Identification of a Selective, Non-Prostanoid EP2 Receptor Agonist for the Treatment of Glaucoma: Omidenepag and its Prodrug Omidenepag Isopropyl

Article abstract of DOI:10.1021/acs.jmedchem.8b00808

EP2 receptor agonists are expected to be effective ocular hypotensive agents; however, it has been suggested that agonism to other EP receptor subtypes may lead to undesirable effects. Through medicinal chemistry efforts, we identified a scaffold bearing a (pyridin-2-ylamino)acetic acid moiety as a promising EP2-selective receptor agonist. (6-((4-(Pyrazol-1-yl)benzyl)(pyridin-3-ylsulfonyl)aminomethyl)pyridin-2-ylamino)acetic acid 13ax (omidenepag, OMD) exerted potent and selective activity toward the human EP2 receptor (h-EP2). Low doses of omidenepag isopropyl (OMDI), a prodrug of 13ax, lowered intraocular pressure (IOP) in ocular normotensive monkeys. OMDI was selected as a clinical candidate for the treatment of glaucoma.

Full text of DOI:10.1021/acs.jmedchem.8b00808

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Article
Identification of a Selective, Non-Prostanoid EP2 Receptor Agonist for the
Treatment of Glaucoma: Omidenepag and its Prodrug Omidenepag Isopropyl
RYO IWAMURA, MASAYUKI TANAKA, EIJI OKANARI, TOMOKO KIRIHARA,
NORIKO ODANI-KAWABATA, NAVEED SHAMS, and KENJI YONEDA
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00808 • Publication Date (Web): 11 Jul 2018
Downloaded from http://pubs.acs.org on July 12, 2018
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Identification of a Selective, NonꢀProstanoid EP2
Receptor Agonist for the Treatment of Glaucoma:
Omidenepag and its Prodrug Omidenepag Isopropyl
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*,†
†
†
‡
‡
Ryo Iwamura, Masayuki Tanaka, Eiji Okanari, Tomoko Kirihara , Noriko OdaniꢀKawabata,
‡
,§
†
Naveed Shams, and Kenji Yoneda
†
Pharmaceuticals Research Laboratory, UBE Industries, Ltd., 1978ꢀ5 Kogushi, Ube, Yamaguchi
7
55ꢀ8633, Japan
‡
R&D Division, Santen Pharmaceutical Co., Ltd., Grand Front Osaka Tower A 4ꢀ20, Ofukacho,
Kitaꢀku, Osaka 530ꢀ8552, Japan
§
R&D Division, Santen Inc., 6401 Hollis Street, Suite 125 Emeryville, California 94608, United
States
ABSTRACT: EP2 receptor agonists are expected to be effective ocular hypotensive agents;
however, it has been suggested that agonism to other EP receptor subtypes may lead to
undesirable effects. Through medicinal chemistry efforts, we identified a scaffold bearing a
(pyridinꢀ2ꢀylamino)acetic acid moiety as a promising EP2ꢀselective receptor agonist. (6ꢀ((4ꢀ
(Pyrazolꢀ1ꢀyl)benzyl)(pyridinꢀ3ꢀylsulfonyl)aminomethyl)pyridinꢀ2ꢀylamino)acetic acid 13ax
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(
omidenepag, OMD) exerted potent and selective activity toward the human EP2 receptor (hꢀ
EP2). Low doses of omidenepag isopropyl (OMDI), a prodrug of 13ax, lowered intraocular
pressure (IOP) in ocular normotensive monkeys. OMDI was selected as a clinical candidate for
the treatment of glaucoma.
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■
INTRODUCTION
Prostaglandin E (PGE ) is a highly bioactive molecule and is associated with various
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biological responses, including inflammation, osteoclastic bone resorption, labor, smooth muscle
1
–3
relaxation, and intraocular pressure (IOP) reduction,
mediated by signaling via four EP
4,5
receptor subtypes (EP1, EP2, EP3, and EP4). Prostaglandin analogs, such as latanoprost, and
their active acid forms are selective FP receptor (prostaglandin F receptor) agonists and one of
2
α
the most potent IOPꢀlowering agents for the treatment of glaucoma. However, IOPꢀlowering
agents with a different, new mechanism of action (MOA) are desired to be developed because
6,7
concomitant treatment can further reduce IOP and be effective in low/nonꢀresponders to FP
8
receptor agonists or when local adverse reactions associated with FP receptor agonists, such as
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iris/eyelid pigmentation and the deepening of upper eyelid sulcus, affect compliance. Ocular
hypotensive agents with new, different MOA from that of FP receptor agonists are expected to
overcome problems associated with FP receptor agonists as stated above. EP2 receptor agonists,
such as butaprost (in its free acid form), have been reported to be selective for the EP2 receptor
10,11
among EP receptor subtypes
and are reported to lower IOP; therefore, they are expected to be
1
2,13
beneficial for the treatment of glaucoma
with better safety profiles than nonꢀselective EP
receptor agonists and different safety profiles from FP receptor agonists. Thus, we explored EP2
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receptor agonists with potent activity and high selectivity to identify a safe compound that is
capable of lowering IOP for the treatment of glaucoma.
14–16
CPꢀ533,536
was selected as the starting compound for the medicinal chemistry efforts
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because: (1) it has been reported as a selective EP2 receptor agonist,
(2) it was expected to be safe, since a clinical trial studying its effectiveness for
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fracture healing was under way, and
(
3) it was presumed that various structural changes could be more accessible than
prostanoid since it has a nonꢀprostanoid structure (Figure 1).
Figure 1. Structures of prostanoids and CPꢀ533,536.
