tables 1
and 2). The broad methylene multiplet H2-2′ (δH 3.47, δC 37.7)
showed 1H-1H COSY coupling with the H2-3′ multiplet (δH 1.42,
δC 31.8). The latter protons showed a 1H-1H COSY coupling with
the H2-4′ triplet (δH 3.59, δC 42.3).
1
Interpretation of the HRMS and H and 13C NMR data of 5
indicated the presence of a 17-O-[N-(phenyl)carbamoyl] side chain
(Tables 1 and 2). The broad doublet H2-3′/7′ (δH 7.47, δC 121.5)
showed COSY coupling with the H2-4′/6′ double-doublet (δH 7.33,
δC 129.1). The latter protons showed coupling in the COSY
spectrum with the H-5′ multiplet (δH 7.15, δC 124.5).
Analysis of the HRMS and 1H and 13C NMR data indicated that
the structure of 6 is closely similar to 5, except that the phenyl
group in 5 was replaced by a benzyl group in the carbamate side
chain (Tables 1 and 2). The methylene doublet H2-2′ (δH 4.39)
showed a 3J HMBC correlation with the carbonyl carbon at δC 153.5
(C-1′), confirming the 17-O-[N-(benzyl)carbamoyl] side chain.
Metastasis is the predominant cause of cancer mortality. The
potential antimetastatic effects of compounds 1-6 against a human
highly metastatic human prostate cancer PC-3M-CT+ cell line
overexpressing calcitonin (CT) were evaluated using two in vitro
models: linear invasion of cells through the Matrigel barrier and
spheroid disaggregation.13a–d Latrunculin A (1) (100 nM) remark-
ably inhibited baseline and CT-stimulated invasion in the linear
Matrigel assay (Figure 1). However, 1 was cytotoxic at doses higher
than 500 nM (data not shown). There is evidence to suggest that
cell migration and invasion involve multiple processes regulated
by various signaling molecules.14a–d The actin cytoskeleton and

398 Journal of Natural Products, 2008, Vol. 71, No. 3
El Sayed et al.

Latrunculin A Inhibits Prostate Tumor Cell InVasion
Journal of Natural Products, 2008, Vol. 71, No. 3 399
Figure 1. Anti-invasive activity of latrunculin A (1) against PC-3M-CT+ cells in a Matrigel assay.
Figure 2. (A) PC3M cells displaying stretched actin fibers with long exon-type extensions (Control). CT (50 nM)-stimulated cells exhibit
long actin-rich extensions resembling filopodia or web-like actin-containing fibers linking these filopodium-like structures (CT). (B) Addition
of latrunculin A (1, 50 nM) resulted in rapid changes in cell morphology and actin distribution as characterized by the remarkable shrinkage
of actin stress fibers (Lat A and Lat A + CT).
actions of latrunculin A (1) with analogues 4-6 at multiple doses.
Analogues 4-6 also attenuated PC-3M-CT+ spheroid disaggre-
gation/cell migration, and this response was dose-dependent in all
three analogues tested. On the basis of the results shown in Figure
3B, analogue 5 was the most potent. Only a dose of 100 nM of
analogue 5 was required to generate inhibition of PC-3M-CT+
spheroid disaggregation/cell migration to the extent of that produced
by 500 nM of 1. Compound 4 required 200 nM to produce the
equivalent effect. Analogue 6 was equipotent to latrunculin A.
Halogenated ethylcarbamates 2 and 3 were inactive, suggesting the
significance of conjugation and extension of the carbamate side
chain. The phenylcarbamate 5 was 2-fold more potent than
benzylcarbamate 6, suggesting the importance of optimum distance
between the aromatic ring and the carbamate functionality for
activity.
Relatively uncontrolled proliferation of cells within a tumor
outstrips the capacity of existing vasculature to supply oxygen. This
results in the formation of hypoxic regions within tumors. The
occurrence of tumor hypoxia is associated with a poor prognosis
in cancer patients.16a No drugs have been approved clinically to
specifically target hypoxic tumor cells. The transcription factor
hypoxia-inducible factor-1 (HIF-1) regulates hypoxia-induced gene
expression in tumor cells that are subjected to hypoxic conditions.16
b
Disruption of HIF-1-mediated hypoxic adaptation/survival reduces
tumor growth in animal models.16c–i HIF-1 inhibitors represent
potential anticancer drug leads that may selectively target hypoxic
tumor masses.16j,k The effects of 1–6 on hypoxia (1% O2)-induced
HIF-1 activation were examined using a T47D human breast
carcinoma cell-based luciferase reporter assay17 and compared to
those observed on 1,10-phenanthroline-induced HIF-1 activation.

