Design and synthesis of novel prostaglandin E2 ethanolamide and glycerol ester probes for the putative prostamide receptor(s)

Article abstract of DOI:10.1016/j.tetlet.2015.01.164

Novel prostaglandin-ethanolamide (PGE₂-EA) and glycerol ester (2-PGE₂-G) analogs were designed and synthesized to aid in the characterization of a putative prostamide receptor. Our design incorporates the electrophilic isothiocyanato and the photoactivatable azido groups at the terminal tail position of the prototype. Stereoselective Wittig and Horner-Wadsworth-Emmons reactions install the head and the tail moieties of the PGE₂ skeleton. The synthesis is completed using Mitsunobu azidation and peptide coupling as the key steps. A chemoenzymatic synthesis for the 2-PGE₂-G is described for first time.

Full text of DOI:10.1016/j.tetlet.2015.01.164

Accepted Manuscript
Design and Synthesis of Novel Prostaglandin E2 Ethanolamide and Glycerol
Ester Probes for the Putative Prostamide Receptor(s)
Erin L. Shelnut, Spyros P. Nikas, David F. Finnegan, Nan Chiang, Charles N.
Serhan, Alexandros Makriyannis
PII:
S0040-4039(15)00214-2
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.01.164
TETL 45834
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 December 2014
20 January 2015
26 January 2015
Please cite this article as: Shelnut, E.L., Nikas, S.P., Finnegan, D.F., Chiang, N., Serhan, C.N., Makriyannis, A.,
Design and Synthesis of Novel Prostaglandin E2 Ethanolamide and Glycerol Ester Probes for the Putative Prostamide
Receptor(s), Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet.2015.01.164
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Design and Synthesis of Novel Prostaglandin E2 Ethanolamide
and Glycerol Ester Probes for the Putative Prostamide
Receptor(s)
Erin L. Shelnuta, Spyros P. Nikasa, David F. Finnegana, Nan Chiangb, Charles N. Serhanb, and
Alexandros Makriyannisa,c,*
aCenter for Drug Discovery and Departments of Chemistry and Chemical Biology and Pharmaceutical
Sciences, Northeastern University, 360 Huntington Ave, 116 Mugar Hall, Boston, MA, 02115, USA.
bCenter for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology,
Perioperative and Pain Medicine, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur (HIM 829),
Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115, USA.
cKing Abdulaziz University, Jeddah, 22254, Saudi Arabia.
*To whom correspondence should be addressed
Corresponding author
Alexandros Makriyannis (Ph.D)
Tel.: +1-617-373-4200; fax: +1-617-373-7493; e-mail: a.makriyannis@neu.edu.

ABSTRACT
Novel prostaglandin-ethanolamide (PGE2-EA) and glycerol ester (2-PGE2-G) analogs were designed
and synthesized to aid in the characterization of a putative prostamide receptor. Our design incorporates
the electrophilic isothiocyanato and the photoactivatable azido groups at the terminal tail position of the
prototype. Stereoselective Wittig and Horner-Wadsworth-Emmons reactions install the head and the tail
moieties of the PGE2 skeleton. The synthesis is completed using Mitsunobu azidation and peptide
coupling as the key steps. A chemoenzymatic synthesis for the 2-PGE2-G is described for first time.
Keywords: Endocannabinoids, Prostamides, Prostaglandin glycerol ester, Lipids

