Marine Cyanobacterial Fatty Acid Amides Acting on Cannabinoid Receptors

Full text of DOI:10.1002/cbic.201200502

DOI: 10.1002/cbic.201200502
Marine Cyanobacterial Fatty Acid Amides Acting on Cannabinoid Receptors
[
a]
[b]
[a]
Rana Montaser, Valerie J. Paul, and Hendrik Luesch*
The fact that lipids are utilized by diverse organisms suggests
[1]
an evolutionarily conserved role of this class of compounds.
Indeed, lipidomics is emerging as a crucial field of research be-
cause of the key role of lipid biomolecules in a wide array of
[
2]
physiological functions. Research has also uncovered some
vital lipid–protein interactions in which lipid molecules bind to
specific protein domains to mediate physiological effects. The
endocannabinoid system is a representative example; here
two characterized G protein-coupled cannabinoid receptors
CB and CB are modulated by endogenous lipids known as
1
2
endocannabinoids. Importantly, this system has been implicat-
ed in various pathophysiologies, including neurodegenerative
[3–7]
diseases, eating disorders, pain, inflammation, and cancer.
Therefore, a better understanding of this system has become
of significant interest, and the cannabinoid receptors are
[
1,5]
viewed as possible targets for different diseases.
The classi-
cal concept that all agonists at a given GPCR induce a similar
repertoire of downstream events is now uncertain, and the
latest experimental evidence supports the existence of ligand-
[3]
specific functional selectivity at the cannabinoid receptors.
Consequently, the identification of new structural scaffolds that
can bind to the cannabinoid receptors remains an essential
tool for digging further into this complex system.
Scheme 1. Structures of the endocannabinoid anandamide and fatty acid
amides from marine cyanobacteria with binding affinities to the cannabinoid
receptors.
Anandamide (N-arachidonoylethanolamine, 1; Scheme 1)
was the first endogenous ligand to be identified among the
From a Lyngbya sample from the Piti Bomb Holes in Guam,
we isolated and then characterized the new fatty acid amide
serinolamide B (5; Scheme 1), a closely related analogue of ser-
inolamide A. Based on structural features, we evaluated the
ability of compound 5 to bind to both human cannabinoid
receptors CB and CB with a functional outcome. The marine
[
8]
endocannabinoid family. The structure of this fatty acid
amide suggested that other natural and synthetic fatty acid
amides might also function as cannabinoid receptor ligands.
Marine cyanobacteria of the genus Lyngbya have a characteris-
tic metabolic profile that is rich in fatty acid amides (in addi-
1
2
[
9]
tion to peptides), and they therefore represent a potential
source of new model compounds that would act on the can-
nabinoid receptors. Support for this assumption comes from
reports of metabolites isolated from Lyngbya samples that can
interact with the cannabinoid receptors. To our knowledge,
only five marine cyanobacterial fatty acid amides with binding
affinities to the cannabinoid receptors have been identified;
cyanobacteria Lyngbya spp. are also well known for the pro-
duction of a large class of fatty acid amides known as malyng-
[13]
amides. More than 30 malyngamide analogues with a broad
spectrum of bioactivities are known. Although malyngamides
usually contain different amine portions and sometimes vary in
the length of the fatty acyl chain, they generally have a unique
and characteristic structural scaffold of an N-substituted amide
of a long-chain 7-methoxy fatty acid with mono-unsaturation
at C4 (Scheme 2). As malyngamides are the most abundant
fatty acid amides found in Lyngbya spp., we examined whether
the malyngamide-type structural features could also bind to
the cannabinoid receptors, although they usually possess more
complicated amine portions and somewhat different fatty acid
side chains from the known endogenous cannabinoids. For
[
10]
[11]
grenadamide (2), semiplenamides A (3), B, and G, and the
[
12]
recently reported metabolite serinolamide A (4) (Scheme 1).
However, none of them has been tested in functional assays
before, so it remains unknown whether those metabolites act
as receptor agonists or antagonists.
[a] R. Montaser, Prof. H. Luesch
[14]
Department of Medicinal Chemistry, University of Florida
this, we tested malyngamide B (6; Scheme 2) for its potential
as a cannabimimetic compound. Interestingly, malyngamide B
can bind to both CB and CB with moderate potencies. Here,
1
600 SW Archer Road, Gainesville, FL 32610 (USA)
E-mail: luesch@cop.ufl.edu
1
2
[b] Dr. V. J. Paul
we report the results of our studies on two marine cyanobac-
terial metabolites; the newly identified analogue serinolami-
de B (5) and a member of a large group of cyanobacterial fatty
acid amides, malyngamide B (6).
