Desmethoxymajusculamide C, a cyanobacterial depsipeptide with potent cytotoxicity in both cyclic and ring-opened forms

Article abstract of DOI:10.1021/np9001674

Cytotoxicity-guided fractionation of the organic extract from a Fijian Lyngbya majuscula led to the discovery of desmethoxymajusculamide C (DMMC) as the active metabolite. Spectroscopic analysis including 1D and 2D NMR, MS/MS, and chemical degradation and derivatization protocols were used to assign the planar structure and stereoconfiguration of this new cyclic depsipeptide. DMMC demonstrated potent and selective anti-solid tumor activity with an IC ₅₀ = 20 nM against the HCT-116 human colon carcinoma cell line via disruption of cellular microfilament networks. A linear form of DMMC was generated by base hydrolysis, and the amino acid sequence was confirmed by mass spectrometry. Linearized DMMC was also evaluated in the biological assays and found to maintain potent actin depolymerization characteristics while displaying solid tumor selectivity equivalent to DMMC in the disk diffusion assay. A clonogenic assay assessing cytotoxicity to HCT-116 cells as a function of exposure duration showed that greater than 24 h of constant drug treatment was required to yield significant cell killing. Therapeutic studies with HCT-116 bearing SCID mice demonstrated efficacy at the highest dose used (%T/C = 60% at 0.62 mg/kg daily for 5 days)

Full text of DOI:10.1021/np9001674

J. Nat. Prod. 2009, 72, 1011–1016
1011
Desmethoxymajusculamide C, a Cyanobacterial Depsipeptide with Potent Cytotoxicity in Both
Cyclic and Ring-Opened Forms
T. Luke Simmons,†,‡ Lisa M. Nogle,‡,§ Joseph Media,⊥ Frederick A. Valeriote,⊥ Susan L. Mooberry,| and
William H. Gerwick*,†,∆
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, UniVersity of California San Diego, La Jolla,
California 92037, College of Pharmacy, Oregon State UniVersity, CorVallis, Oregon 97331, Josephine Ford Cancer Center, Henry Ford
Hospital, Detroit, Michigan 48202, Southwest Foundation for Biomedical Research, San Antonio, Texas 78245, and Skaggs School of
Pharmacy and Pharmaceutical Science, UniVersity of California San Diego, La Jolla, California 92037
ReceiVed March 12, 2009
Cytotoxicity-guided fractionation of the organic extract from a Fijian Lyngbya majuscula led to the discovery of
desmethoxymajusculamide C (DMMC) as the active metabolite. Spectroscopic analysis including 1D and 2D NMR,
MS/MS, and chemical degradation and derivatization protocols were used to assign the planar structure and
stereoconfiguration of this new cyclic depsipeptide. DMMC demonstrated potent and selective anti-solid tumor activity
with an IC50 ) 20 nM against the HCT-116 human colon carcinoma cell line via disruption of cellular microfilament
networks. A linear form of DMMC was generated by base hydrolysis, and the amino acid sequence was confirmed by
mass spectrometry. Linearized DMMC was also evaluated in the biological assays and found to maintain potent actin
depolymerization characteristics while displaying solid tumor selectivity equivalent to DMMC in the disk diffusion
assay. A clonogenic assay assessing cytotoxicity to HCT-116 cells as a function of exposure duration showed that
greater than 24 h of constant drug treatment was required to yield significant cell killing. Therapeutic studies with
HCT-116 bearing SCID mice demonstrated efficacy at the highest dose used (%T/C ) 60% at 0.62 mg/kg daily for 5
days).
Actin is a ubiquitous and highly conserved protein found in either
globular (G-actin monomer) or filamentous (F-actin polymer) forms.
A tightly controlled dynamic equilibrium exists between actin
filaments and monomers and is essential for cell growth and
division, as well as cell signaling, motility, and tertiary cellular
structure.1 Transformed cancer cells undergo distinct changes in
actin cytoskeletal organization and protein regulation, which
contribute to the abnormal growth characteristics of tumor cells.
