Malyngamide 4, a New Lipopeptide from the Red Sea Marine Cyanobacterium

Phytochemistry Letters 6 (2013) 183–188

Contents lists available at SciVerse ScienceDirect

Phytochemistry Letters

jo urnal homepage: www.elsevier.com/locate/phytol

Malyngamide 4, a new lipopeptide from the Red Sea marine cyanobacterium

Moorea producens (formerly Lyngbya majuscula)

a b, c d

Lamiaa A. Shaala , Diaa T.A. Youssef *, Kerry L. McPhail , Mohamed Elbandy

Natural Products Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia

Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, United States

Clinical Nutrition Department, Applied Medical Science Faculty, Jazan University, Jazan, Saudi Arabia

A R T I C L E I N F O A B S T R A C T

Article history: In our continuing effort to discover new drug leads from Red Sea marine organisms, a sample of the

Received 19 November 2012

marine cyanobacterium Moorea producens (previously Lyngbya majuscula) was investigated. Bioassay-

Received in revised form 28 December 2012

directed puriﬁcation of a tumor cell-growth inhibitory fraction of the organic extract of the Red Sea

Accepted 16 January 2013

cyanobacterium afforded a new compound, malyngamide 4 (1), together with ﬁve previously reported

Available online 13 February 2013

compounds, malyngamide A (2) and B (3), (S)-7-methoxytetradec-4(E)-enoic acid (lyngbic acid, 4),

aplysiatoxin (5) and debromoaplysiatoxin (6). Assignment of the planar structures of these compounds

Keywords:

was based on extensive analysis of one- and two-dimensional NMR spectra and high-resolution mass

Red Sea cyanobacterium

spectrometric data. The isolated compounds were evaluated for their inhibitory activity against three

Moorea producens

cancer cell lines. In addition, the antibacterial activity of the compounds against Mycobacterium

Malyngamides 4, A and B and lyngbic acid

Aplysiatoxin and debromoaplysiatoxin tuberculosis H37Rv ATCC 27294 (H37Rv) was evaluated. Lyngbic acid (4) was the most active against M.

In vitro tumor growth inhibition tuberculosis, while malyngamides 4 (1) and B (3) moderately inhibited the cancer cell lines. The other

In vitro inhibition of Mycobacterium compounds were deemed inactive at the test concentrations.

1. Introduction (Appleton et al., 2002; Suntornchashwej et al., 2007). It is believed

that these malyngamides come either from cyanobacteria eaten by

Marine cyanobacteria are a proliﬁc source of diverse classes of the sea hares (Appleton et al., 2002; Gallimore and Scheuer, 2000;

secondary metabolites (Blunt et al., 2012). Marine members of the Suntornchashwej et al., 2007) or from an association of the red alga

genus Lyngbya (recently reclassiﬁed as Moorea) (Engene et al., 2012) with cyanobacteria (Kan et al., 1998). Two new compounds,

continue to provide a structurally diverse array of compounds with malyngamides 2 and 3, were recently reported from the marine

different bioactivities including peptides (Balunas et al., 2010; Blunt cyanobacteria Lyngbya sordida (Malloy et al., 2011) and Lyngbya

et al., 2012; Gutierrez et al., 2008; Hooper et al., 1998; Nogle et al., majuscula (Gunasekera et al., 2011).

2001; Orjala and Gerwick, 1996; Taniguchi et al., 2010), macrolides Chemically, malyngamides are amide derivatives of a fatty acid

(Blunt et al., 2012; Klein et al., 1997, 1999; Luesch et al., 2002; Pereira (mostly, 7-methoxytetradec–4(E)–enoic acid = lyngbic acid) with a

et al., 2010; Tan et al., 2002; Teruya et al., 2009) and malyngamides variety of amine-substituted moieties. Members of the malynga-

(Blunt et al., 2012; Cardellina et al., 1978, 1979; Gross et al., 2010; mide family display diverse biological activity including cancer cell

Gunasekera et al., 2011; Malloy et al., 2011). cytotoxicity (Appleton et al., 2002; Gunasekera et al., 2011; Gross

To date, more than 30 malyngamides have been reported with et al., 2010; Kwan et al., 2010; Malloy et al., 2011; Suntornch-

the majority from ﬁeld collections of members of the genus ashwej et al., 2007), anti-inﬂammatory (Appleton et al., 2002;

Lyngbya. However, two malyngamides, M and N, have been Malloy et al., 2011), anti-malarial (Suntornchashwej et al., 2007),

reported from the red alga Gracilaria coronopifolia (Kan et al., 1998). anti-tubercular (Suntornchashwej et al., 2007), ichthyotoxic

In addition, malyngamides O and P were isolated from the sea hare (Orjala et al., 1995) and nitric oxide-inhibiting activity (Malloy

Stylocheilus longicauda (Gallimore and Scheuer, 2000), while the et al., 2011), toxicity to crayﬁsh (Kan et al., 2000) and inhibition of

sea hare Bursatella leachii provided malyngamides X and S the MyD88-dependent pathway (Villa et al., 2010).

