Discovery of Potent and Orally Active Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Inhibitors as a Potential Therapy for Diabetic Macular Edema

Article abstract of DOI:10.1021/acs.jmedchem.5b01930

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) is considered to be a promising therapeutic target for several inflammation-associated diseases. Herein, we describe the discovery of a series of pyrimidone derivatives as Lp-PLA₂ inhibitors. Systematic structural modifications led to the identification of several pyrimidone compounds with promising in vitro inhibitory potency and pharmacokinetic properties. Compound 14c, selected for in vivo evaluation, demonstrated decent pharmacokinetic profiles and robust inhibitory potency against Lp-PLA₂ in Sprague-Dawley (SD) rats. Furthermore, 14c significantly inhibited retinal thickening in STZ-induced diabetic SD rats as a model of diabetic macular edema (DME) after oral dosing for 4 weeks. Taken together, these results suggested that 14c can serve as a valuable lead in the search for new Lp-PLA₂ inhibitors for prevention and/or treatment of DME.

Full text of DOI:10.1021/acs.jmedchem.5b01930

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Article
Discovery of Potent and Orally Active Lipoprotein-Associated Phospholipase
2
2
A (Lp-PLA) Inhibitors as a Potential Therapy for Diabetic Macular Edema
Xinde Chen, Kai Wang, Wenwei Xu, Quanxin Ma, Minli Chen,
Lili Du, Mingguang Mo, Yi-Ping Wang, and Jianhua Shen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01930 • Publication Date (Web): 29 Feb 2016
Downloaded from http://pubs.acs.org on February 29, 2016
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Discovery of Potent and Orally Active Lipoprotein-Associated
Phospholipase A (Lp-PLA ) Inhibitors as a Potential Therapy for
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Diabetic Macular Edema
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1†
1†
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Xinde Chen , Kai Wang , Wenwei Xu , Quanxin Ma , Minli Chen , Lili Du ,
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Mingguang Mo , Yiping Wang *, Jianhua Shen *
1
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China
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Laboratory Animal Research Center, Zhejiang Chinese Medical University, Hangzhou
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10053, China
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ABSTRACT:
Lipoproteinꢀassociated phospholipase A (LpꢀPLA ) is considered to be a promising
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therapeutic target for several inflammationꢀassociated diseases. Herein, we describe the
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discovery of a series of pyrimidone derivatives as LpꢀPLA inhibitors. Systematic
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structural modifications led to the identification of several pyrimidone compounds with
promising in vitro inhibitory potency and pharmacokinetic properties. Compound 14c,
selected for in vivo evaluation, demonstrated decent pharmacokinetic profiles and robust
inhibitory potency against LpꢀPLA in SpragueꢀDawley (SD) rats. Furthermore, 14c
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significantly inhibited retinal thickening in STZꢀinduced diabetic SD rats as a model of
diabetic macular edema (DME) after oral dosing for 4 weeks. Taken together, these
results suggested that 14c can serve as a valuable lead in the search for new LpꢀPLA2
inhibitors for prevention and/or treatment of DME.
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INTRODUCTION
Lipoproteinꢀassociated phospholipase A (LpꢀPLA ) is a vascular specific enzyme that
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circulates mainly with lowꢀdensity lipoprotein in human plasma. It is produced by
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inflammatory cells, and is able to hydrolyze oxidized modified phosphatidylcholine to
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generate two products, oxidized free fatty acids and lysophosphatidylcholine (lysoꢀPC),
both products are proꢀinflammatory mediators and are considered to promote
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inflammatory responses. Extensive studies have shown that LpꢀPLA plays a key role
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ꢀ11
in the development and progression of atherosclerosis,
and LpꢀPLA has long been
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considered a promising therapeutic target for atherosclerosis. Additionally, it was
reported that inhibition of LpꢀPLA had beneficial effects on the functional integrity of
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the blood−brain barrier (BBB) in a diabetic and hypercholesterolemia porcine model,
and another study demonstrated that inhibition of LpꢀPLA might be protective against
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the hyperglycemiaꢀrelated compromise of the blood−retinal barrier (BRB) in Brown
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Norway rats. As a result of these observations, inhibition of LpꢀPLA has been
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suggested to be a potential therapeutic target for neurodegenerative diseases and diabetic
macular edema (DME).