As a consequence of altered microfilament dynamics, cancer cells
have enhanced capacity for tissue adhesion, tumorigenesis, and an
increased ability to metastasize.2,3 Metastatic tissue invasion
involves a form of cellular motility usually termed “amoeboid
motility”, a process driven by cycles of actin polymerization, cell
adhesion, and acto-myosin contraction.4 These cellular processes
therefore offer logical targets for the development of new anticancer
drugs.
A large number of secondary metabolites have been isolated from
marine invertebrates and microorganisms that show promising
anticancer activities.5,6 Several of these, such as jasplakinolide,
hectochlorin, and doliculide, are known to stimulate actin poly-
merization, while others such as latrunculin and various trisoxazole-
containing macrolides display actin depolymerization properties.7-11
The mechanism of action has been studied in some detail for
jasplakinolide and doliculide, which both cause cell cycle arrest at
the G2/M phase by inducing actin hyperpolymerization and ag-
gregation of the resultant F-actin. Additionally, both compounds
competitively displace a fluorescent phalliodin derivative from the
actin polymer.7 Interestingly, hectochlorin displayed many of the
same effects on actin polymerization but is unable to displace
fluorescent phalliodin from the polymerized protein.8 The cyclic
depsipeptides majusculamide C and dolastatins 11 and 12 also show
promising cytotoxic activity by arresting cells at cytokinesis by
modulating microfilament polymerization in a dose- and time-
dependent manner.12,13 Conversely, latrunculin and the trisoxazole
macrolides inhibit polymerization and nucleotide (ATP) exchange,
respectively, ultimately causing microfilament depolymerization and
cell death.9,10
Here we report the discovery of desmethoxymajusculamide C
(DMMC, 1), a new cyclic depsipeptide with potent actin depoly-
merization properties in both the native cyclic (1) and ring-opened
forms (2). Potent cytotoxicity was observed in initial screening of
the organic extract of a Fijian collection of the cyanobacterium
Lyngbya majuscula. Bioassay-guided isolation led to DMMC (1)
as the active constituent. Structure elucidation by extensive chemical
and spectroscopic approaches revealed 1 to be an analogue in a
well-known class of bioactive depsipeptides (Figure 3). DMMC,
like its structural relatives, displays potent activity against a variety
of cancer cell lines by the disruption of cytoskeletal actin microfila-
ment networks. Moreover, we found that the ring-opened form of
DMMC (2) maintains equivalent efficacy and solid tumor selectivity
as the cyclic molecule (1). These findings raise significant and
intriguing questions regarding the active form of the many
biologically active cyclic (depsi)peptides reported in the literature
to date.
Results and Discussion
* To whom correspondence should be addressed. E-mail: wgerwick@
ucsd.edu.
Isolation and Structure Determination of DMMC (1). The
marine cyanobacterium L. majuscula was collected from Yanuca
Island, Fiji, and repetitively extracted with 2:1 CH2Cl2/CH3OH to
yield 5.5 g of crude extract. A portion of the extract (5.3 g) was
sequentially fractionated by vacuum liquid chromatography (VLC)
over silica gel, C18 SPE (7:3 CH3OH/H2O), and RP HPLC to yield
27.5 mg of pure 1, isolated as a light yellow glassy oil. Cytotoxic
assay of fractions at each stage of the chromatography identified 1
to be the most active compound in the extract.
† Scripps Institution of Oceanography.
‡ These two authors contributed equally to the work (T.L.S. for reisolation
and production of compounds 1 and 2 for biological assay, some
stereoanalysis, and molecular modeling; L.M.N. for initial isolation, planar
structure, and some stereoanalysis).
§ Oregon State University.
⊥ Henry Ford Hospital.
| Southwest Foundation for Biomedical Research.
∆ Skaggs School of Pharmacy and Pharmaceutical Sciences.
10.1021/np9001674 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/02/2009

1012 Journal of Natural Products, 2009, Vol. 72, No. 6
Simmons et al.