Smithsonian Marine Station
Fort Pierce, FL 34949 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200502.
2
676
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ChemBioChem 2012, 13, 2676 – 2681

1
13
Table 1. H and C NMR spectroscopy data for serinolamide B (5) in
CDCl
3
at 600 MHz.
[
a]
Unit
C/H #
d
C
d
H
(J [Hz])
HMBC
fatty acid
1
172.9 qC
2, 3, 19, NH
3b, 4
2, 4, 5
2
3
3
4
5
6
7
8
1
1
1
36.4 CH
28.4 CH
2
2
2.27 dd (6.6, 2.1)
2.28 d (6.9)
2.23 m
a
b
127.8 CH 5.39 m
132.1 CH 5.49 m
3a, 3b, 5, 6
3b, 4, 6, 7
1.96 ddd (7.7, 7.5, 6.8) 4, 5, 7, 8
32.3 CH
29.3 CH
–15 29.4 CH
2
2
2
2
2
3
1.32 m
1.25 m
1.25 m
1.29 m
6, 8
6, 7
17, 18
16, 18
16, 17
[
b]
6
7
8
31.8 CH
22.4 CH
13.9 CH
0.88 t (6.8)
serinol ether 19
50.3 CH 4.07 m
20a, 21a, 21b, NH
19, 21a, 21b
2
2
2
2
2
0a
64.2 CH
73.6 CH
59.2 CH
2
2
3
3.82 dd (11.2, 4.1)
3.66 brd (11.2)
3.58 dd (9.3, 4.2)
3.53 dd (9.3, 4.2)
3.36 s
0b
1a
1b
2
19, 21a, 20b, 22
21a, 21b
NH
6.16 d (6.8)
[
a] Protons showing long-range correlation to indicated carbon. [b] Over-
lapping peaks.
Scheme 2. General and specific structures of malyngamides.
fatty acid chain between C4 and C5 (Scheme 1, Table 1). Finally,
to complete the molecular formula suggested by the MS data,
the number of methylene groups forming the fatty acid chain
was assigned to construct an 18-carbon mono-unsaturated
fatty acid.
A cyanobacterial sample from Guam was extracted three
times with EtOAc/MeOH. Solvent partitioning of the organic
extract yielded 7.2 g of a semipolar nBuOH fraction that was
then fractionated by silica gel chromatography. Compound 5
was purified by reversed-phase HPLC from a silica column frac-
The absolute configuration of the chiral center in the serinol
moiety was determined through Jones oxidation of the pri-
mary alcohol to its corresponding carboxylic acid, followed by
acid hydrolysis to liberate O-Me serine. Enantioselective analy-
sis revealed an S configuration of the amino acid and conse-
quently an R configuration in the parent compound. Addition-
ally, the double-bond geometry was assigned as trans based
on the chemical shifts of the adjacent methylene groups C3
[
15]
tion that also contained pitipeptolides. The molecular formu-
la C H NO was established by high-resolution electrospray
2
2
43
3
ionization mass spectrometry (HRESIMS; m/z 370.3316
+
[
M+H] ). NMR profiles of this compound were characteristic of
a fatty acid derivative, and the typical chemical shifts of
a mono-unsaturated fatty acid chain were prominent (Table 1):
[12,16]
and C6
and the absence of NOESY correlations between
1
13
H and C NMR spectra showed a number of overlapping
methylene groups (d ꢀ1.2 ppm and d ꢀ23 ppm), a carbonyl
the two olefinic protons. While we were investigating the bio-
logical activity of this metabolite, the Gerwick group reported
H
C
[12]
1
(
d =172.9 ppm), an a-methylene group (d =2.27 ppm and
the closely related analogue 4 (Scheme 1). Notably, H and
C
H
13
d =36.4 ppm), a terminal methyl group (d =0.88 ppm and
C chemical shifts and the stereochemical assignments for 5
C
H
dC =13.9 ppm), and characteristic olefinic methines (d =
match those reported for 4.
