Asymmetric synthesis of novel N-(1-phenyl-2,3-dihydroxypropyl) arachidonylamides and evaluation of their anti-inflammatory activity

Article abstract of DOI:10.1016/j.lfs.2012.06.040

Aims: To design and synthesize novel N-(1-phenyl-2,3-dihydroxypropyl) arachidonylamides and evaluate their analgesic and anti-inflammatory potential. Main methods: The murine macrophage cell line RAW 264.7 has been widely used as a model for inflammatory responses in vitro. Our model consists of cultured monolayers of RAW 264.7 cells in which media concentrations of 15-deoxy-Δ^13,14-PGJ₂ (PGJ) are measured by ELISA following LPS (10 ng/ml) stimulation and treatment with 0.1, 0.3, 1.0, 3.0 and 10 μM concentrations of the compounds. Key findings: Our data indicate that several of our compounds have the capacity to increase production of PGJ and may also increase the occurrence of programmed cell death (apoptosis). Significance: Thus these agents are potential candidates for the therapy of conditions characterized by ongoing (chronic) inflammation and its associated pain.

Full text of DOI:10.1016/j.lfs.2012.06.040

Life Sciences 92 (2013) 506–511
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Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
Asymmetric synthesis of novel N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamides
and evaluation of their anti-inflammatory activity
Padmanabha V. Kattamuri a, Rebecca Salmonsen b, Catherine McQuain b, Sumner Burstein b,
,
⁎
a,c,⁎⁎
Hao Sun c, Guigen Li
a
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA
b
c
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA
Institute of Chemistry and Biomedical Sciences, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2012
Accepted 27 June 2012
Aims: To design and synthesize novel N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamides and evaluate
their analgesic and anti-inflammatory potential.
Main methods: The murine macrophage cell line RAW 264.7 has been widely used as a model for inflamma-
tory responses in vitro. Our model consists of cultured monolayers of RAW 264.7 cells in which media con-
centrations of 15-deoxy-Δ13,14-PGJ2 (PGJ) are measured by ELISA following LPS (10 ng/ml) stimulation and
treatment with 0.1, 0.3, 1.0, 3.0 and 10 μM concentrations of the compounds.
Key findings: Our data indicate that several of our compounds have the capacity to increase production of
PGJ and may also increase the occurrence of programmed cell death (apoptosis).
Significance: Thus these agents are potential candidates for the therapy of conditions characterized by ongoing
(chronic) inflammation and its associated pain.
Keywords:
Cannabinoid receptor
Anandamide
Arachidonyl amide
15-Deoxy-Δ13,14-PGJ2 (PGJ) stimulation
Apoptosis
© 2012 Elsevier Inc. All rights reserved.
Introduction
1993). In addition to this, structure–activity relationship (SAR) studies
of anandamide analogs have provided insights into the stereoelectronic
Arachidonylethanolamide (anandamide, A) (Fig. 1) was isolated
from porcine brain and was identified as a putative endogenous
ligand for the cannabinoid receptor. This identification was mainly
based on the ability of anandamide to inhibit both the specific binding
of a tritiated cannabinoid ligand to synaptosomal membranes and
the electrically evoked twitch response of the mouse vas deferens
(Dewane et al., 1992). Further research in this area has shown that
the pharmacological activity of anandamide, when administered in
vivo, parallels that of other cannabinoid receptor agonists (Fride
and Mechoulam, 1993). Furthermore, like other cannabimimetic
agents, anandamide was found to be capable of inhibiting forskolin
stimulated adenylate cyclase both in neuroblastoma cell lines that
naturally express cannabinoid receptors and in cells transfected with
plasmids carrying cannabinoid receptor DNA (Vogel et al., 1993).
Anandamide has also been found to exhibit cross-tolerance with
(−)−Δ9-tetrahydrocannabinol (Δ9-THC) in the mouse vas deferens
and to bind, albeit with a lower affinity to a second type of cannabinoid
receptor expressed in the periphery (Pertwee et al., 1993; Munro et al.,
requirements for interaction with the CB1 receptor (Yao et al., 2008;
Abadji et al., 1994; Ryan et al., 1997; Adams et al., 1995; Khanolkar et
al., 1996; Lin et al., 1998; Sheskin et al., 1997; Goutopoulos et al.,
2001). Likewise, 2-arachidonylglycerol (2-AG, B) (Fig. 1) which is an
endogenous cannabinergic ligand that interacts with both CB1 and CB2
receptors, was found to possess various biological activities, such as
binding to CB1 and CB2 receptors, inhibition of adenylyl cyclase in
mouse spleen cells, and induction of hypothermia, reduction of sponta-
neous activity, analgesia and immobility in mice. 2-Arachidonylglycerol
(2-AG) acts as a cannabinergic agonist, and the structure of 2-AG is rec-
ognized by the cannabinoid receptors (CB1 and CB2) (Vadivel et al.,
2011). Anandamide possesses only a moderate affinity for the receptor
(Ki=78 nM) and it has a short metabolic half-life (Abadji et al., 1994).