Recently, we reported about the identiﬁcation of cytotoxic

cyclic depsipeptides from Red Sea ﬁeld collections and laboratory

cultures of a marine cyanobacterium Leptolyngbya sp. (Thornburg

* Corresponding author. Tel.: +966 548535344; fax: +966 26951696.

E-mail addresses: [email protected], [email protected] (Diaa T.A. Youssef). et al., 2011). In continuation of our efforts to identify drug leads

184 L.A. Shaala et al. / Phytochemistry Letters 6 (2013) 183–188

O 11' 10'

O N O O N O O 7' 8' O 5' 6' O O 1 1' 4' 14 N N 3' O O

13' Cl 12' Cl

Malyngamide 4 (1) Malyngamide A (2)

OH O N O O O N O O

Cl OH

Malyngamide B (3) Lyngbic acid (4)

HO HO

O O O Br O O O

O O O O O O O O

OH OH OH OH

Aplysiatoxin (5) Debromoaplysiatoxin (6)

Fig. 1. Structure of Compounds 1–6.

from Red Sea cyanobacteria, we have investigated the active experiments delineated an aliphatic chain consistent with a 7–

fraction of a lipophilic extract of a new ﬁeld collection of the methoxytetradec–4(E)-enoic acid (fragment A, Fig. 2) (Cardellina

marine cyanobacterium Moorea producens (previously L. majus- et al., 1979; Kwan et al., 2010; Moore, 1981). The E conﬁguration of

cula). Bioassay-directed fractionation of the active fractions the C-4/C-5 oleﬁn was assigned based on the large coupling

resulted in isolation of a new malyngamide, malyngamide 4 (1), constant value (J4,5 = 15.5 Hz) (Kan et al., 2000). The absolute

together with several previously reported compounds including conﬁguration at C-7 in 1 was determined by comparison of the

malyngamide A (2) (Cardellina et al., 1979), malyngamide B (3) optical rotation value ([a]D = 12.5) for the hydrolytic product of 1

(Cardellina et al., 1978), lyngbic acid (4) (Cardellina et al., 1978), with that for lyngbic acid (4), suggesting that both compounds

aplysiatoxin (5) (Yoshinori and Scheuer, 1974) and debromoaply- have 7S conﬁguration (Gunasekera et al., 2011; Kwan et al., 2010).

siatoxin (6) (Yoshinori and Scheuer, 1974) (Fig. 1). In this paper, we The chemical shifts of the signals for the chloromethylene moiety

0 0

report the structure determination of compounds 1–6 and results (C-2 /C-3 ) ﬂanked by two methylenes (fragment B, Fig. 2) are in

of assays to test their growth inhibition of three cancer cell lines good agreement with other malyngamides containing this

and Mycobacterium tuberculosis H37Rv. structural motif (Cardellina et al., 1979; Kwan et al., 2010; Moore,

1981). Furthermore, chemical shifts for isolated oleﬁnic methine

0 0

2. Results and discussion CH-6 (d 6.87/93.7) and the HMBC-correlated amidic carbon C-7

(164.8, qC), together with chemical shifts for quaternary oleﬁnic C-

0 0

The molecular formula of malyngamide 4 (1) was assigned as 5 (d 173.8) and HMBC-correlated methoxy H3-12 (d 3.76/56.2),

C28H43ClN2O5 from the HRFABMS peak at m/z 523.2947 [M + H] , supported the assignment of fragment C (Fig. 2) and are again in

requiring eight degrees of unsaturation. The IR spectrum showed good agreement with literature values (Cardellina et al., 1979;

absorptions for the amidic carbonyls at 1660 and 1640 cm and Kwan et al., 2010; Moore, 1981). Finally, the assignment of the N-

1 1 13

for the C-N at 1600 cm . The H and C NMR data for 1 (Table 1) substituted pyrrol-2-one substructure (fragment D, Fig. 2) was

1 13

supported its malyngamide nature with a typical partial structure accomplished by considering HSQC-correlated H/ C chemical

of the common 7-methoxytetradec–4(E)–enoic acid (lyngbic acid) shift values, and COSY and HMBC experiments. COSY correlations

0 0

(Cardellina et al., 1979; Kwan et al., 2010; Moore, 1981). Complete between H-9 (d 7.28, 1H) and H-10 (d 6.16, 1H), which in turn

