Potential anti-inflammatory actions of the elmiric (lipoamino) acids

Article abstract of DOI:10.1016/j.bmc.2007.03.026

A library of amino acid-fatty acid conjugates (elmiric acids) was synthesized and evaluated for activity as potential anti-inflammatory agents. The compounds were tested in vitro for their effects on cell proliferation and prostaglandin production, and compared with their effects on in vivo models of inflammation. LPS stimulated RAW 267.4 mouse macrophage cells were the in vitro model and phorbol ester-induced mouse ear edema served as the principal in vivo model. The prostaglandin responses were found to be strongly dependent on the nature of the fatty acid part of the molecule. Polyunsaturated acid conjugates produced a marked increase in media levels of i15-deoxy-PGJ₂ with minimal effects on PGE production. It is reported in the literature that prostaglandin ratios in which the J series predominates over the E series promote the resolution of inflammatory conditions. Several of the elmiric acids tested here produced such favorable ratios suggesting that their potential anti-inflammatory activity occurs via a novel mechanism of action. The ear edema assay results were generally in agreement with the prostaglandin assay findings indicating a connection between them.

Full text of DOI:10.1016/j.bmc.2007.03.026

Bioorganic & Medicinal Chemistry 15 (2007) 3345–3355
Potential anti-inflammatory actions of the elmiric (lipoamino) acids
d
b
a
Sumner H. Burstein,a,c, Jeffrey K. Adams, Heather B. Bradshaw, Cristian Fraioli,
*
Ronald G. Rossetti,c Rebecca A. Salmonsen,a John W. Shaw,d J. Michael Walker,b
Robert E. Zipkind and Robert B. Zurierc
aDepartment of Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA
bThe Gill Center for Biomolecular Science and the Department of Psychological and Brain Sciences, Indiana University,
Bloomington, IN, USA
cDepartment of Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA
dBIOMOL International LP, Plymouth Meeting, PA 19462, USA
Received 9 January 2007; revised 23 February 2007; accepted 8 March 2007
Available online 13 March 2007
Abstract—A library of amino acid-fatty acid conjugates (elmiric acids) was synthesized and evaluated for activity as potential anti-
inflammatory agents. The compounds were tested in vitro for their effects on cell proliferation and prostaglandin production, and
compared with their effects on in vivo models of inflammation. LPS stimulated RAW 267.4 mouse macrophage cells were the
in vitro model and phorbol ester-induced mouse ear edema served as the principal in vivo model. The prostaglandin responses were
found to be strongly dependent on the nature of the fatty acid part of the molecule. Polyunsaturated acid conjugates produced a
marked increase in media levels of i15-deoxy-PGJ2 with minimal effects on PGE production. It is reported in the literature that pros-
taglandin ratios in which the J series predominates over the E series promote the resolution of inflammatory conditions. Several of
the elmiric acids tested here produced such favorable ratios suggesting that their potential anti-inflammatory activity occurs via a
novel mechanism of action. The ear edema assay results were generally in agreement with the prostaglandin assay findings indicating
a connection between them.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Acid congeners of anandamide or lipoamino acids, here
named elmiric acids, exist as endogenous substances and
may have a role in regulating tissue levels of ananda-
mide.1 This hypothesis received support from a study
that showed that such a substance, N-arachidonylgly-
cine (NAGly, Fig. 1), is indeed an endogenous constitu-
ent of many tissues and occurs at higher concentrations
Figure 1. The structures of the endocannabinoid anandamide and its
endogenous analog, N-arachidonylglycine (NAGly). Several amino
than anandamide.2 An interesting property of NAGly is
its potent inhibitory effect on FAAH, the enzyme pri-
marily responsible for the termination of anandamide
action via hydrolysis. As could be expected, NAGly
treatment in both in vitro and in vivo models leads to
a robust increase in anandamide concentrations.3 It
was also observed that several of the elmiric acids pro-
foundly reversed the prostaglandin (PG) profile from a
acid–fatty acid combinations have been found in tissue extracts.2
pro inflammatory to an anti-inflammatory state in a cell
culture model. This has formed the basis for a hypothe-
sis on the mechanism for the potential anti-inflamma-
tory action of these compounds. At this point, it is
assumed that these two effects occur independently of
one another, however, the possibility that the amidase
inhibition and PG ratio effects are somehow interrelated
cannot be ruled out.
Keywords: Endocannabinoid; Anti-inflammatory; Ear edema;
Prostaglandin.
*
The older literature on this topic is mainly concerned
with lipoamino acids of bacterial origin.4–8 These
Corresponding author. Tel.: +1 508 856 2850; e-mail:
sumner.burstein@umassmed.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.026

3346
S. H. Burstein et al. / Bioorg. Med. Chem. 15 (2007) 3345–3355
involve amino acid conjugation with complex and unu-
sual fatty acids, and little is known about their function
in bacteria. More recently, attention has been given to
the lipoamino acids present in mammalian species in
part because of their possible relationships to the endo-
cannabinoids. The origin of NAGly in vivo is not well
understood, however, it is an endogenous substance
found in rat brain and other sites that occurs in amounts
greater than the closely related endocannabinoid,
anandamide.2 In this same report, it was suggested that
NAGly might have analgesic properties similar to those
reported for anandamide9,10 but would be inactive in as-
says for psychotropic action such as the ring test.11 The
latter was in agreement with a report showing a lack of
affinity by NAGly for the cannabinoid receptor, CB1.12
This activity profile is reminiscent of that observed for
THC-11-oic acid and its analog ajulemic acid.13 Based
on preliminary data, NAGly appears to be the endoge-
nous ligand for the orphan G-protein-coupled receptor
GPR18.14,15 Like anandamide, NAGly is also a sub-
strate for COX-2 giving rise to amino acid conjugates
of the prostaglandins.16 Other amino acid conjugates
have been found in diverse tissues17 and N-arachido-
noyl-L-serine has recently been isolated from rat17 and
from bovine brain and reported to have vasodilatory
effects in rat mesenteric arteries.18 Finally, NAGly inhib-
its the glycine transporter, GLYT2a, which is expressed
in pre synaptic glycinergic neurons.19
a potent and efficacious molecule for the treatment of
conditions characterized by acute and chronic
inflammation.