Table 1. 1H and 13C NMR Data for DMMC (1) Recorded in
CDCl3 at 400 and 100 MHz, Respectively
residue
Map
position
δC
δH, mult., J (Hz)
HMBCa
1
172.8
2
3
42.7 2.76, qd (7.0, 1.6) 1, 3
51.4 4.52, m
4 (NH)
5
6
7
7.07, d (10.1)
10.0 1.10, d (7.0)
26.3 1.56, m;1.48, m
11.2 0.93, t (7.4)
173.0
3, 8
1, 2, 3
2, 3, 7
3, 6
Ala
Ibu
8
9
48.5 4.45, m
7.76, d (7.9)
15.7 1.07, d (7.9)
172.3
8, 11
9, 11, 12
8, 9
10 (NH)
11
12
13
55.1
14
210.3
15
16 (NH)
17
18
19
52.0 4.92, m
7.32, d (6.4)
22.5 1.53, s
21.7 1.48, s
19.3 1.16, d (6.8)
168.0
14, 19
14, 15, 20
12, 13, 14, 18
12, 13, 14, 17
14, 15
N-Me Phe 20
21
61.0 5.23, m
20, 23, 24, 30
22 (N)
23
35.7 3.31, dd (14.0, 6.3) 20, 21, 24, 25/29
2.89, dd (14.0, 8.2)
137.0
Figure 1. Molecular structures of cyclic DMMC (1) and its linear
hydrolysis product (2).
24
25/29
26/28
27
129.7 7.25, m
129.0 7.28, m
127.2 7.20, t (6.9)
29.5 2.99, s
23, 24, 26/28, 27
24, 25/29
25/29
30
21, 31
N-MeVal 31
170.4
32
58.3 4.79, d (10.6)
31, 34, 35, 36, 37, 38
33 (N)
34
35
36
37
27.3 2.22, m
18.7 0.33, d (6.4)
18.6 0.74, d (6.5)
29.5 2.97, s
169.5
32, 35, 36
32, 34, 36
32, 34, 35
32, 38
Gly
38
39
40.8 4.62, dd (17.6, 7.9) 38, 41
3.47, d (17.6)
Figure 2. CID MS fragmentation pattern of derivative 2.
40 (NH)
41
7.58 d (7.9)
41
N-MeIle
171.3
42
61.4 4.91, m
41, 44, 45, 47, 48, 49
43 (N)
44
45
46
47
33.0 2.11, m
25.2 1.46, m;1.11, m
10.3 0.89, m
15.8 1.03, d (7.3)
30.6 3.22, s
44
44, 45
42, 44, 45
42, 49
48
Gly
49
170.2
50
41.0 4.44, m
3.57, dd (15.9, 4.6)
7.36, t (5.5)
49, 52
51 (NH)
52
49, 50
Hmpa
170.7
53
54
55
56
78.6 5.21, m
37.6 2.07, m
24.0 1.47, m;1.24, m
11.9 0.90, m
15.4 0.90, m
1, 52, 54, 55, 57
53, 54, 56
54, 55
53, 54
57
a Proton showing correlation to indicated carbon resonance.
bands at 1739 and 1641 cm-1 for ester and amide functionalities,
respectively, these data indicated a peptide-type natural product.
1
The H and 13C NMR spectra for 1 were well dispersed in CDCl3
(Table 1 and Supporting Information) and allowed the construction
of nine partial structures by 2D NMR (COSY, HMBC), accounting
for all atoms in the molecular formula for 1. Six standard amino
acids were deduced as alanine (Ala), N-methylvaline (N-MeVal),
N-methylphenylalanine (N-MePhe), N-methylisoleucine (N-MeIle),
and two glycine (Gly) units. Two additional residues were similarly
deduced as the R-hydroxy acid 2-hydroxy-3-methylpentanoate
(Hmpa) and the ꢀ-amino acid 3-amino-2-methylpentanoate (Map).
The ninth and final moiety, deduced mainly from HMBC and
Figure 3. Representative cyclic depsipeptides related to DMMC
(1) obtained from Lyngbya majuscula or the predatory sea hare
Dolabella auricularia.
Desmethoxymajusculamide C (1) possessed a molecular formula
of C49H78N8O11, as determined by HRFABMS (obsd m/z [M + H]+
955.5867; calcd for m/z 955.5868). Combined with IR absorption

Cyanobacterial Depsipeptide with Potent Cytotoxicity
Journal of Natural Products, 2009, Vol. 72, No. 6 1013
Table 2. Cytotoxicity of 1 and 2 against a Panel of Cancer Cell
Lines
applied to the filter disk. As noted above, bioassay-directed
fractionation yielded DMMC (1) as the most active pure compound.
