Pharmacophore elucidation of phosphoiodyn A - Potent and selective peroxisome proliferator-activated receptor β/δ agonists with neuroprotective activity

Article abstract of DOI:10.1016/j.bmcl.2016.03.028

We report the pharmacophore of the peroxisome proliferator-activated receptor δ (PPARδ) agonist natural product phosphoiodyn A is the phosphonate core. Synthesis of simplified phosphonate esters 13 and 15 provide structurally novel, highly selective and potent PPARδ agonists (EC₅₀ = 78 and 112 nM, respectively). Further, both compounds demonstrate significant neuroprotective activity in an in vitro cellular model indicating that phosphonates may be an effective novel scaffold for the design of therapeutics for the treatment of neurodegenerative disorders.

Full text of DOI:10.1016/j.bmcl.2016.03.028

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1889–1893
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pharmacophore elucidation of phosphoiodyn A – Potent and
selective peroxisome proliferator-activated receptor b/d agonists with
neuroprotective activity
a
b
a
b
a,c,
⇑
Nihar Kinarivala , Ji Ho Suh , Mina Botros , Paul Webb , Paul C. Trippier
a
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA
The Methodist Hospital Research Institute, Genomic Medicine Program, Houston, TX 77030, USA
Center for Chemical Biology, Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the pharmacophore of the peroxisome proliferator-activated receptor d (PPARd) agonist natu-
ral product phosphoiodyn A is the phosphonate core. Synthesis of simplified phosphonate esters 13 and
Received 8 January 2016
Revised 7 March 2016
Accepted 8 March 2016
Available online 9 March 2016
15 provide structurally novel, highly selective and potent PPARd agonists (EC50 = 78 and 112 nM, respec-
tively). Further, both compounds demonstrate significant neuroprotective activity in an in vitro cellular
model indicating that phosphonates may be an effective novel scaffold for the design of therapeutics for
the treatment of neurodegenerative disorders.
Keywords:
PPAR agonist
Ó 2016 Elsevier Ltd. All rights reserved.
Neuroprotection
Neurodegeneration
Pharmacophore identification
Peroxisome proliferator-activated receptors (PPARs) are a fam-
ily of nuclear hormone receptors and ligand-activated transcrip-
tion factors that play significant roles in diverse cellular
PPARd agonism may be a pharmacological mechanism of action
for the design of drugs to treat PD.
1
3
Current PPARd agonists (Fig. 1) include GW0724 (1) and the clo-
sely related analog GW501516 (2) with EC50 values of 1.0 and
1.1 nM, respectively coupled with 1000-fold selectivity for
1,2
processes including metabolism and differentiation. PPARs exist
as three different isoforms – , and d (also known as b) expressed
at different levels in a range of cell types including microglia, astro-
a, c
1
4
PPARd. Bioisosteric replacement of the allylic sulfur in 2 with
3
15
cytes and neurons. The PPAR
a
and PPAR
c
receptors are validated
selenium also provided a selective PPARd agonist. KD3010 (3),
4
therapeutic targets in multiple disease states including diabetes,
is a potent and highly selective PPARd agonist with EC50 of
inflammatory diseases,5 neurodegeneration
6,7
and cancer. The
8
1.5 nM, which was scheduled for phase II trials in 2009 for dia-
16
1
7
role of the PPARd isoform has not been fully explored due to its
ubiquitous expression and until recently, lack of selective chemical
probes. However, PPARd is emerging as a promising pharmacolog-
ical target for the treatment of a range of neurodegenerative dis-
eases including Alzheimer’s Disease,9 Multiple sclerosis,
betes mellitus.
A structure containing a benzisoxazole ring
known as ‘compound 20’ (4) was found to have an EC50 = 25 nM
1
0
with activity at PPAR
a and PPARc > 10 lM. The benzothiazole
ring-containing compound (5) was found to have a PPARd EC50 of
10
18
0.39 lM with activity at PPARa and PPARc > 10 lM. Further
1
1
12
Huntington’s Disease and Parkinson’s Disease (PD).
compounds containing a trisubstituted isoxazole ring system, illus-
PPARd is known to be expressed in the nucleus of dopaminergic
neurons with striatal PPARd levels increasing immediately follow-
ing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) insult in
trated by LCI765 (6) display potent PPARd agonist activity with
19,20
EC50 of 70 nM with EC50 for PPAR
a
and PPAR
c
> 10
l
M.
A ser-
ies of para-alkylthiophenoxyacetic acid derivatives were shown to
be potent and selective PPARd agonists with the most potent com-
1
3
a murine model of PD, compared to wild type control mice.
2
1
Treatment of mice with the PPARd agonist GW0742 (1) reduced
MPTP-induced loss of dopaminergic neurons while PPARd
heterozygous mice showed no neuroprotection, suggesting that
pound (7) displaying an EC50 of 8.6 nM. Phenoxyacetic acid
derivatives provided a series of compounds with excellent selectiv-
ity and potency for PPARd, compound (8) has a PPARd EC50 of 3 nM
2
2
with EC50 for PPARa and PPARc > 100 lM.
These known PPARd agonists possess high structural similarity,
all containing a terminal carboxylic acid. Recently a structurally
⇑
Corresponding author.
E-mail address: paul.trippier@ttuhsc.edu (P.C. Trippier).
http://dx.doi.org/10.1016/j.bmcl.2016.03.028
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0

