Lipids DOI 10.1007/s11745-014-3949-9

ORIGINAL ARTICLE

Mooreamide A: A Cannabinomimetic Lipid from the Marine Cyanobacterium Moorea bouillonii Emily Mevers • Teatulohi Matainaho • Marco Allara’ • Vincenzo Di Marzo • William H. Gerwick

Received: 1 May 2014 / Accepted: 26 August 2014 Ó AOCS 2014

Abstract Bioassay-guided fractionation of a collection of Moorea bouillonii from Papua New Guinea led to the isolation of a new alkyl amide, mooreamide A (1), along with the cytotoxic apratoxins A–C and E. The planar structure of 1 was elucidated by NMR spectroscopy and mass spectrometry analysis. Structural homology between mooreamide A and the endogenous cannabinoid ligands, anandamide, and 2-arachidonoyl glycerol inspired its evaluation against the neuroreceptors CB1 and CB2. Mooreamide A was found to possess relatively potent and selective ligand binding activity to CB1 (K1 = 0.47 lM) versus CB2 (K1 [ 25 lM). This represents the most potent

Electronic supplementary material The online version of this article (doi:10.1007/s11745-014-3949-9) contains supplementary material, which is available to authorized users. E. Mevers  W. H. Gerwick (&) Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093, USA e-mail: [email protected] E. Mevers Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, USA T. Matainaho Discipline of Pharmacology, School of Medicine and Health Sciences, University of Papua New Guinea, National Capital District, Port Moresby, Papua New Guinea M. Allara’  V. Di Marzo Institute of Biomolecular Chemistry, National Research Council, 80078 Pozzuoli, Italy W. H. Gerwick Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USA

marine-derived CB1 ligand described to date and adds to the growing family of marine metabolites that exhibit cannabinomimetic activity. Keywords Marine natural product  Mooreamide  Cyanobacteria  Cannabinoid  Acyl amide  HPLC  NMR Abbreviations 2-AG 2-Arachidonoyl glycerol AEA Anandamide CDCl3 Deuterated chloroform CH2Cl2 Dichloromethane CNS Central nervous system COSY Correlation spectroscopy ECS Endocannabinoid system EtOAc Ethyl acetate ESIMS Electrospray ionization mass spectrometry FT Fourier transform GPCR G protein-coupled receptor HMBC Heteronuclear multiple bond correlation HPLC High performance liquid chromatography HR High resolution HSQC Heteronuclear single quantum correlation IR Infrared LR Low resolution MeCN Acetonitrile MeOH Methanol NMR Nuclear magnetic resonance NOESY Nuclear Overhauser effect spectroscopy NP Normal phase OLS Olefin synthase RP Reverse phase SAM S-Adenosyl methionine SCUBA Self-contained underwater breathing apparatus SPE Solid phase extraction

123

Lipids

UV Wt

Ultraviolet Weight

2'

HO

1'

H N

O

21

1

HO

16

2'

20 7

Mooreamide A (1)

Introduction Marine organisms have been an exceptionally prolific source of both structurally intriguing and biological active natural products [1–3]. In recent years, several structurally modified marine metabolites have successfully been approved to treat numerous human disease conditions, such as pathogenic viruses [Ara-V (sponge)], cancer [Ara-C (sponge), trabectedin (sea squirt), halaven (sponge), brentuximab vedotin (cyanobacteria)], and pain [prialt (cone snail)], with many more in various stages of clinical trial [4]. One subset of a rather intriguing biomedical target are neuroreceptors, more specifically, the cannabinoid receptors. There are two known subtypes of this G protein-coupled receptor (GPCR), CB1 and CB2, which are primarily localized in the central nervous (CNS) and immune systems, respectively [5]. The endocannabinoid system (ECS) is known to have a host of biological functions including regulation of appetite [6], development [7], learning and memory [8], and its pharmacological manipulation has been suggested to be useful for the management of pain [9], cancer [10], and diabetes [7]. Two endogenous ligands for CB1 are anandamide (AEA) and 2-arachidonoyl glycerol (2-AG). These consist of arachidonic acid with either an ethanolamine (AEA) or glycerol (2-AG) moiety in amide or ester linkage, respectively. Over the past several years, our laboratory, as well as others, have isolated a number of cyanobacterial metabolites with striking structural resemblance to these known endocannabinoids, thus, revealing that these prokaryotes are a rich source of these bioactive lipids [12, 13]. In the current investigation, a collection of a filamentous marine cyanobacterium from Pigeon Island, Papua New Guinea in May 2005 yielded a chromatography fraction exhibiting potent toxicity against both brain and pancreatic cancer cell lines and was, thus, chosen for further evaluation. Subsequent bioassay-guided fractionation yielded apratoxins A–C and E as the cancer cell toxins [11], along with a new acyl amide, mooreamide A (1) (Fig. 1). The structure of this new co-metabolite was determined by NMR and mass spectroscopy methods, and based on its structure, it was evaluated for cannabinomimetic activity and shown to be a selective CB1 receptor binding ligand.