2. Chemistry
Several of the elmiric acids were synthesized in the uni-
versity laboratories using the procedures described
below and shown in Scheme 1. Briefly this involved con-
densation of the acid chloride with the amino acid
methyl ester in the presence of triethylamine followed
by saponification with lithium hydroxide to yield the
elmiric acid. However, the majority of the compounds
came from a commercially available library of endocan-
nabinoids obtained from Biomol International LP
(Plymouth Meeting, PA 19462) that contains sixty
examples, half of which would fall into the category of
elmiric acids. Table 1 is a partial list of these com-
pounds. They are composed of glycine, L-alanine, and
c-aminobutyric acid (GABA) conjugates with ten differ-
ent naturally occurring long-chain fatty acids ranging
from palmitic to docosahexaenoic acid.
3. Results and discussion
3.1. Effects on cell proliferation
We have defined the term elmiric acids as compounds
that conform to the general structure shown in Figure
2 for which a short hand nomenclature system is pro-
posed. Using this system N-arachidonylglycine would
be written as: EMA-1 (20:4). EMA stands for elmiric
acid; each amino acid constituent is assigned a number,
for example, 1 = glycine; 2 = alanine, etc. The identity of
the acyl substituent is indicated in parentheses; for
example, (20:4) = arachidonoyl; (16:0) = palmitoyl, etc.
This system would also accommodate any number of
unnatural amino acids. We are proposing this nomen-
clature to simplify the naming of these compounds; it
has not been approved or adopted by any official body.
Initially, the library shown in Table 1 was screened for
anti-proliferative activity in an in vitro assay using the
mouse macrophage cell line RAW264.7 as the drug tar-
get. Table 2 shows the results of this study in which each
compound was tested at three concentrations, 0.1, 1.0,
and 10 lM, and compared with vehicle treated cells as
a control. Based on these limited data, it is not possible
to assign a rank order for this group of substances, how-
ever, it is clear that many produced around 90% reduc-
tion of cell numbers with increasing concentrations
between 1.0 and 10 lM. In the glycine series this
amounted to 6 out of 10 compounds, whereas in the
L-alanine series 9 out of 10 compounds were inhibitory
at 10 lM. A cross series comparison showed general
agreement with one exception, namely N-palmitoyl gly-
cine, which was inactive while its L-alanine counterpart
showed 89% inhibition at 10 lM. Palmitoyl glycine is
an abundant component of the lipid fraction from rat
skin and has significant effects on calcium levels in F11
cells, considered to be a model for dorsal root ganglion
neurons (Bradshaw et al., unpublished data). It also can
reduce heat stimulated firing of nociceptive neurons.
The results obtained with the c-aminobutyric acid
(GABA) conjugates were similar to most of the other
elmiric acids tested (data not shown).
In this report, we have screened in vitro a library of
elmiric acids consisting of glycine and L-alanine conju-
gates with a series of ten fatty acids. The objective was
to determine whether any of these compounds are can-
didates for further study as potential anti-inflammatory
agents. Positive findings would support further studies
in which the amino acid moieties would be expanded
not only to include naturally occurring molecules but
also synthetic examples. The ultimate goal is to discover
O
C
R2
C
3.2. Anti-inflammatory action in vitro
COOH
R1
N
H
R3
Data obtained with selected examples of elmiric acids
from our library are shown in Table 3. These were cho-
sen to provide a variety of fatty acid conjugates with
either glycine or alanine to test for a possible struc-
ture–activity relationship. With the latter, examples of
both enantiomers were included to detect possible
Figure 2. The general structure for the elmiric acids. This name has
been chosen to designate the entire family of molecules that includes
both the naturally occurring lipoamino acids such as NAGly and
synthetic analogs where R1 is a long chain alkyl group, and R2 and R3
can be a wide range of substituents.

S. H. Burstein et al. / Bioorg. Med. Chem. 15 (2007) 3345–3355
3347
R2
C
R2
C
long-chain fatty acid
chloride/triethylamine
methanol/HCl
H
H
N
H
COOH
R2
N
H
COOCH3
R3
R3
R2
LiOH/THF
R1CO
R1CO
C
C
N
COOH
N
COOCH3
H
R3
H
R3
Scheme 1. General synthetic procedure for the elmiric acids.
Table 1. Library of compounds
Several literature reports suggest that an elevation of tis-
sue concentrations of 15d-PGJ2 is associated with the
resolution of an inflammatory condition.20–23 This is
thought to come about through the binding and activa-
tion of the transcription factor PPAR-c followed by
increased expression of anti-inflammatory factors.
Previous studies in our laboratory showed that the syn-
thetic cannabinoid analog ajulemic acid elevates i15d-
PGJ2 in similar models, that is, LPS-challenged fibro-
blast-like synovial cells (Stebulis et al. unpublished
data).