The same zone value of the HCT-116 and H125 cell lines was
achieved with 117 ng of 1, indicating an approximately 20-fold
purification from crude extract to obtain DMMC. Interestingly, the
linear form of DMMC (2) retained full potency in this assay and
gave the same clearing zone at 35 ng/disk.
IC50 determinations were carried out for both 1 and 2 against
four cells lines as shown in Table 2. HCT-116 was the most
sensitive cell line, with IC50 values of 20 and 16 nM, respectively.
The H-460 cell line was about 5-fold less sensitive, the MDA-
MB-435 cell line was over 10-fold less sensitive, and Neuro-2A
cells were resistant to both compounds.
IC50 (µM)
cyclic DMMC (1) linear DMMC (2)
HCT-116
human colon carcinoma
H-460
human large cell lung carcinoma
MDA-MB-435
human carcinoma
Neuro-2A
0.020
0.063
0.22
0.016
0.094
0.23
murine neuroblastoma
>1.0
>1.0
Actin microfilament disruption assays were conducted to further
understand the mechanism of action for compounds 1 and 2. Cyclic
(1) and linear (2) DMMC at 52 nM caused the complete loss of
filamentous (F)-actin coincident with dramatic changes in cell
morphology when tested against A-10 cells (Figure 4). The effects
were specific for microfilaments, as there was no evidence of
microtubule loss at these drug concentrations. Binuclear cells were
present, consistent with inhibition of the actin-dependent process
of cytokinesis. Evidence of apoptosis and the breakdown of nuclei
into apoptotic bodies were prevalent at 52 nM, and altered cellular
morphology accompanied total disruption of the microfilament
network at this concentration.
Energy-minimized molecular modeling of both compounds 1 and
2 under aqueous conditions as well as under hydrophobic cell
membrane conditions showed that the two molecules behave
differently in these two environments. Using semiempirical opti-
mized potentials for liquid simulations (OPLS) modeled at pH 7.2,
comparisons with the literature, was constructed as 4-amino-2,2-
dimethyl-3-oxo-pentanoate (Dmop).14
Sequencing of the amino and hydroxy acids in DMMC was
accomplished by HMBC (Table 1) and supported by CID MS
fragmentation of the base hydrolysis product of 1 (Figure 2). The
configuration of the stereogenic centers found in 1 was determined
by chiral HPLC and Marfey’s analysis. Fortunately, the doubling
1
and broadening of several specific H and 13C NMR signals, as
observed for dolastatin 12, lyngbyastatin 1, and others, was not
present in the spectra recorded for DMMC (1), thus suggesting the
presence of a single C-15 epimer.12
Cytotoxicity of Cyclic (1) and Linear DMMC (2). The
original extract (WHG-1338) was identified as solid tumor selective
and very potent.17 The zone differential between the three human
tumor cell lines HCT-116 human colon cancer, H125 human lung
cancer, and MCF-7 human breast cancer and the CCRF-CEM
human leukemia was 500 zone units when 2.3 µg of extract was
Figure 4. Effects of cyclic (1) and linear (2) DMMC on the actin cytoskeleton of A-10 cells. After 24 h of drug exposure, the cells
were fixed, permeabilized, and exposed to the microfilament-staining reagent TRITC-phalloidin (visualized as red). (A) Control cells
were treated with vehicle alone. (B) Cells treated with DMMC (1) at 52 nM caused complete loss of the cellular microfilament
network and generated binucleated cells (not shown). At 10 nM DMMC, no microfilament loss was observed in A-10 cells. (C)
Treatment of cells with linearized DMMC (2) at 52 nM also induced the complete disruption of cellular microfilament networks and
generated binucleated cells (not shown).

1014 Journal of Natural Products, 2009, Vol. 72, No. 6
Simmons et al.