Materials and Methods General Experimental Procedures UV spectra were obtained on a Beckman Coulter DU-800 spectrophotometer, and IR spectra were obtained using a

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O HO

N H

HO HO

Anandamide (2) O

O 2-Arachidonoylglycerol (3)

Fig. 1 Structures of mooreamide A (1), anandamide (2), and 2-arachidonoylglycerol (3)

Nicolet IR-100 FT-IR spectrophotometer using KBr plates. NMR spectra were recorded with solvent peaks as internal standard (dC 77.0, dH 7.26 for CHCl3) on a Varian Unity 500 MHz spectrometer (500 and 125 MHz for 1H and 13C NMR, respectively), a Varian VNMRS (Varian NMR System) 500 MHz spectrometer equipped with a cold probe (500 and 125 MHz for 1H and 13C NMR), and a Bruker 600 MHz spectrometer equipped with a 1.7 mm MicroCyroProbe (600 and 150 MHz for 1H and 13C NMR). LR- and HR-ESIMS were obtained on ThermoFinnigan LCQ Advantage Max and Thermo Scientific LTQ Orbitrap-XL mass spectrometers, respectively. All solvents were either distilled or of HPLC quality. Cyanobacterial Collection, Extraction and Isolation The producing cyanobacterium PNG 5/19/05-8 was collected by self-contained underwater breathing apparatus (SCUBA) in 10–30 feet of water where it was found growing tangled within the coral Stylophora pistillata off Pigeon Island, Papua New Guinea, in May 2005. The cyanobacterium was separated from the coral by hand and was previously identified as Moorea bouillonii by 16S rRNA sequencing [14–16]. The biomass (37.9 g, dry wt) was extracted with 2:1 CH2Cl2/MeOH to afford 1.2 g of dried extract and was subsequently fractionated by silica gel VLC using a stepwise gradient solvent system of increasing polarity starting from 100 hexanes to 100 % MeOH (nine fractions, A-I). The fraction eluting with 75 % EtOAc/MeOH (fraction H) was separated further by a 1 g RP SPE using a stepwise gradient solvent system of decreasing polarity starting from 50 MeOH/H2O to 100 % CH2Cl2. Fractions 3 and 4, which eluted with 70 % MeOH/ H2O and 80 % MeOH/H2O, respectively, were combined and further purified using RP HPLC [4l Synergi Fusion, 60 % MeCN/H2O for 30 min, followed by 70 % MeCN/ H2O for 15 min and then 100 % MeCN for 20 min at 3 mL/min to produce six fractions (1–6)]. The final step in the purification employed NP HPLC [5l Luna, holding

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90 % hexanes/EtOAc for 5 min, then changing to 100 % EtOAc over 15 min at 3 mL/min to produce another eight fractions (A–H)] yielding 0.7 mg (a 0.06 % yield from crude extract) of pure mooreamide A (1), and an impure mixture of apratoxin A-C and D. Mooreamide A (1): pale yellow oil; UV (MeOH) kmax (log e) 230 (4.12) nm; IR (neat) vmax 3,399, 2,924, 2,853, 1,657, 1,543, 1,441, 1,383, 1,265, 1,197, 1,076, 738 cm-1; 1 H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Table 1; HRESIMS [M ? H]? m/z 390.3006 (calcd for C24H40NO3 390.3003). CB1 and CB2 Receptor Binding Assays CB1 and CB2 displacement assays were carried out following previously reported techniques, which are outlined below [17]. Membranes from HEK-293 cells stably transfected with human recombinant CB1 receptor (Bmax = 2.9 pmol/