H
5
.39 ppm, d =127.8 ppm and d =5.49, d =132.1 ppm). Addi-
Given the structural similarity of serinolamide A (4) to the
endocannabinoids anandamide (1) and 2-arachidonoyl glycer-
ol, it was tested for binding to the human cannabinoid recep-
tors CB and CB ; it appeared to possess more than fivefold se-
C
H
C
tionally, the compound appeared to be a fatty acid amide; the
amide proton at d =6.16 ppm showed HMBC correlation to
H
the carbonyl carbon as well as a COSY correlation to a methine
1
2
proton at 4.07 ppm (d =50.3 ppm). Three oxygenated groups
lectively for the CB receptor, with a moderate binding affinity
1
C
[12]
were also identified based on their chemical shifts; two meth-
(K =1.3 mm). As the only structural difference between the
i
ylene groups (d =3.58, 3.53 ppm, d =73.6 ppm; and d =
two serinolamide analogues is a secondary amide in 5 instead
of the tertiary amide in 4, we evaluated the cannabimimetic
activity of serinolamide B. This compound can also bind to
both CB and CB receptors with moderate to weak binding
H
C
H
3.66, 3.82 ppm, d =64.2 ppm) and a methoxy group (d =
C H
3
.36 ppm and d =59.2 ppm); the latter showed HMBC correla-
C
tion to the first oxygenated methylene group (d =73.6 ppm;
C
1
2
Table 1). Further analysis of COSY, TOCSY, and HMBC data al-
lowed the amine part of this molecule to be constructed as
a monomethyl serinol and located the olefinic system in the
affinities (Figure 1A, Table 2). However, 5 showed an opposite
trend in binding affinities compared to 4, as it exhibited a mod-
erate affinity and higher selectivity for CB (K =5.2 mm) over
2
i
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2677

malyngamide structural features
were not probed before for can-
nabinoid receptor interactions.
Accordingly, we were interested
in testing a representative ana-
logue so as to determine
whether this molecular architec-
ture is capable of possessing
cannabimimetic effects. We ob-
tained malyngamide B (6) from
our marine natural products li-
brary and tested it for CB and
1
CB2 binding. Interestingly,
6
appeared to possess moderate
binding affinities to both recep-
tors with K values of 3.6 mm for
i
CB1 and 2.6 mm for CB2 (Fig-
ure 1A, Table 2). Those results
are noteworthy because 6 has
a more complex amine portion
than the endocannabinoids and
other cyanobacterial fatty acid
amides that possess the same
biological activity (Schemes 1
and 2).
It has not yet been deter-
mined whether the marine cya-
nobacterial fatty acid amides
that can bind to the cannabi-
noid receptors act as agonists
or antagonists. In order to clari-
fy this, we tested the functional
response induced by the bind-
ing of 5 and 6 to the cannabi-
Figure 1. A) Binding of compounds 5 (*) and 6 (
(
&
) as well as HU-210 (~) to the receptors CB
(left) and CB
1 2
right), represented as percent inhibition of the binding of a radioactive ligand. B) Simplified representation of the
cannabinoid receptors and the consequences of agonist binding. C) Effect of compounds 5 (top) and 6 (bottom)
on forskolin-induced cAMP accumulation. A higher level of cAMP produces a higher luminescence reading (RLU).
noid receptors. Cannabinoid receptors are G protein-coupled
receptors that are functionally coupled to the inhibition of ad-
enylyl cyclase and subsequent inhibition of cAMP accumula-
Table 2. The affinities of compounds 4 and 5 for the cannabinoid recep-
tors (K) and consequent functional effects on cAMP accumulation (EC50).
i
[6]
tion (Figure 1B). Compounds 5 and 6 were able to inhibit for-
skolin-stimulated cAMP accumulation through both CB1 and
CB2 receptors with moderate potencies (Figure 1C, Table 2);
this proves that those metabolites act as cannabinoid receptor
agonists. Intriguingly, serinolamide B (5) appeared to be more
CB
1
CB
2
[
a]
[a]
Compound
K
i
[mm]
EC50 [mm]
K
i
[mm]
EC50 [mm]
5
6
16.4
3.6
11.8
5.3
–
5.2
2.6
0.0011
–
1.8
8.8
–
[
b]
HU-210
CP 55940
0.00069
–
[c]
0.00024
0.00036
CB -receptor-selective in the binding as well as the functional
2
[
a] Results from cAMP functional assays. EC50 is the agonist concentration
assays, but malyngamide B (6) appeared to bind to both recep-
tors similarly with comparable functional outcomes (Figure 1C,
Table 2).
required to cause half-maximal inhibition of forskolin-induced cAMP accu-
mulation. [b] Positive control for binding assays. [c] Positive control for
functional assays.