Since CPꢀ533,536 contains a sulfonamide group in the center of the molecule, the structures of
the phenoxyacetic acid moiety, the pyridinꢀ3ꢀylsulfonyl moiety, and the tertꢀbutylphenyl moiety
can be independently changed. Retrosynthetic analysis indicated that the burden of structural
change of the phenoxyacetic acid moiety is greater than that of the pyridinꢀ3ꢀylsulfonyl and tertꢀ
butylphenyl moieties (Figure 2). However, the following was considered in terms of activities: In
the structural comparison between CPꢀ533,536 and butaprost, the phenoxyacetic acid and tertꢀ
butylphenyl moieties of CPꢀ533,536 were estimated to correspond to the α and ω chain moieties,
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respectively, of prostaglandins (Figure 1). Because the ω chain moiety of prostaglandins has
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been reported to be derivatized to various structures,
the tertꢀbutylphenyl moiety of CPꢀ
533,536 was also predicted to be transformable into various structures. However, we
hypothesized that the transformation of the phenoxyacetic acid moiety is likely to exert a large
effect on in vitro activity because it is a critical substructure containing a carboxy group
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4,25
potentially involved in enthalpyꢀdriven binding.
On the basis of these presumptions, our medicinal chemistry strategy involved the following
two steps (Figure 2):
STEP 1 Explore chemotypes with activities higher than that of the starting compound by
transforming the phenoxyacetic acid moiety of CPꢀ533,536 into a novel
substructure.
STEP 2 Transform the pyridinꢀ3ꢀylsulfonyl and tertꢀbutylphenyl moieties using
chemotype(s) containing a highly active novel substructure(s) identified in STEP
1
, and optimize the structure.
The attainment of STEP 1 objective was a key to this strategy to produce novel compounds with
high activities and enabled the tertꢀbutylphenyl moiety to be transformed into a wide range of
structures. Herein, we describe the synthesis and detailed structure−activity relationship (SAR)
leading to the discovery of omidenepag (OMD, 13ax) and omidenepag isopropyl (OMDI).
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Figure 2. Medicinal chemistry strategy adopted in this study and retrosynthetic analysis.
■ RESULTS AND DISCUSSION
Chemistry. For exploring the phenoxyacetic acid moiety (STEP 1), the synthesis was basically
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conducted through the route shown in Scheme 1. By reaction of compound 1a as the common
intermediate with the building block of the phenoxyacetic acid moiety corresponding to each
compound and inducing the phenoxyacetic acid moiety as appropriate, target compounds were
obtained. Specific routes of synthesis for each compound are shown in Scheme 2–11.
Scheme 1. General Synthetic Route to Explore the Phenoxyacetic Acid Moiety (STEP 1)
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The benzofuryl derivatives 5 and 9 were obtained through the synthetic routes shown in
Scheme 2. Ethyl 6ꢀmethylbenzofuranꢀ2ꢀcarboxylate (2) and ethyl 4ꢀmethylbenzofuranꢀ2ꢀ
carboxylate (6) were converted to compounds 3 and 7, respectively, through the bromination of
the benzylic methyl group by a photoꢀinduced radical reaction (step a); compounds 3 and 7 were
converted to precursor compounds 4 and 8, respectively, by reaction with compound 1a under
basic conditions (step b); and compounds 4 and 8 were converted to compounds 5 and 9,
respectively, through basic hydrolysis (step c).
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Scheme 2. Synthetic Routes for Benzofuryl Derivatives 5 and 9
CO Et
CO Et
CO Et
CO H
2
2
2
2
O
O
O
O
a
b
c
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N
Br
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O
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N
N
1a
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O
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O
CO Et
CO Et
CO Et
CO H
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2
a
b
c
HN
S
Br
N
S
O
N
S
O
O
N
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N
N
1a
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a
Reagents and conditions: (a) NBS, hν, 1,2ꢀdichloroethane, 0.5–1 h, 92% for 3 and 86% for 7;
b) K CO , DMF, rt, 5 h to overnight, 83% for 4 and 84% for 8; (c) NaOH, EtOH/H O/THF for 5
(
2
3
2
and EtOH/H O for 9, rt, 3 h, 58% for 5 and 62% for 9.
2
Compound 13aa, one of the aminopyridyl derivatives, was obtained through the synthetic
26
26–28
route shown in Scheme 3. Compound 10
was converted by reaction with compound 1a
under basic conditions to compound 11 (step a), which was then converted to the precursor
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compound 12aa by hydrogenolysis (step b). The precursor compound 12aa was converted to
aminopyridyl derivative 13aa by acidic deprotection (step c).
a
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Scheme 3. Synthetic Route for Aminopyridyl Derivative 13aa
a
Reagents and conditions: (a) K CO , DMF, rt, 3.5 h, 85%; (b) PdꢀC, H , Et N, EtOH, rt, 1 h,
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31%; (c) HCl, CH Cl /1,4ꢀdioxane, rt, 16 h, quantitative yield.
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Compound 19, a regioisomer of aminopyridyl derivative 13aa, was obtained through the
synthetic route shown in Scheme 4. Compound 15, which was obtained by protecting the amino
group of compound 14 with a tertꢀbutoxycarbonyl (Boc) group (step a), was converted by
reaction with tertꢀbutyl 2ꢀbromoacetate under basic conditions to compound 16 (step b). After
the deprotection of the tertꢀbutyldimethylsilyl (TBDMS) group (step c), compound 17 was
obtained. Compound 17 was converted by reaction with compound 1a under Mitsunobu reaction
conditions to the precursor compound 18 (step d), which was then converted to compound 19 by
acidic deprotection (step e).
a
Scheme 4. Synthetic Route for Compound 19
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Boc
NH
Boc
N
Boc
Boc
N
H
N
N
NH2
N
N
CO2t-Bu
N
N
CO2t-Bu
N
CO2t-Bu
N
CO2H
a
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d
e
HCl salt
OTBDMS
OTBDMS
OTBDMS
OH
17
HN
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Reagents and conditions: (a) Boc O, pyridine/tꢀBuOH/AcOEt, rt, overnight, 62%; (b) (i) 15,
NaH, DMF, rt → 0 °C, 20 min, (ii) BrCH CO tꢀBu, 0 °C → rt, 6 h, 87%; (c) TBAF, THF, 0 °C
rt, 4.5 h, 96%; (d) TMAD, P(nꢀBu) , THF, rt, overnight, 78%; (e) HCl, CH Cl /1,4ꢀdioxane,
rt, overnight, quantitative yield.