Chemical name
EMA name
Palmitoyl glycine
Oleoyl glycine
Linoleoyl glycine
EMA-1 (16:0)
EMA-1 (18:1)
EMA-1 (18:2)
EMA-1 (c-18:3)
EMA-1 (20:2)
EMA-1 (c-20:3)
EMA-1 (20:4)
EMA-1 (20:5)
c-Linolenoyl glycine
Eicosa-11Z,14Z-dienoyl glycine
Dihomo-c-linolenoyl glycine
Arachidonoyl glycine
Eicosapentaenoyl glycine
Docosatetra-7Z,10Z,13Z,16Z-enoyl glycine EMA-1 (22:4)
Docosahexaenoyl glycine
EMA-1 (22:6)
Palmitoyl L-alanine
Oleoyl L-alanine
Linoleoyl L-alanine
L-EMA-2 (16:0)
L-EMA-2 (18:1)
L-EMA-2 (18:2)
L-EMA-2 (c-18:3)
L-EMA-2 (20:2)
L-EMA-2 (c-20:3)
L-EMA-2 (20:4)
L-EMA-2 (20:5)
L-EMA-2 (22:4)
The murine macrophage cell line RAW 264.7 has been
used widely as a model for inflammatory responses
in vitro. Moreover, we and others have reported data
on cannabinoid induced responses in these cells.24–26
LPS is a potent inducer of inflammatory responses
including, the arachidonic acid cascade, and has been
used in a wide variety of models including some used
to evaluate drug candidates. Our model consisted of a
cultured monolayer of RAW cells in which media con-
centrations of prostaglandins were measured by ELISA
following LPS stimulation. The use of immunoassay
procedures always raises questions of specificity. There-
fore, we have indicated that we measured immunoreac-
tive prostaglandin as defined by the cross reactivities of
the antiserum used by the manufacturer. It is also neces-
sary to monitor PGE2 concentrations in our model since
they may increase as well. In view of the fact that PGE2
is a pro inflammatory eicosanoid, a sharp increase in its
level following drug treatment would possibly offset any
benefit resulting from an increase in PGJ concentration.
It is suggested that PGJ/PGE ratio measurements may
be a useful initial test for anti-inflammatory activity.
c-Linolenoyl L-alanine
Eicosa-11Z,14Z-dienoyl L-alanine
Dihomo-c-linolenoyl L-alanine
Arachidonoyl L-alanine
Eicosapentaenoyl L-alanine
Docosatetra-7Z,10Z,13Z,16Z-enoyl
L-alanine
Docosahexaenoyl L-alanine
L-EMA-2 (22:6)
Palmitoyl GABA
Oleoyl GABA
Linoleoyl GABA
EMA-9 (16:0)
EMA-9 (18:1)
EMA-9 (18:2)
EMA-9 (c-18:3)
EMA-9 (20:2)
EMA-9 (c-20:3)
EMA-9 (20:4)
EMA-9 (20:5)
c-Linolenoyl GABA
Eicosa-11Z,14Z-dienoyl GABA
Dihomo-c-linolenoyl GABA
Arachidonoyl GABA
Eicosapentaenoyl GABA
Docosatetra-7Z,10Z,13Z,16Z-enoyl GABA EMA-9 (22:4)
Docosahexaenoyl GABA EMA-9 (22:6)
stereoselectivity. For comparison, the widely used
NSAID naproxen, a COX inhibitor, was included. All
of the elmiric acids were screened at two concentrations,
1 and 10 lM. A clear pattern emerged from this experi-
ment in which compounds with saturated fatty acids
have little or no effect on the ratio of PGJ/PGE, while
all of those with polyunsaturated fatty acids induced
large increases. The control substance naproxen failed
to cause a measurable increase even at a concentration
of 50 lM. This suggests a novel mechanism of action
for the active elmiric acids. A modest degree of stereo-
selectivity was observed between the alanine containing
D and L-EMA-2 (20:4) isomers; interestingly, the D iso-
mer was somewhat more active. Unlike the cell prolifer-
ation assay shown above, a possible structure–activity
pattern seems to be present, suggesting that the PGJ/
PGE ratio in RAW cells may be a useful preliminary
screening procedure to identify anti-inflammatory activity.
3.3. Anti-inflammatory effects in vivo
In vivo data on a small group of the elmiric acids have
been obtained and are shown in Figures 3–7. These
specific examples were chosen on the basis of their
responses in the in vitro assay for the PGJ/PGE in
mouse macrophages (Table 3). Both active and inactive
examples were selected to determine whether or not an
SAR could be demonstrated in these whole animal mod-
els. Several examples containing unnatural amino acids
were also tested to study the effect of steric factors.
The in vivo assay models used have been previously
described in a variety of applications including proof
of principle studies for anti-inflammatory agents.27–32
However, complete dose response studies and a much
larger series of compounds will be needed to confirm
the findings shown here.

