2
M.-J. Blanco et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
O
OH
functional activity (hEP4 IC50 = 96.7 nM). However, the corre-
sponding 4-hydroxy-piperidine analog 4f, showed an improved
activity profile (hEP4 IC50 = 5.6 nM; hWB IC50 = 123 nM), similar
to meta-carboxylic acid analog 2. Noteworthy, a linear correlation
between binding or functional activity and hWB activity was not
apparent. Since the hWB assay uses a native tissue matrix, addi-
tional parameters such as protein binding and cell-type influence
the response.
NH
N
OH
O
Incorporating saturated heterocycles into this scaffold led to
favorable increases in the sp3 fraction going from 0.17 (2) to 0.41
(4d). Additional physicochemical properties and ligand efficiencies
are presented in Table 2. Compounds 3–4 are characterized by
MW < 450, cLogD < 3, and LEAN values maintained or decreased
(0.30–0.24) relative to 2. These physicochemical parameters were
targeted to achieve improvements in pharmacokinetic profiles.
Selected compounds were assessed for solubility. Compounds 3c,
4d and 4f showed increased solubility at pH 6 relative to 2.
Demonstrating potent functional (hEP4 IC50 = 5.6 nM) and hWB
(123 nM, >10-fold increase vs. 1) activity and with a sp3 fraction of
0.38, compound 4f was selected for additional characterization.
Compound 4f showed well-behaved rat and dog PK profiles, char-
acterized by low clearances and volumes of distribution and by
acceptable oral bioavailabilities (Table 3).
Compound 4f showed exquisite selectivity toward other EP
receptors with no detectable binding for EP1, EP2 or EP3 (Table 4).
In addition, 4f was active in the rat EP4 functional assay (rEP4
IC50 = 12 nM) and a suitable candidate for study in animal models
of pain and inflammation.
Compound 4f was tested in the monoiodoacetic acid (MIA)
model of joint pain. The injection of MIA into the knee joint of rats
produces an acute inflammatory insult that develops into chronic
degeneration of the tissues in the injected joint and pain. This pain
can be measured via differential weight bearing of the hind legs
using an incapacitance tester.20 Efficacy is assessed by the ability
of a test compound to normalize weight distribution indicating
reduction of joint pain. The MIA model has been extensively
described in the literature21 and has been used to demonstrate
pain reversal for a variety of mechanisms with compounds show-
ing efficacy at plasma exposures comparable to clinically effective
human22 exposures.
2
Figure 2. Compound 2.
further optimization of these scaffolds to identify high quality
compounds suitable for clinical studies.
Recently, there has been interest in the medicinal chemistry
community to track calculated physicochemical properties13–16
such as molecular weight, topological polar surface area, rotatable
bonds, hydrogen bond donors and acceptors, and molecular com-
plexity as measured by amount of carbon bond saturation. This last
parameter is defined by fraction sp3 (Fsp3), where Fsp3 = number
of sp3 hybridized carbons/total carbon count.17 Saturation allows
the preparation of topologically more complex molecules without
substantially increasing molecular weight. The introduction of
out-of-plane substituents could also increase receptor–ligand com-
plementarity. Importantly, a connection has been noted between
Fsp3 and the probability of successfully progressing compounds
into clinical evaluation. Toward this end, we describe here opti-
mization studies of our previously identified EP4 antagonist scaf-
fold focused on increasing the sp3 character and reducing the
overall aromaticity of the compounds.
One important feature of this research is the use of a human
whole blood assay (hWB)18 to measure preclinical potency and
effective target engagement biomarker to facilitate clinical transla-
tion.19 In this assay, compounds are evaluated for their ability to
reverse the inhibitory effects of PGE2 on LPS-induced TNF
a pro-
duction in a concentration-dependent manner. The human whole
blood assay provides a measure of EP4 antagonist function in a
clinically relevant tissue.
To enhance the sp3 character of our compounds, we replaced
the benzylic alcohol of 2 with saturated heterocyclic groups
(Table 2). For example, incorporation of a 3-hydroxymethyl-piper-
idine group yielded compound 3a and a substantial decrease in
functional hWB activity. This negative effect was more pronounced
with the 4-hydroxy-4-methyl-piperidine analog 3b. However, the
corresponding des-methyl analog 3c demonstrated hWB activity
comparable to clinical benchmark 1.
In the case of the para-benzoic acid derivatives (4a–f), similar
activity trends were observed. Hydroxymethyl-piperidine 4a
showed a modest increase in hWB activity, trending slightly better
than the meta analog 3a. Replacing the piperidine in 4a by pyrro-
lidine to give 4b was detrimental. The less polar 4-di-fluoro piper-
idine analog 4c showed potent antagonism (hEP4 IC50 = 1.6 nM)
but only modest hWB activity (1160 nM). Introducing a 4-meth-
oxy-piperidine substituent led to 4e and a significant decrease in
In the experiment shown in Figure 3, rats were injected intra-
articularly with 0.3 mg MIA in 50
ll saline into the right knee with
50 l saline injected into the left knee on day 0. Twelve days later,
l
the rats were randomized and dosed orally with either vehicle;
compound 4f at 0.3, 1, 3 or 10 mg/kg; or the positive control
non-steroidal anti-inflammatory drug (NSAID) diclofenac at
5 mg/kg. Efficacy was measured using incapacitance testing
30 min post dose. Compound 4f dose dependently inhibited differ-
ential weight bearing versus vehicle at 3 and 10 mg/kg as did the
NSAID diclofenac.
Compound 4f was also tested for the potential to block inflam-
mation in the rat adjuvant induced arthritis (AIA) model. This
model has been well-described in the literature23 and has been
used to demonstrate efficacy versus inflammation for a variety of
Table 1
PK profile of compound 2 in rat
Compd
HTSA solubility,a
(mg/mL)
iv CL
(mL/min/kg)
po AUC
po Cmax
(ng/mL)b
T1/2
(h)
%Fb,c
(ng * h/mL)b
2
0.242
2.2
8520
1640
2.1
22
a
High Throughput Solubility Assay (HTSA), assessment of aqueous solubility at pH = 6. Samples prepared in DMSO were dried for 12 h. The powder or film was re-dissolved
in the solvent at pH 6 and DMSO control at 2 mM target concentration. Samples are stirred for 20 h and filtered through a 0.7
assay for concentration against DMSO standard curve.
lm GF filter. The filtrate was analyzed by HPLC
b
Dose of 1 mg/kg iv.
c
Dose of 5 mg/kg po.