Unlike atherosclerosis and wellꢀknown neurodegenerative diseases, DME receives
less attention by researchers in the pharmaceutical industry. However, it is a severe
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diabetic eye complication, and is the leading cause of visual loss in diabetic patients. It
occurs as a direct consequence of disruption of the BRB, which leads to increased
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accumulation of fluid in the macular. Currently available pharmacotherapies for DME
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are antiꢀVEGF drugs and corticosteroids, both which are delivered by somewhat risky
and inconvenient intravitreal injection. Additionally, both antiꢀVEGF drugs and
corticosteroids therapies have a percentage of nonresponders, and are associated with
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risks of visual loss. Therefore, it is obvious that there is an urgent need to further
develop riskless and effective drug candidates for DME treatment. Most recently, a
phaseꢀII trial revealed that oral administration of darapladib (a LpꢀPLA inhibitor) was
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safe and well tolerated, and demonstrated modest improvements in macular edema and
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vision acuity in centerꢀinvolved DME patients. These results further indicated that
LpꢀPLA inhibitors might serve as potential therapeutic agents for the treatment of DME.
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Several LpꢀPLA inhibitors have been discovered over the past decade (Figure 1),
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and can be separated into two categories based on their structures: nonꢀpyrimidone
compounds, and pyrimidone derivatives. Nonꢀpyrimidone compounds include
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19, 20
21, 22
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βꢀlactams, oximes,
amides of xanthurenic acid,
and carbamates, however,
none of these compounds were reported to have efficacy in vivo. Pyrimidone derivatives
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include the compound darapladib, which was developed by GSK. Although darapladib
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is the most advanced LpꢀPLA inhibitor, it suffers from rather high lipophilicity (clogP,
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8.33) and molecular weight (MW, 666.78), and it missed its `primary end points in two
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phaseꢀIII trials focusing on coronary heart disease.
Examination of the patent
literatures suggested that GSK’s researchers have shifted their efforts away from
darapladib analogues in favor of a series of pyrimidone compounds with simplified
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9ꢀ36
structures, as exemplified by pyrimidone derivatives 1–3.
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Figure 1. Representative structures of LpꢀPLA inhibitors
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Our research group has focused on the discovery of LpꢀPLA inhibitors for many
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years. We previously reported a series of imidazole and triazole derivatives as analogues
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of darapladib.
Very recently, we discovered
a
novel series of
imidazo[1,2ꢀa]pyrimidine derivatives (Figure 1), which have favorable pharmacokinetic
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properties and demonstrated robust inhibitory efficacy against LpꢀPLA in vivo.
2
However, given the high attrition rate of drug candidates, the discovery of additional
candidates with different chemotypes is still needed.
Inspired by the excellent results of the imidazo[1,2ꢀa]pyrimidine derivatives, we then
concentrated our attention on the discovery of novel LpꢀPLA inhibitors with simplified
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structures. As part of our continued efforts, we describe here the design of a series of
LpꢀPLA inhibitors based on a pyrimidone scaffold (Figure 2). On the basis of the
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structure−activity relationship (SAR) results of our previously reported
imidazo[1,2ꢀa]pyrimidine derivatives, and assuming that the left hand side of the newly
designed pyrimidone compounds adopt a similar binding mode to that of the
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imidazo[1,2ꢀa]pyrimidine derivatives, we suggested that subtle changes in the left hand
side of the pyrimidone compounds would also cause a large shift in potency similar to the
imidazo[1,2ꢀa]pyrimidine derivatives. Given the challenges of aligning potency and
pharmacokinetic properties in a drug candidate, we expected to further define another
structural region so that inhibitory potency and pharmacokinetic properties could be
modulated. After careful analysis of the structure, we opted to focus our attention on the
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right hand side of the structure (R and R ).
Figure 2. General structure of the pyrimidone derivatives
Herein, we describe the design, synthesis, and systematic structural modifications of
the pyrimidone derivatives as LpꢀPLA inhibitors. These efforts resulted in the
2
identification of compound 14c, which displayed significant efficacy in a rat model of
DME.
CHEMISTRY
The synthesis of derivatives 9a−d is summarized in Scheme 1. A nucleophilic
substitution
reaction
between
4ꢀchloroꢀ3ꢀ(trifluoromethyl)phenol
and
3
,4,5ꢀtrifluorobenzaldehyde in the presence of K CO yielded intermediate 4, and
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subsequent reduction of 4 using NaBH provided intermediate 5. A nucleophilic
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substitution reaction between 5 and 6a–b in the presence of NaH gave intermediates 7a–
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b, which were converted to 8a–b using NaNO . Compounds 9a–d were obtained by
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substitution of 8a–b with R I using Cs CO as a base.
