Identification and biological activity of 6-alkyl-substituted 3-methyl-pyridine-2-carbonyl amino dimethyl-benzoic acid EP4 antagonists

Article abstract of DOI:10.1016/j.bmcl.2016.03.041

Continued SAR optimization of a series of 3-methylpyridine-2-carbonyl amino-2,4-dimethyl-benzoic acid led to the selection of compound 4f for clinical studies. Compound 4f showed an IC₅₀ of 123 nM for inhibition of PGE2-induced TNFα reduction i

Full text of DOI:10.1016/j.bmcl.2016.03.041

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and biological activity of 6-alkyl-substituted 3-methyl-
pyridine-2-carbonyl amino dimethyl-benzoic acid EP4 antagonists
⇑
Maria-Jesus Blanco , Tatiana Vetman, Srinivasan Chandrasekhar, Matthew J. Fisher, Anita Harvey,
Steven L. Kuklish, Mark Chambers, Chaohua Lin, Daniel Mudra, Jennifer Oskins, Xu-Shan Wang,
Xiao-Peng Yu, Alan M. Warshawsky
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Continued SAR optimization of a series of 3-methylpyridine-2-carbonyl amino-2,4-dimethyl-benzoic acid
Received 26 January 2016
Revised 10 March 2016
Accepted 11 March 2016
Available online xxxx
led to the selection of compound 4f for clinical studies. Compound 4f showed an IC50 of 123 nM for inhi-
bition of PGE2-induced TNFa reduction in an ex vivo LPS-stimulated human whole blood assay (showing
>10-fold increase over clinical compound CJ-023,423). Pharmacokinetic profile, selectivity and in vivo
efficacy comparing 4f to NSAID diclofenac in the monoiodoacetic acid (MIA) pain model and adjuvant
induced arthritis (AIA) inflammatory model are included.
Keywords:
Ó 2016 Elsevier Ltd. All rights reserved.
Prostaglandin E2 (PGE2)
EP4 antagonist
Clinical candidate
Pain
Inflammation
An increasing amount of preclinical data supports the potential
therapeutic value of prostaglandin E receptor 4 (EP4) antagonists
across several indications.1 Most scientific evidence suggests that
a selective EP4 antagonist could be an effective inflammatory pain
relieving drug with GI tolerability2 better than NSAIDs and COX-2
inhibitors, the current standard of care. Importantly, a safer cardio-
vascular safety profile could be envisioned with EP4 antagonists
since they do not interfere directly with the biosynthesis of prosta-
glandin E (PGE2) and other prostanoids, such as prostacyclin and
thromboxane. EP4 receptor antagonists may also have therapeutic
application in the treatment of migraine3,4 since EP4 receptors are
involved in PGE2-mediated cerebral vascular dilatation, an impor-
tant factor in migraine pain. Overexpression of COX2 in various
tumor types leads to increased levels of PGE2 suggesting that
blocking PGE2 signaling with a selective EP4 antagonist during
cancer therapies may have benefits for patients in the oncology
setting.1 The EP4 receptor has also been reported to play a signifi-
cant role in a number of neurodegenerative5 diseases, such as mul-
tiple sclerosis and Alzheimer’s disease where PGE2 is involved.
CJ-023,4236,7 was first reported in 20028 as a potent and selec-
tive EP4 antagonist for the treatment of pain and inflammation,
arthritis, and oncology (Fig. 1). It was the first selective EP4 recep-
tor antagonist evaluated in clinical studies for the treatment of
N
N
O
O
N
S
N
N
H
O
H
1
Figure 1. Clinical compound CJ-023,423.
osteoarthritis. This compound achieved a positive proof-of-concept
in chronic inflammatory pain2 (OA pain). More recently, CJ-
023,423 (by the name of grapiprant9) has been reported to be in
development for the control of OA pain and inflammation in com-
panion animals. Grapiprant has demonstrated a good safety profile
and efficacy in canines.10
Due to the interest in modulating the biological effects of PGE2,
multiple companies have reported medicinal chemistry efforts in
this field. We have previously described our studies toward the
discovery of selective EP4 antagonists with acceptable potency
and pharmacokinetic (PK) profile for evaluating preclinical
in vivo efficacy. During the course of our research, initial hits were
modified to improve potency and overall properties by (1) intro-
ducing a carboxylic acid group onto the dimethylbenzamide moi-
ety11; and by (2) modifying the central bicyclic aromatic ring to a
3-methyl-pyridine ring12 (Fig. 2, Table 1). In this Letter, we disclose
⇑
Corresponding author. Tel.: +1 317 433 3612.