400 Journal of Natural Products, 2008, Vol. 71, No. 3
El Sayed et al.
Figure 3. (A) Spheroid disaggregation of PC-3M-CT+ cells using 500 nM latrunculin A (1). (B) Spheroid disaggregation of PC-3M-CT+
cells comparison of different doses of carbamates 4–6 with the activity of latrunculin A (1).
Table 3. Virtual Binding Affinity of Compounds 1-6 for
Rabbit Muscle R-Actin Complexa
compound
score
crash
polar
1
2
3
4
5
6
6.24
4.37
4.60
5.93
6.12
3.89
-0.96
-2.52
-3.38
-2.80
-1.54
-2.97
1.14
0.00
0.02
0.01
0.01
2.46
a Using SYBYL 7.3.4, SurFlex-Dock 2.1.
Figure 4. Effects of latrunculin A (1) and 6 on hypoxia-induced
HIF-1 activation under hypoxic conditions in T47D breast carci-
noma cells.
Compound 1 inhibited hypoxia-induced activation (IC50 6.7 µM,
Figure 4) and 1,10-phenanthroline-induced HIF-1 activation (IC50
25 µM, data not shown). Compound 6 weakly inhibited hypoxia-
induced HIF-1 activation (IC50 29 µM). None of the other
compounds (2-5) exerted greater than 50% inhibition of hypoxia-
induced HIF-1 activation at the highest concentration tested (30
µM). The effects of 1–6 on the expression of luciferase from a
control construct were examined in a T47D cell-based reporter assay
and hypoxic cell viability/proliferation in a sulforhodamine B-based
cell viability assay. None of the latrunculin analogues significantly
inhibited the activity of a constitutively expressed pGL3 control
reporter or significantly suppressed T47D cell viability under
experimental conditions (less than 20% inhibition at the concentra-
tion of 30 µM). Therefore, latrunculin-based actin depolymerization
disruptors appear to have the ability to selectively inhibit hypoxia-
induced HIF-1 activity in tumor cells at concentrations below those
evident for the inhibition of other critical cellular processes. The
role of actin in HIF-1 signaling has never been documented. These
results represent the first observation of the ability of actin inhibitors
to inhibit HIF-1 activation at concentrations that also inhibit actin
polymerization, and these findings may indicate that actin polym-
erization plays a vital role in HIF-1 signaling.
To further correlate the anti-invasive activity with the actin
binding affinity, docking of 1–6 toward rabbit muscle R-actin was
implemented using the SYBYL 7.3.4 and SurFlex-Dock programs.
Docking results were expressed in three functions: total score, crash,
and polar. The total score was expressed in -log(Kd) units to
represent binding affinities. Crash is the degree of inappropriate
penetration by the ligand into the protein and of interpenetration
between ligand atoms that are separated by rotatable bonds. Crash
scores close to 0 are favorable. Negative numbers indicate penetra-
tion. The polar score is the contribution of the polar non-hydrogen
bonding interactions to the total score and may be useful for
excluding docking results that involve no hydrogen bonds. The
docking procedure was validated using the same conditions to dock
Figure 5. Detailed view of docked structure 5 with the correspond-
ing rabbit muscle R-actin interacting amino acids.
latrunculin A (1) into the binding pocket of R-actin. The docking
simulation resulted in a very close model to the crystallographic
structure.9,10 The docking data are summarized in Table 3.
The parent compound, latrunculin A (1), as well as the phenyl-
carbamate (5), showed the highest scores and molecular interaction,
consistent with the observed anti-invasive activity (Table 3, Figure
5). The carbonyl oxygen of the carbamoyl moiety in 5 makes a
strong H-bonding interaction with the guanidine amino group of
arginine (ARG) 210 of actin (Figure 5). A similar interaction has
been observed for carbamates 3 and 6. Therefore, ARG 210 seems
to play a central role in the binding of latrunculin A (1) and
analogues within the actin monomers.9,10 Although structure 6
showed anti-invasive activity, the scoring was the lowest among
latrunculin A derivatives. Such deviation could be attributed to the
activity of structure 6 on protein target(s) other than actin.