3348
S. H. Burstein et al. / Bioorg. Med. Chem. 15 (2007) 3345–3355
Table 2. Effects of elmiric acids on the proliferation of mouse macrophage RAW cells
Acyl group
Concd*
Glycine series
SD
L-Alanine series
SD
Mean
T/C**
Mean
T/C**
DMSO + LPS
Palmitoyl
0.0
0.1
1.0
10
4866
5120
5524
5108
326
480
207
851
1.000
1.052
1.135
1.050
3889
4541
4670
432
369
249
248
46
1.000
1.168
1.201
0.111
Oleoyl
0.1
1.0
10
5172
5557
425
239
269
6
1.063
1.142
0.087
3637
4533
422
339
535
45
0.935
1.166
0.109
Linoleoyl
0.1
1.0
10
5979
4595
713
179
1793
24
1.229
0.944
0.147
3955
4233
621
236
635
55
1.017
1.088
0.160
c-Linolenoyl
0.1
1.0
10
5435
5700
6602
157
190
1.117
1.171
1.357
4247
4557
2696
636
173
511
1.092
1.172
0.693
1935
Eicosa-11Z,14Z-dienoyl
Dihomo-c-linolenoyl
Arachidonoyl
0.1
1.0
10
2945
5738
609
982
179
3
0.605
1.179
0.125
3641
4397
605
612
280
4
0.936
1.131
0.156
0.1
1.0
10
5486
5563
647
494
122
20
1.127
1.143
0.133
4326
4680
637
487
389
12
1.112
1.203
0.164
0.1
1.0
10
5630
6156
591
245
68
1.157
1.265
0.121
4781
4735
2110
530
398
1.229
1.218
0.543
20
1327
Eicosapentaenoyl
Docosatetra-7Z,10Z,13Z,16Z-enoyl
Docosahexaenoyl
0.1
1.0
10
5659
5161
5937
282
771
367
1.163
1.061
1.220
4270
4798
4672
758
518
222
1.098
1.234
1.201
0.1
1.0
10
5131
5310
3574
282
127
655
1.055
1.091
0.734
4668
4415
618
382
734
22
1.200
1.135
0.159
0.1
1.0
10
5708
5675
2103
387
288
888
1.173
1.166
0.432
4452
4176
1010
419
251
153
1.145
1.074
0.260
Ninty-six-well plates were seeded with 500 RAW cells/100 ll media/well. The cells were incubated, for 24 h at 37 ꢁC and 5% CO2; removed from the
incubator and a 1 ll solution of each of the above compounds added with an N of 4. After 1 h, 1 ll LPS (1 lg/ml) was added to each well and the cells
returned to the incubator for 48 h. Cell numbers were determined with the CellTiter-Glo assay kit (Promega). Luminometer units are linearly
proportional to the number of cells in each well and the values shown are means of four wells.
*
Final concentration in culture media (lM).
Ratio of drug treated/DMSO treated (1.0 ll).
**
Suppression of chemically induced paw edema in mice
is a long-standing assay for assessment of an agent’s
potential anti-inflammatory activity.33–39,32 In prior
studies, several cannabinoids40 and a number of NSA-
IDs tested in this model exhibited inhibitory effects on
chemically induced edema. Figure 3 shows a single
dose comparison of three glycine derivatives in this
assay with arachidonate as the edema inducer. Some
suppression was seen with the polyunsaturated com-
pounds, however the effects were barely statistically
significant. Thus, further studies using this assay were
not carried out. Induction of inflammation by carra-
geenan in the paw edema assay gave an SAR different
than that predicted by the prostaglandin ratio screen
with EMA-1 (16:0) being a potent anti-inflammatory
agent (data not shown). At this time we cannot ex-
plain these differences, however, it seems clear that
the nature of the assay will influence the relative activ-
ities of the elmiric acids in reducing an inflammatory
reaction.
Figure 4 shows several examples of the effects of elmiric
acids in the subcutaneous pouch assay using two meth-
ods for inducing an inflammatory response, namely, a
combination of cytokines in (A) and carrageenan in
(B). The subcutaneous air pouch simulates a synovium
lined joint space by providing a blind connective tissue
cavity without a mesothelial basement membrane; the
pouch lining has the two cell types (fibroblasts and mac-
rophage-like cells) common to synovia.41–43 While the
precision of the assay was improved over the paw ede-
ma, an unexpected discrepancy arose with the palmitoyl
derivatives when carrageenan was the inducer. There is
no obvious explanation for this finding and further stud-
ies are needed to address this question. Thus, pending a
better understanding of this issue, it was decided not to
use this assay.
In Figure 5 we show the results of a preliminary exper-
iment comparing two methods, using either arachi-
donic acid or phorbol ester, for inducing ear edema

S. H. Burstein et al. / Bioorg. Med. Chem. 15 (2007) 3345–3355
Table 3. Effects of elmiric acids on the ratio of iPGJ/iPGE in RAW cell media
3349
Treatment
Concd
iPGJ2
SD
iPGE2
SD
J/E ratio
DMSO + LPS
Naproxen
Naproxen
—
2
136
57
84
51
38
44
120
106
75
3
1
1
6
9
10
50
1
70
119
1
Naproxen
81
2
1
EMA-1 (16:0)
EMA-1 (16:0)
EMA-1 (18:0)
EMA-1 (18:0)
EMA-1 (18.3)
EMA-1 (18:3)
EMA-1 (20:4)
EMA-1 (20:4)
EMA-1 (20:5)
EMA-1 (20:5)
D-EMA-2 (16:0)
D-EMA-2 (16:0)
L-EMA-2 (16:0)
L-EMA-2 (16:0)
D-EMA-2 (20:4)
D-EMA-2 (20:4)
L-EMA-2 (20:4)
L-EMA-2 (20:4)
L-EMA-2 (22:6)
L-EMA-2 (22:6)
219
401
215
194
103
128
121
126
127
289
273
425
126
355
256
319
251
232
124
269
241
652
229
437
23
12
20
1
2
10
1
3
201
159
159
26
1
10
1
1
5042
80205
40407
97830
1729
44729
281
1672
7534
10331
9805
249
6
39
277
147
229
13
125
1
10
1
19
125
180
14
46
35
18
21
16
5
10
1
10
1
8848
46
10
1
3079
261
3936
55
9
1
10
1
4177
1496
28224
2345
41059
3156
62176
1352
427
17
12
104
9
10
1
5201
252
27
18
92
22
153
10
1
3834
317
62
13
142
10
12969
Forty-eight-well plates were seeded with 20,000 RAW cells/500 ll media and incubated for 20 h at 37 ꢁC and 5% CO2. After washing, 500 ll of serum
free RPMI media was added to each well. Cells were then treated with 5 ll in DMSO of each of the elmiric acids; final concentrations of agents were 1
or 10 lM (N = 3). After 30 min, 5 ll LPS (1 lg/ml) was added to each well and the incubation continued for 2 h. Media were then harvested,
centrifuged at 800g, and 50 ll from each tube assayed by ELISA with iPGE (Cayman) and iPGJ2 (Assay Design) enzyme immunoassay kits.