Figure 5. Lowest energy conformations of DMMC (1) and its base-opened seco-acid form (2): top, structures modeled for aqueous conditions
at pH 7.2 (e.g., cytoplasm mimicking) using the optimized potentials for liquid simulations (OPLS) parameter set; bottom, structures modeled
for hydrophobic conditions (e.g., cell membrane-mimicking) using the semiempirical parametrized model 3 (PM3).18-20
Figure 6. Clonogenic dose-response curve of HCT-116 human
colon cancer cells exposed to DMMC (1) for 2 h (squares), 24 h
(diamonds), and 168 h (triangles) in vitro.
Figure 7. Therapeutic assessment of DMMC (1) against HCT-116
human colon cancer cells growing subcutaneously in SCID mice.
Drug was administered daily for 5 days intravenously [untreated
control (diamonds), 0.31 mg/kg/day (triangles), 0.62 mg/kg/day
(squares)].
the linear molecule 2 takes on a “double hairpin-like” conformation,
while in the hexadecane parametrized model 3 (PM3) calculations,
compound 2 “unwinds” to a nearly linear conformation (Figure 5).18
By contrast, native DMMC (1) maintains a similar conformation
under both modeling conditions. One possible interpretation of these
data, based on the differences in the lowest energy conformations
of 1 and 2 as well as their biological activity profiles, is that natural
product 1 functions as a pro-drug that crosses the cell membrane
and is then cleaved by cellular esterases found in the cytosol, to
yield the highly bioactive linearized form of the molecule (2).
Alternatively, it is possible that linear DMMC (2) is as active as
the cyclic form (1) because it adopts an active conformation similar
to DMMC, but only on the surface of actin, its biomolecular target.
A clonogenic concentration-survival study was conducted for
compound 1 to both define the effect of exposure duration on
cytotoxicity and to provide a guide for determining the most
effective dose schedule for the subsequent therapeutic assess-
ment.17 As shown in Figure 6, clonogenic survival of HCT-116
cells was determined at three different exposure durations: 2 h, 24 h,
and continuous (168 h or 7 day), as a function of drug concentration.
The concentration for an exposure duration that yields a surviving
fraction of 10% (tS10) was determined: 2S10 > 10.5 µM; 24S10 > 10.5
µM; 168S10 ) 2.1 nM. These results indicate that in order to observe
a therapeutic effect the concentration of DMMC with HCT-116
cells in vivo needed to be >10.5 µM at either the 2 or 24 h time
point if a single, bolus dose was administered, or continuously at
>2.1 nM if a chronic (7 day) dosing was given.
The clonogenic results were sufficiently encouraging to design
a therapeutic efficacy trial of DMMC (1) using HCT-116 tumor
cells in SCID mice. We first determined the maximum tolerated
dose (MTD) to be approximately 2.5 mg/kg for SCID mice.
Unfortunately, we did not have sufficient drug to carry out a 5-day
therapeutic trial at 2.5 mg/kg/day. Because we needed two doses
of drug to carry out the trial, and given the limitation of DMMC
availability, we chose doses of 0.62 and 0.31 mg/kg/day. The results
are presented in Figure 7 and show that the tumor growth rate for
the lower dose schedule was identical to the untreated control, while
the higher dose yielded a %T/C value of 60% at 17 days and
indicates that efficacy was found at about one-fourth of the
maximum tolerated dose. A further 30 mg of DMMC (1) is needed

Cyanobacterial Depsipeptide with Potent Cytotoxicity
Journal of Natural Products, 2009, Vol. 72, No. 6 1015
with EtOAc. The organic layer was dried under N2 and purified using
HPLC (17:3 CH3OH/H2O) to give the base hydrolysis product 2 in
>98% yield and gave the following FABMS (3-nitrobenzyl alcohol):
m/z 973 (35), 803 (25), 675 (41), 630 (52), 505 (30), 469 (100), 356
(66), 342 (54), 299 (82), 244 (31).
for an adequate trial against HCT-116 at doses closer to the MTD;
if found effective, evaluations should be repeated both in a multiple
treatment schedule and against other tumor types.