Experimental Section
General Experimental Procedures. Measurements of optical
rotation were carried out on a Rudolph Research Analytical Autopol
III polarimeter. IR spectra were recorded on a Varian 800 FT-IR

Latrunculin A Inhibits Prostate Tumor Cell InVasion
Journal of Natural Products, 2008, Vol. 71, No. 3 401
spectrophotometer. The 1H and 13C NMR spectra were recorded in
CDCl3, using TMS as an internal standard, on a JEOL Eclipse NMR
spectrometer operating at 400 MHz for 1H and 100 MHz for 13C. The
HREIMS experiments were conducted at the University of Michigan
on a Micromass LCT spectrometer. TLC analysis was carried out on
precoated silica gel 60 F254 500 µm TLC plates, using the developing
systems n-hexane-EtOAc (1:1) or CHCl3-MeOH (9:1). For column
chromatography, silica gel 60 (particle size 63–200 µm) or Bakerbond
octadecyl (C18), 40 µm, was used. For Sephadex LH-20 column
chromatography, n-hexane-CHCl3 (1:3), CHCl3, and CHCl3-MeOH
(9:1) systems were used. For column chromatography, silica gel 70–230
mesh was used.
Biological Material. The sponge Negombata magnifica Kelly
Borges and Vacelet (order Poecilosclerida, suborder Mycalina, family
Podospongiidae) was collected as red, long, finger-like strips by scuba
from the sand-covered bottom at -10 to -15 m at Hurghada, on the
Egyptian Red Sea coast, in June 2003.18,19 The sponge was identified
by Mr. Tamer Helmy, Suez Canal University. A voucher specimen
(03RS3) was deposited in the Department of Basic Pharmaceutical
Sciences, College of Pharmacy, University of Louisiana at Monroe,
Louisiana.
medium, which consisted of 90% basal RPMI medium and 10%
conditioned medium from the cultures of PC-3M-CT+ cells expressing
constitutively active Gas protein. The incubation was carried out for
24 h, after which the Matrigel (along with noninvading cells) was
scraped off with cotton swabs, and the outer side of the insert was
fixed and stained using Diff Quick staining (Dade Behring Diagnostics,
Aguada, Peurto Rico). The number of cells migrated on the outer bottom
side of the insert was counted under the microscope in six or more
randomly selected fields (magnification: 100×). Final results are
expressed as mean ( SEM per 100× field. Each experiment was
performed in triplicate, and the experiment was repeated twice.13a–d
Growth Correction. Since some cell lines can exhibit high
proliferation rates, it is likely that the cells migrating during the
early part of the 24 h incubation period could proliferate during the
remaining period of incubation, leading to a slight overestimation
of the final results. To correct this probability, the growth rate of
PC-3M cells was determined under identical culture conditions.
Twenty-five thousand cells were plated at hourly intervals in six-
well dishes and cultured with/without CT (50 nM) for 1–24 h. Mean
percent increase in the cell number was determined at the end of
the incubation period by counting the net increase in the number of
cells. The relative CT-induced increase of the pooled results of all
time points was found to be 1.19 (vehicle control ) 1). This
correction was applied to the results of invasion assays.13b,15d,e
Spheroid Disaggregation Assay. Spheroids were prepared from
single cell suspension of prostate cell lines as described before.13b,d
In brief, 5 × 104/mL cells in RPMI 1640 serum-free medium were
placed on 96-well low-attachment tissue culture plates. The plates
were rocked on a gyrorotatory shaker in a CO2 incubator at 37 °C
for 2 days, at the end of which the spheroids measuring 150–300
µm in diameter (∼4 × 104 cells/spheroid) were formed. A single
spheroid was then placed in the center of each well of an
extracellular matrix (ECM)-coated 24-well microplate in 200 mL
of serum-free medium. From previous studies, it was determined
that 1 h is an appropriate time for spheroids to begin adhering to an
ECM. Thus t ) 0 was set as 1 h from initial plating, so that if the
plate was not disturbed, the spheroids would not move from their
location at the time of plating. Spheroids were photographed digitally
at t ) 0, cultured at 37 °C for 48 h, and then rephotographed. The
spheroids were then fixed, stained with Diff-Quik (Dade Behring,
Newark, DE), and examined under light microscopy. The diameter
of the area covered with cells migrated from the spheroids was
measured in a microscope calibrated with a stage and ocular
micrometer. The radial distance of migration was calculated after
subtraction of the mean initial spheroidal diameter at t ) 0. Values
shown represent the average percent increase in surface area of
spheroids.