Prostaglandin concentrations are in pg/ml.
due to the transient nature of the induction produced
by arachidonic acid. Nevertheless a favorable compar-
ison between EMA-1 (20:4) and dexamethasone, a po-
tent anti-inflammatory agent, was demonstrated (A).
All subsequent studies used phorbol ester as the induc-
ing agent. In (B) ear edema was induced by the topical
application of phorbol ester in acetone. EMA-1 (20:4)
gave a comparable reduction in ear thickness of about
40–50% as that obtained in the arachidonate model.
The response obtained with EMA-23(20:4) was similar
suggesting that substituted amino acids do not lose
activity.
The four examples of elmiric acids tested at three
doses shown in Figure 6 suggest that the anti-inflam-
matory response is sensitive to both fatty acid and
amino acid structure. Comparing 6B and 6D, namely,
oleoyl and arachidonoyl show a preference for greater
unsaturation in reducing ear edema. When 6A and 6B
are compared it appears that the substitution pattern
at the a position can be an important factor. How-
ever, in 6C and 6D, the two a disubstituted arachido-
noyl examples are active as are the compounds shown
in Figure 5.
Figure 3. Effects of the elmiric acids in the mouse paw edema model.
The compounds were dissolved in safflower oil and administered orally
at 30 mg/kg. After 60 min, 50 ll of the 0.5% arachidonate in pH 8.3
carbonate buffer was injected into the plantar surface of the left
(ipsilateral) hind paw of each mouse. The right (contralateral) paws
were injected with 50 ll of the buffer vehicle. This step was done with
mice under deep halothane anesthesia. After 2 h, mice were euthanized
with halothane, both hind paws severed at the ankle joint and the
weights recorded. *Values shown are means SEM p < 0.05. Plots and
statistical analyses were done using Prism by GraphPad.
The data in Figure 7 were obtained using dimethylaceta-
mide rather than acetone as the formulation vehicle.
This was necessary due to poor solubility of the fully sat-
urated elmiric acids in acetone. As predicted by the re-
sults of the prostaglandin ratio screen, the palmitoyl
(7B) and steroyl (7C) congeners showed little or no
activity as anti-inflammatory agents. The results in 7A
in the mouse. Both procedures yielded useful data,
however, we found that the phorbol ester data were
more precise and more easily obtained. This may be

3350
S. H. Burstein et al. / Bioorg. Med. Chem. 15 (2007) 3345–3355
from cell membranes and the activity of specific terminal
synthases on the prostaglandin endoperoxide, PGH2.
This process is depicted in Scheme 2. The dehydration
product of PGD2, 15d-PGJ2, antagonizes inflammatory
signaling pathways mediated by the transcription fac-
tors NF j-B, AP-1, and STAT, suppresses production
of matrix metalloproteinases (MMPs) and other media-
tors of inflammation, prevents joint damage in rats with
adjuvant arthritis, and generally acts to facilitate resolu-
tion of inflammation serving as a negative regulator of
inflammation.
The mechanisms responsible for the anti-inflammatory
and joint protective properties of the synthetic cannabi-
noid ajulemic acid are currently being studied in our lab-
oratory.46 Although the elmiric acids are structurally
dissimilar to ajulemic acid, there are certain similarities
in their actions. In preliminary experiments, addition
of ajulemic acid to human fibroblast-like synovial cells
(FLS) in vitro increased arachidonic acid release and
COX-2 expression with a subsequent marked increase
in15d-PGJ2 production without an appreciable change
in PGE2 synthesis (Stebulis et al. unpublished findings).
The stimulatory effect of ajulemic acid could be reduced
by pretreatment with the CB2 antagonist SR144728 but
not with the CB1 antagonist SR141716a. These remark-
able results are potentially important in light of accumu-
lating evidence that the timing of COX-2 expression
may determine its potential to promote either initiation
or resolution of inflammation by differential production
of prostaglandins, depending on the stage and progres-
sion of the inflammatory response.47 Moreover, we have
observed that EMA-1 (20:4) causes the mobilization of
free arachidonic acid in RAW cells from sources other
than the elmiric acid (unpublished data). These consid-
erations provided the rationale for the current studies
on the effects of the elmiric acids on prostaglandin ratios
described above.
