Novel tail and head group prostamide probes

Article abstract of DOI:10.1016/j.bmcl.2015.01.064

We report the design and synthesis of novel prostaglandin-ethanolamide (PGE₂-EA) analogs containing head and tail group modifications to aid in the characterization of a putative prostamide receptor(s). Our synthetic approach utilizes Horner-Wadsworth-Emmons and Wittig reactions to construct the head and the tail moieties of the key PGE₂ precursor, which leads to the final products through a peptide coupling, Swern oxidation and HF/pyridine assisted desilylation. The synthesized analogs were shown not to interact significantly with endocannabinoid proteins and recombinant EP1, EP3 and EP4 receptors and suggest a yet to be identified prostamide receptor as their site(s) of action.

Full text of DOI:10.1016/j.bmcl.2015.01.064

Accepted Manuscript
Novel Tail and Head Group Prostamide Probes
David F. Finnegan, Erin L. Shelnut, Spyros P. Nikas, Nan Chiang, Charles N.
Serhan, Alexandros Makriyannis
PII:
S0960-894X(15)00085-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.01.064
BMCL 22404
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 December 2014
22 January 2015
26 January 2015
Please cite this article as: Finnegan, D.F., Shelnut, E.L., Nikas, S.P., Chiang, N., Serhan, C.N., Makriyannis, A.,
Novel Tail and Head Group Prostamide Probes, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://
dx.doi.org/10.1016/j.bmcl.2015.01.064
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Novel Tail and Head Group Prostamide Probes
David F. Finnegana, Erin L. Shelnuta, Spyros P. Nikasa, 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

ABSTRACT
We report the design and synthesis of novel prostaglandin-ethanolamide (PGE2-EA) analogs containing
head and tail group modifications to aid in the characterization of a putative prostamide receptor(s). Our
synthetic approach utilizes Horner-Wadsworth-Emmons and Wittig reactions to construct the head and
the tail moieties of the key PGE2 precursor, which leads to the final products through a peptide coupling,
Swern oxidation and HF/pyridine assisted desilylation. The synthesized analogs were shown not to
interact significantly with endocannabinoid proteins and recombinant EP1, EP3 and EP4 receptors and
suggest a yet to be identified prostamide receptor as their site(s) of action.
KEY WORDS: Prostamides, Cyclooxygenase-2, Endocannabinoids

The endocannabinoid system has broad physiological function and is involved in pain, weight
management, neuroprotection and the immune system.1 This is accomplished by modulating the levels
of endocannabinoids, one of which, anandamide (AEA, Figure 1), is produced on demand and
metabolized by a number of enzymes including COX-2,2 fatty acid amide hydrolase (FAAH),3,4 N-
acylethanolamide acid amidase (NAAA)5 and monoacylglycerol lipase (MGL).6 Enzymatic
transformation of AEA by COX-2 leads to the production of a new family of endogenous substances
namely prostamides (prostaglandin-ethanolamides, PG-EA) with distinct biological activities from either
AEA or prostaglandins.7 Biological testing of the PGE2, PGF2α and PGD2 related prostamides exhibited
physiological properties that ranged from contraction of feline iris to the relaxation of rabbit jugular
vein.8 Although a number of studies related to their biological activity have been reported, the full
picture regarding their mechanism of action and their molecular targets remains to be elucidated.8-10
Yu and co-workers identified the first prostamide (PGE2-EA, Figure 1), during studies of anandamide
deactivation.11 In their studies, arachidonic acid ethanolamide was submitted to both COX-1 and COX-2
to test whether this reaction is involved in the biotransformation of a significant portion of AEA. They
showed that AEA was not a substrate for COX-1 but underwent oxidative cyclization with COX-2 to
produce a PGE2 related prostamide.2 Earlier, amide derivatives of prostaglandins were studied and
shown to be inactive in a number of receptor specific preparations.12,13 The drug Latanoprost (Figure 1),
14,15
a PGF2α ester derivative approved to treat glaucoma was reported to act as a pro-drug for PGF2α
while the receptors for the related amide (Bimatoprost) and the PGF2α-EA, have been identified as
heterodimers of the wild type FP receptor and one of its splice variants.10 It has also been reported that
possibly the actions of PGE2 and PGD2 related prostamides may be associated with district yet
unidentified receptors.8,16 Further work after the endogenous nature of prostamides was established
revealed a marked difference in their activity compared to the prostaglandins.16-18