Conclusions
Absolute Configuration of the Standard Amino Acid Residues
in 1. Approximately 1.0 mg of 1 was hydrolyzed with 6 N HCl (Ace
high-pressure tube, microwave, 1.0 min), and the hydrolysate was
evaporated to dryness and resuspended in H2O (50 µL). A 0.1%
1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (L-Marfey’s reagent) solu-
tion in acetone (50 µL) and 20 µL of 1 N NaHCO3 were added, and
the mixture was heated at 40 °C for 1 h. The solution was cooled to
room temperature, neutralized with 10 µL of 2 N HCl, and evaporated
to dryness. The residue was resuspended in H2O (50 µL) and analyzed
by reversed-phase HPLC (LiChrospher 100 C18, 5 µm, UV detection
at 340 nm) using a linear gradient of 9:1 50 mM triethylammonium
phosphate (TEAP) buffer (pH 3.1)/CH3CN to 1:1 TEAP/CH3CN over
60 min. The retention times (tR, min) of the derivatized residues in the
hydrolysate of 1 matched L-Ala (22.1; D-Ala, 26.9), N-Me-L-Val (36.5;
N-Me-D-Val, 39.7), and N-Me-L-Phe (38.5; N-Me-D-Phe, 39.2). Given
that only N-Me-L-Ile and N-Me-L-allo-Ile were commercially available,
the D-Marfey’s reagent was used to make N-Me-D-Ile and N-Me-D-
allo-Ile chromatographic equivalents. The retention time of the Ile
derivative from the hydrolysate matched that of N-Me-L-Ile (40.7 min;
N-Me-L-allo-Ile, 41.2 min; N-Me-D-Ile, 44.2 min; N-Me-D-allo-Ile, 44.7
min).
Absolute Configuration of the Nonstandard Amino- and Hy-
droxy-Acid Residues in 1. Determination of the absolute configuration
for the 3-amino-2-methylpentanoic (Map), 2-hydroxy-3-methylpen-
tanoic (HMPA), and 4-amino-2,2-dimethyl-3-oxopentanoic acid (Ibu)
residues was accomplished using a combination of Marfey’s method
and chiral HPLC. Authentic chromatographic standards for the Map
and Ibu residues were obtained as gifts from the laboratory of R. E.
Moore, Department of Chemistry, University of Hawaii. In both cases,
approximately 1.0 mg of 1 was hydrolyzed with 6 N HCl (Ace high-
pressure tube, microwave, 1.0 min), and the hydrolysate was evaporated
to dryness and resuspended in H2O (50 µL). A 0.1% 1-fluoro-2,4-
dinitrophenyl-5-L-alaninamide (Marfey’s reagent) solution in acetone
(50 µL) and 20 µL of 1 N NaHCO3 were added, and the mixture was
heated at 40 °C for 1 h. The solution was cooled to room temperature,
neutralized with 10 µL of 2 N HCl, and evaporated to dryness. The
residue was resuspended in H2O (50 µL) and analyzed by reversed-
phase HPLC (LiChrospher 100 C18, 5 µm, UV detection at 340 nm)
using a linear gradient of 9:1 50 mM triethylammonium phosphate
(TEAP) buffer (pH 3.1)/CH3CN to 4:6 TEAP/CH3CN over 60 min.
Retention times, under these conditions, for the hydrolysate of 1
indicated 2S,3R-Map (47.0 min; 2R,3S-Map, 36.5 min; 2S,3S-Map, 37.0
min; 2R,3R-Map, 28.9 min) and 4S-Ibu (34.2 min; 4R-Ibu, 32.8 min,
respectively).
Preparation and Chiral Analysis of HMPA. L-Ile (100 mg, 0.75
mmol) was dissolved in 50 mL of 0.2 N perchloric acid (0 °C). To this
was added a cold (0 °C) solution of NaSO3 (1.4 g, 20 mmol) in 20 mL
of H2O with rapid stirring. With continued stirring the reaction mixture
was allowed to reach room temperature until evolution of N2 subsided
(∼30 min). The solution was then brought to boil for 3 min, cooled to
room temperature, and saturated with NaCl. The mixture was then
extracted with Et2O and dried under vacuum. The three other stereoi-
somers 2R,3R-HMPA, 2R,3S-HMPA, and 2S,3R-HMPA were synthe-
sized in a similar manner from D-Ile, D-allo-Ile, and L-allo-Ile,
respectively.22 A portion of the resultant oil was dissolved in aqueous
2 mM CuSO4 buffer for HPLC. The retention time [Chirex-D, isocratic
system (85:15) 2 mM CuSO4: MeCN] of the natural product hydrolysate
matched that for 2S,3S-HMPA (17.1 min; 2R,3S-HMPA, 11.8 min;
2S,3R-HMPA, 9.2 min; and 2R,3R-HMPA, 21.2 min, respectively).