2
2
2
→
3 2 2
Compound 25, the other regioisomer of aminopyridyl derivative 13aa, was obtained through
the synthetic route shown in Scheme 5. (4ꢀNitropyridinꢀ2ꢀyl)methanol (20) was converted by
reaction with compound 1a under Mitsunobu reaction conditions to compound 21 (step a). After
the subsequent reduction of the nitro group (step b) and the substitution of the amino group with
a Boc group and then with an acetic acid tertꢀbutyl ester under basic conditions (steps c and d),
the precursor compound 24 was obtained, which was converted to compound 25 by acidic
deprotection (step e).
a
Scheme 5. Synthetic Route for Compound 25
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Reagents and conditions: (a) TMAD, P(nꢀBu) , THF, rt, 4 h, 94%; (b) Zn dust, CH CO H, 60
3
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°
C, 1 h 20 min, 98%; (c) (i) 22, NaHMDS, THF, 0 °C, 10 min, (ii) Boc O, 0 °C, 1 h 20 min,
2
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2
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HCl, CH Cl /1,4ꢀdioxane, rt, overnight, 88%.
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2
Compound 28, with an NꢀmethylꢀNꢀ(pyridinꢀ2ꢀyl)aminoacetic acid substructure corresponding
to the compound obtained by methylating the amino nitrogen atom in the aminopyridyl moiety of
aminopyridyl derivative 13aa, was obtained through the synthetic route shown in Scheme 6.
Compound 12aa (the precursor compound of aminopyridyl derivative 13aa), after the
deprotection of the Boc group at the amino nitrogen atom by reaction with paraꢀtoluenesulfonic
acid monohydrate (PTSA•H O) in ethanol, formed compound 26 (step a), which was then
2
methylated at the amino nitrogen atom by reaction with methyl iodide under basic conditions to
give compound 27 (step b), which was converted to compound 28 by acidic deprotection (step
c).
a
Scheme 6. Synthetic Route for Compound 28
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Reagents and conditions: (a) PTSA•H O, EtOH, 60 °C → 65 °C, 16.5 h, 82%; (b) (i) 26, NaH,
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DMF, 0 °C → rt, 15 min, (ii) MeI, 0 °C → rt, 1 h, 32%; (c) HCl, CH Cl /1,4ꢀdioxane, rt,
overnight, quantitative yield.
2
2
The indolyl derivative 33 with a (1Hꢀindolꢀ1ꢀyl)acetic acid substructure was obtained through
the synthetic route shown in Scheme 7. 1Hꢀindoleꢀ6ꢀcarbaldehyde (29) was converted by
reaction with tertꢀbutyl 2ꢀbromoacetate under basic conditions to compound 30 (step a), which
was then reduced at the aldehyde group by reaction with NaBH to give compound 31 (step b).
4
Compound 31 was converted to the indolyl derivative 33 (steps c and d) according to the
procedures similar to those of steps d and e in Scheme 4.
a
Scheme 7. Synthetic Route for Indolyl Derivative 33
a
Reagents and conditions: (a) (i) 29, NaH, DMF, 0 °C, (ii) BrCH CO tꢀBu, 0 °C → rt, overnight,
2
2
9
1%; (b) NaBH , EtOH, rt, 1 h, 85%; (c) TMAD, P(nꢀBu) , THF, rt, overnight, 78%; (d) HCl,
4
3
CH Cl /1,4ꢀdioxane, rt, overnight, 9.9%.
2
2
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9
Compound 41, with a 3ꢀ(pyridinꢀ2ꢀylamino)propionic acid substructure having one more
carbon atom in its carbon chain than the aminopyridyl derivative 13aa, was obtained through the
synthetic route shown in Scheme 8. The amino nitrogen of 6ꢀmethylpyridinꢀ2ꢀamine (34) was
substituted with a propionic acid tertꢀbutyl ester and then with a Boc group to give compound 36
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(steps a and b). Compound 36 was oxidized at the pyridyl nitrogen atom by reaction with metaꢀ
chloroperoxybenzoic acid (mꢀCPBA) to give compound 37 (step c), which was converted to
compound 38 through a rearrangement reaction (step d) and then converted to compound 39
through the hydrolysis of the acetoxy group (step e). Compound 39 was converted to compound
4
1 (steps f and g) according to the procedures similar to those of steps d and e in Scheme 4.
a
Scheme 8. Synthetic Route for Compound 41
a
Reagents and conditions: (a) CH =CHCO tꢀBu, 2,5ꢀdiꢀtertꢀbutylbenzeneꢀ1,4ꢀdiol, 100 °C, 2 d,
2
2
1
1%; (b) Boc O, DMAP, N,Nꢀdiisopropylethylamine, tꢀBuOH, rt, overnight, 50%; (c) mꢀCPBA,
2
CH Cl , rt, 4.5 h, quantitative yield; (d) Ac O, 110 °C, 1 h, 66%; (e) LiOH•H O, THF/H O, 60
2
2
2
2
2
°C, 13.5 h, 85%; (f) TMAD, P(nꢀBu) , THF, rt, 2 h, 86%; (g) HCl, CH Cl /1,4ꢀdioxane, rt,
3
2
2
overnight, 75%.