It is known that anandamide signaling is terminated by the
enzyme fatty acid amide hydrolase (FAAH), which catalyzes
anandamide hydrolysis. Therefore, one emerging pharmaco-
logical approach to augment endocannabinoid activity is di-
CB1 (K =16.4 mm). Notably, the endocannabinoid 1 shows
i
[21]
higher selectivity for CB (K for CB =32 nm; K for CB =
rected towards FAAH inhibition. Thus, we tested the ability
of metabolites 5 and 6 to inhibit this enzyme. However, no
considerable inhibitory effects were detected for either com-
pound at 10 and 100 mm.
1
i
1
i
2
[
17]
1
.9 mm), thus suggesting that the presence of a secondary
rather than a tertiary amide is not the main determinant for re-
ceptor selectivity.
Malyngamides have been reported with different biological
activities, including cytotoxic, anti-inflammatory, and quorum-
It has been repeatedly shown that cannabimimetic com-
[1,7,22]
pounds can also mediate anti-inflammatory responses.
From that perspective, we tested the ability of compounds 5
[
18–20]
sensing actions.
Yet, to the best of our knowledge, the
2
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2012, 13, 2676 – 2681

and 6 to exert anti-inflammatory effects in lipopolysaccharide
LPS)-induced murine macrophages RAW 264.7. Serinolamide B
showed a weak effect with an IC >25 mm; however, malyng-
receptor agonists implies that they can mediate certain physio-
logical effects through this pathway, and therefore opens more
research avenues. Several malyngamides have been subjected
to total chemical syntheses and some well-established synthet-
(
50
amide B was more potent, inhibiting NO production with an
[25]
IC50 of 6.2 mm without affecting cellular viability at up to
ic routes are already available, these can assist structural op-
timization efforts towards more potent analogues.
25 mm. Although the evidence suggests that the anti-inflamma-
tory effects of some cannabimimetic compounds are mediated
through cannabinoid receptors, particularly CB , it is questiona-
2
Experimental Section
ble whether this is also the case with malyngamide B. Notably,
Mukhopadhyay et al. showed that no detectable CB receptors
2
General experimental procedures: The optical rotation was mea-
sured on a PerkinElmer 341 polarimeter. UV and optical activity
were measured on a SpectraMax M5 (Molecular Devices), and IR
data were obtained on a PerkinElmer Spectrum One FTIR Spec-
trometer. The H and 2D NMR spectra were recorded on a Bruker
Avance II 600 MHz spectrometer. All spectra were obtained in
were apparent in RAW 264.7 cells unless they were stimulated
[
23]
by LPS. Therefore, the effect of 6 on LPS-induced inflamma-
tion is probably not totally mediated through the cannabinoid
receptors, as it was able to prevent the early stimulation by
1
LPS. Further experiments with CB -receptor-selective antago-
2
CDCl3 by using residual solvent signals (d =7.26 ppm, d =
H
C
nists or CB -receptor-deficient cells will help ascertain the pres-
2
7
7.16 ppm) as internal standards. HSQC and HMBC experiments
ence or absence of a cannabinoid-receptor-mediated anti-in-
flammatory effect for 6.
1
1
were optimized for JCH =145 Hz and JCH =7 Hz, respectively.
HRMS data were recorded on an Agilent LC-TOF mass spectrome-
ter equipped with an APCI/ESI multimode ion source detector in
positive-ion mode. LC-MS data were obtained by using an API
3200 triple quadrupole MS (Applied Biosystems) equipped with
a Shimadzu LC system.