1 1 0

analysis of the 2D NMR experiments including H– H COSY, HSQC couples with H2-11 (d 4.44, 2H) established a spin system that

and HMBC established the spin systems within 1 and supported could be placed in a heterocyclic ring based on the presence of two-

the presence of four sub-structures including C-1 to C-14 and three-bond HMBC correlations from H signals for all three

0 0 0 0 13 0

(Fragment A), C-1 to C-4 (Fragment B), C-5 to C-7 (fragment C) positions to the C signal for amidic C-8 (dC 170.0, Table 1). Key

0 0

and C-8 to C-11 (fragment D) (Table 1 and Fig. 2). The assignments HMBC correlations (Table 1 and Fig. 3) established the connectivity

of the spin-coupling systems and the protonated carbons within 1 of the subunits of 1 (Table 1 and Fig. 2). For example HMBC of H2-

1 1 1 0 0

were easily deduced from H– H correlations in the COSY and JCH 1 /C-1 and H3-13 /C-1 supported the connection of fragments A

correlations in the HSQC, respectively (Table 1). In addition, the H and B. In addition, the connectivity of fragment B with C was

0 0

NMR and edited HSQC experiments showed resonances for two supported by a H-4 /C-5 HMBC cross-peak. Finally, an HMBC

0 0 0

methoxy groups at d 3.31/56.5 (CH3-15) and 3.76/56.2 (CH3-12 ), correlation from H2-11 to C-7 secured the connection of fragment

and one N-methyl at d 2.90/34.0 (CH3-13 ), together with C and D. Detailed assignment of all observed HMBC correlations is

resonances for ﬁve quaternary carbons including three amidic shown in Table 1 and Fig. 3, and supported the assignment of the

0 0

carbonyls at d 172.8 (NC = O, C-1), 134.4 (C-2 ), 173.8 (C-5 ), 164.8 quaternary carbons in 1. Thus, the planar structure of malynga-

0 0 1

(NC = O, C-7 ) and 170.0 (NC = O, C-8 ). The H NMR and COSY mide 4 could be deﬁned as shown.

L.A. Shaala et al. / Phytochemistry Letters 6 (2013) 183–188 185

Table 1

1 13

H (500 MHz) and C (125 MHz) NMR assignments for Malyngamide 4 (1) in CDCl3.

a 1 1 b

Position dC, mult. dH, mult. (J in Hz) H– H COSY HMBC

1 172.8, qC

2 34.0, CH2 2.42, t (7.3) H-3 C-1, C-3, C-4

3 28.3, CH2 2.32, m C-1, C-2, C-4, C-5

4 131.4, CH 5.51, dt (15.5, 3.6) H2-2, H-5 C-3, C-5

5 127.1, CH 5.45, dt (15.5, 3.6) C-3, C-4, C-6

6 36.4, CH2 2.17, m H-5, H-7 C-4, C-5, C-7, C-8

7 80.7, CH 3.13, m H2-6, H-8 C-5, C-9, C-15

8 33.3, CH2 1.43, m H-7, H2-9 C-6, C-7

9 25.6, CH2 1.26, br s H2-8, H2-10

10 29.7, CH2 1.26, br s

11 29.3, CH2 1.26, br s

12 31.8, CH2 1.26, br s

13 22.6, CH2 1.26, br s H3-14

14 14.1, CH3 0.87, t (7.0) H2-13 C-12, C-13

15 56.5, CH3 3.31, s C-7

0 0 0 0

1 45.5, CH2 4.34, s C-1, C-2 , C-3 , C-4 0

2 134.4, qC

0 0 0 0

3 117.6, CH 6.08, s H2-4 C-2 , C-4

0 0 0 0

4 35.8, CH2 3.54, d (1.3) H-3 C-2 , C-5 0

5 173.8, qC

0 0 0

6 93.7, CH 6.87, s C-5 , C-7 0

7 164.8, qC 0

8 170.0, qC

0 0

9 146.0, CH 7.28, td (5.6, 2.0) H-10

0 0 0 0

10 127.9, CH 6.16, td (5.6, 2.0) H-9 , H2-11 C-8

0 0 0 0 0

11 51.2, CH2 4.44, t (2.0) H-10 C-7 , C-9 , C-10

0 0

12 56.2, CH3 3.76, s C-5

0 0

13 34.0, CH3 2.90, s C-1, C-1

Multiplicity was assigned from a multiplicity-edited HSQC experiment.

b 3

HMBC experiments were optimized for a JCH of 8.0 Hz.

compounds 2 and 4, all four compounds were more than 100 times

less potent than the positive control, anticancer agent doxorubicin

O O N N (Table 2). In addition, the growth inhibitory activity of compounds

1–6 against M. tuberculosis H Rv was evaluated in an in vitro assay.