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a
Scheme 1. Synthesis of Compounds 9a−d
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a
Reagents and conditions: (a) K CO , DMF, 120 °C, 2 h; (b) NaBH , EtOH, rt; (c) NaH,
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THF; (d) NaNO , HAc,rt; (e) ) R I, DMF, Cs CO
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The synthesis of compounds 13a–p is outlined in Scheme 2. Methylation of
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ꢀchlorouracil using CH I provided intermediate 10. A nucleophilic substitution reaction
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between intermediate 10 and R H gave intermediates 11a–p, which were converted to
chlorineꢀsubstituted intermediates 12a–p using POCl . Compounds 13a–p were obtained
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by treating 12a–p with intermediate 4 through a nucleophilic substitution reaction.
Morpholine derivatives 14a–e were synthesized using a method similar to that described
for compounds 13a–p starting from intermediate 12h and the corresponding benzyl
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alcohols (synthesis according to reference ).
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a
Scheme 2. Synthesis of Compounds 13a−p
O
O
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b
c
d
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=
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m
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e
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o
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d R2=
O
h R2=
O
l R2=
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p
O
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Reagents and conditions: (a) CH I, K CO , DMSO, rt; (b) R H, EtOH, 70 °C; (c) POCl ,
3
2
3
3
70 °C; (d) NaH, THF, rt; (e) m-CPBA, DCM, rt.
RESULT AND DISCUSSION
SAR, Metabolic Stability, and Caco-2 Permeability. Compounds were first
evaluated for their inhibitory activity against recombinant human LpꢀPLA (rhLpꢀPLA )
2
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at two concentrations (100 nM and 10 nM) in vitro, and rhLpꢀPLA activity was
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measured using 2ꢀthioꢀPAF as the substrate. IC50 values against rhLpꢀPLA for those
2
compounds with an acceptable inhibitory activity (>50% at 10 nM) in initial assessments
were tested to confirm their inhibitory potency. Compounds with acceptable potency
were also evaluated for their metabolic stabilities in human and rat liver S9 fractions, and
Cacoꢀ2 permeability tests were conducted to assess the penetration properties of those
compounds with favorable potency and metabolic stabilities.
Our initial SAR efforts were directed toward the introduction of substituents at the
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ꢀposition and 2ꢀposition of the pyrimidone moiety to examine their tolerability for the
potency (Table 1). Interestingly, the first prepared compound, 9a, with methyl at the 1ꢀ
and 2ꢀpositions displayed single digit nanomolar potency. Replacement of the methyl
group at the 1ꢀposition with bulkier substituents, such as ethyl (9b) or propyl (9c),
resulted in a decrease in potency, whereas introduction of a bulkier group to the
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ꢀposition, such as methoxy (9d), dimethylamine (13a) or diethylamine (13b), resulted in
comparable potency with the methyl derivative 9a. These results suggested that the
2ꢀposition of the pyrimidone moiety might possess great tolerability for a variety of
substituents. Therefore we focused our structural modifications at this position. Given the
excellent potency and synthetic accessibility of the amine derivatives 13a and 13b, we
sought to further explore the inhibitory potency and pharmacokinetic properties of the
amine series.
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Table 1. Preliminary SAR of the R and R substituents
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inhibition against
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compd
rhLpꢀPLA
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IC （nM）
R
R
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00 nM
10 nM
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a
Me
Et
Me
Me
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75
88
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2.6
8.0
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9c
Pr
Me
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d
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OMe
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4.2
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3b
darapladib
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a
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a
Reported IC = 0.25 nM.
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To obtain a preliminary understanding of the pharmacokinetic properties of this
amine series, the metabolic stabilities of 13a and 13b were evaluated. As shown in Table
, although 13a and 13b both displayed a low intrinsic clearance in human liver S9
fractions, a high and modest intrinsic clearance in rat liver S9 fractions was observed for
3a and 13b, respectively. Therefore, further structural modifications of these compounds
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were aimed at improving their metabolic stabilities in rat liver S9 fractions. We
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postulated that the poor metabolic stabilities of 13a and 13b might have resulted from
rapid Nꢀdealkylation of the dimethylamine and diethylamine groups. Cyclopropyl was
then introduced to replace one of the methyl groups of the dimethylamine in 13a, the
resulting compound 13c displayed unexpected higher intrinsic clearance when compared
with 13a. More polar functional groups were also introduced to evaluate their effect on
metabolic stabilities, the resulting methoxyethylamine 13d and ethyl ester 13e both
maintained favorable potency, but displayed rather different metabolic stabilities in rat
liver S9 fractions. Compound 13d with a methoxyethylamine group displayed rather poor
stability in rat liver S9 fractions, whereas ethyl ester 13e was quite stable in both human
and rat liver S9 fractions. Additionally, 13e displayed modest permeability, with a Papp
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ꢀ6
value of 0.32 × 10 cm/s (apical side to basolateral side, A to B) and an efflux ratio of
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.19 in a Cacoꢀ2 permeability assay.