Some malyngamides can also reduce NO accumulation
under similar anti-inflammatory assay conditions; malyngami-
de F acetate (7) and malyngamide 2 (8; Scheme 2) have IC
50
[
20]
[19]
values of 7.1 and 8 mm, respectively. Acetate 7 was shown
to possess a distinctive cytokine profile and appeared to selec-
Extraction and isolation: The sample of the marine cyanobacteri-
um Lyngbya majuscula (recollection of UOG strain VP627) was col-
lected at Piti Bomb Holes, Guam, in February 2000. A voucher
sample (voucher specimen number EC025) has been preserved at
the Smithsonian Marine Station at Fort Pierce, FL. The freeze-dried
organism was extracted with EtOAc/MeOH (1:1, 3ꢁ) to give
a crude organic extract (35.5 g), which was partitioned between
hexanes and 80% aqueous MeOH. After the methanolic phase had
[
20]
tively inhibit the MyD88-dependent pathway. Notably, 7 and
share common structural features such as oxidized cyclohex-
8
yl rings, whereas compound 6 has a significantly different
amine entity (Scheme 2). To our knowledge, this is the first
report of such activity for an anti-inflammatory malyngamide
with the pyrrolidone ring in the amine portion rather than the
common six-membered cyclic ketone or lactone.
been dried, the residue was partitioned between nBuOH and H O.
2
We also tested the cytotoxic effects of compounds 5 and 6
against cancer cells. Serinolamide B failed to show significant
cytotoxicity against HT-29 colon adenocarcinoma and MCF7
breast cancer cell lines at up to 100 mm. It is important to
point out here that serinolamide A showed some cytotoxic
The concentrated nBuOH residue (7.2 g) was subjected to flash
chromatography over silica gel, eluting with increasing gradients
of iPrOH in CH Cl , and finally with MeOH. The fraction eluting
2 2
with 4% iPrOH/CH Cl2 was fractionated on a semi-preparative
2
reversed-phase HPLC column (YMC-Pack ODS-AQ, 250ꢁ10 mm,
ꢁ1
[
12]
5 mm, 2 mLmin ; UV detection at 220/254 nm) by using a linear
properties in a different cell line. In contrast, malyngamide B
gradient of MeOH/H O (75–100% aqueous MeOH over 30 min, and
[
24]
2
is known as a feeding deterrent and, in our hands, was cyto-
toxic to HT-29 cells with an IC50 value of 26 mm, but it remains
unclear if its cannabimimetic activity contributes to this cyto-
toxic effect.
then 100% MeOH for 10 min) to afford ten fractions. Repurification
[26]
[15]
of five fractions yielded pitiprolamide and pitipeptolides. Com-
pound 5 eluted as a single peak (fraction 10) at t =28.8 min.
R
20
Serinolamide B (5): colorless, amorphous solid; [a] =ꢁ7.9 (c=
D
In this report, we have identified the new cannabimimetic
marine cyanobacterial fatty acid amide serinolamide B (5). Seri-
1
13
0
.075, CHCl ); H NMR, C NMR and HMBC data, see Table 1; IR
3
(
film): n =3290, 3077, 2955, 2920, 2851, 1641, 1542, 1465,
max
ꢁ1
nolamide B with a secondary amide had higher CB receptor
2
1377 cm ; HRESI/APCIMS: m/z calcd for C H NO : 370.3316
[M+H] , found: 370.3324.
22
44
3
+
selectivity and lower cytotoxicity than its analogue with a terti-
ary amide. In agreement, other reports showed that several
Jones oxidation and enantioselective amino acid analysis by
HPLC/MS: Compound 5 (1 mg) was dissolved in acetone (1 mL),
then freshly prepared Jones reagent (CrO in diluted H SO , 50 mL)
structural features can increase affinity for the CB receptor, in-
2
cluding an E double bond at position 4, an amide proton, and
3
2
4
[
1,22]
additional substituents in the amine part.