Compound 47 was obtained according to the procedure similar to that for compound 19
described in Scheme 4.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 9. Synthetic Route for Compound 47
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) TBDMSCl, imidazole, DMF, rt, overnight, 99%; (b) BrCH CO tꢀ
Bu, K CO , DMF, rt, 1 h, 86%; (c) TBAF, THF, 0 °C → rt, 4 h, 96%; (d) TMAD, P(nꢀBu) ,
THF, rt, 2 h, 81%; (e) HCl, CH Cl /1,4ꢀdioxane, rt, overnight, 90%.
2
2
2
3
3
2
2
For the exploration of the pyridinꢀ3ꢀylsulfonyl and tertꢀbutylphenyl moieties, the synthesis of
aminopyridyl derivatives represented by general formula 13 were efficiently conducted through
the routes shown in Scheme 10 and 11 using key intermediates, such as tertꢀbutyl (tertꢀ
butoxycarbonyl(6ꢀ(hydroxymethyl)pyridinꢀ2ꢀyl)amino)acetate
(48),
tertꢀbutyl
(tertꢀ
butoxycarbonyl(6ꢀ((4ꢀtertꢀbutylbenzyl)aminomethyl)pyridinꢀ2ꢀyl)amino)acetate (49), and tertꢀ
butyl
(tertꢀbutoxycarbonyl(6ꢀ((pyridinꢀ3ꢀylsulfonyl)aminomethyl)pyridinꢀ2ꢀyl)amino)acetate
Precursor compounds 12 obtained through synthetic steps a, b, or c were converted to
2
6–28
(51).
aminopyridyl derivatives 13 by acidic deprotection (step d). Compound 48 was converted to
precursor compound 12 by reaction with compound 1 under Mitsunobu reaction conditions (step
a). Compound 49 was converted to precursor compound 12 by reaction with sulfonyl chloride 50
under basic conditions (step b). Compound 51 was converted to precursor compound 12 by
reaction with compound 52 under basic conditions or with compound 53 under Mitsunobu
reaction conditions (step c). The synthetic steps to produce each aminopyridyl derivative are
shown in Table 1.
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a
Scheme 10. General Synthetic Routes for Aminopyridyl Derivatives 13
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) TMAD, P(nꢀBu) , THF, rt; (b) Et N, CH Cl rt; (c with 52) (i) 51,
3
3
2
2,
NaH, DMF, 0 °C → rt, (ii) 52, 0 °C → rt; (c with 53) TMAD, P(nꢀBu) , THF, rt; (d) HCl,
3
CH Cl /1,4ꢀdioxane, rt.
2
2
Table 1. Synthetic Routes for Aminopyridyl Derivatives 13ab–ba
synthetic
route
1
2
compound
R
R
form
a
13ab
13ac
13ad
13ae
methyl
phenyl
4ꢀtertꢀbutylphenyl
4ꢀtertꢀbutylphenyl
4ꢀtertꢀbutylphenyl
4ꢀtertꢀbutylphenyl
HCl salt
HCl salt
HCl salt
HCl salt
a, d
a, d
a, d
b, d
pyridinꢀ2ꢀyl
2ꢀfluorophenyl
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3af
3ag
3ah
2ꢀchlorophenyl
3ꢀfluorophenyl
3ꢀchlorophenyl
4ꢀfluorophenyl
4ꢀchlorophenyl
4ꢀtertꢀbutylphenyl
4ꢀtertꢀbutylphenyl
4ꢀtertꢀbutylphenyl
4ꢀtertꢀbutylphenyl
4ꢀtertꢀbutylphenyl
HCl salt
HCl salt
HCl salt
HCl salt
HCl salt
HCl salt
HCl salt
HCl salt
HCl salt
HCl salt
free form
b, d
b, d
b, d
b, d
b, d
b, d
a, d
a, d
c, d
a, d
c, d
c, d
c, d
a, d
a, d
c, d
c, d
c, d
c, d
c, d
a, d
c, d
1
1
1
1
3ai
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3aj
1
3ak
3al
3am
3an
4ꢀmethoxyphenyl 4ꢀtertꢀbutylphenyl
1
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
3ꢀtertꢀbutylphenyl
1
4ꢀtertꢀbutylcyclohexyl
1
4ꢀtrifluoromethylphenyl
4ꢀ(1ꢀmethylcyclopropyl)phenyl
4ꢀ(1ꢀethylcyclopropyl)phenyl
1
3ao
3ap
3aq
1
1
4ꢀ(1ꢀisopropylcyclopropyl)phenyl free form
1
1
3ar
4ꢀ(1ꢀethylcyclobutyl)phenyl
biphenylꢀ4ꢀyl
free form
HCl salt
HCl salt
free form
free form
HCl salt
free form
free form
HCl salt
HCl salt
3as
1
3at
3au
3av
3aw
3ax, OMD pyridinꢀ3ꢀyl
4ꢀphenylbutyl
1
4ꢀcyclohexylphenyl
4ꢀ(pyridinꢀ2ꢀyl)phenyl
4ꢀ(pyridazinꢀ4ꢀyl)phenyl
4ꢀ(pyrazolꢀ1ꢀyl)phenyl
4ꢀ(1,2,4ꢀtriazolꢀ1ꢀyl)phenyl
benzofuranꢀ2ꢀyl
1
1
1
13ay
13az
13ba
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ3ꢀyl
benzo[b]thiophenꢀ2ꢀyl
a
Synthetic route involves synthetic steps for each aminopyridyl derivative in Scheme 10.