1
890
N. Kinarivala et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1889–1893
Figure 1. Structure and activity of known PPARd agonists and the natural products phosphoiodyn A and B.
novel phosphonate-containing polyacetylene (9) was isolated from
12 afforded the deprotected phosphonate diester derivate 15 in
good yield.
a Korean marine sponge, named phosphoiodyn A the compound
2
3
has a PPARd EC50 of 23.7 nM with >200-fold selectivity. The
structurally similar phosphate-containing compound phosphoio-
dyn B (10), lacking the P–C bond of phosphoiodyn A, was also iso-
lated from the same marine sponge yet possessed no PPARd agonist
To determine if the phosphonate group is a prerequisite for
activity, or if a carboxylic acid bioisostere (formed by in vitro
hydrolysis of the parent ester) could also provide significant PPARd
agonism in this class of compound, as in known PPARd agonists
(Fig. 1), we synthesized ethyl ester derivative 19. 3-(Benzyloxy)
propan-1-ol (16) was obtained in good yield by exposing 1,3-pro-
pane diol to standard protecting conditions. Oxidation of 16 with
Jones reagent provided carboxylic acid 17 in moderate yield. Ester-
ification to provide ethyl ester 18 and subsequent deprotection of
the benzyl group by hydrogenation yielded bioisostere 19 in good
overall yield.
activity at a concentration of 10 lM. The P–C bond of phosphoio-
dyn A therefore plays a critical role in the bioactivity of this com-
pound, pointing to the phosphonate moiety as the pharmacophore
for PPARd agonism.
We herein describe a structurally novel scaffold for the design
of potent and selective PPARd agonists developed by pharma-
cophore elucidation of phosphoiodyn A.
Synthesis of the phosphonate core of phosphoiodyn
A
The four phosponate analogs 13, 14, 15 and 19 were evaluated
(Scheme 1) was accomplished via an Arbuzov reaction between
for their selectivity to act as agonists of the PPARa, PPARd and
bromalkane 11 and triethylphosphate to install the P–C bond of
protected phosphonate 12.24 Acid catalyzed hydrolysis of one or
both phosphonate esters with concomitant deprotection of the
benzyl moiety could be achieved by careful control of reaction con-
ditions affording access to phosphonate monoester (13) as a mix-
ture of two isoforms after 10 h reflux and phosphonic acid (14)
after 75 h reflux. Hydrogenation of benzyl protected phosphonate
PPAR receptors (Fig. 2). A dual-luciferase reporter assay was
c
employed to determine the agonist effect of each phosphonate
analog compared with no treatment control and a known potent
and selective agonist for each of the PPAR isoforms. None of the
phosphorous-containing compounds showed activity at the PPAR
receptor while the ester bioisostere 19 displayed potent activity,
greater than that of the PPAR agonist WY-14643 (Fig. 2A).
a
a