The synthesis of pure 2-AG is problematic because of the migra-
tion of the arachidonyl group from the secondary to the primary
hydroxyl group, resulting in the formation of the more stable
1-arachidonyl glycerol. Nearly all known methods for the synthesis
of cannabinoid receptor ligands reported so far suffer from extended
reaction times, harsh conditions for the removal of protecting groups
as well as extensive work up procedures and purification methods
(Vadivel et al., 2011). According to the literature, some of the research
groups reported the design and synthesis of anandamide analogs
with high metabolic stability, with the aim of improving the affinity
of the anandamide ligand forthe receptor (Abadji et al., 1994;
Mahadevan and Razdan, 2005; Razdan and Mahadevan, 2002).
⁎
Correspondence to: S. Burstein, Department of Biochemistry and Molecular
Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605,
USA. Tel.: +1 508 856 2850; fax: +1 508 856 2003.
⁎⁎ Correspondence to: G. Li, Department of Chemistry and Biochemistry, Texas Tech
University, Lubbock, TX 79409, USA. Tel.: +1 806 742 3015; fax: +1 806 742 1289.
E-mail addresses: sumner.burstein@umassmed.edu (S. Burstein),
Guigen.Li@ttu.edu (G. Li).
0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2012.06.040

P.V. Kattamuri et al. / Life Sciences 92 (2013) 506–511
507
Fig. 1. The structures of anandamide, arachidonylglycerol and N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamide.
There are several reports in the literature showing that, structural
modifications on the arachidonyl side chain resulted in changes in
the receptor affinity. According to these reports, complete saturation,
or replacement of the alkenes with alkynes, resulted in the complete
loss of receptor affinity. Also, when the arachidonyl chain was
substituted with other fatty acid chains, with ω-olefinic bonds and
with a trans double bond, obvious reduction in affinity for CB1 was ob-
served (Yao et al., 2008). The affinity of anandamide for CB1 can be sig-
nificantly enhanced by substituting the terminal pentyl group with a
1,1-dimethyl moiety, as has also been observed with (−)–Δ9-THC
(Seltzman et al., 1997). Anandamide analogs of variable chain lengths
in which the terminal carbon is functionalized with a phenyl, substitut-
ed phenyl, or heterocyclic rings showed that the stereochemical features
of the anandamide tail may have a big impact on the ligand's affinity for
CB1 (Yao et al., 2008). Taking all the above factors into consideration, we
envisioned that the introduction of an aromatic ring and two hydroxyl
groups in the main carbon chain of arachidonyl amide may have a signif-
icant effect on CB1 or CB2 affinity and potency. The present work de-
scribes the synthesis and evaluation of anti-inflammatory activity of
N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamide (C) (Fig. 1) and
its analogs.
synthesis scheme. The synthesis of N-(1-phenyl-2,3-dihydroxypropyl)
arachidonylamides is illustrated in Scheme 1.
(2) Isopropyl cinnamic ester. Cinnamic acid (10.00 g, 67.50 mmol) was
dissolved in isopropanol (100 mL) and thionyl chloride (8.37 mL,
114.74 mmol) was added dropwise with constant stirring at room tem-
perature. After the completion of addition, the reaction mixture was
heated to reflux over night. Then, the reaction mixture was brought to
room temperature and an excess amount of isopropanol was removed
under reduced pressure. The crude reaction mixture was dissolved in
dichloromethane and washed with saturated sodium bicarbonate
solution followed by water (3×100 mL). The organic layer was separat-
ed, dried over anhydrous MgSO4 and concentrated under reduced pres-
sure. The product is a yellow, viscous, oily liquid (12.4 g, 65.18 mmol,
97% yield).