Figure 4. Effects of elmiric acids in the mouse subcutaneous pouch
assay. Subcutaneous pouches were generated in male CD-1 mice (30–
35 g) by injection of air on two–three successive occasions. (a) Drugs
were given orally in safflower oil (50 ll) at a dose of 20 mg/kg. After
60 min, cytokines TNF-a and IL1-b were injected into the pouches
with mice under halothane anesthesia. After 90 min, mice were
euthanized with halothane, cells harvested and counted. (b) One hour
after oral dosing at the indicated levels, 2% carrageenan was injected
into the pouches. The pouch contents were allowed to incubate for 4 h
following which the mice were euthanized with halothane and the cells
harvested and counted. Plots and statistical analyses were done using
Prism by GraphPad. N = 4 mice/group. p < 0.05 by ANOVA.
4. Summary and conclusions
suggest a possible biphasic response, however, this needs
a more detailed study before such a conclusion is made.
As positive controls,44 indomethacin (20 mg/ml), pheni-
done (40 mg/ml), and dexamethasone (1 mg/ml) were
studied using the conditions described in Figure 7.
Phenidone, a lipoxygenase inhibitor, had no effect,
whereas indomethacin and dexamethasone produced
some decrease in ear thickness (data not shown) demon-
strating a favorable comparison for the elmiric acids
with established anti-inflammatory agents.
The goal of this project was to discover a structure–
activity relationship for the elmiric (lipoamino) acids
as potential anti-inflammatory agents. To this end, a
screening procedure was developed to allow the selec-
tion of molecules for testing in intact animal models
for anti-inflammatory activity. The procedure is based
on the concept that prostaglandins can mediate either
pro or anti-inflammatory actions depending on their
molecular composition. While PGE2 is dominant during
the development of inflammation, prostaglandins of the
J series are dominant during the resolution of an inflam-
matory condition. Thus, compounds that promote the
synthesis of the latter without significantly raising the le-
vel of the former could be considered as good candidates
for the treatment of inflammation. The findings reported
here support this hypothesis. Specifically, elmiric acids
with unsaturated structures generally produced a favor-
able prostaglandin ratio and were effective in reducing
in vivo responses to pro inflammatory agents. Analogs
with saturated structures did not enhance the J prosta-
glandins and had only minor effects in vivo. These
3.4. Possible anti-inflammatory mechanisms
In animal models of acute versus chronic inflammation
(carrageenan-induced pleurisy and collagen induced
arthritis, respectively), COX inhibition suppresses
endogenous 15d-PGJ2 production and prolongs the
inflammatory phase, whereas administration of
15d-PGJ2 has been shown to promote resolution of
inflammation.45,21 Prostaglandins released during an
inflammatory response derive from the sequential
activity of COX-2 on free arachidonic acid released

S. H. Burstein et al. / Bioorg. Med. Chem. 15 (2007) 3345–3355
3351
Figure 5. Reduction of arachidonic acid (a) phorbol ester (b) induced ear edema in the mouse. CD-1 male mice between 34 and 40 g were treated as
described in Section 5. Treatment was initiated by the application of vehicle (10 ll), dexamethasone (10 lg/ear) or EMA-1 (20:4), and EMA-23 (20:4)
(200 lg/ear) in 10 ll acetone. Values shown are expressed as the differences between the ipsilateral (induced) and contralateral (vehicle control) ears in
microns. Paired t-test results were: p < 0.02 for vehicle versus dexamethasone and *p < 0.007 for vehicle versus EMA-1 (20:4). Plots and statistical
analyses were done using Prism by GraphPad. N = 8. Note: EMA-23 = 1-aminocyclopropane-carboxylic acid.
a
b
130
110
90
130
110
90
*
70
70
50
50
0.0
2.2
6.7
20
0.0
2.2
6.7
20
L-EMA-2 (18:1) mg/ml
EMA-22 (18:1) mg/ml
c
d
130
110
90
130
110
90
*
*
*
*
70
70
50
50
0.0
2.2
6.7
20
0.0
2.2
6.7
20
EMA-25 (20:4) mg/ml
EMA-22 (20:4) mg/ml
Figure 6. Dose related reduction of phorbol ester induced ear edema in the mouse. Treatment was initiated by the application of vehicle (10 ll
acetone) or drug in 10 ll acetone to the ipsilateral ears and only acetone to all contralateral ears. Thirty minutes after drug treatment, 10 ll of the
PMA (phorbol ester) solution in acetone was applied to all ipsi lateral ears and 10 ll of acetone to all of the contralateral ears. After 4 h, all ear
thicknesses were measured using a digital micrometer. Data are expressed as the differences between the ipsi and contralateral ears in microns. Plots
and statistical analyses were done using Prism by GraphPad. N = 4; p < 0.05. Note: EMA-22 = 1,1-dimethylglycine; EMA-25 = 1-aminocyclohexane-
carboxylic acid. *p < 0.05.

3352
S. H. Burstein et al. / Bioorg. Med. Chem. 15 (2007) 3345–3355
a
b
160
140
120
100
80
160
140
*
120
100
80
*
60
60
40
40
0.0
4.4
13.3
40
0.0
1.1
3.3
10
EMA-1(20:4) mg/ml
EMA-1 (16:0) mg/ml
c 160
d
160
140
120
100
80
140
120
100
80
*
60
60
40
40
0.0
2.2
6.7
20
0.0
4.4
13.3
40
L-EMA-2 (18:0) mg/ml
EMA-8(16:0) mg/ml
Figure 7. Dose related reduction of phorbol ester induced ear edema in the mouse. All of the conditions were the same as in Fig. 6 except that the
drugs were dissolved in dimethylacetamide (DMA) instead of acetone. *p < 0.05. Note: EMA-8 = methionine.