E-mail address: blanco_maria@lilly.com (M.-J. Blanco).
http://dx.doi.org/10.1016/j.bmcl.2016.03.041
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Blanco, M.-J.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.041

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.
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M.-J. Blanco et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 2
In vitro data and calculated properties
O
OH
O
OH
O
HO
NH
NH
NH
N
R
N
N
R
O
OH
O
O
2
3a-c
4a-f
b
Compd
R
hEP4 functional
cAMP IC50 (nM)
hWB IC50
(nM)
MW
F sp3
cLogDc
LEANd
hEP4 cAMP
HTSA solubilitye
(mg/mL)
a
1
2
11.7 + 8 (6)
2.36 1.4 (5)
1520 + 1030 (82)
38.7 17.1 (9)
492
390
0.27
0.17
3.08
1.55
0.23
0.30
0.977
0.242
OH
N
3a
14.2 (1)
2890 2830 (3)
398
0.4
0.81
0.27
—
N
3b
87.9 4.2 (2)
—
398
0.41
0.59
0.24
—
OH
N
3c
19.5 8.8 (2)
1450 880 (3)
383
0.38
0.31
0.28
0.784
OH
OH
N
4a
4b
16.5 1.3 (2)
84 21 (2)
1450 407 (2)
—
398
383
0.4
0.89
0.45
0.27
0.25
—
—
N
0.38
OH
N
N
N
4c
4d
4e
1.61 0.6 (4)
39.8 36 (4)
1160 723 (3)
3600 765 (3)
403
398
0.38
0.41
2.14
0.67
0.30
0.25
—
F
F
0.817
OH
O
96.7 36.6 (2)
5.62 2.8 (8)
—
398
383
0.41
0.38
1.03
0.39
0.24
0.30
—
N
4f
123 81 (8)
0.762
OH
a
Data is expressed as the geometric mean standard deviation; (n) = number of independent determinations. The standard deviation is calculated by the delta method,
being SDlogIC50 ꢀ geometric mean ꢀ ln(10). For additional details on the assay please see Ref. 25.
b
Data is expressed as the geometric mean standard deviation; (n) = number of independent determinations. The standard deviation is calculated by the delta method,
being SDlogIC50 ꢀ geometric mean ꢀ ln(10). For additional details on the assay please see Ref. 18.
c
cLogD at pH = 7.4, calculated using Marvin and calculator plugin freeware (www.chemaxon.com, ChemAxon Kft, Budapest, Hungary).
d
LEAN is a metric for ligand efficiency defined as ꢁlog(IC50)/number of non-hydrogen atoms.
e
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
Table 3
PK profiling for compound 4f
Species
iv/po dose (mg/kg)
po AUC (ng * h/mL)
po Cmax (ng/mL)
po T1/2 (h)
iv CL (mL/min/kg)
Vdss (L/Kg)
F (%)
Rat
Dog
1/5
1/5
9670
6320
3100
1870
5.1
5.1
3
10
0.34
1.73
34
75
mechanisms and compounds including those that target prosta-
glandin pathways such as NSAIDs. Efficacy is measured as the abil-
ity of a test compound to inhibit paw swelling.
In the experiment shown in Figure 4, rats had their paw widths
measured and were then inoculated with 0.25 mg of adjuvant
intradermal at the base of the tail to induce adjuvant disease
(day 0). Eleven days post inoculation paw width of both right
and left paws was measured again and the percent change from
the day 0 reading was calculated. This measurement along with
the body weight of the rats was used to randomize the animals.