(3) (2R,3S)-isopropyl 3-(((benzyloxy)carbonyl)amino)-2-hydroxy-3-
arylpropanoate. (2R,3S)-isopropyl 3-(((benzyloxy)carbonyl)amino)-
2-hydroxy-3-phenylpropanoate was prepared in accordance with
the procedure described in the literature (Li et al., 1996). The product
was obtained as a white solid.
(4) Benzyl ((1S,2R)-2,3-dihydroxy-1-arylpropyl)carbamate. (2R,3S)-
isopropyl 3-(((benzyloxy)carbonyl)amino)-2-hydroxy-3-phenyl-
propanoate (0.8 g, 2.23 mmol) was dissolved in absolute ethanol
(50 mL) and stirred at room temperature. To the stirring reaction mix-
ture, sodium borohydride (0.254 g, 6.71 mmol) was added slowly and
stirring continued overnight at room temperature. The progress of reac-
tion was monitored by TLC. After confirming the completion, EtOH was
removed under reduced pressure, and water was added to the crude
product, which was extracted with ethyl acetate. The product was puri-
fied by column chromatography and isolated as a white solid (0.51 g,
1.69 mmol, 76% yield).
Materials and methods
Chemicals
All the cinnamic acids were purchased from Sigma Aldrich chem-
ical company and Acros Organics. All the solvents were obtained from
Acros Organics, Mallinckrodt Chemicals and Fisher Scientific. Lithium
aluminum hydride (LAH) and palladium on activated carbon was
obtained from Acros Organics. Thionyl chloride (SOCl2) was obtained
from Sigma Aldrich chemical company. Triethylamine was obtained
from Acros Organics. Arachidonic acid and palmitic acid were pur-
chased from Nu-Chek Prep. Inc. (Elysian, MN). Chemicals for the
amino hydroxylation reaction were obtained from Sigma Aldrich
and Macron chemical companies.
(5) (2R,3S)-3-amino-3-arylpropane-1,2-diol. The CBZ group present in
(4) was removed by the treatment with Pd/C in MeOH under hydro-
gen atmosphere. Compound (4) (0.33 g, 1.09 mmol) was placed in a
round bottomed flask and under inert atmosphere Pd/C (0.033 g, 10%
by weight of the starting material) catalyst was added. To the resulting
mixture, anhydrous MeOH (10 mL/g of the starting material) was
slowly added under argon. Then, after degassing the round bottomed
flask containing the reaction mixture, a hydrogen balloon was attached
to the flask and was stirred overnight. After confirming the completion
of reaction, Pd/C was filtered off on a celite pad and the filtrate was
concentrated under reduced pressure. The crude product was purified
Reagents
Chemical preparation of chiral arachidonyl amides and related analogs
A series of N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamides
were synthesized and characterized by using cinnamic acid or
substituted cinnamic acids as starting materials. Sharpless asym-
metric aminohydroxylation (AA) is the key step involved in the

508
P.V. Kattamuri et al. / Life Sciences 92 (2013) 506–511
Scheme 1. General procedure for the synthesis of N-(1-aryl-2,3-dihydroxypropyl)arachidonylamide and its analogs.
by column chromatography using methanol/dichloromethane solvent
mixture. The product was obtained as an off-white solid (0.095 g,
0.568 mmol, 52% yield).
Procedure for stimulation of prostaglandin J adapted from (Burstein
et al., 2012)
Cells used were RAW 264.7 and were obtained from ATCC. The
base medium is Gibco DMEM with 10% fetal bovine serum added.
Cells are grown in a T-75 flask in 15 mL of medium; medium is re-
placed on day four and sub cultured on day seven. Cells are removed
by scraping without the aid of trypsin. A sub cultivation ratio of
1:3–1:6 was used. ELISA assay kits for PGJ were obtained from
Assay Designs, Inc. (Ann Arbor, MI). The identity of the PGJ analyte
in the culture medium was confirmed by mass spectrometry (Wood
and Makriyannis, data not shown). Treatments were carried out in
48 well plates with 50,000 cells/500 μl DMEM+FCS media/well and
TNFa (10 nM) added. Cells were incubated for 20 h at 37 °C and 5%
CO2. Media were changed to 500 μl of serum free DMEM, treated
for 2 h and 100 μM removed for assays. NAgly [10 lM] control;
16,300 pg/mL. DMSO control: b16.0 pg/mL. N=4.