Cl O O 37

Compound 4 displayed signiﬁcant inhibitory activity (65%) against

A B C D M. tuberculosis H37Rv at a concentration of 12.5 mg/mL, whereas

compounds 1 (17% inhibition), 2 (18% inhibition) and 3 (10%

Fig. 2. Signiﬁcant HMBC correlations connecting subunits of 1. inhibition) displayed much weaker inhibition of mycobacterial

growth at the same concentration.

The following known compounds were identiﬁed through 3. Experimental

acquisition of spectroscopic data (1D, 2D NMR and MS) and

comparison of these data with those reported in the literature: 3.1. General experimental procedures

malyngamide A (2) (Cardellina et al., 1979), malyngamide B (3)

(Cardellina et al., 1978), lyngbic acid (4) (Cardellina et al., 1978), UV spectra were measured on a Hitachi 300 Spectrophotome-

aplysiatoxin (5) (Yoshinori and Scheuer, 1974) and debromoaply- ter. Optical rotation was recorded using a Rudolph Autopol III

siatoxin (6) (Yoshinori and Scheuer, 1974).

Compounds 1–6 were evaluated in vitro for growth inhibition of

Table 2

lung carcinoma (A549), colorectal carcinoma (HT29), and breast Cancer cell line inhibition by 1–6.

adenocarcinoma (MDA-MB-231) cells (Table 2). Compounds 1–4

Fraction/Compound Cell line [GI50 (mM)]

exhibited variable activity with GI50 values (the concentration

MDA-MB-231 A549 HT-29

required to achieve 50% growth inhibition of the cells) in the

a a a

micromolar range and no selectivity between the cell lines tested. Crude extract 2.0 0.2 2.0

a a a

While compounds 1 and 3 were slightly more cytotoxic than CH2Cl2-extract 1.0 0.1 1.0

a a a

Fraction 6 0.05 0.10 1.00

a a a

Fraction 7 0.05 0.01 0.01

a a a

Fraction 8 0.05 0.05 0.05

Compound 1 44 40 50

O O N Compound 2 75 88 70

O Compound 3 52 45 60

O Compound 4 65 66 63

N Compound 5 NT NT NT

Compound 6 NT NT NT

O b

Doxorubicin 0.30 0.35 0.40

COSY Cl

HMBC NT = not tested.

Values are expressed in mg/mL.

Fig. 3. COSY and HMBC correlations for 1. Positive antiproliferative control.

186 L.A. Shaala et al. / Phytochemistry Letters 6 (2013) 183–188

polarimeter (USA). The IR spectra were obtained on a Nicolet 380 air-dried. Sulforhodamine B was then extracted in 10 mM trizma

FT-IR instrument. NMR spectra were obtained in CDCl3 on Varian base solution and the absorbance measured at 490 nm. Results are

Innova 500 Spectrometers at 500 MHz for H NMR and 125 MHz expressed as GI50, the concentration that causes 50% inhibition in

for C NMR. NMR chemical shifts are expressed in parts per cell growth after correction for cell count at the start of the

million (ppm) referenced to residual CHCl3 solvent signals (d 7.25 experiment (NCI algorithm). Doxorubicin and DMSO (solvent)

1 13

for H and d 77.23 for C). Positive ion FAB mass spectrometric were used as the positive and negative controls in this assay. Prism

data were obtained with a Micromass Q-ToF equipped with leucine 3.03 from GraphPad was used for the statistical analysis of the cell

enkaphalin lockspray, using m/z 556.2771 [M + H] as a reference growth inhibition results.

mass. Optical rotation was measured on a JASCO digital

Polarimeter. For column chromatography, silica gel (Merck, 70- 3.4.2. Evaluation of antimycobacterial activity

230 mesh ASTM) and Sephadex LH-20 (Pharmacia) were used. Pre- The identiﬁed compounds 1–6 were submitted to the

coated silica gel 60 F-254 plates (Merck) were used for TLC. Tuberculosis Antimicrobial Acquisition and Coordinating Facility

(TAACF) of the Southern Research Institute for biological studies

3.2. Biological material with M. tuberculosis H37Rv. In vitro evaluation of antimycobacterial

activities of the compounds was performed according to the

The marine cyanobacterium M. producens was collected from Alamar Blue Assay (Collins and Frazblau, 1997) described brieﬂy

the Red Sea by hand at 1 m depth near Jeddah, Saudi Arabia. The below.

cyanobacterium was identiﬁed by Dr. Ali Gaballa, Faculty of

Science, Suez Canal University. A voucher sample was kept at 3.4.2.1. Alamar blue susceptibility test (MABA) (Collins and Frazblau,