Access to prostamide analogs with improved pharmacological properties, such as enhanced stability
toward hydrolytic and oxidative pathways, would greatly aid in understanding their molecular
mechanism of action. In this communication we describe the synthesis of a set of novel aromatic tailed
prostamide probes basing our design on the chemical structures of the endogenous PGE2-EA and the
drugs Bimatoprost and Latanoprost. Because PGE2-EA is the major product of prostamide
biosynthesis,11 the E2 structure was selected as the template in our design. As with earlier work on
anandamide probes (e.g. AM356, Figure 1) we included in our synthesis analogs with R- or S-methyl
groups at the ethanolamide moiety of the prototype to increase the stability of the compound toward
enzymatic hydrolysis (Scheme 4).19 The aromatic tail of the drugs Bimatoprost and Latanoprost was
included because it maintains biological activity while it is expected to have enhanced stability for P450
induced ω-oxidation when compared to the n-pentyl tail of the PGE2 prototype (Scheme 3). Biological
testing results indicate that the analogs reported here do not exhibit significant interactions with the
proteins of the endocannabinoid system. Also, in vitro testing of a representative analog employing
electrical cell-substrate impedance sensing system revealed that the compound is inactive at the
recombinant EP1, EP3 and EP4 receptors.
The synthetic approach we used as outlined below is a modification of the Corey prostaglandin
synthesis20,21 and begins with the Fisher esterification of commercially available acid 1a to form methyl
ester 2a in 56% yield (Scheme 1).22 Compound 2a was converted to the β-keto-phosphonate 3a by
exposure to dimethyl(lithiomethyl)phosphonate which is generated in situ from dimethylphosphonate
and n-BuLi (82% yield).23 Submitting phosphonate 3a and commercially available (-)-Corey-
aldehyde/lactone 4 to the Masamune-Roush modification of the Horner-Wadsworth-Emmons reaction
yielded the key enone 5a in 92% yield.24 Under the experimental conditions used this reaction gave the
1
E geometrical isomer exclusively, as confirmed by H NMR data (3JCH=CH = 15.8 Hz). Reduction of

compound 5a followed two different paths (Scheme 2). The first uses (R)-2-methyl-CBS-
oxazaborolidine and catechol borane to provide alcohols 6a and 6b in 65% yield with an 1:5 enrichment
in favor of the naturally occurring diastereomer 6a.25 Alternatively, standard Luche reduction procedures
could be used to furnish alcohols 6a and 6b in higher yield (87%) with an enrichment ratio of 1:1.6.26
Subsequently, the two diastereomers were separated by flash column chromatography on silica gel. It
should be noted that assignment of the absolute stereochemistry at C15 of the alcohols 6a and 6b was
based on our work with close related analogs where the “Mosher’s method” was used.27 With the
individual compounds 6a and 6b in hand, the synthesis could be completed (Scheme 3). Thus, alcohol
6a was protected with TBSOTf to provide lactone 7a in 79% yield.28 Standard DIBAL-H reduction gave
lactol 8a in 82% yield. This compound underwent a Wittig olefination reaction to yield acid 9a as a
single geometrical isomer under the experimental conditions used (68% yield).14,29 Intermediate 9a was
coupled with the 2-(tert-butyldimethylsilyloxy)ethanamine30 to give compound 10a (58% yield) which
was subsequently oxidized to ketone 11a in excellent yield (89%) using Swern conditions.31 The
synthesis was completed by global desilylation of ketone 11a with hydrofluoric acid/pyridine to give the
final product 12a (AM6905) in 49% yield.27 Similarly, the 15R-isomer (12b, AM6909, Scheme 3) was
synthesized in six steps starting from alcohol 6b. Following the completion of AM6905 and AM6909
various analogs were synthesized to allow for a study of these intriguing compounds. Thus, readily
available 3-(4-iodophenyl)propionic acid32 was converted to the key 15S alcohol 6c in four steps through
the Luche reduction approach (Schemes 1 and 2). Subsequently, the iodo-substituted tail analog 12c was
synthesized from 6c in six steps (Scheme 3). The intermediate prostaglandin acid derivatives 9a, 9b and
9c served as the starting points for the synthesis of the head group modified analogs 15a, 15b and 15c
through our well established three step process which involves amide coupling, Swern oxidation and
HF/pyridine assisted desilylation (Scheme 4).