(6) N-(1-Aryl-2,3-dihydroxypropyl)arachidonylamide. To the solution of
arachidonic acid (0.063 g, 0.21 mmol) in anhydrous dichloromethane
(10 mL), oxalyl chloride (36 μL, 0.41 mmol) dissolved in dichloro-
methane (1 mL) was added slowly under stirring. N,N-dimethyl-
formamide (1 drop) was added next. The reaction mixture was
stirred at room temperature for 1 h and concentrated to give crude
arachidonyl chloride (Casida et al., 2010). This crude arachidonyl chlo-
ride was dissolved in 5 mL of THF and added slowly dropwise to an
ice-cold solution of (5) (0.044 g, 0.26 mmol) and triethylamine
(45 μL, 0.31 mmol) in 15 mL of THF. The temperature of the reaction
mixture was slowly brought to room temperature and stirred over-
night. Progress of the reaction was monitored by TLC. After confirming
the completion of the reaction, the reaction solution was diluted with
hexanes and the triethylamine hydrochloride salt was filtered. The
filtrate was concentrated, dissolved in chloroform, washed with
water, dried over anhydrous magnesium sulfate and concentrated to
give the crude product as viscous pale yellow oil (Casida et al., 2010).
The product was purified using column chromatography (0.090 g,
0.19 mmol, 75% yield).
Results
Anti‐inflammatory activity (PGJ stimulation)
The murine macrophage cell line RAW 264.7 has been widely used
as a model for inflammatory responses in vitro. LPS is a potent induc-
er of inflammatory responses including the arachidonic acid cascade;
in a wide variety of models, some of which have been used to
N-((1S,2R)-2,3-dihydroxy-1-phenylpropyl)palmitamide
The procedure for the synthesis of palmitoyl chloride and
N-((1S,2R)-2,3-dihydroxy-1-phenylpropyl)palmitamide (E) is the
same as described above for the synthesis of compound (6). Compound
(5) (0.040 g, 0.23 mmol), palmitoyl chloride (0.0986 g, 0.35 mmol) and
triethyl amine (40 μL, 0.28 mmol) were used for this reaction. The final
product N-((1S,2R)-2,3-dihydroxy-1-phenylpropyl)palmitamide (E)
was obtained as a white solid after purification using column chroma-
tography (0.075 g, 0.18 mmol 77% yield).
The compound characterization data and NMR spectra of all
the compounds discussed above are provided in the supporting
information.
Fig. 2. Structure of the compound tested for PGJ stimulation.

P.V. Kattamuri et al. / Life Sciences 92 (2013) 506–511
509
Fig. 3. Structures of the compounds tested for PGJ stimulation in RAW cells.
evaluate drug candidates. Thus, our model consisted of cultured
Discussion
monolayers of RAW 264.7 cells in which media concentrations of
prostaglandins were measured by ELISA following LPS (10 ng/mL)
stimulation and treatment with 0.1, 0.3, 1.0, 3.0 and 10 μM concentra-
tions of the test compounds for 2 h (Figs. 2–3). To confirm the identi-
ty of the analyte by an independent method, mass spectral analysis of
selected samples was performed using procedures previously done
(Burstein et al., 2002).
The use of immunoassay procedures always raises questions of
specificity. Therefore, we have indicated that we measured immuno-
reactive prostaglandin as defined by the cross reactivities of the anti-
serum used by the manufacturer.1 The major cross reactants (>1%)
are all precursors of the analyte and would not substantially change
the conclusions on anti-inflammatory action of our compounds
since the precursors all show anti-inflammatory actions. Regardless
of the particular species that the assay detects, the preliminary results
suggest that an empirical relationship exists between the ELISA data
and the in vivo anti-inflammatory effects of the analogs tested thus
far (Burstein, 2008) (Table 1).
Based on recent reports, it is suggested that members of the elmiric
acid (EMA) family are candidates for drugs to treat various inflammato-
ry conditions (Burstein et al., 2007). In the process of our ongoing effort
to design and prepare novel analgesic and anti-inflammatory agents, we
developed a mechanism based on in vitro assay for screening libraries of
EMAs for potential anti-inflammatory activity based on their stimulatory
action on PGJ levels. It is noteworthy that, in contrast to the nonsteroidal
anti-inflammatory drugs (NSAIDS), the elmiric acids are not COX-2 in-
hibitors. Thus, it is expected that their side effect profile, if any, would
be different.
Several literature reports indicate that an elevation of tissue con-
centrations of PGJ is associated with the resolution of an inflammato-
ry condition (Gilroy et al., 2003, 2004). This is believed to come about
through the binding and activation of the transcription factor PPAR-γ
followed by increased expression of anti-inflammatory factors.