5. Experimental
5.1. General chemical methods
All materials were obtained from commercial sources
and used without further purification. For TLC, Silica
Gel 60 F254 plates from Merck were used with detec-
tion by UV light or iodine vapor chamber. Acid chlo-
rides were obtained from Nu-chek Prep, Inc. (Elysian,
MN). Amino acids and esters were from Sigma–
Aldrich.
5.2. General procedure for synthesis
Compounds not available commercially in the Biomol
library were prepared as follows.
Esterification: The amino acid (200 mg) is dissolved in
30 ml of methanol saturated with HCl and refluxed
Scheme 2. Putative mechanism of action for the elmiric acids. During
an inflammatory response, cells are activated to release arachidonic
for 6 h. The solvent is then removed under vacuum
and the crude product dissolved in ethyl acetate and
partitioned with saturated bicarbonate to remove
traces of unreacted amino acid. Conjugation: the ester
is dissolved in methylene chloride/triethylamine and
cooled in ice. Commercially available fatty acid chlo-
ride (100 mg) (Nucheck) in methylene chloride is added
and the mixture allowed to react for 4 h on ice. The
mixture is then quenched on ice and extracted with
ethyl acetate. After washing with dilute HCl, saturated
bicarbonate, and water, the solution is dried over so-
dium sulfate and evaporated to a clear oil. Saponification:
acid. Activity of the bi-functional enzyme COX-2 oxidizes arachidonic
acid to PGG2 and a subsequent peroxidase reaction yields PGH2.
Then, under the influence of terminal synthases, PGD2 and PGE2 are
formed. PGD2 is then converted to the PGJ series in an unregulated
manner. Resolution of inflammation occurs when the PGJ series
predominates over the PGE series. The elmiric acids, which are not
COX-2 inhibitors, apparently up regulate PGD synthase without
affecting PGE synthase.
observations can now be applied to the search for drug
candidates in larger libraries of elmiric acids and possi-
bly in other families of compounds.

S. H. Burstein et al. / Bioorg. Med. Chem. 15 (2007) 3345–3355
3353
the ester is dissolved in THF and treated under nitro-
gen with an equal volume of 1 N LiOH for 24 h at
room temperature with stirring. Silica gel chromatogra-
phy is used to purify the crude product whose identity
is confirmed by mass spectral and NMR analysis.
5.2.6. Enantiomeric alanine analogs. Chirally pure ala-
nine enantiomers, D-EMA-2 and L-EMA-2, were pre-
pared as previously reported by Cascio et al.48
5.2.7. Pharmacological assays.
5.2.7.1. Cell proliferation assay. Cell numbers were
estimated using a commercially available kit (Promega).
The CellTiter-GloTM Luminescent Cell Viability Assay
kit used here is based on the measurement of ATP,
which signals the presence of metabolically active
cells.49,50 The assay uses luciferase as the detection en-
zyme because of the absence of endogenous luciferase
activity in mammalian cells. An equal volume of CellT-
iter-GloTM Reagent is added to the cell culture and lumi-
nescence is measured. The light signal is proportional to
the amount of ATP present, which correlates with the
number of viable cells present. The VeritasTM Microplate
Luminometer used detects as little as 1.5 · 10–15 mol
ATP and values are linear from 760 fg to 5.1 ng of
ATP. The supplier’s instructions were followed in our
studies reported here.
5.2.1. N-Arachidonoyl-1,1-dimethylglycine; EMA-22
(20:4). Waxy low melting off-white solid. TLC: 10%
methanol/methylene chloride; Rf 0.42. 1H NMR
(250 MHz CDCl3) d 5.93 (br s, 1H), 5.25–5.45 (m,
8H), 2.72–2.90 (m, 6H), 2.18–2.29 (t, 2H), 2.00–2.17
(m, 4H); 13C NMR (CDCl3) d 176.3, 174.6, 130.8,
129.3, 121.1, 128.9, 128.5, 128.3, 128.0, 127.7, 77.6,
77.4, 77.2, 76.9, 36.2, 31.7, 29.5, 27.5, 26.7, 25.9, 25.4,
25.2, 22.8, 14.3, 0.23; HRMS calcd C24H39NO3
(M+H); 390.3003, found; 390.3008 (D mass À0.0005).
5.2.2. N-Arachidonoyl-1-aminocyclopropane carboxylic
acid; EMA-23 (20:4). Waxy low melting off-white solid.
TLC: 10% methanol/methylene chloride; Rf 0.44. 1H
NMR (250 MHz CDCl3) d 5.95 (s, 1H), 5.25–5.45 (m,
8H), 2.72–2.90 (m, 6H), 1.98–2.32 (m, 6H), 1.66–1.80
(dt, 2H), 1.57–1.65 (m, 2H), 1.25–1.46 (m, 6H), 1.14–
1.22 (m, 2H), 0.83–0.96 (t, 3H); 13C NMR (CDCl3) d
175.1, 130.8, 129.2, 129.1, 128.9, 128.5, 128.4, 128.1,
127.7, 35.9, 31.7, 29.5, 27.5, 26.8, 25.9, 25.8, 25.4, 22.8,
18.2, 14.3. HRMS calcd C24H37NO3 (M+H); 388.2846,
found; 388.2855 (D mass À0.0009).
5.2.7.2. Measurement of the ratio of iPGJ/iPGE in
RAW cell media. Forty-eight-well plates were seeded
with 20,000 RAW cells/500 ll media and incubated for
20 h at 37 ꢁC and 5% CO2. After washing, 500 ll of ser-
um-free RPMI media was added to each well. Cells were
treated with 5 ll in DMSO of each of the elmiric acids;
final concentrations 1 or 10 lM (N = 3). After 30 min,
5 ll LPS (1 lg/ml) was added to each well and the incu-
bation continued for 2 h. Media were then harvested,
centrifuged, and 50 ll from each tube assayed by ELISA
with iPGE (Cayman) and iPGJ2 (Assay Design) enzyme
immunoassay kits.