Introduction
Prostamides are a class of endogenous eicosanoids resulting from the cyclooxygenase-2 (COX-2)
metabolism of the endocannabinoid anandamide (AEA).1,2 The biosynthesis of prostamides is similar to
that of the prostaglandins and involves stimulus-induced phospholipase release of anandamide,
oxygenation by COX-2 to a hydroxyl-endoperoxide, and finally enzymatic conversion by one of the
prostaglandin synthases.1 While basal levels of the precursor AEA are not sufficient to produce
significant concentrations of prostamides, it is likely that physiological conditions exist, as in
inflammatory or infectious conditions, where anandamide release coincides with COX-2 induction.2,3
The involvement of prostamides in inflammatory settings suggests that they constitute a novel class of
lipid mediators, modulation of which could lead to new anti-inflammatory approaches.4,5
Since their discovery in 1997,6 the effects of prostamides have been studied on various systems known
to recognize endocannabinoid and prostaglandin classes of compounds. Prostamide E2 (PGE2-EA) has
shown little to no affinity for cannabinoid receptors CB1 and CB2 and little to no activity at the
endocannabinoid metabolizing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol
lipase (MGL).7,8 While the prostamides have been shown to possess the ability to activate the human
prostaglandin receptors (hEP), it is with 100-1,000-fold lower affinity than the respective
prostaglandins, making prostaglandin receptors an unlikely target for the prostamides.9 Additionally, in
ex vivo functional assays on isolated tissue preparations from guinea pig known to contain prostaglandin
EP2 and EP3 receptors, PGE2-EA activity was found to be approximately 10-fold higher than those
determined by the purified hEP2 and hEP3 receptor radioligand binding assay and argues for the
existence of putative selective receptors within these tissues.9 Moreover, the lack of affinity of
prostamides at prostaglandin, cannabinoid receptors and the endocannabinoid enzymes FAAH and MGL
suggests that there may be more substantial interactions with other biological targets. A more detailed

understanding of the physiological roles of these novel compounds can be greatly enhanced by the
availability of unique pharmacological reagents and is the motivating factor for the work presented here.
The current study presents a multiple step synthesis of PGE2-EA analogs that allows for convergent
functionalization of both the head and/or tail groups and provides potentially useful probes for the
characterization of the prostamide biochemical system.10 Because PGE2-EA is the major product of
prostamide biosynthesis, analogs of the E2 configuration were selected for synthesis.6 The initial set of
compounds incorporates azido and isothiocyanato functionalities at the terminal tail position as affinity
probes for covalent binding to the putative prostamide receptor (Figure 1). Upon photoirradiation to a
nitrene intermediate, the azido moiety can attach to reactive residues in the vicinity of the binding site.11-
13
Similarly, the electrophilic isothiocyanato group is capable of reacting with nucleophilic amino acid
residues including thiol, imidazole, or amino groups that are unprotonated under physiological
conditions.14-16 These covalent probes allow for the future isolation and characterization of putative
protein targets involved in prostamide function. In earlier work we have shown that introducing steric
hindrance and/or chirality adjacent to the amide nitrogen atom in endocannabinoids leads to analogs
with enhanced stability towards hydrolyzing enzymes without any reduction in bioactivity.17 Thus, in
our rational design of PGE2-EAs we have included the R- and S-2-methyl ethanolamide and
cyclopropylamide moieties as head group variations (Figure 1).
2-Glycerol ester and 15R isomers were also incorporated in this set of compounds to explore their
effects on metabolic stability and bioactivity. 2-Arachidonoylglycerol (2-AG) is an additional key
representative of another class of endocannabinoids and was found to be subject to the same
biosynthetic pathway via COX-2 to the prostaglandin glycerol esters.18 For this reason the respective 2-
PGE2-G analog was included in this series of targeted analogs. Also, while the 15S isomer is the native
prostanoid configuration, the 15R isomer mimics the stereochemistry of the aspirin-triggered lipoxins