mg protein) or human recombinant CB2 receptor (Bmax = 6.0 pmol/mg protein) were incubated with the high affinity ligand [3H]-CP-55 940 (0.4 nM/Kd = 0.11 nM and 0.53 nM/Kd = 0.19 nM, respectively, for the CB1 and CB2 receptors). This radioactive ligand was displaced with 10 lM WIN 55212-2 as the heterologous competitor to calculate non-specific binding (Ki values 8.8 and 0.9 nM, respectively, for the CB1 and CB2 receptor). All compounds were tested following the procedure described by the manufacturer (Perkin Elmer, Italia). Displacement curves were generated by incubating drugs with [3H]-CP-55,940, for 90 min at 30 °C. Ki values were calculated by applying the Cheng-Prusoff equation to the IC50 values obtained from GraphPad for the displacement of the bound radioligand by increasing concentrations of the test compound. Data are means of at least three independent experiments.

Results 1

13

Table 1 H and CDCl3 Position

daC

1

167.2

2 3

C NMR assignments for mooreamide A (1) in dH (J in Hz)b

HMBCc

COSYb

122.0

5.74, d (11.7)

1, 4

3, 4

145.9

6.03, dt (11.5, 7.7)

1, 4, 5

2, 4

4

28.6

2.70, dt (7.5, 6.4)

1, 2, 3, 5

2, 3, 5

5

32.0

2.12, m

3, 4

4, 6, 7

6

129.2

5.43, m

4, 5, 7, 8

5, 8

7

130.9

5.43, m

5, 6, 8

5, 8

8

31.1

2.12, m

6, 7, 11

4, 6, 7

9

29.5

1.25, m

8, 11

10

28.8

1.31, m

8, 11

11

39.8

2.05, m

8, 12, 13, 21

12

136.0

13

124.8

5.78, d (11.0)

11, 14, 15, 21

14

14

126.6

6.22, dd (15.1, 11.0)

12, 13, 16

13, 15

15 16

132.5 32.9

5.57, dt (15.1, 7.0) 2.07, m

13, 16, 17 14, 15, 17

15, 16 15, 18

17

28.7

1.39, m

15, 16, 18

18

33.7

2.04, m

17, 19, 20

17, 19

19

139.1

5.82, dt (10.3, 6.5)

17, 18, 20

18, 20

20a

114.2

4.99, d (17.0)

18

19

4.93, d (10.3)

18

19

20b 21

16.5

1.72, s

11, 13

10

52.1

4.00, dt (7.2, 4.1)

20

NH

63.9

3.85, m

10

OH

NH

6.25, m

1

10

OH

2.50, bs

0

2 /3

0

a

125 MHz for

b

13

500 MHz for 1H NMR, and COSY

c

600 MHz for HMBC

C NMR

20

The cyanobacterium Moorea bouillonii was collected by SCUBA in 10-30 feet of water where it was found growing tangled within the coral Stylophora pistillata off Pigeon Island, Papua New Guinea, in May 2005. The isopropanolpreserved collection was repetitively extracted (CH2Cl2/ MeOH, 2:1) and fractionated using normal-phase vacuum liquid chromatography (VLC). Previously, a middle polarity fraction from this extract exhibited potent molluscicidal activity and yielded the polyglycosylated macrolide, cyanolide A [18]. A more polar fraction eluting with 25 % MeOH/EtOAc exhibited potent toxicity against brain and pancreatic cancer cell lines and was, thus, chosen for further fractionation. Using a combination of reverse-phase solid phase extraction (SPE), along with both normal and reverse-phase HPLC, yielded 0.7 mg of mooreamide A (1), a pale yellow oil, along with the previously identified apratoxins A-C and E. The mixture of the four apratoxins was subsequently found to explain the cancer cell toxicity of this fraction. HR-ESIFTMS of 1 yielded an [M ? H]? peak at m/z 390.3006, indicating a molecular formula of C24H39NO3 and requiring six degrees of unsaturation (calcd for C24H40NO3 390.3003). IR spectroscopy suggested the presence of a carbonyl along with NH and/or OH functionalities with strong absorption bands at 1,657 and 3,399 cm-1. The 13C NMR spectrum suggested that the carbonyl was present as an amide or ester functionality with an observed shift at dC 167.2, and also indicted the presence of five olefinic bonds (dC 145.9, 139.1, 136.0, 132.5, 130.9, 129.2, 126.6, 124.8, 122.0, and 114.2), thus, accounting for all six degrees of unsaturation. The 1H NMR spectrum supported the presence of both NH and OH protons resonating at dH 6.25 (1H) and 2.50 (2H),