Cell Proliferation/Viability Assay. Human breast carcinoma T47D
cells were grown in DMEM/F12 medium with L-glutamine (Medi-
atech) supplemented with 10% (v/v) fetal calf serum (FCS, Hyclone),
50 units mL-1 penicillin G (Na salt), and 50 µg mL-1 streptomycin
sulfate (referred to as “Pen/Strep”) (Invitrogen) in a humidified
atmosphere (5% CO2/95% air) at 37 °C. Exponentially grown cells
were plated at a density of 30 000 cells per well into 96-well tissue
culture plates (Corning) in a volume of 100 µL of DMEM/F12
medium with 10% FCS and Pen/Strep. The cells were incubated at
37 °C overnight.17,20 Test compounds were diluted in DMEM/F12
medium with Pen/Strep and added in a volume of 100 µL per well.
Following a 30 min incubation, the compound treatment continued
for another 16 h (or 48 h) at 37 °C under hypoxic (5% CO2/1%
O2/94% N2) or normoxic (5% CO2/95% air) conditions. Cell
proliferation/viability (performed in triplicate) was determined as
previously described.17,20 The absorbance at 515 nm was measured
on a BioTek Synergy HT microplate reader with correction
wavelength at 690 nm. The data were normalized to the untreated
control. The following formula was used to calculate % inhibition
of cell proliferation/viability: % inhibition ) 1 - OD515(treated)/
OD515(control).
Extraction and Isolation. The frozen sponge (6 kg) was coarsely
minced and extracted with CHCl3 (5 × 1000 mL) at room temperature.
The CHCl3 extract was then concentrated under vacuum and subjected
to liquid chromatography, as described elsewhere, to afford 1, 1.2 g
(0.0002%).1,4,11
Preparation of Carbamates 2–6. To solutions of 50 mg of 1 in
toluene (2 mL) were added 59 µL of 2-chloroethyl isocyanate, 60 µL
of 2-bromoethyl isocyanate, 60 µL of 3-chloropropyl isocyanate, 40
µL of phenyl isocyanate, or 36 µL of benzyl isocyanate, respectively,
and separately mixed with 10 µL of Et3N. Each solution was separately
stirred at room temperature for 1 h (12 h for benzyl isocyanate only).
Water (10 mL) was then added, and the product of each reaction mixture
was extracted with EtOAc. Each EtOAc extract was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. Crude
products were then purified by column chromatography on silica gel
60 using EtOAc-n-hexane, 1:9, to give compounds 2 (20.0 mg, 40%
Rf 0.60, silica gel, CHCl3-MeOH, 9.5:0.5) and 4 (13.2 mg, 26.4%, Rf
0.64), or EtOAc-n-hexane, 2:8, to give compounds 3 (13.3 mg, 26.6%,
Rf 0.57) and 6 (19.4 mg, 38.8%, Rf 0.55), or CHCl3-MeOH, 9.5:0.5,
to afford compound 5 (13.6 mg, 27.2%, Rf 0.51).
17-O-[N-(2-Chloroethyl)carbamoyl]latrunculin A (2): colorless
oil; [R]25 +48.8 (c 0.51, CHCl3); IR νmax (neat) 3564, 3331,
D
2953–2856, 1761, 1708, 1680, 1542, 1355, 1164 cm-1
;
13C and H
1
NMR see Tables 1 and 2, respectively; HRESIMS m/z 549.1808 (calcd
for C25H35ClN2O6SNa, 549.1802 [M + Na]+).
17-O-[N-(2-Bromoethyl)carbamoyl]latrunculin A (3): colorless
oil; [R]25 +30.0 (c 0.55, CHCl3); IR νmax (neat) 3563, 3327,
D
2995–2857, 1735, 1713, 1560, 1356, 1301, 1128 cm-1
;
13C and H
1
NMR see Tables 1 and 2, respectively; HRESIMS m/z 593.1306 (calcd
for C25H35BrN2O6SNa, 593.1297 [M + H]+).