(ATL), shown to have a leading role in mediating inflammation resolution.19 This COX-2 mediated
metabolite of AA not only exhibits anti-inflammatory and pro-resolving properties, but in its 15R
configuration also increases potency and metabolic stability over that of the native 15S compounds.20
Thus, representative analogs with the 15R configuration were included in the targeted set of compounds
synthesized in this study.
Results and discussion
Our overall synthetic strategy incorporates Corey’s prostaglandin synthesis21 along with an additional
protecting group at the tail terminal carbon. It is essential that the protective scheme allow for complete
selectivity in removal of the terminal protecting group to avail further functionalization at this site. In
the initial synthesis, triisopropylsilyl (TIPS) groups at the 11 and 15 secondary alcohol positions and a
tert-butyldimethylsilyl (TBS) group at the primary terminal position were chosen for this purpose.
The synthesis begins with the commercial starting material, benzoyl protected Corey’s lactone aldehyde
1 (Scheme 1), undergoing a Horner-Wadsworth-Emmons reaction to give the E-enone 3 exclusively as
1
confirmed by H NMR analysis (3JCH=CH = 15.8 Hz) and in near quantitative yield. The phosphonate
reagent 2 used here is prepared by reaction of n-butyllithium with dimethyl methyl phosphonate
followed by ring opening of ε-caprolactone and protection of the terminal hydroxyl with TBS chloride
(details are given under Supporting information).22 Asymmetric reduction of 3 to enol 4 using Corey’s
oxazaborolidine catalyst23 was attempted, however enantiomeric excess never exceeded 30%.
Alternatively, we elected to perform a non-stereoselective Luche reduction,24,25 isolate both 15S and 15R
isomers (86% overall yield), and carry on the remainder of the synthesis in parallel. It should be noted
that determination of the exact stereochemistry at C15 in the two isomers 15S (less polar, Rf = 0.55, 60%
AcOEt in hexanes) and 15R (more polar, Rf = 0.45, 60% AcOEt in hexanes) was based on the synthesis

of the respective Mosher esters (see supporting information for details) followed by comparative
1
1
analysis of their H NMR spectral data.26,27 Our H NMR configurational correlations are in agreement
with previous assignments done on prostaglandin intermediates with a straight chain where the less polar
compound was determined to be the 15S secondary alcohol and the more polar compound was found to
be the 15R.28 Hydrolysis of the benzoyl group (89-91% yields) followed by TIPS protection of the
secondary free hydroxyls (90-93% yields) establishes the last of our strategic protecting groups for
intermediate 6. Reduction of the lactone to the lactol using DIBAL-H (93% yield) is followed by a
Wittig reaction to introduce the head carboxylic acid chain with the desired Z-stereochemistry at the
newly formed double bond (73-78% yields). Key intermediate 8 avails the choice of establishing either
the head or tail moieties first. In our case, we decided to first incorporate the tail moiety and were thus
able to synthesize several head group analogs.
Modifications at the tail aimed at incorporating azido and isothiocyanato functionalities are shown in
Schemes 2 and 3. Upon exposure to TMSCHN2 in MeOH, the acid 8 is converted to its methyl ester 9
(92-93% yields) and the 9S-hydroxy on the ring is oxidized to the ketone 10 (96% yield) with Dess-
Martin periodinane buffered with pyridine to avoid potential elimination of the 11-hydroxy substituent
to form the α,β-unsaturated ketone. The terminal hydroxy is then selectively deprotected using
AcOH:THF:H2O (3:1:1) to give 11 in high yields (91-93%). At this point we have established a path to
synthesize the key primary alcohol 11 which allows the further functionalization of the terminal tail
carbon. In the current study we focused on putative affinity probes incorporating azido and
isothiocyanato groups. A modified Mitsunobu reaction provides the subsequent conversion to azide 12
in 91% yield (IR: s, 2093 cm-1). Throughout the synthesis certain conditions, specifically those
encountered during the amide coupling, led to the formation of the undesirable α,β-unsaturated ketone
by dehydration of the 11-hydroxyl group. To prevent this, we reduced the 9-keto back to its hydroxyl