Rats were dosed with vehicle and compound 4f at doses of 1, 3
(Mycobacterium tuberculosis H37 RA) in 100 ll of mineral oil
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4
M.-J. Blanco et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 4
Selectivity profile for 4fa
hEP4 FB binding Ki (nM)
hEP1 binding Ki (nM)
hEP2 binding Ki (nM)
hEP3 binding Ki (nM)
hEP4 cAMP rel IC50 (nM)
5.6 2.8 (8)
rEP4 cAMP rel IC50 (nM)
12
58 39 (10)
>17500 (2)
>18900 (4)
>14000 (4)
a
Data is expressed as the geometric mean standard deviation; (n) = number of independent determinations. The standard deviation is calculated by the delta method,
being SDlogIC50 ꢀ geometric mean ꢀ ln(10).
25
Day 12 post-MIA injecꢀon
20
*
*
15
*
10
5
0
Vehicle compd 4f compd 4f compd 4f compd 4f diclofenac
0.3 mg/kg 1.0 mg/kg 3.0 mg/kg
10.0
mg/kg
5.0 mg/kg
Figure 3. Dose response study with compound 4f in the MIA model with pain
measured 30 min post dose The data are presented as mean SEM where group size
is n = 5. Statistical comparison to vehicle: Dunnett’s test (*p <0.05). Vehicle is 10%
Acacia in water plus 0.05% antifoam.
Figure 5. X-ray structure of compound 4f.
significantly inhibited increases in paw inflammation (swelling)
compared to vehicle at a dose of 30 mg/kg as did the NSAID
diclofenac.
40
% increase in paw swelling
Compound 4f was found to have no inhibitory activity up to
10 lM in vitro against several CYP enzymes (Table 5). Therefore,
35
30
25
20
15
the risk of drug–drug interactions with 4f was expected to be
low. Compound 4f was also evaluated at Cerep for broader selectiv-
ity against a panel of 13 receptors, four ion channels, one trans-
porter, and one enzyme, and showed no significant activity on
any of the targets at 10
l
M.24 Finally 4f had no activity up to
100
l
M against hERG in a [3H]-astemizole binding assay.
*
*
The X-ray structure of 4f was determined (Fig. 5). The amide
carbonyl oxygen is anti to the pyridine nitrogen. The slight twist
of the piperidine ring and nearly ninety degree twist of dimethyl
phenyl ring out of plane of the pyridine carboxamide core con-
tributed to the rich topology of this compound and potentially con-
tributor to improved physicochemical profile.
10
5
0
Vehicle
diclofenac compd 4f
10mg/kg
compd 4f
10mg/kg
compd 4f
30mg/kg
3mg/kg
The synthesis of compound 4f is described in Scheme 1. Hydrol-
ysis of methyl 6-chloro-3-methyl-pyridine-2-carboxylate 5 using
KOH led to carboxylic acid 6. Amide coupling of 6 with methyl 4-
amino-3,5-dimethyl-benzoate (8) was performed as previously
reported.11,12 4-Hydroxy-piperidine was combined with 7 under
microwave irradiation to give the ester derivative of 4f. Hydrolysis
under basic conditions afforded 4f in good yield. The analogs
shown in Table 2 were synthesized in a similar manner.25
Figure 4. Dose response study with compound 4f in the AIA model to assess
inhibition of increase in paw swelling. The data are presented as mean SEM where
group size is n = 8. Statistical comparison to vehicle: Dunnett’s test (*p <0.05).
Vehicle is 10% Acacia in water plus 0.05% antifoam.
Table 5
Additional profiling data for 4f
In conclusion, directed SAR optimization of 6-aryl-substituted-3-
methyl-pyridine-2-carbonyl-amino-2,4-dimethyl-benzoic acid (2)
to increase sp3 character led to 6-alkyl-substituted analog 4f. Com-
pound 4f displayed an IC50 of 123 nM for inhibition of PGE2-induced
Assay
Results
CYP1A2, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CYP2D6,
CYP3A4 inhibition
CEREP mini-panel (with hERG) and PG ala carte (DP1, FP, No significant
IP, TP, COX-1/2) activity
IC50 > 10
all
lM for
TNFa reduction in an ex vivo LPS-stimulated human whole blood
assay, a >10-fold increase in potency over clinical compound CJ-
023,423 in the same assay. Furthermore, compound 4f showed a
PK profile compatible with an oral administration, and robust
in vivo efficacy compared to NSAID diclofenac in the MIA model to
assess pain relief and in the AIA model to assess reduction of inflam-
mation. In vitro selectivity assessment indicated that 4f was highly
selective for EP4 versus other EP receptors as well as a broader panel
of targets. These observations contributed to the selection of 4f as a
clinical candidate.
and 10 mg/kg. The nonsteroidal anti-inflammatory drug (NSAID)
diclofenac was included at a dose of 10 mg/kg as a positive control.