17-O-[N-(3-Chloropropyl)carbamoyl]latrunculin A (4): colorless
oil; [R]25 +50.3 (c 0.47, CHCl3); IR νmax (neat) 3564, 3335,
D
2953–2856, 1740, 1704, 1545, 1356, 1288, 1163 cm-1
;
13C and H
1
NMR see Tables 1 and 2, respectively; HRESIMS m/z 563.1953 (calcd
for C26H37ClN2O6SNa, 563.1959 [M + H]+).
17-O-[N-(Phenyl)carbamoyl]latrunculin A (5): colorless oil; [R]25
D
+23.0 (c 0.14, CHCl3);IR νmax (neat) 3570, 3286, 2927–2853, 1710,
1601, 1680, 1554, 1446, 1296, 1160 cm-1 13C and 1H NMR see Tables
;
1 and 2, respectively; HRESIMS m/z 563.2200 (calcd for C29H36-
N2O6SNa, 563.2192 [M + Na]+).
17-O-[N-(Benzyl)carbamoyl]latrunculin A (6): colorless oil; [R]25
D
+46.3 (c 0.55, CHCl3); IR νmax (neat) 3564, 3334, 2953–2857, 1730,
1703, 1680, 1542, 1455, 1357, 1282, 1166 cm-1 13C and 1H NMR see
;
Tables 1 and 2, respectively; HRESIMS m/z 577.2360 (calcd for
C30H38N2O6SNa, 577.2348 [M + Na]+).
Invasion Assay. These experiments were conducted in 24-well,
two-compartment, Matrigel invasion chambers (Becton Dickinson,
Bedford, MA).13a–d The PC-3M-CT+ cells were grown exponentially
under serum-starved conditions for 24 h [basal RPMI medium contain-
ing no serum or growth factors but containing 0.1% BSA, 10 mM
HEPES, 4 mM L-glutamine, 100 IU mL-1 penicillin G, and 100 mg
mL-1 streptomycin]. The cells were then harvested and seeded at a
density of 25 × 103 cells per well in the upper insert of the Matrigel
invasion chamber. The lower chamber received the chemoattractant
Cell-Based Reporter Assay for HIF-1 Activity. The transfection,
compound treatment, exposure to hypoxic conditions (5% CO2/1%
O2/94% N2), normoxic conditions (5% CO2/95% air), and a hypoxia
mimetic (10 µM 1,10-phenanthroline), and luciferase activity
determination were performed as previously described.20 Emetine
was used as a positive control (IC50 0.11 µM).
Molecular Modeling. Docking and scoring modeling studies were

402 Journal of Natural Products, 2008, Vol. 71, No. 3
El Sayed et al.
2006, 20, 1894–1911. (c) Chien, J.; Ren, Y.; Wang, Y. Q.; Bordelon,
W.; Thompson, E.; Davis, R.; Rayford, W.; Shah, G. Mol. Cell.
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performed using SYBYL 7.3.4 (Tripos Discovery Informatics, St.
Louis, MO) installed on a Dell desktop workstation equipped with
a 1.86 GHz Intel Xeon processor and the Red Hat Enterprise Linux
(version 4) operating system. The 3D coordinates of rabbit muscle
R-actin complex with latrunculin A (1) were retrieved from the
Protein Data Bank (PDB code: 1esv). The selected structure is of
the best 3D resolution (2.0 Å). The protein structure was utilized in
subsequent docking experiments without energy minimization.
Explicit water molecules were removed from the structure. Chemical
structures of 1-6 were drawn in SYBYL 7.3.4 and assigned
Gasteiger partial charges and energy minimized using Energy Force
Field. Docking simulations of structures 1-6 were carried out using
SurFlex-Dock (version 2.1). SurFlex-Dock identifies the active site
of the protein and constructs a docking target (protomol) to which
molecules match. Protmol was generated by the ligand-based
method, setting the Threshold and Bloat parameters as default value
(0.5 and 0.0, respectively).
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Acknowledgment. This investigation was made possible through
the support of NIH grant number P20RR16456 from the BRIN Program
of the National Center for Research Resources, grant R01CA96534
(G.V.S.), and grant CA98787 (D.G.N., Y.-D.Z.). Its contents are solely
the responsibility of the authors and do not necessarily represent the
official views of NIH. Louisiana Board of Regents grant LEQSF(2007-
08)-ENH-TR-80 is acknowledged for the support of the purchase of
the NMR probe.
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NP070587W