N. Kinarivala et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1889–1893
1891
size and simple chemical scaffold allowing it to fit and bind equally
well onto all three PPAR receptors. The phosphonate ester is there-
fore the critical structural moiety of this class of compounds
required to impart selectivity towards the PPARd receptor.
We next determined the potency of each phosphonate deriva-
tive to act as an agonist of PPARd (Table 1.) Phosphonic acid 14
showed an EC50 value >100
lM confirming its inactivity. Ester bioi-
sostere 19 was a weak PPARd agonist with an EC50 value >1
lM
that was not further defined due to the promiscuous nature of this
compound between PPAR receptors. The two selective PPARd ago-
nists, phosphonate esters 13 and 15, displayed potent EC50 values
of 0.078 lM and 0.112 lM, respectively (Fig. S3, Supporting
Scheme 1. Synthesis of the phosphonate pharmacophore of phosphoiodyn A and
derivatives.
Compounds 13, 15 and 19 all show potent activity at the PPARd
receptor, while phosphonic acid 14 displays little activity above
the negative control (Fig. 2B). Activity at the PPAR
not observed in any of the compounds except for ester bioisostere
9, which displayed activity approximately half that of rosiglita-
zone (Rosi), a potent and selective PPAR agonist (Fig. 2C).
c receptor was
1
c
These data reveal that phosphonate esters 13 and 15 are selec-
tive agonists of the PPARd receptor (additional selectivity assays
for both compounds where performed, Figs. S1 and S2, Supporting
information) with the potential to possess high potency. Phospho-
nic acid 14 lacking the phosphonate ester group is inactive across
all three PPAR isoforms suggesting that a di- or at least a mono-
phosphonate ester is required to retain activity. Without the ethyl
ester masking the phosphonate a highly charged species will result
at physiological pH and in in vitro media. Such a hydrophilic moi-
ety (clogP = À2.9) will be highly hindered in passing through the
cell membrane to reach the PPAR target and desolvation of the
compound represents an energetically disfavored event impeding
binding to the PPAR receptor.25
The ethyl ester bioisostere 19 is a promiscuous pan-PPAR iso-
form agonist. Hydrolysis of the ethyl ester can be expected to occur
at physiological pH and in in vitro media providing a terminal car-
boxylic acid that is a commonly encountered moiety in the PPARd
agonists depicted in Figure 1. The promiscuity of compound 19
between the various PPAR receptors may be attributed to its small
Figure 2. Selectivity of phosphonate derivatives 13, 14, 15 and 19 to act as agonists
of (A) PPAR , (B) PPARb/d, and (C) PPAR . Rosi = rosiglitazone. Results represent the
mean of three experiments ± SEM.
a
c