Table 1
Induction of apoptosis
Stimulation of PGJ production by compound C in RAW cells.
Conc. C (μM)
OD
Cell #
PGJ (pg/mL)
SEM
PGJ/103 cells
Based on the results of the PGJ stimulation response from previous
studies, studies for the induction of apoptosis were done using these
analogs.
We speculate that these effects are due to programmed cell death
or apoptosis rather than necrosis. An important consequence of apo-
ptosis in certain cell types including macrophages is the resolution of
chronic inflammation.
0
0.284
0.287
0.274
0.271
0.266
0.253
39,800
40,200
38,300
38,000
37,300
35,400
0
0
0
0
0
0
0
0
0
0.1
0.3
1.0
3.0
10
1376
8831
15,534
172
413
1220
36.2
238
439
Experimental protocol (see Materials and methods section).
Based on these results, analogs of the compound C were synthesized and PGJ stimulation
studies were done.
Experimental protocol
• Seed 2 (12-well) plates (24 wells total) with 150,000 RAW cells/mL
10% FBS medium (DMEM).
Table 2
• Add 10 μL of LPS (100 μL/10 mLs) to all wells.
• Incubate the flasks for 18 h at 37 °C and 5% CO2.
• Wash cells with serum free DMEM, then add 1 mL serum free
DMEM to wells.
Stimulation of prostaglandin J3 production by compounds C, D and E in RAW cells.
Values shown represent the ratio of analog treated/NAGly control (NAGly=1.00).
NT = not tested.
Conc. (μM)
C
D
E
• Add treatments (10 μL) to each well as shown in Table 2 and incu-
bate for 3 h.
• Scrape wells with cell scraper and spin in centrifuge 1350 rpm for
5 min. Remove 800 μL supernate and triturate remaining 200 μL.
Take 20 μL and add 20 μL of 1/5 trypan blue stain. Count in hemocy-
tometer, 4 corner squares and center square (N=3).
0.10
0.30
0.74
1.00
2.20
3.00
6.70
10.00
20.00
30.00
60.00
0.00
0.02
NT
0.62
NT
3.56
NT
6.33
NT
0.00
NT
0.00
NT
0.016
NT
0.029
NT
0.00
NT
NT
0.00
NT
0.00
NT
0.00
NT
0.00
NT
2.18
NT
6.83
NT
NT
1
Cross reactivities (Assay Designs, Ann Arbor, MI): 15-deoxy-Δ12,14-PGJ2 100%; PGJ2
49.2%; delta12-PGJ2 5.99%; PGD2 4.92%; arachidonic acid 0.03%; b0.01%: PGF2alpha/
9alpha, 11beta-PGF2alpha/PGE2/thromboxane B2/2-arachidonylglycerol/anandamide.
Experimental protocol (see Materials and methods section).

510
P.V. Kattamuri et al. / Life Sciences 92 (2013) 506–511
Fig. 4. Increase in trypan blue exclusion by C: possible occurrence of apoptosis.
Previous studies showed that the synthetic cannabinoid analog
ajulemic acid elevates PGJ in similar models, i.e. fibroblast-like syno-
vial cells (Stebulis et al., 2008). More recent data demonstrated that
a similar response can be observed with N-arachidonyl glycine in
vivo (Burstein et al., 2011).
Health grant R21DA031860-01, Bethesda, MD. Its contents are solely
the responsibility of the authors and do not necessarily represent
the official views of the National Institute on Drug Abuse and the
National Institutes of Health.
The concentration of PGJ, formed from the dehydration of PGD2, in-
creases during the resolution phase of chronic inflammation, and acts as
a brake on inflammation by, among other actions, inducing apoptosis of
inflammatory cells. Programmed cell death (apoptosis) of inflammato-
ry cells is an important part of the resolution process. Inflammatory and
synovial cells from joints of patients with rheumatoid arthritis are resis-
tant to apoptosis, which interferes with resolution of acute inflamma-
tion and leads to chronic inflammation and joint tissue injury. Our
data indicate that several of our compounds have the capacity to in-
crease production of PGJ and possibly also increase the occurrence of
apoptosis (Fig. 4). The parent compound N-arachidonylglycine has re-
cently been reported to induce apoptosis in macrophages (Takenouchi
et al., 2012). Thus, these agents (analogs) are potential candidates for
the therapy of conditions characterized by ongoing (chronic) inflamma-
tion and its associated pain.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.lfs.2012.06.040.
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