Testing analogues
was added. The reaction mixture was stirred at room temperature
for 1 h. The reaction was then quenched by the addition of a few
drops of iPrOH, and the mixture was filtered through a pad of
celite. The reaction mixture was dried down under nitrogen, and
the residue was redissolved in water and partitioned between
water and EtOAc three times. The organic layer was dried under ni-
trogen, and the crude product was purified by HPLC (YMC-Pack
4
and 5 side by side under the same experimental conditions
will unequivocally clarify this comparison. We also show that
malyngamide B (6) also possesses cannabimimetic properties;
this provides new insight into the biological activities of ma-
lyngamides, the most abundant marine fatty acid amide class
in Lyngbya spp. This finding introduces a new structural lead
to the cannabimimetic field from the marine environment, and
should foster the cannabimimetic evaluation of further ana-
logues. Additionally, our finding that both metabolites act as
ꢁ1
ODS-AQ, 250ꢁ10 mm, 5 mm, 2 mLmin ; UV detection at 220/
2
00 nm) using a MeOH/0.05% aq. TFA linear gradient (75–100%
aqueous MeOH over 20 min, then 100% MeOH for 10 min) to give
the oxidized compound (0.7 mg) at t =27.7 min (68% yield). Then,
R
ChemBioChem 2012, 13, 2676 – 2681
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
2679

HCl (6n, 400 mL) was added to the product (100 mL), and the mix-
ture was stirred at 1108C overnight. The reaction mixture was
dried, reconstituted in water (100 mL) and subjected to HPLC/MS
enantioselective analysis. For the standards, (S)-O-Me-Ser standard
Griess reagent. Briefly, sulfanilamide (1% w/v) in phosphoric acid
(5% v/v; 50 mL) was added to the cell culture supernatant (50 mL),
and the culture was incubated for 5 min at room temperature in
the dark, then naphthylethylenediamide·HCl (0.1% w/v, 50 mL) was
added. After 5 min of incubation at room temperature in the dark,
the absorbance of the reaction mixture was measured at 540 nm
by using a microplate reader. Assays were run in duplicate. Nitrite
quantification was determined relative to a nitrite standard curve
(0–100 mm).
(
0.3 mg; Waterstone Technology, Carmel, IN, USA) was subjected to
partial epimerization to obtain the (R)-O-Me Ser standard. The com-
pound was dissolved in water (80 mL), then triethylamine (32 mL)
and acetic anhydride (32 mL) were added. The reaction mixture was
stirred at 608C for 1 h and then dried down. The residue was redis-
solved in HCl (6n, 100 mL), and the solution was stirred at 1108C
overnight, then dried again. The S/R enantiomer ratio obtained
from the partial epimerization reaction was 9:1. Standards as well
as the test compound were subjected to HPLC/MS chiral analysis
Cell viability assays: Cells were propagated and maintained in
Dulbecco’s modified Eagle medium (Invitrogen) supplemented
with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) at
3
78C under humidified air with 5% CO . Cells were seeded in 96-
2
(
MRM monitoring) under the following conditions: CUR 10, CAD
well plates (MCF7: 10500 cells per well; HT-29: 11000 cells per
well). After 24 h, cells were treated with various concentrations of
the test compound or solvent control (1% EtOH). After 48 h of
incubation, cell viability was measured by using MTT (Promega)
according to the manufacturer’s instructions.
medium, IS 5500, TEMP 600, GS1 55, GS2 55, positive-ion mode,
MRM pair [120!74], t : (S)-O-Me-Ser: 10.6 min, (R)-O-Me-Ser:
R
1
5.4 min, oxidized moiety from compound 5 (10.6 min).
Cannabinoid CB /CB receptor binding assays: Assays were done
1
2
by Caliper Life Sciences (Hanover, MD, USA). Human recombinant
CB (B =1.5 pmol per mg protein) or CB (B =8 pmol per mg
1
max
2
max
3
protein) receptors were expressed in HEK-293 cells. [ H]CP-55940 Acknowledgements
(
0.5 nm) was used as the radioligand (K for CB =0.6 nm and for
d
1
CB =4.2 nm). HU-210 (1 mm) was used as nonspecific binding de-
2
This research was supported by the National Institutes of Health,
NIGMS Grant P41M086210. This is contribution 893 from the
Smithsonian Marine Station.
terminant (K values of 1.1 and 3.0 nm for CB and CB , respective-
i
1
2
ly). Reactions were carried out in Tris·HCl buffer (50 mm, pH 7.4)
containing EDTA (2.5 mm), MgCl (5 mm), and BSA (0.1%) at 308C
2
for 90 min. The reaction was terminated by rapid vacuum filtration
onto glass fiber filters. Radioactivity trapped onto the filters was
determined by liquid scintillation spectrometry and compared to
control values in order to ascertain any interactions of the test
compound with the CB or CB binding sites.
Keywords: amides
cannabimimetics · malyngamides · marine cyanobacteria
·
anti-inflammatory
agents
·
1
2
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1] J. Gertsch, Planta Med. 2008, 74, 638–650.
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1
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2
[
2
2
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4
dures. Briefly, cells were seeded in 96-well plates (3ꢁ10 cells per
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Received: August 3, 2012
Published online on November 9, 2012
ChemBioChem 2012, 13, 2676 – 2681
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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