1
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N. Kinarivala et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1889–1893
Table 1
Agonist activity at the PPARd receptor and clogP calculation of compounds 1, 9, 10,
the PPARd receptor, affords 80% cell survival to abort etoposide-
induced apoptosis.
1
3–15 and 19
The ability of phosphonate esters 13 and 15 to abort serum star-
vation-induced apoptosis in neuron-like PC12 cells was evaluated
to eliminate the possibility that compounds of this structural class
act to simply block the binding of etoposide to topoisomerase II/
DNA thereby preventing apoptosis rather than eliciting neuropro-
tection (Fig. 3B). Both monoester 13 (PPARd EC50 = 78 nM) and die-
ster 15 (PPARd EC50 = 112 nM) provided 77% and 78% cell survival
respectively compared with 55% cell survival with no treatment
and 82% cell survival upon flupirtine treatment.
This data demonstrates that potent and selective phosphonate
ester PPARd agonists elicit neuroprotection against toxic insult in
a neuron-like PC12 cell line. Both phosphonate esters demonstrate
highly similar levels of neuroprotective effect despite compound
13 being a more potent PPARd agonist than compound 15. This
may be due to a minimum threshold of agonism of PPARd required
to display neuroprotection that both of these compounds, and
others (Fig. 1) exceed. Indeed at a treatment concentration of
b
Compound
clogPa
EC50 PPARb/d (
lM)
1
9
1
1
1
1
1
(GW0742)
5.16
4.56
5.51
À0.2
À2.9
0.22
À0.1
0.001
2
3
0.024
2
3
0
>10
3
4
5
9
0.078
>100
0.112
>1
a
Calculated by ChemBioDraw 14.0.
Concentration required to produce 50% response.
b
information). Phosphonate monoester 13 mirrors the phosphonate
moiety of phosphoiodyn A and shows potent activity suggesting
that the free alcohol or charged oxygen functionality plays a role
in hydrogen bonding within the PPARd receptor. When the free
oxygen is masked as an ester (15) activity is diminished from
7
8 nM to 112 nM.
1 lM in both assays the more potent compound 13 provides
When the mono-ester is hydrolyzed to form phosphonic acid
4 the PPARd EC50 value is beyond 100 lM confirming inactivity
greater protective effect than the less active PPARd agonist 15.
In conclusion, the results of this study demonstrate that the
phosphonate moiety of phosphoiodyn A is the pharmacophore of
this bioactive natural product. Simplified phosphonates 13 and
15 based on the phosphoiodyn A structure are highly potent and
selective agonists of the PPARd receptor. Inclusion of ester moieties
that confer greater lipophilicity would be expected to provide
more potent compounds, allowing cell wall penetration and
greater enthalpy gains in binding to the receptor. Phosphonates
are capable prodrug moieties known to be able to penetrate the
blood–brain barrier, with the NMDA antagonist and phosphonic
acid perzinfotel showing efficacy in in vivo models of neuropathic
1
posited to be due to the high hydrophilicity of this compound.
When the clogP values of compounds 14, 13 and 9 are calcu-
lated (À2.9, À0.2 and 4.56, respectively) a clear increase in
potency is observed in correlation with increasing lipophilicity.
This observation suggests the dialkyne chain of the phosphoio-
dyn A natural product plays an auxophoric role, enhancing
lipophilicity to allow cell membrane penetration and possibly
taking part in hydrophobic interactions within the PPARd
receptor.
Having identified two potent and selective PPARd agonists we
evaluated their utility as neuroprotective agents. We utilized the
cell viability agent PrestoBlueÒ to determine the protective ability
of compounds 13 and 15 to abort the etoposide-induced apoptosis
of neuron-like PC12 cells (Fig. 3A).26
2
8
pain. Phosphonates 13 and 15 display potent in vitro neuropro-
tective activity and represent novel chemical scaffolds for potential
drug discovery in neurodegenerative diseases.
The phosphonate esters 13 and 15 both display significant
neuroprotective activity over a wide concentration range. Both
compounds showed maximum activity at a concentration of
Acknowledgments
This work was supported financially by Texas Tech University
Health Sciences Center School of Pharmacy (P.C.T.). We thank the
Shimadzu Center for Advanced Analytical Chemistry at The Univer-
sity of Texas at Arlington for HRMS experiments.
3 lM with phosphonate mono-ester 13 (PPARd EC50 = 78 nM)
providing 110% cell viability, and di-ester 15 (PPARd EC50 = 112 -
nM) providing 116% cell viability compared with only 52% cell
viability with no treatment. Cell viability values of >100% sug-
gest that these compounds may play a role in triggering prolif-
eration or neurogenesis. These data also indicate that the
compounds are non-toxic at this concentration. The clinically
employed non-opioid analgesic flupirtine is known to be neuro-
protective and was employed as a positive control.27 At a con-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.03.
0
28. These data include MOL files and InChiKeys of the most
centration of 3 lM, flupirtine, which has no reported activity at
important compounds described in this article.
Figure 3. Protective ability of phosphonate esters 13 and 15 to (A) abort etoposide-induced apoptosis and (B) abort serum starvation-induced apoptosis in neuron-like PC12
cells. Data is the mean of three experiments ± SD.

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