Compounds 13bb and 13bc, belonging to aminopyridyl derivatives, were obtained through the
27,28
27,28
synthetic route shown in Scheme 11.
Compound 12a
was synthesized using compounds
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4
8 and 1, as shown in Scheme 10, under the conditions same to step a in Scheme 10. Compound
2a was converted to the precursor compounds 12bb and 12bc by reaction with appropriate
1
boronic acids under Suzuki–Miyaura reaction conditions (step a), which were then converted to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compounds 13bb and 13bc, respectively, by acidic deprotection (step b).
a
Scheme 11. Synthetic Route for Aminopyridyl Derivatives 13bb and 13bc
a
Reagents and conditions: (a) boronic acid, Pd(OAc) , tricyclohexylphosphine, K PO ,
2
3
4
toluene/H O, 100 °C, 94% for 12bb and 84% for 12bc; (b) HCl, CH Cl /1,4ꢀdioxane, rt, 89% for
1
2
2
2
3bb and 93% for 13bc.
Structure−Activity Relationship Study. Exploration of the Phenoxyacetic Acid Moiety.
We explored the phenoxyacetic acid moiety of CPꢀ533,536, STEP 1 of the medicinal chemistry
strategy described above, using a chemotype with a (4ꢀtertꢀbutylbenzyl)(pyridinꢀ3ꢀ
ylsulfonyl)amino moiety. Our initial design aimed at enhancing the in vitro activity of the
compound by fixing the conformation of the phenoxyacetic acid moiety. Statistical analysis
using the Cambridge Structural Database (CSD) suggested that the following characteristics are
29–32
associated with the conformation of the phenoxyacetic acid moiety
(Figure 3 and S2):
1
. The distribution of torsional angle 1 in the phenoxyacetic acid moiety has a clear
preference for approximately 0° or 180°. In contrast, the orthoꢀunsubstituted phenethyl
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
moiety has a different distribution, with a maximum at approximately 90° (Figure S1
in the Supporting Information), owing to steric constraints. These analysis results
2
suggest that the phenoxy oxygen atom has sp orbital characteristics and adopts a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
planar orientation of torsional angle 1 owning to resonance stabilization.
2
. The preferred conformations include one in which the phenoxy and carboxy groups are
contained in the same plane (planar geometry; torsional angle 1 approximately 0° or
1
80°, torsional angle 2 approximately 180°) and one in which the carboxy group
extrudes from the plane of the phenoxy group (skewed geometry; torsional angle 1
approximately 0° or 180°, torsional angle 2 approximately 90°); among these two, the
former conformation is preferred.
We hypothesized that the phenoxyacetic acid moiety bound to the hꢀEP2 receptor has the
conformation equivalent to either of the planar or skewed geometry; consequently, we designed
new compounds mimicking these two preferred conformers throughout the exploration of the
phenoxyacetic acid moiety.
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Figure 3. Preferred conformations of the phenoxyacetic acid moiety based on statistical analysis
using CSD. The torsional angle of C–O–CH –CO H (torsional angle 2) is plotted against that of
2
2
CH=C–O–CH (torsional angle 1). The two islands plotted in the upper graph region represent
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the conformers with planar geometry and those in the middle graph region represent the
conformers with skewed geometry.
We designed a structural mimetic of the planar geometry (the estimated most preferred
conformation of the phenoxyacetic acid moiety) by transforming the phenoxyacetic acid moiety
into benzofuranꢀ2ꢀcarboxylic acid, and fixed the conformation (Figure 4). The planar geometry
of the phenoxyacetic acid moiety includes two different conformations with a torsional angle 1
of approximately 0° or 180°, corresponding to two possible regioisomers (5 and 9) of the
benzofuranꢀ2ꢀcarboxylic acid moiety. The binding affinity of the regioisomer 9 for the hꢀEP2
receptor was comparable to that of CPꢀ533,536 (Table 2) and higher than that of the other
regioisomer 5. The binding affinity of 9 encouraged us to further optimize the benzofuryl
derivative, and the tertꢀbutylphenyl moiety of 9 was derivatized, resulting in more potent
compounds (data not shown). However, chemotypes containing benzofuranꢀ2ꢀcarboxylic acid
generally appeared to be highly crystalline with limited solubility in solvents, including water.
Therefore, we explored the phenoxyacetic acid moiety to identify chemotypes with enhanced in
vitro activities and without solubility limitations before advancing to STEP 2. The SAR of 5 and
9
suggested that the phenoxyacetic acid moiety of the bound conformer was equivalent to 9 in
terms of the geometry of torsional angle 1 and was applied to the subsequent medicinal
chemistry efforts.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Design of benzofuryl derivatives: structural mimetics of the planar geometry (the
estimated most preferred conformation of the phenoxyacetic acid moiety) and fixing the
conformation.
Table 2. Binding Affinity of CPꢀ533,536 and Benzofuryl Derivatives for hꢀEP2 Receptor
a
compound
K [nM]
i
CPꢀ533,536
16
433
15
5
9
a
11
The binding assay was carried out in compliance with the method of Abramovitz et al. The
50
3
IC value, the concentration of test compound required to replace 50% of the [ H]prostaglandin
E2 bound to hꢀEP2 receptor expressed on a membrane fraction of HEK293 cells (ESꢀ562ꢀM,
Euroscreen), was calculated on the basis of the results from two or four data points of a single
experiment, performed in duplicate, and the inhibition constant (K value) was determined.
i
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9
The limited solubility of chemotypes containing benzofuranꢀ2ꢀcarboxylic acid may be
attributed in part to their rigid bicyclic structures introduced to reduce rotatable bonds and fix
their conformation. Therefore, we tried to transform the phenoxyacetic acid moiety within the
scope of the monocyclic structures without increasing the rigidity of the molecule. To enhance in
vitro activity, we applied nitrogenꢀscan (Nꢀscan) and basicity enhancement strategies in which
we transformed the phenoxy group in the phenoxyacetic acid moiety into an aminopyridyl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3,34
group.