Department of Natural Products, Faculty of Pharmacy, King 1997). Antimicrobial susceptibility testing was performed in

Abdulaziz University under the registration code No. 2012-LM1. black, clear-bottomed, 96-well microtiter plates (black view

plates; Packard Instrument Company, Meriden, Conn.) in order to

3.3. Extraction and isolation of the compounds minimize background ﬂuorescence. Outer perimeter wells were

ﬁlled with sterile water to prevent dehydration in experiment

The freeze-dried cyanobacterium M. producens (50 g) was wells. Initial test compound dilutions were prepared in either

extracted at room temperature with a mixture of MeOH-CH2Cl2 DMSO or distilled deionized water, and subsequent twofold

(4:1). The combined extracts were evaporated under reduced dilutions were performed in 0.1 mL of 7H9GC (no Tween 80) in the

pressure to give a greenish organic extract. The extract was microtiter plates. BACTEC 12B-passaged inocula were initially

partitioned between 60% MeOH-H2O (500 mL) and CH2Cl2 diluted 1:2 in 7H9GC, and 0.1 mL was added to wells. Subsequent

(3 150 mL). The crude CH2Cl2 partition showed growth determination of bacterial titers yielded 1 10 CFU/mL in plate

inhibitory activity against the cancer cell lines tested wells for H37Rv. Frozen inocula were initially diluted 1:20 in

(Table 2). This active CH2Cl2 extract (610 mg) was subjected BACTEC 12B medium followed by 1:50 dilution in 7H9GC.

to a ﬂash silica gel column eluted with a solvent gradient of n- Addition of 1/10 mL to wells resulted in ﬁnal bacterial titers of

hexane-CH2Cl2-acetone to afford 10 fractions. Fractions 6, 7 and 2.0 10 CFU/mL. Wells containing drug only were used to detect

8 displayed potent growth inhibitory activity against the cell autoﬂuorescence of compounds. Additional control wells con-

lines tested (Table 2). Therefore, these fractions were chosen for sisted of bacteria only (B) and medium only (M). Plates were

further separation. Fraction 6 (eluted with 20% MeOH in CH2Cl2, incubated at 37 8C. Starting at day 4 of incubation, 20 mL of 10 x

120 mg) was subjected to HPLC puriﬁcation on a semiprepara- AlamarBlue solution (Alamar Biosciences/Accumed, Westlake,

tive HPLC column (Cosmosil AR II, 5 mm, 250 10 mm) using Ohio) and 12.5 mL of 20% Tween 80 were added to one B well and

60% ACN-H2O at a ﬂow rate of 2 mL/min to give compounds 1 one M well, and plates were reincubated at 37 8C. Wells were

(2.2 mg), 3 (7.5 mg), 4 (6.6 mg) and 5 (3.2 mg). Similarly, observed at 12 and 24 h for a color change from blue to pink and

fractions 7 (eluted with 30% MeOH in CH2Cl2) and 8 (eluted for a reading of 50,000 ﬂuorescence units (FU). Fluorescence was

with 40% MeOH in CH2Cl2) were combined and the residue measured in a Cytoﬂuor II microplate ﬂuorometer (PerSeptive

(100 mg) was subjected to HPLC puriﬁcation (Cosmosil AR II, Biosystems, Framingham, Mass.) in bottom-reading mode with

5 mm, 250 10 mm, 60% ACN-H2O, 2 mL/min) to give com- excitation at 530 nm and emission at 590 nm. If the B wells

pounds 2 (5.5 mg), 5 (3.0 mg) and 6 (3.5 mg). became pink by 24 h, reagent was added to the entire plate. If the

well remained blue or 50,000 FU was measured, additional M

3.4. Biological evaluation of compounds 1–6 and B wells were tested daily until a color change occurred, at

which time reagents were added to all remaining wells. Plates

3.4.1. Cancer cell growth inhibition assay were then incubated at 37 8C, and results were recorded at 24 h

Three cancer cell lines were used in this assay, namely lung post reagent addition. Visual MICs were deﬁned as the lowest

carcinoma (A549, ATCC CCL-185), colorectal carcinoma (HT29, concentration of drug that prevented a color change. For

ATCC HTB-38), and breast adenocarcinoma cell (MDA-MB-231, ﬂuorometric MICs, a background subtraction was performed on

ATCC HTB-26). The cancer cell lines were obtained from American all wells with a mean of triplicate M wells. Percent inhibition was

Type Culture Collection (ATCC). The cell lines were maintained in deﬁned as {1 (test well FU/mean FU of triplicate B wells) x 100}.

RPMI medium supplemented with 10% fetal calf serum (FCS), The lowest drug concentration effecting an inhibition 90% was

2 mM L-glutamine and 100 U/mL penicillin and streptomycin, at considered the MIC.