123

Lipids

respectively, and a number of olefins with ten protons resonating between dH 4.90 and 6.30 along with one deshielded vinyl methyl at dH 1.71. Analysis of 2D NMR data for 1 (COSY, HSQC, HMBC, and NOESY) led to the identification of three partial structures (a–c). The first (a) was comprised of two hydroxy groups (dH 2.50 9 2), one amide proton (dH 6.25), two methylenes (dH 3.85 9 2) and a methine (dH 4.00). From 1H-1H COSY correlations, it was clear that this fragment was an amino glycerol moiety and this was further supported by the symmetry seen in both the 1H and 13C NMR data (Table 1). The second spin system (b) consisted of 11 carbons arrayed in a linear fashion, beginning with an a,b-unsaturated ketone, followed by two methylene groups in the c and d positions. Adjacent to the distal methylene, was a disubstituted carbon–carbon double bond followed by four consecutive methylene groups. The third and final fragment (c) consisted of the remaining ten carbons, and included a conjugated diene that was substituted at one end with a vinyl methyl group, followed by three consecutive methylene groups, and a terminal carbon–carbon double bond. The three partial structures were assembled by two key HMBC correlations. Partial structures a and b were linked by a correlation from the NH proton of a to the a,bunsaturated ketone in b (NH to C-1). A second key HMBC correlation was between the vinyl methyl in partial structure c to the distal methylene in b (H-21 to C-10), thus, completing the planar structure of mooreamide A (1) (Fig. 2). The configurational assignments of the four internal olefins were determined by a combination of NOE correlations, 3J coupling, and 13C NMR analysis. The configuration of the tri-substituted olefin between C-12/C-13 was determined from an NOE correlation between H-21 (dH 1.72) and the olefinic proton on C-14 (dH 6.22), establishing the configuration of the double bond as E. The configuration of the olefins between C-2/C-3 and C-14/C-

H N

HO

c

HO a

HO

O

H N

CH3 b

O

HO COSY

HMBC

ROESY

Fig. 2 Partial structures and select 2D NMR data for mooreamide A (1)

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15 were determined using 3J coupling analysis. The measured coupling constant between C-2/C-3 was 11.7 Hz, indicative of Z configuration, whereas the coupling constant between C-16/C-17 was 15.1 Hz, which is indicative of E configuration [19]. Determining the configuration of the final olefin between C-6/C-7 was more challenging because both vinyl protons and adjacent methylenes were overlapped, thus, preventing the use of either 3J coupling constant analysis or NOE correlations. Thus, the distinctive carbon shifts of the two allylic methylenes (C-5 and C-8) adjacent to the olefin were used to infer its geometry [20, 21]. Both methylene groups showed downfield-shifted carbon resonances (C-5, dC 32.0; C-8, dC 31.1), which is indicative of an E configured olefin. Due to the isolation of the highly cytotoxic apratoxins from the same fraction that yielded mooreamide A (1), and the limited quantity of 1 available, we did not evaluate its cytotoxic activity [11]. However, based on structure homology, between 1 and the two endocannabinoid ligands, anandamide (2), and 2-arachidonoylglycerol (3), it was evaluated against both cannabinoid receptors CB1 and CB2. Compound 1 exhibited reasonably potent affinity for CB1 with a Ki value of 0.47 lM but no affinity for CB2 (Ki [ 25 lM) and, thus, appears to be relatively selective for CB1 ([50-fold). This is the strongest affinity for CB1 thus far measured from any marine metabolite that, due to chemical similarities with either cannabinoids or endocannabinoids, has ever been tested on these receptors [13, 22–24].