Because of their structural and functional proximity with the endocannabinoid system all synthesized
compounds (12a-12c and 15a-15c) were tested for their inhibitory activities on three key
endocannabinoid enzymes namely, FAAH, MGL, and NAAA as well as for their binding affinities at
CB1 and CB2 cannabinoid receptors using procedures we described earlier.30,33 Our testing results
(Table 1) revealed that all analogs reported here have no significant inhibitory activity exhibiting IC50s
greater than 100 µM for all three endocannabinoid deactivating enzymes. The testing results for FAAH
are consistent with those reported earlier for the endogenous prototype PGE2-EA.8 In addition, all
analogs exhibited weak binding affinities for both the CB1 and CB2 cannabinoid receptors with Ki
values greater than 620 nM, which is in agreement with earlier studies on PGE2-EA derivatives.34
To assess whether AM6905, a representative of this new class of PGE2-EA analogs, has direct
interactions with specific prostaglandin receptors or may interact with distinct functional proteins, we
next carried out in vitro screening assays using recombinant EP1, EP3 and EP4 receptors in an electrical
cell-substrate impedance sensing system (ECIS). The prototypes PGE2 and PGE2-EA were also tested
for comparison (see supporting information and Table 2). ECIS can be used to investigate the signal
transduction of G protein-coupled receptors such as the EP receptors.35 G proteins (Gs, Gi, or Gq)
coupling to the receptor give diverse characteristic profiles of impedance change upon ligand binding.
Measurement of cell impedance gave a rapid change in impedance in these cells that was PGE2
dependent, conversely, AM6905 and PGE2-EA by direct comparison did not activate either the EP1,
EP3, or EP4 receptors each expressed separately in CHO cells.
In summary, we have described the synthesis of six prostamide analogs with structural modifications at
the tail, and at the head group of the endogenous prototype PGE2-EA. These novel compounds may be
considered as hybrid ligands with contributions from both the endocannabinoid and prostaglandin
biochemical systems. However, our biological testing results indicate that these analogs do not exhibit

significant interactions with the proteins of the endocannabinoid system. Also, further in vitro testing of
a representative analog employing electrical cell-substrate impedance sensing system revealed that the
compound is inactive at the prostaglandin EP1, EP3 and EP4 receptors. These results which parallel
those observed with PGE2-EA suggest a yet to be identified target protein as their site(s) of action.9 We
are now planning to utilize these new compounds as probes for the characterization of such a target.
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 final compounds and methods associated with this
article can be found, in the online version.
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Table 1: Enzyme inhibition data and cannabinoid receptor binding affinities for synthesized compounds.
Compd
Enzyme inhibition data
(IC50, µM)
Cannabinoid receptor
binding affinities (Ki, nM)
CB1 (rat)
with PMSF
without PMSF
>1,000
>1,000
~740
FAAH
human
rat
MGL
human
NAAA
human
CB2
mouse
human
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>100µM ~100µM >100µM
>100µM
>100µM >100µM >100µM
>100µM
>100µM >100µM >100µM
>100µM
>100µM >100µM >100µM
>100µM
>100µM >100µM >100µM
>100µM
>100µM >100µM >100µM
>100µM
12a
12b
12c
15a
15b
15c
~740
~620
~830
~730
~770
~950
>1,000
>1,000
>1,000
PMSF: Phenylmethanesulfonyl fluoride.
Table 2: Changes of impedance for PGE2, PGE2-EA, and compound 12a using recombinant EP1, EP3
and EP4 receptors.
Compd
Changes of impedance
(Ω)
EP1
no
EP2
no
EP3
no
12a
no
yes
no
yes
no
yes
PGE2-EA
PGE2

Figure 1: Chemical structures of the endogenous AEA and PGE2-EA and their synthetic derivatives
AM356, Bimatoprost and Latanoprost.
Scheme 1: Synthesis of the key enones 5a and 5b.

Scheme 2: Reduction of enones 5a and 5b to introduce the 15S- and 15R- stereochemistry.

Scheme 3: Synthesis of the tail modified prostamide analogs AM6905, AM6909 and AM6910.

Scheme 4: Synthesis of the head group modified analogs AM6906, AM6907 and AM6908.

Graphical abstract