37 8C and 5% CO2. Triplicate cultures were incubated for 72 h in the

presence or absence of test compounds (at ten concentrations 3.4.2.2. Statistical analysis. All analyses were performed with the

ranging from 10 to 0.0026 mg/mL). For quantitative estimation of program SAS (SAS Institute Inc., Cary, N.C.). Correlation coefﬁcients

cancer cell growth inhibition, the colorimetric sulforhodamine B were deﬁned according to Spearman for ranked data analysis and

(SRB) method was used (Skehan et al., 1990). Pearson for raw data analysis to determine the differences between

Briefly, the cells were washed twice with PBS, fixed for 15 min the BACTEC system and the MABA, either fluorometrically or

in 1% glutaraldehyde solution, rinsed twice in PBS, and stained in visually, for determination of MIC (Steel and Torrie, 1980).

0.4% SRB solution for 30 min at room temperature. The cells were A general linear model procedure using analysis of variance of

then rinsed several times with 1% acetic acid solution and ranked data was performed for each of the three variables of MIC

L.A. Shaala et al. / Phytochemistry Letters 6 (2013) 183–188 187

References

generation to determine signiﬁcant differences between the

replicate comparisons of the four experiments. Tukey’s

Appleton, D.R., Sewell, M.A., Berridge, M.V., Copp, B.R., 2002. A new biologically

Studentized range test was also used for pairwise post hoc

active malyngamide from a New Zealand collection of the sea hare Bursatella

comparison of variable analysis of ranked measure for signiﬁcant leachii. J. Nat. Prod. 65, 630–631.

Balunas, M.J., Linington, R.G., Tidgewell, K., Fenner, A.M., Uren˜a, L-D., Togna, G.L.,

differences among techniques of MIC determination and for

Kyle, D.E., Gerwick, W.H., 2010. Dragonamide E, a modiﬁed linear lipopeptide

differences between techniques for individual antimicrobial agents

from Lyngbya majuscula with antileishmanial activity. J. Nat. Prod. 73, 60–66.

for each bacterial strain. Signiﬁcance was determined at P 0.05. Blunt J.W., Copp, B.R., Keyzers, R.A., Munro, M.H.G., Prinsep, M.R., 2012. Marine

natural products. Nat. Prod. Rep. 29, 144–222, and previous reports of this

series.

3.4.2.3. Bacterial strains and growth conditions. M. tuberculosis

Cardellina II, J.H., Dalietos, D., Marner, F.-J., Mynderse, J.S., Moore, R.E., 1978. (-)-

H37Rv ATCC 27294 (H37Rv), was obtained from the American Trans-7(S)-methoxytetradec-4-enoic acid and related amides from the marine

Type Culture Collection (Rockville, Md.). For the ﬁrst three (of cyanophyte Lyngbya majuscula. Phytochemistry 17, 2091–2095.

Cardellina II, J.H., Marner, F.J., Moore, R.E., 1979. Malyngamide A, a novel chlorinated

four) replicate experiments, H37Rv inocula were ﬁrst passaged in

metabolite of the marine cyanophyte Lyngbya majuscula. J. Am. Chem. Soc. 101,

radiometric 7H12 broth (BACTEC 12B; Becton Dickinson Diag- 240–242.

nostic Instrument Systems, Sparks, Md.) until the growth index Collins, L., Frazblau, S.G., 1997. Microplate alamar blue assay versus BACTEC 460

system for high-throughput screening of compounds against Mycobacterium

(GI) reached 800 to 999. For the fourth replicate experiment,

tuberculosis and Mycobacterium avium. Antimicrob. Agents Chemother. 41,

H37Rv was grown in 100 mL of Middlebrook 7H9 broth (Difco, 1004–1009.

Detroit, Mich.) supplemented with 0.2% (vol/vol) glycerol (Sigma Engene, N., Rottacker, E.C., Kasˇtovsky´ , J., Byrum, T., Choi, H., Ellisman, M.H.,

Koma´rek, J., Gerwick, W.H., 2012. Moorea producens gen. nov., sp. nov. and

Chemical Co., Saint Louis. Mo.), 10% (v/v) OADC (oleic acid,

Moorea bouillonii comb. nov. tropical marine cyanobacteria rich in bioactive

albumin, dextrose, catalase; Difco), and 0.05% (v/v) Tween 80

secondary metabolites. Int. J. Syst. Evol. Microbiol. 62, 1171–1178.

(Sigma). The complete medium was referred to as 7H9GC-Tween. Gallimore, W.A., Scheuer, P.J., 2000. Malyngamides O and P from the sea hare

Stylocheilus longicauda. J. Nat. Prod. 63, 1422–1424.