counterpart and reintroduced it again after the amide coupling step. This strategy is essentially utilized
as a protection/deprotection approach during the incorporation of the azido moiety and was found to be
advantageous over the use of additional 9-OH protection groups that would require selective
deprotection. Thus, Luche reduction of the ring ketone (83-89% yields) is followed by hydrolysis of the
methyl ester 13 (NaOH, THF/H2O) to give the precursor acid 14 in 84-85% yields.
Varying the head moieties begins with the free acid (14) coupling to the amine derivatives to give the
desired amide analogs (Scheme 3). As we reported earlier for the synthesis of novel anandamide
analogs, the carbonyldiimidazole (CDI) activation procedure worked well and provided these
compounds in excellent yields (88-92%).29,30 The Dess-Martin periodinane/pyridine system oxidizes the
9-hydroxy back to the ketone 16 (97-98% yields). The desilylation step leading to azides 17 proved to be
challenging because of the enhanced stability of the TIPS group at the 11-hydroxy position. After
considerable experimentation we found that the 48% HF/pyridine reagent works effectively and
provides the azido analogs 17a-17d in acceptable yields (45-48%) along with unidentified byproducts.
Subsequently, the isothiocyanato compounds 18a and 18b were isolated after treatment of the respective
azido-ethanolamides 17a and 17b with carbon disulfide in the presence of triphenylphosphine (94-96%
yields, IR: s, 2090 cm-1). For the synthesis of the n-pentyl chain tail analogs 23 (Scheme 4) where no
selectivity in removal the silyl protecting groups is required, the full synthesis was carried out using
TBS protection for both the 11- and 15-hydroxyls. In the final step, removal of all three silyl groups was
carried out smoothly with 1% HCl in 95% MeOH and produced the amides 23 in short reaction time (5
min) and high yields (80-83%). The intermediate ketones 22a-22c were synthesized with high chemical
efficiency from the precursor acid 20 which was in turn synthesized in three steps from commercially
available 19 following previously reported procedures.31 It should be noted that an earlier synthesis of
23a (no yields reported) involved exposure of PGE2 to ethyl chloroformate and treatment of the in situ

generated mixed carbonic anhydride to (S)-(+)-amino-2-propanol.7 Carbonyldiimidazole mediated
coupling of acid 20 with readily available 1,3-divaleric glycerol32 gave the triglyceride 24 in 82% yield
(Scheme 5). Dess-Martin oxidation of the free hydroxy on the cyclopentane ring (97% yield) was
followed by the deprotection of both silyl protecting groups (79% yield). Finally, the precursor
triacylglycerol 26 was conveniently deprotected in ethanol with Candida rugosa lipase type VII to give
the desired 2-PGE2-G (27) in 46% yield. To the best of our knowledge the chemoenzymatic synthesis of
this endogenous substance is reported here for the first time and parallels those we reported earlier for
the endocannabinoid lipid 2-AG and its analogs.33,34 Importantly, our approach provides the 2-PGE2-G
1
essentially free of 1(3)-acylglycerol rearrangement byproducts as confirmed by H NMR data. In a
previous study on the metabolism of prostaglandin glycerol esters the 2-PGE2-G was the minor product
which was synthesized as a mixture with the 1(3) regioisomer in a ratio 1: 9.35
The involvement of prostaglandins as pro-inflammatory lipid mediators has long been recognized.
However, more recent studies have established that certain prostaglandins, specifically PGE2 and PGD2,
trigger eicosanoid class switching from pro-inflammatory to local acting pro-resolving lipid mediators.36
However, the role of this new class of prostaglandin related amides and ester is not well understood. In
this preliminary study the actions of a representative analog within the series, the 15S hydroxy, 20-
isothiocyanato compound 18a was assessed in vivo using a widely employed model of inflammation,
namely zymosan A-stimulated murine peritonitis.37 Prostaglandin PGE2 was also tested for comparison.
The results of administration on cell numbers of both polymorphonuclear neutrophils (PMN) and
monocytes are illustrated in Figure 2. We observe that while both PGE2 and 18a effectively reduce PMN
infiltrates in this inflammatory model, 18a was slightly more efficacious.
In summary, a successful synthetic strategy has been established for the construction of a focused family
of prostamide analogs containing head and tail modifications to aid in the characterization of a putative

prostamide receptor. This synthesis incorporates the strategic use of silyl protecting groups for selective
deprotection and functionalization of the ω terminus. Azide and isothiocyanate functionalities are
introduced at this tail position to serve as affinity probes designed to covalently attach to target proteins
associated with this prostamide thus aiding in their identification. In a preliminary biological study we
found that the isothiocyanato compound 18a effectively reduces PMN infiltrates in the zymosan A-
stimulated murine peritonitis model of inflammation and is more efficacious than PGE2. A more detailed
biochemical characterization of the compounds reported here is currently being explored.
Acknowledgments
This work was supported by grants from the National Institutes of Health, DA09158 (A.M), DA07215
(A.M), T32 DA07312 (A.M), DA03801 (A.M) and P01GM095467 (C.N.S), R01-GM038765 (C.N.S).
Supplementary data
Experimental procedures, characterization data for compounds and murine peritonitis. These data can be
found in the online version.
References and notes
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Figure 1: PGE2-EA and 2-PGE2-G analogs synthesized.