Determination of IC50 Values for 1 and 2 against HCT-116 Cells.
Concentration-cell number studies (IC50 assay) were carried out against
HCT-116 cells. These cells were grown in 5 mL culture medium
(RPMI-1640 + 15% FBS containing 1% penicillin-streptomycin and
1% glutamine) at 37 °C and 5% CO2 at a starting concentration of 5 ×
104 cells/T25 flask. On day 3, cells were exposed to different
concentrations of the drug. Flasks were incubated for 120 h (5 days)
in a 5% CO2 incubator at 37 °C, and the cells were harvested with
trypsin, washed once with HBSS, and then resuspended in HBSS and
DMMC (1), a new cyclic depsipeptide from the marine cyano-
bacterium L. majuscula, was isolated through a cancer cell
cytotoxicity assay-directed process. As such, it represents the newest
member of the majusculamide/lyngbyastatin natural product group
and extends our knowledge of the range of methylation patterns
possible within this skeletal class. DMMC displays both a mech-
anism and potency of biological activity that is consistent with the
most active members in this structural class.21 Remarkably, we
observed a similar high level of cytotoxic activity in the ring-
opened, linear form of this compound with IC50 values equivalent
to those of the parent structure. Thus, in vitro cellular and limited
in vivo therapeutic studies indicate the potential for DMMC, and
possibly its linearized form, in cancer treatment.
The majusculamide/lyngbyastatin structure class displays inter-
esting features from a biosynthetic perspective. The first and most
obvious is the variable degree of methylation on the mixed PKS/
NRPS backbone. As illustrated in Figure 3, there are five sites of
variable C-, N-, and O-methylation, which give rise to the metabolite
diversity found in this structural class. While some of the variability
can be attributed to promiscuity of amino acid incorporation during
NRPS chain extension (e.g., Tyr/Phe, Ile/Leu, Map/Ampa), ad-
ditional variability derives from variable methylation of the tyrosine
oxygen as well as the amide nitrogen of several amino acid residues.
As such, a growing appreciation of structure-activity relationships
in this metabolite class is developing from these naturally occurring
analogues and, combined with the antitumor efficacy studies
performed herein, should stimulate additional synthetic efforts to
more completely evaluate this group of natural product for useful
properties.
Experimental Section
General Experimental Procedures. Optical rotation was measured
on a Perkin-Elmer 243 polarimeter, UV recorded on a Waters Millipore
Lambda-Max model 480 LC spectrophotometer, and IR recorded on a
Nicolet 510 Fourier transform IR spectrophotometer. All NMR data
were recorded on Bruker AM400 (Table 1) and DRX300 MHz
(Supporting Information) spectrometers, with the solvent CDCl3 used
as an internal standard (δC 77.0, δH 7.26). Chemical shifts are reported
in ppm, and coupling constants (J) are reported in Hz. The FAB mass
spectrum and CID mass spectrum were recorded on Kratos MS50TC
and Perkin-Elmer Sciex API3 mass spectrometers, respectively. HPLC
isolation of 1 was performed using Waters 515 HPLC pumps, and all
solvents were either freshly distilled or purchased as HPLC grade.
Cyanobacterial Collection and Identification. The marine cyano-
bacterium Lyngbya majuscula was collected from the Kauviti Reef of
Yanuca Island, Fiji, on February 16, 2000. The specimen was identified
morphologically by WHG (voucher specimen available as collection
number VKR-16/Feb/00-05). The material was stored in 2-propanol
at reduced temperature until extraction.
Isolation of DMMC (1). Approximately 300 g dry weight of the
cyanobacterium was repetitively extracted with 2:1 CH2Cl2/CH3OH to
yield 5.5 g of crude extract. A portion of the extract (5.3 g) was
fractionated by vacuum liquid chromatography (VLC) over silica gel.