5.2.3. N-Arachidonoyl-1-aminocyclohexane carboxylic
acid; EMA-25 (20:4). Waxy low melting off-white solid.
TLC: 10% methanol/methylene chloride; Rf 0.48. 1H
NMR (250 MHz CDCl3) d 5.57 (s, 1H), 5.25–5.45 (m,
8H), 2.72–2.90 (m, 6H), 2.23–2.34 (t, 2H), 1.85–2.20
(m, 8H), 1.52–1.80 (m, 6H), 1.20–1.48 (m, 8H), 0.83–
0.93 (t, 3H); 13C NMR (CDCl3) d 175.4, 175.3, 130.8,
129.4, 128.9, 128.8, 128.6, 128.3, 128.0, 127.7, 77.6,
77.4, 77.2, 76.9, 60.2, 42.2, 36.2, 32.2, 31.7, 29.5, 27.4,
26.7, 25.9, 25.6, 25.2, 22.8, 21.5, 14.3.
5.2.7.3. The mouse paw edema model. The compounds
were dissolved in safflower oil and administered orally to
male CD-1 mice that weighed between 35 and 40 g.
After 60 min, 50 ll of the 0.5% arachidonate in pH 8.3
carbonate buffer was injected into the plantar surface
of the left (ipsilateral) hind paw of each mouse. The
right (contralateral) paws were injected with 50 ll of
the buffer vehicle. This step was done with mice under
deep halothane anesthesia. After 2 h, mice were eutha-
nized with halothane and both hind paws severed at
the ankle joint, placed in a tared weighing pan, and
the weights recorded. Plots and statistical analyses were
done using Prism by GraphPad.
HRMS calcd C27H43NO3 (M+H); 430.3316, found;
430.3320 (D mass À0.0004).
5.2.4. N-Steroyl-L-alanine; L-EMA-2 (18:0). Crystalline,
white solid; mp 103–105 ꢁC. 1H NMR (400 MHz
CDCl3) 5.97–5.99 (1H), 4.53–4.58 (1H), 2.22–2.26
(2H), 1.61 (2H), 1.45–1.47 (3H), 1.25–1.30 (29H),
0.86–0.89 (3H); 13C NMR (CDCl3) d 175.0, 174.5,
48.6, 36.6, 32.2, 29.9, 29.89, 29.83, 29.69, 29.60,
29.53, 29.40, 25.7, 22.9, 17.9, 14.4. HRMS calcd
C21H41NO3 (M+H); 356.3159, found; 356.3187 (D mass
À0.0028).
5.2.7.4. The mouse subcutaneous pouch assay. Subcu-
taneous pouches were generated in male CD-1 mice
(30–35 g) by injection of air on two or three successive
occasions. Drugs were given orally in safflower oil
(50 ll) at a dose of 20 mg/kg. One hour later, inflamma-
tion is induced by injection into the pouch cavity of
10 ng rHuIL-1-b plus 0.25 ng rHuTNF-a in 3 ml of
1% carboxymethylcellulose. Inflammation is quantified
90 min later by determination of pouch exudate leuko-
cyte counts. Alternatively, after 60 min, 2% carrageenan
was injected into the pouches. The pouch contents are
allowed to incubate for 4 h following which the mice
were euthanized with halothane and the cells harvested
and counted. Plots and statistical analyses were done
5.2.5. N-Palmitoyl methionine; EMA-8 (16:0). Crystal-
line white solid; mp 75–77 ꢁC. 1H NMR (400 MHz
CDCl3) 6.34–6.36 (1H), 4.67–4.72 (1H), 2.56–2.60
(2H), 2.11–2.22 (3H), 2.02–2.10 (1H), 1.62–1.65 (2H),
1.25–1.29 (25H), 0.86–0.89 (3H); 13C NMR (CDCl3) d
174.8, 174.6, 77.6, 77.4, 77.2, 76.9, 52.2, 36.7, 32.2,
31.0, 30.3, 29.94, 29.92, 29.89, 29.86, 29.72, 29.60,
29.55, 29.45, 25.8, 22.9, 15.7, 14.4; HRMS calcd
C21H41NO3S (M+H); 388.2880, found; 388.2899 (D
mass À0.0019).

3354
S. H. Burstein et al. / Bioorg. Med. Chem. 15 (2007) 3345–3355
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feed and tap water. Treatment was initiated by the appli-
cation of vehicle (10 ll acetone) or drug in 10 ll acetone
to the ipsilateral ears and acetone to all contralateral
ears. After 30 min, ear edema was induced by the topical
application of 10 ll of free arachidonic acid (20 mg/ml)
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Phorbol ester induced ear edema. Conditions were as
above up until the induction of edema. Thirty minutes
after drug treatment, 10 ll of the PMA (phorbol ester)
solution in acetone was applied to all ipsilateral ears and
10 ll of acetone to all of the contralateral ears. After
4 h, all ear thicknesses were measured using a digital
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This publication was made possible by Grants DA17969
(SHB) and DA13691 (RBZ) from National Institute on
Drug Abuse, National Institutes of Health, Bethesda,
MD. Its contents are solely the responsibility of the
author and do not necessarily represent the official views
of the National Institute on Drug Abuse. We thank Dr.
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