Figure 2: Comparisons for PGE2 and 18a in reducing leukocytes in murine peritonitis.
Polymorphonuclear neutrophils (PMN) and monocytes were enumerated following injection with
1µg/mouse compound i.v., after 5 min initiation of peritonitis with 1mg zymosan A. After 4 hrs,
leukocytes were obtained via lavages and leukocytes were identified and enumerated using light
microscopy as well as fluorescence-activated cell sorting.

Scheme 1: Synthesis of the C-8 and C-12 chain appendages.
Reagents and conditions: (a) 2, NaH, THF, 0oC, 1 h, then 1, 0oC to r t, 2.5 h, 100%; (b) CeCl3, NaBH4,
MeOH, 0oC, 30 min, then chromatographic separation of 4a and 4b, 86% overall yield; (c) K2CO3,
THF/MeOH, 50oC, 2.5 h, 91% for 5a and 89% for 5b; (d) TIPSOTf, 2,6-lutidine, CH2Cl2, 0oC, 1h, 90%
for 6a and 93% for 6b; (e) DIBAL-H, CH2Cl2, -78oC, 30 min, 93% for 7a and 7b; (f) Br Ph3P-(CH2)4-
COOH, t-BuOK, THF, 0oC, 30 min, then 7a or 7b, 1 h, 73% for 8a and 78% for 8b.

Scheme 2: Synthesis of the functionalized azido tail.
Reagents and conditions: (a) TMSCHN2, Et2O, MeOH, r t, 15 min, 93% for 9a and 92% for 9b; (b)
Dess-Martin periodinane, pyridine, CH2Cl2, 0oC to r t, 1 h, 96% for 10a and 10b; (c) AcOH:THF:H2O
(3:1:1), r t, overnight, 93% for 11a and 91% for 11b; (d) Zn(N3)2-Py, DIAD, PPh3, toluene, r t, 7 h, 91%
for 12a and 12b; (e) CeCl3, NaBH4, THF/MeOH, 0oC, 1 h, 83% for 13a and 89% for 13b; (f) NaOH,
THF/H2O, r t, 24 h, 85% for 14a and 84% for 14b.

Scheme 3: Final steps to the azido and isothiocyanato substituted prostamide probes.
Reagents and conditions: (a) carbonyldiimidazole, CH2Cl2, r t, 30 min, then R-NH2, 3h, 88-92%; (b)
Dess-Martin periodinane, pyridine, CH2Cl2, 0oC to r t, 1 h, 97-98%; (c) 48% HF, pyridine, THF/MeCN,
0oC to r t, overnight, 45-48%; (d) CS2, PPh3, THF, r t, 48 h, 94-96%.

Scheme 4: Synthesis of the n-pentyl chain tail analogs.
Reagents and conditions: (a) carbonyldiimidazole, CH2Cl2, r t, 30 min, then R-NH2, 3h, 87-90%; (b)
Dess-Martin periodinane, pyridine, CH2Cl2, 0oC to r t, 1 h, , 89-91%; (c) 1% HCl, aqueous MeOH, 5
min, 80-83%.

Scheme 5: Chemoenzymatic synthesis of the glycerol ester analog.
Reagents and conditions: (a) HO-CH[CH2-O-C(O)-(CH2)3CH3]2, carbonyldiimidazole, CH2Cl2, 0oC to r
t, 24 h, 82%; (b) Dess-Martin periodinane, pyridine, CH2Cl2, 0oC to r t, 1 h, , 97%; (c) 1% HCl, aqueous
MeOH, r t, 5 min, 79%; (d) Candida rugosa lipase, EtOH, 2 h, 46%.

Graphical abstract