On day 15 post adjuvant injection the rats were euthanized with-
out further dosing and paw width measured again. The percent
increase in mean paw thickness from day 11 to day 15 was then
calculated as a measure of paw swelling which is indicative
of the amount of inflammation in the paws. Compound 4f
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M.-J. Blanco et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
O
O
O
O
O
NH2
O
MeO
O
OH
N
Cl
(a)
(c, d)
HO
O
N
Cl
8
O
–O
K +
N
Cl
N
H
N
N
N
H
(b)
5
6
7
4f
Scheme 1. Synthetic route to compound 4f. Reagents and conditions: (a) KOH, IPA 85%; (b) BOP-Cl, DIEA, DMF, room temperature, 16 h, 78%; (c) NMP, 4-hydroxypiperidine,
150 °C, 4 h, 65%; (d) 1 N NaOH, 1:1 THF/MeOH, 77%.
12. Blanco, M. J.; Vetman, T.; Chandrasekhar, S.; Fisher, M. J.; Harvey, A.; Chambers,
Acknowledgements
M.; Lin, C.; Mudra, D.; Oskins, J.; Wang, X. S.; Yu, X. P.; Warshawsky, A. M.
Bioorg. Med. Chem. Lett. 2016, 26, 931.
13. Leeson, P. D.; Springthorpe, B. Nat Rev Drug Disc. 2007, 6, 881.
14. Desai, P. V.; Raub, T. J.; Blanco, M. J. Bioorg. Med. Chem. Lett. 2012, 22, 6540.
15. Ritchie, T. J.; MacDonald, S. J. F. J. Med. Chem. 2014, 57, 7206.
16. Ritchie, T. J.; MacDonald, S. J. F. Drug Discovery Today 2009, 14, 1011.
17. Lovering, F.; Bikker, J.; Humblet, C. J. Med. Chem. 2009, 52, 6752.
We would like to thank Jaclyn Barrett (Eli Lilly) for analytical
support and solubility data. We gratefully acknowledge the contri-
butions by Benjamin A Diseroad and Gregory A Stephenson (Eli
Lilly) to obtain a crystal structure of compound 4f.
18. Blood is collected from normal volunteer donors into sodium heparin
vacutainer tubes. Donors have not taken NSAIDs or celecoxib within 48 h or
glucocorticoids within 2 weeks of the donation. AU tubes/donor are pooled
into 50 mL Falcon conical centrifuge tubes and 98 pL/well is distributed into
96-well tissue culture plates (Falcon 3072). Compounds are diluted into DMSO
to 100ꢀ final and I pL/well in triplicate is added to the blood to give 7 point
concentration response curves. The blood is pretreated with the compounds at
37 °C, in a 5% CO2 humidified atmosphere, for 30 min, after which I pL/well of a
solution of I mg/mL of lipopolysaccharide (LPS) (Sigma 0111:B4) in 0.2 mg/mL
bovine serum albumin (BSA)/PBS+/ꢁ1 mM PGE2 (Cayman 14010) is added to
give a final LPS concentration of 10 pg/mL+/ꢁ10 nM PGE2. The plates are
incubated for 20–24 h at37 °C in a 5% CO2 humidified atmosphere. The plates
are centrifuged at 1800ꢀg, 10 min at 22 °C, in an Eppendorf 5810R centrifuge.
Plasma is removed from the cell layer and is transferred to v-bottom
Supplementary data
The crystal structure of compound 4f has been deposited at the
Cambridge Crystallographic Data Centre with a deposition number
CCDC 1459066. Supplementary data (CEREP panel data) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2016.03.041.
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24. See Supplemental data.
25. Synthesis protocols, compound characterization, and assay protocols have
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Please cite this article in press as: Blanco, M.-J.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.041