Cultures were incubated in 500 mL nephelometer ﬂasks on a

Gross, H., McPhail, K.L., Goeger, D.E., Valeriote, F.A., Gerwick, W.H., 2010. Two

rotary shaker (New Brunswick Scientiﬁc, Edison, N.J.) at 150 rpm

cytotoxic stereoisomers of malyngamide C, 8-epi-malyngamide C and 8-O-

and 37 8C until they reached an optical density of 0.4–0.5 at acetyl-8-epi-malyngamide C, from the marine cyanobacterium Lyngbya majus-

cula. Phytochemistry 71, 1729–1735.

550 nm. Bacteria were washed and suspended in 20 mL of

Gunasekera, S.P., Owle, C.S., Montaser, R., Luesch, H., Paul, V.J., 2011. Malyngamide 3

phosphate-buffered saline and passed through a ﬁlter of 8 mm

and cocosamides A and B from the marine cyanobacterium Lyngbya majuscula

pore size to eliminate clumps. The ﬁltrates were aliquoted, stored from Cocos Lagoon, Guam. J. Nat. Prod. 74, 871–876.

at 80 8C, and used within 30 days. Gutierrez, M., Suyama, T.L., Engene, N., Wingerd, J.S., Matainaho, T., Gerwick, W.H.,

2008. Apratoxin D, a potent cytotoxic cyclodepsipeptide from Papua New

Guinea collections of the marine cyanobacteria Lyngbya majuscula and Lyngbya

3.5. Spectral data

sordida. J. Nat. Prod. 71, 1099–1103.

Hooper, G.J., Orjala, J., Schatzman, R.C., Gerwick, W.H., 1998. Carmabins A and B,

new lipopeptides from the Caribbean cyanobacterium Lyngbya majuscula. J. Nat.

3.5.1. Compound 1 (Malyngamide 4)

Prod. 61, 529–533.

Pale yellow oil; [a] 19.68 (c 0.018, MeOH); UV (MeOH) l

D max Kan, Y., Fujita, T., Nagai, H., Sakamoto, B., Hokama, Y.J., 1998. Malyngamides M and N

(log e) 267 (4.35), 210 (4.21) nm; IR (ﬁlm) 3320, 2923, 2845, 1660, from the Hawaiian red alga Gracilaria coronopifolia. J. Nat. Prod. 61, 152–155.

1 1 Kan, Y., Sakamoto, B., Fujita, T., Nagai, H., 2000. New malyngamides from the

1640 (br), 1600, 1544, 1455, 1370, 1256, 1123, 1061 cm ; H and

13 + Hawaiian cyanobacterium Lyngbya majuscula. J. Nat. Prod. 63, 1599–1602.

C NMR see Table 1; HRFABMS m/z 523.2947 [M + H] (calcd.

Klein, D., Braekman, J.C., Daloze, D., Hoffmann, L., Demoulin, V., 1997. Lyngbyalo-

523.2938 for C28H44 ClN2O5, D + 1.1 ppm). side, a novel 2,3,4-tri-O-methyl-6-deoxy-a-mannopyranoside macrolide from

Lyngbya bouillonii (Cyanobacteria). J. Nat. Prod. 60, 1057–1059.

Klein, D., Braekman, J.C., Daloze, D., Hoffmann, L., Castillo, G., Demoulin, V., 1999.

4. Conclusion

Madangolide and laingolide A, two novel macrolides from Lyngbya bouillonii

(Cyanobacteria). J. Nat. Prod. 62, 934–936.

Kwan, J.C., Teplitski, M., Gunasekera, S.P., Paul, V.J., Luesch, H., 2010. Isolation and

Six compounds, malyngamide 4 (1), malyngamide A (2),

biological evaluation of 8-epi-malyngamide C from the Floridian marine cya-

malyngamide B (3), lyngbic acid (4), aplysiatoxin (5) and

nobacterium Lyngbya majuscula. J. Nat. Prod. 73, 463–546.

debromoaplysiatoxin (6) were isolated and identiﬁed from a Luesch, H., Yoshida, W.Y., Harrigan, G.G., Doom, J.P., Moore, R.E., Paul, V.J., 2002.

new collection of the Red Sea marine cyanobacterium M. Lyngbyaloside B, a new glycoside macrolide from a Palauan marine cyanobac-

terium, Lyngbya sp. J. Nat. Prod. 65, 1945–1948.

producens. The isolated compounds were evaluated for cancer

Malloy, K.L., Villa, F.A., Engene, N., Matainaho, T., Gerwick, L., Gerwick, W.H., 2011.