The fraction eluting with 100% EtOAc was further chromatographed
by C18 SPE (7:3 CH3OH/H2O) followed by RP HPLC (Phenomenex
Sphereclone 5 µm ODS column, 17:3 CH3OH/H2O) to yield 27.5 mg
of pure 1.
Desmethoxymajusculamide C (1): colorless glassy oil; [R]22D -104
(c 1.86, CH2Cl2); IR (neat) 3315, 2966, 2934, 2877, 1739, 1641, 1519,
1460, 1410, 1286 cm-1 1H and 13C NMR data, see Table 1 and
;
Supporting Information; HRFABMS m/z [M + H]+ 955.5867 (calcu-
lated for C49H79N8O11, 955.5868).
Base Hydrolysis of 1. Approximately 6 mg of 1 was suspended in
2 mL of a 1:1 CH3OH/0.5 M NaOH solution and allowed to stand
overnight at room temperature. The CH3OH was removed by evaporat-
ing under N2 and the mixture neutralized with HCl and then extracted

1016 Journal of Natural Products, 2009, Vol. 72, No. 6
Simmons et al.
counted using a hemocytometer. The results were normalized to an
untreated control and IC50 values determined using Prism 4.0.
DMMC (1) Clonogenic Dose-Response Analysis against HCT-
116 Cells. Concentration- and time-survival studies were carried out
with HCT-116 cells seeded at 200 or 20 000 cells in 60 mm dishes.
DMMC (1) was added to the medium (RPMI + 10% FBS) to a final
concentration of 10 µg/mL () 10.5 µM) and 10-fold dilutions thereof.
At either 2 or 24 h, the drug-containing medium was removed and
fresh medium without drug was added. For continuous exposure to
drug, the medium containing the compounds remained in contact with
the cells for the entire incubation period (168 h). The dishes were
incubated for 7 days, the medium was removed, and the colonies were
stained with methylene blue. Colonies containing 50 cells or more
were counted. The results were normalized to an untreated control.
Plating efficiency for the untreated cells was about 90%.
Therapeutic Assessment of 1. The in vivo therapeutic assessment
trial was carried out using the HCT-116 human colon tumor model as
previously described.23 Individual mouse body weights for each
experiment were within 5 g, and all mice were over 17 g at the start of
therapy. The mice were supplied food and water ad libidum. SCID
mice were pooled, implanted subcutaneously with 106 tissue-derived
tumor cells, and pooled again before distribution to treatment and
control groups (5 mice per group). Treatment with 1 started 1 day after
tumor inoculation. Mice were sacrificed after 30 days had elapsed from
tumor inoculation. Tumor weights were estimated using two-
dimensional caliper measurements done three times per week using
the following formula: tumor weight (mg) (a × b2)/2, where a and b
are the tumor length and width, respectively, in mm. The median tumor
weight was calculated as an indication of antitumor effectiveness. The
parameter, %T/C, was determined after each measurement and the
minimum value reported as therapeutic efficacy. Compound 1 was
prepared as a stock solution in DMSO, diluted 1:1 (v/v) with
Cremophor-propylene glycol (40:60 v/v), and further diluted at least
10-fold with saline before injection. Drug was prepared at 0.05 and
0.25 mg/mL for intravenous administration as a bolus injection, daily
for 5 days, in 0.25 mL volumes via the tail vein, corresponding to
0.62 and 0.31 mg/kg/day, respectively.
spectrometry and the Chemistry Department, OSU for NMR spectros-
copy. This work was supported by NIH grant CA100851.
Supporting Information Available: FT-IR, MALDI-TOF-MS, 1H
1
1
1
NMR, 13C NMR, H-1H COSY, H-13C HMBC NMR, and H-13C
meHSQC NMR of DMMC (1) in CDCl3 at either 300 MHz (1H) or 75
MHz (13C). This material is available free of charge via the Internet at
http://pubs.acs.org.
References and Notes
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Microfilament-Disrupting Activity of Compounds 1 and 2. Cyclic
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Acknowledgment. We thank B. Marquez and T. Okino for collection
of the source cyanobacterium and the country of Fiji for permission to
make these collections, and A. Risinger for help with data assembly.
We thank the Environmental Health Sciences Center at OSU for mass
NP9001674