The phenyl group was transformed into a pyridyl group to yield different regioisomers
and the phenoxy oxygen atom was transformed into –NH– to enhance a basicity of a pyridyl
nitrogen atom; that is, the Nꢀscan was performed on the pyridyl nitrogen atom of compounds
3
5
having a pyridylaminoacetic acid substructure (Figure 5). 13aa, a compound with a (pyridinꢀ2ꢀ
ylamino)acetic acid substructure substituted at the 6ꢀposition of the pyridyl group, showed higher
hꢀEP2 receptor agonist activity than CPꢀ533,536, while 19 and 25 (regioisomers around the
pyridyl nitrogen atom) showed substantially lower in vitro activity, demonstrating that the
location of the pyridyl nitrogen atom is highly involved in hꢀEP2 receptor agonist activity (Table
3
).
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2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. (a) Design of the chemotypes with aminopyridyl substructures based on the Nꢀscan
and basicity enhancement strategies. The phenyl group was transformed into a pyridyl group for
the Nꢀscan and the phenoxy oxygen atom was transformed into –NH– to enhance a basicity of
the pyridyl nitrogen atom. (b) Comparison of the pK values between methoxypyridines and
a
3
5
aminopyridines in water at 25 °C. Replacement of the –OMe in each methoxypyridine with a –
NH enhances the basicity of the pyridyl nitrogen atom.
2
Table 3. In Vitro hꢀEP2 Receptor Agonist Activity of Compounds with Aminopyridyl,
Indolyl and Aminothiazolyl Substructures
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1
1
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1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
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4
4
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4
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4
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5
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5
5
5
5
6
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
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5
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7
8
9
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1
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
compound
EC [nM]
50
CPꢀ533,536
17
2.8
1
3aa
1
2
2
9
5
8
>1000
>1000
283
33
41
47
>1000
83
360
a
Measurement of in vitro hꢀEP2 receptor agonist activity was carried out in compliance with the
36
method of Wilson et al. The concentration of test compound required to increase the amount of
cAMP to 50% of the maximum increase (EC50 value) in HEK293 cells expressing hꢀEP2
receptor (ESꢀ562ꢀC, Euroscreen) was calculated on the basis of the results from at least seven
data points of a single experiment, performed in duplicate.
Statistical analysis using the CSD indicated that the following characteristics were associated
with the conformation of the (pyridinꢀ2ꢀylamino)acetic acid moiety (Figure 6 and S3):
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
1
. The amino nitrogen atom in the aminopyridyl moiety could also have sp orbital
characteristics, and could adopt a planar orientation of torsional angle 1, as with the
phenoxy oxygen atom.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
. The preferred conformations include one in which the aminopyridyl and carboxy
groups are contained in the same plane (planar geometry; torsional angle 1
approximately 0° or 180°, torsional angle 2 approximately 180°) and one in which the
carboxy group extrudes from the plane of the aminopyridyl group (skewed geometry;
torsional angle 1 approximately 0° or 180°, torsional angle 2 approximately 90°).
Although it exhibits a conformation similar to that seen in the phenoxyacetic acid moiety,
statistics obtained from the CSD indicated a difference in abundance between the planar and
skewed geometries; the former geometry is preferred in the phenoxyacetic acid, while the
abundance of the two geometries is comparable in the (pyridinꢀ2ꢀylamino)acetic acid. Because it
was speculated that the enhanced activity of 13aa resulted from the contribution of the
conformer with skewed geometry, we substituted the amino group in the aminopyridyl moiety
with a methyl group to increase the proportion of conformers with skewed geometry. The
substitution of the amino group with a methyl group leads to a preference for skewed geometry
due to pseudo 1,2ꢀstrain between the carboxy and methyl groups and pseudo 1,3ꢀstrain between
the carboxy group and the pyridyl nitrogen atom or CH (Figure 6). 28, a compound obtained by
substituting the amino group in the aminopyridyl moiety with a methyl group, was found to have
reduced hꢀEP2 receptor agonist activity. Furthermore, 33, a compound in which the 2ꢀ
(
methylamino)pyridyl group was replaced with an indolyl group to fix the conformation so that
the skewed geometry with the same torsional angle 1 to that in 9 is dominant, was found to have
further reduced hꢀEP2 receptor agonist activity (Table 3). On the basis of the above SAR and
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CSD analysis, the bound conformer was suggested to be in its planar geometry with regard to the
(pyridinꢀ2ꢀylamino)acetic acid moiety.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Preferred conformations of the (pyridinꢀ2ꢀylamino)acetic acid moiety based on the
statistical analysis using CSD. (a) Scatterplot for (pyridinꢀ2ꢀylamino)acetic acid, (NꢀmethylꢀNꢀ
(pyridinꢀ2ꢀyl)amino)acetic acid, and (NꢀmethylꢀNꢀphenylamino)acetic acid substructures in the
CSD. The analysis for a (NꢀmethylꢀNꢀphenylamino)acetic acid substructure was appended as
reference because of insufficient data for the (NꢀmethylꢀNꢀ(pyridinꢀ2ꢀyl)amino)acetic acid
substructure. The torsional angle of C–N(H or Me)–CH –C (torsional angle 2) is plotted against
2
that of CH=C–N(H or Me)–CH (torsional angle 1). The islands plotted in the upper graph
2
region represent the conformers with planar geometry and those in the middle graph region
represent the conformers with skewed geometry. (b) Pseudo 1,2ꢀstrain and pseudo 1,3ꢀstrain in
the Nꢀunsubstituted/substituted (pyridinꢀ2ꢀylamino)acetic acid moiety.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Further transformations were conducted at the (pyridinꢀ2ꢀylamino)acetic acid moiety of 13aa
to improve in vitro activity as follows: elongation of the acetic acid moiety to adjust the position
of the carboxy group and the isosteric replacement of the pyridyl group. However, 41 (with a
oneꢀcarbonꢀlonger alkyl spacer to adjust the location of the carboxy group) and 47 (containing
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(thiazolꢀ2ꢀylamino)acetic acid, an estimated isostere of (pyridinꢀ2ꢀylamino)acetic acid) were
found to have weaker hꢀEP2 receptor agonist activities than 13aa (Table 3).