Discussion The new lipid metabolite, mooreamide A (1), was isolated from a collection of Moorea bouillonii that was found growing on the tropical coral Stylophora pistillata. This collection has been extraordinarily fruitful as numerous structurally intriguing metabolites have previously been isolated, such as several apratoxins, lyngbyabellins [31], and cyanolide A [14–16]. Mooreamide A is structurally distinct from each of these other compound classes as it consists of a modified fatty acid tail and an amino glycerol amide linkage. The planar structure of 1 was determined by NMR and other spectroscopic techniques, and the configurational analysis of each of the olefins utilized a variety of methods, including, 3J coupling, NOE, and 13C NMR chemical shift analysis. This structure adds to a growing family of acyl amide metabolites from cyanobacteria. Most of them possess interesting biological activities. Biosynthetically, the C1-C20 chain is likely assembled by a polyketide synthase (PKS). Four of the five olefins (C2/C3, C6/C7, C12/C13, and C14/C15) occur in between the predicted acetate units, and, therefore, the modules

Lipids

responsible for each are predicted to lack the enoylreductase domain [25]. However, the terminal olefin (C19/C20) must be formed in a different manner as it involves both carbons of a single acetate unit. The mechanism for formation of this functionality may be similar to that involved in forming the terminus of the jamaicamides that involves a fatty acid type desaturase [26, 27]. Other known mechanisms to form terminal olefins, such as the olefin synthase (OLS) in the curacin A pathway, introduce terminal olefins at the carboxyl terminus and, thus, are not likely involved in forming the terminal olefin in mooreamide A [28, 29]. The C-21 methyl branch is predicted to be attached to C-2 of an acetate group and, thus, likely derives from S-adenosyl methionine (SAM). The amino glycerol ‘‘head group’’ may derive from the amino acid serine, in which case the carboxylic acid could be reduced to a primary alcohol as part of a reductive offloading [30]. Mooreamide A was found to be a moderately strong ligand for the CB1 receptor (Ki 0.47 lM), with [50-fold selectivity over the CB2 receptor, and, to the best of our knowledge, the highest affinity CB1 ligand thus far identified from a marine source (e.g., the first sub-micromolar ligand). For example our previous reports on acyl amides serving as CB1 ligands, such as semiplenamides A, B, and G (Ki’s ranging from 17 to 20 lM) and serinolamide A (Ki 1.3 lM), demonstrated these all to be of low to moderate potency [22–24]. Similarly, the Luesch group has reported that serinolamide B is a modestly potent CB2 selective ligand (Ki 5.2 lM) with agonist properties, whereas malyngamide B is a moderately potent non-selective CB ligand (CB1 Ki 3.6 lM; CB2 Ki 2.6 lM) [13]. Comparison of these various acyl amides and their relative potency as CB1 or CB2 ligands suggests that the increased affinity of mooreamide relative to the serinolamides may be due to the fact that both oxygen functional groups in the amine head group are free alcohols, the lipid tail has increased unsaturation (five olefins in mooreamide A versus one olefin in both serinolamides) and is two carbons longer with a methyl branch, or a combination of these structural attributes. There has been a long debate on the evolution of cannabinomimetic compounds vs. that of CB1 and CB2 receptors. While the former are found in several terrestrial plants, the most famous and richest being by definition Cannabis sativa, as well as in animals as primitive as the coelenterate Hydra vulgaris, cannabinoid receptor orthologues are found only in vertebrates and in the chordate Ciona intestinalis [32]. Thus, high affinity cannabinoid receptor ligands appeared in evolution much earlier than cannabinoid receptors, as supported by the current finding, and strengthens the hypothesis that these ligands may have functions not related to the modulation of CB1 and CB2 orthologues. Nevertheless, because compounds such as

mooreamide A are unlikely to be metabolized by endocannabinoid catabolic enzymes, they might represent useful pharmacological and therapeutic tools. In this sense, further studies are required using functional assays of CB1 receptor activity to assess whether mooreamide A behaves as an agonist or an antagonist. Acknowledgments We thank the government of Papua New Guinea for the permission to collect the cyanobacterial specimens. We thank the UCSD Chemistry and Biochemistry mass spectrometry facilities for their analytical services. The 500 MHz NMR 13C XSens cold probe was supported by NSF CHE-0741968. We also acknowledge The Growth Regulation & Oncogenesis Training Grant NIH/NCI (T32A009523-24) for a fellowship to E. M., and NIH Grants (CA100851, NS053398) for support of the research.

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