cell toxicity and inhibition of M. tuberculosis. Malyngamides 4

Malyngamide 2, an oxidized lipopeptide with nitric oxide inhibiting activity

(1) and B (3) displayed moderate inhibition of the cancer cell from a Papua New Guinea marine cyanobacterium. J. Nat. Prod. 74,

95–98.

lines, while malyngamide A (2) was weakly active. Lyngbic acid

Moore, R.E., 1981. In: Scheuer, P.J. (Ed.), Marine Natural Products: Chemical and

(4) was inactive against all cell lines. On the other hand, lyngbic

Biological Perspectives, Vol. IV. Academic Press, New York, pp. 1–52.

acid was the only active compound, which signiﬁcantly Nogle, L.M., Okino, T., Gerwick, W.H., 2001. Antillatoxin B, a neurotoxic lipopeptide

from the marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 64,

inhibited the growth of M. tuberculosis. Other compounds

983–985.

were inactive or very weakly active at the same concentration.

Orjala, J., Nagle, D., Gerwick, W.H., 1995. Malyngamide H, an ichthyotoxic amide

This study clearly demonstrates the potential biosynthetic possessing a new carbon skeleton from the Caribbean cyanobacterium Lygnbya

majuscula. J. Nat. Prod. 58, 586–589.

capabilities of members of the marine cyanobacterial genus

Orjala, J., Gerwick, W.H., 1996. Barbamide, a chlorinated metabolite with mollus-

Moorea to produce different secondary metabolites of diverse

cicidal activity from the Caribbean cyanobacterium Lyngbya majuscula. J. Nat.

biosynthetic origins, which have been reported from other Prod. 59, 427–430.

Pereira, A.R., McCue, C.F., Gerwick, W.H., 2010. Cyanolide A, a glycosidic macrolide

Moorea species worldwide.

with potent molluscicidal activity from the Papua New Guinea cyanobacterium

Lyngbya bouillonii. J. Nat. Prod. 73, 217–220.

Acknowledgment Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T.,

Bokesch, H., Kenney, S., Boyd, M.R., 1990. New colorimetric cytotoxicity assay

for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107–1112.

This work was funded by the Deanship of Scientiﬁc Research

Steel, R.G.D., Torrie, J.H., 1980. Principles and procedures of statistics. A biometrical

(DSR), King Abdulaziz University, Jeddah, under grant no. (322/

approach, 2nd ed. McCraw-Hill, New York, NY 272–284 and 550–551.

166/1432). The authors, therefore, acknowledge with thanks DSR Suntornchashwej, S., Suwanborirux, K., Koga, K., Isobe, M., 2007. Malyngamide X:

the ﬁrst (7R)-lyngbic acid that connects to a new tripeptide backbone from the

technical and ﬁnancial support. In vitro antimycobacterial evalua-

Thai sea hare Bursatella leachii. Chem. Asian J. 2, 114–122.

tions were provided by the U.S. Tuberculosis Antimicrobial

Tan, L.T., Ma´rquez, B.L., Gerwick, W.H., 2002. Lyngbouilloside, a novel glycosidic

Acquisition and Coordinating Facility (TAACF), NIAID/NIH under macrolide from the marine cyanobacterium Lyngbya bouillonii. J. Nat. Prod. 65,

925–928.

the Research and Development Contract No. 1540.

188 L.A. Shaala et al. / Phytochemistry Letters 6 (2013) 183–188

Taniguchi, M., Nunnery, J.K., Engene, N., Esquenazi, E., Byrum, T., Dorrestein, P.C., and E and Ibu-epidemethoxylyngbyastatin 3, from a Red Sea Leptolyngbya

Gerwick, W.H., 2010. Almyramide A, a cyclic depsipeptide from a Palmyra Atoll cyanobacterium. J. Nat. Prod. 74, 1677–1685.

collection of the marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 73, Villa, F.A., Lieske, K., Gerwick, L., 2010. Selective MyD88-dependent pathway

393–398. inhibition by the cyanobacterial natural product malyngamide F acetate Eur.

Teruya, T., Sasaki, H., Kitamura, K., Nakayama, T., Suenaga, K., 2009. Biselyngbyaside, J. Pharmacol. 629, 140–146.

a macrolide glycoside from the marine cyanobacterium Lyngbya sp. Org. Lett. Yoshinori, K., Scheuer, P.J., 1974. Aplysiatoxin and debromoaplysiatoxin, constitu-

11, 2421–2424. ents of the marine mollusk Stylocheilus longicauda (Quoy and Gaimard, 1824). J.

Thornburg, C.C., Thimmaiah, M., Shaala, L.A., Hau, A.M., Malmo, J.M., Ishmael, J.E., Am. Chem. Soc. 7, 2245–2246.

Youssef, D.T.A., McPhail, K.L., 2011. Cyclic depsipeptides, grassypeptolides D