These findings suggested that in terms of the exploration of the phenoxyacetic acid moiety of
CPꢀ533,536, the conformational preference for planar geometry (the putative bound conformer)
and the introduction of a nitrogen atom at a specific site would effectively enhance hꢀEP2
receptor agonist activity, and the chemotype with a (pyridinꢀ2ꢀylamino)acetic acid substructure
would be a particularly effective EP2 receptor agonist. The higher hꢀEP2 receptor agonist
activity of 13aa may be due to the physicochemical properties of the pyridyl nitrogen atom
(typically, the characteristics of a hydrogenꢀbonding acceptor) and also to the tendency that a 2ꢀ
2
aminopyridyl group has a high level of sp hybridization at the amino nitrogen atom (i.e., a high
level of planarity). We subsequently advanced to STEP 2 of the medicinal chemistry strategy and
sequentially transformed the structure of the pyridinꢀ3ꢀylsulfonyl and tertꢀbutylphenyl moieties
using the chemotype containing a (pyridinꢀ2ꢀylamino)acetic acid substructure to further enhance
in vitro activity.
Exploration of the pyridinꢀ3ꢀylsulfonyl moiety. We attempted to transform the pyridinꢀ3ꢀ
ylsulfonyl moiety to enhance in vitro hꢀEP2 receptor agonist activity using a chemotype with a
(6ꢀ((4ꢀtertꢀbutylbenzyl)aminomethyl)pyridinꢀ2ꢀylamino)acetic acid moiety (Table 4). While
1
13ac with the phenyl group and 13ad with the pyridinꢀ2ꢀyl group as R showed slightly reduced
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9
and equivalent agonist activities as compared with 13aa, respectively; 13ab with the methyl
1
group as R showed a reduced agonist activity as compared with 13aa. Thus, the transformation
1
of R was focused on the optionally substituted aromatic groups. Different substituents were
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
introduced into the phenylsulfonyl group of 13ac, and the steric environment was assessed. The
results showed that substitution at the orthoꢀ or metaꢀposition relative to the sulfonyl group with
F or Cl led to a reduction in agonist activity, suggesting a small steric environment around the
orthoꢀ and metaꢀpositions (13ae–ah). 13ai and 13aj, compounds in which the paraꢀposition
relative to the sulfonyl group was substituted with F and Cl, respectively, were found to be
stronger than and equipotent to 13ac, respectively, in terms of agonist activity, but substitution
with a methoxy group (a bulkier group) led to a reduction in agonist activity (13ak). Thus, the
steric environment was not estimated to be extensive around the paraꢀposition either. The
transformation of the pyridinꢀ3ꢀylsulfonyl group to a pyridinꢀ2ꢀylsulfonyl or a 4ꢀ
fluorophenylsulfonyl group was found to maintain potent in vitro hꢀEP2 receptor agonist
activity, as shown in Table 4. Because pyridinꢀ3ꢀylsulfonyl group, the substructure in CPꢀ
5
33,536 that was under clinical development at the time, was expected to pose a limited safety
risk and because no safety data were available for compounds containing a pyridinꢀ2ꢀylsulfonyl
or a 4ꢀfluorophenylsulfonyl group in humans, we selected a pyridinꢀ3ꢀylsulfonyl group from
among these three functional groups, with comparable agonist activity, for further structural
optimization.
Table 4. In Vitro hꢀEP2 Receptor Agonist Activity of Aminopyridyl Derivatives 13aa–ak;
Optimization of the Pyridinꢀ3ꢀylsulfonyl Moiety
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
a
compound
13aa
R
EC [nM]
50
pyridinꢀ3ꢀyl
2.8
13ab
13ac
methyl
91
4.3
2.7
45
phenyl
13ad
13ae
pyridinꢀ2ꢀyl
2ꢀfluorophenyl
2ꢀchlorophenyl
3ꢀfluorophenyl
3ꢀchlorophenyl
4ꢀfluorophenyl
4ꢀchlorophenyl
4ꢀmethoxyphenyl
13af
>1000
6.1
13ag
1
3ah
620
2.7
1
3ai
1
3aj
4.4
1
3ak
19
a
Measurement of in vitro hꢀEP2 receptor agonist activity was carried out in compliance with the
36
method of Wilson et al. The concentration of test compound required to increase the amount of
cAMP to 50% of the maximum increase (EC50 value) in HEK293 cells expressing hꢀEP2
receptor (ESꢀ562ꢀC, Euroscreen) was calculated on the basis of the results from at least seven
data points of a single experiment, performed in duplicate.
Exploration of the tertꢀbutylphenyl moiety. The optimization of the structure regarding in
vitro hꢀEP2 receptor agonist activity was attempted by transforming the tertꢀbutylphenyl moiety
using a chemotype with a (6ꢀ((pyridinꢀ3ꢀylsulfonyl)aminomethyl)pyridinꢀ2ꢀylamino)acetic acid
moiety, which has both a (pyridinꢀ2ꢀylamino)acetic acid substructure (as selected in the
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“
Exploration of the phenoxyacetic acid moiety” section) and a pyridinꢀ3ꢀylsulfonyl group (as
selected in the “Exploration of the pyridinꢀ3ꢀylsulfonyl moiety” section) (Table 5). The tertꢀ
butylphenyl moiety could be transformed into various structures as expected. It was confirmed
that a paraꢀposition on the phenylene group was found to be favorable for substitution on the
basis of the agonist activity of 13al. The assessment of the transformation of the phenylene
moiety showed that compounds retaining the original 1,4ꢀphenylene group exhibited more potent
agonist activity than those with 1,4ꢀcyclohexylene and nꢀbutylene groups (13aa vs. 13am and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3as vs. 13at). Some derivatives mimicking a tertꢀbutyl group showed agonist activity
comparable to 13aa, but none of them were superior (13an–ar), whereas the agonist activity of
13as (in which the tertꢀbutyl group is transformed into a phenyl group) tended to be more potent
than 13aa. Therefore, we then focused on the transformation of the tertꢀbutylphenyl moiety into
an aromatic system, such as optionally substituted biaryl or fused bicyclic aromatic groups. The
agonist activity of 13bb and 13bc, compounds in which the 4'ꢀposition of the biphenyl group of
1
3as was substituted with F and Cl, respectively, was reduced with increasing substituent size,
and a reduction in agonist activity was also observed in 13au with a 4ꢀcyclohexylphenyl group,
suggesting the constrained steric environment around this moiety. The transformation of the tertꢀ
butylphenyl moiety into an aromatic system containing a hetero aryl group worked as expected
to obtain compounds with potent agonist activity (13av–ba). Especially, 13ax (OMD), which
contains a pyrazolꢀ1ꢀyl group, was found to exhibit the most potent and more than 10 times
higher hꢀEP2 receptor agonist activity than CPꢀ533,536. 13ax was expected to be effective at
low concentrations because of its potent EP2 receptor agonist activity and underwent further
assessments as described below.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. In Vitro hꢀEP2 Receptor Agonist Activity of Aminopyridyl Derivatives 13al–bc;
Optimization of the tertꢀButylphenyl Moiety
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
a
compound
13aa
R
EC [nM]
50
4ꢀtertꢀbutylphenyl
2.8
13al
3ꢀtertꢀbutylphenyl
109
649
119
2.6
4.5
18
13am
13an
4ꢀtertꢀbutylcyclohexyl
4ꢀtrifluoromethylphenyl
4ꢀ(1ꢀmethylcyclopropyl)phenyl
4ꢀ(1ꢀethylcyclopropyl)phenyl
4ꢀ(1ꢀisopropylcyclopropyl)phenyl
4ꢀ(1ꢀethylcyclobutyl)phenyl
biphenylꢀ4ꢀyl
13ao
13ap
13aq
13ar
13
1
3as
3at
3bb
3bc
3au
3av
3aw
2.0
118
35
1
4ꢀphenylbutyl
1
4'ꢀfluoroꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl
4'ꢀchloroꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl
4ꢀcyclohexylphenyl
1
75
1
29
1
4ꢀ(pyridinꢀ2ꢀyl)phenyl
4ꢀ(pyridazinꢀ4ꢀyl)phenyl
1.6
307
1.1
43
1
1
3ax, OMD 4ꢀ(pyrazolꢀ1ꢀyl)phenyl
3ay
4ꢀ(1,2,4ꢀtriazolꢀ1ꢀyl)phenyl
1
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2
3
4
5
6
7
8
9
1
3az
3ba
Measurement of in vitro hꢀEP2 receptor agonist activity was carried out in compliance with the
benzofuranꢀ2ꢀyl
5.6
1
benzo[b]thiophenꢀ2ꢀyl
3.4
a
36
method of Wilson et al. The concentration of test compound required to increase the amount of
cAMP to 50% of the maximum increase (EC50 value) in HEK293 cells expressing hꢀEP2
receptor (ESꢀ562ꢀC, Euroscreen) was calculated on the basis of the results from at least seven
data points of a single experiment, performed in duplicate.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Improvement in the membrane permeability of 13ax (OMD) and further biological
evaluation. Through the medicinal chemistry efforts described above, 13ax, a compound with
potent hꢀEP2 receptor agonist activity, was selected as the most promising active form. An
evaluation of the binding affinity for hꢀEP receptor subtypes, namely hꢀEP1, hꢀEP2, hꢀEP3, and
hꢀEP4, revealed that 13ax binds strongly and with a high selectivity to the hꢀEP2 receptor (Table
6
), based on which it was assumed that 13ax has better safety profiles than nonꢀselective EP
receptor agonists. The in vitro ADMET properties of 13ax are summarized in Tables 7, 8, S1, S3,
and S4. 13ax showed slow metabolic rate in human liver microsome and was expected to pose a
low risk of drugꢀdrug interaction as shown by the cytochrome P450 (CYP) inhibition and
induction assay. Meanwhile, the cell membrane permeability of 13ax was presumed to be
ꢀ7
insufficient: the Cacoꢀ2 cell permeability rate of 13ax was in the order of 1 × 10 cm/s at pH 7.4
and 6.5 apically, and the permeability rate measured with parallel artificial membrane
ꢀ6
permeability assay (PAMPA) for 13ax was 0.9 × 10 cm/s. The lipophilic corneal epithelium
3
7
represents a major barrier to the absorption of IOPꢀlowering eye drops into the eyes. Therefore,
improvements to cell membrane permeability are considered necessary to facilitate effective
IOPꢀlowering activity following ocular administration. Generally, cell membrane permeability
can be enhanced by increasing the lipophilicity of a compound, and the corneal epithelium is
38
negatively charged under physiological conditions. Therefore, a prodrug approach to masking
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