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Table 2. SAR, Metabolic Stability, and Caco-2 Permeability of Analogues 13a–n
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%
inhibition against
rhLpꢀPLA2
liver S9 stability
(mL/min/kg)
Cacoꢀ2 permeability
A to B
2
compd
IC50 （nM）
R
1
00 nM
90
10 nM
88
human
rat
efflux ratio
ꢀ
6
(10 cm/s)
1
3a
3b
3.9
5.04
82.80
0.65
1.57
1
88
77
88
93
4.2
4.0
1.2
9.87
0.00
25.5
41.90
123
1
3c
100
100
1
3d
107
1
3e
100
85
96
48
57
89
1.1
9.68
0.00
5.69
4.09
13.8
3.11
0.32
0.08
0.01
1.39
1.19
12.40
32.30
0.77
1
3f
1
3g
3h
94
8.3
1.6
21.80
25.40
1
100
15
13i
1
3j
100
100
100
96
77
88
89
76
83
83
91
3.5
1.1
2.2
4.6
1.8
1.8
1.7
65.5
20.90
1.49
0.00
3.26
49.1
57.5
1138
184
1
3k
13l
5.20
23.6
13.7
1198
83.4
0.28
0.09
0.43
1.90
0.61
0.60
1
3m
1
3n
97
13o
90
1
3p
99
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In a parallel strategy to improve the metabolic stabilities, several cyclic amines were
introduced as cyclic substituents often display better metabolic stabilities when compared
with noncyclic substituents. As shown in Table 2, both the pyrrolidine analogue 13f and
the piperidine analogue 13g were rather stable in human and rat liver S9 fractions,
indicating that cyclic substituents at the 2ꢀposition actually had beneficial effects on the
metabolic stabilities of these pyrimidone analogues. However, 13f and 13g demonstrated
somewhat reduced potency, and unexpected low permeability with a high efflux ratio of
12.4 and 32.3, respectively. A real breakthrough was made by the morpholine analogue
10
11
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13
14
15
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18
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29
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43
44
45
46
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60
1
3h, which showed increased inhibitory potency and similar metabolic stability, and gave
a significant improvement in permeability (efflux ratio, 0.77) when compared with the
piperidine analogue 13g. Thus, we hypothesized that introduction of hydrogenꢀbond
acceptor(s) to the cyclic substituents at the 2ꢀposition was beneficial for both inhibitory
potency and permeability. The beneficial effect of hydrogenꢀbond acceptor(s) on potency
was further verified by the distinctly different potency of compounds 13i and 13j.
Compound 13j with a 1ꢀmethylpiperazine group demonstrated comparable potency with
the morpholine analogue 13h, whereas the 4ꢀmethylpiperidine analogue 13i was almost
inactive. On the basis of these results, we therefore undertook a systematic exploration of
a series of cyclic substituents containing hydrogenꢀbond acceptor(s).
As shown in Table 2, replacement of the morpholine in 13h with a
4ꢀmethoxypiperidine group (13k) resulted in compound that displayed a rather high
intrinsic clearance in rat liver S9 fractions. Introduction of 4ꢀfluoropiperidine (13l) or
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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
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4
4
4
4
5
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5
5
5
5
5
5
5
6
4
,4ꢀdifluoropiperidine (13m) resulted in compounds with excellent potency and
metabolic stabilities, but the Cacoꢀ2 permeability of these two compounds was relatively
low. The 1ꢀmethylpiperazine analogue 13j was also rather unstable in both human and rat
liver S9 fractions. Cyclopropyl was introduced to replace the methyl of
1ꢀmethylpiperazine in 13j to block the possible Nꢀdemethylation metabolic pathway. The
resulting compound 13n turned out to be quite stable, and the Cacoꢀ2 permeability of this
compound was modest. Replacement of the oxygen atom of the morpholine in 13h with a
sulfur atom resulted in thiomorpholine 13o, although this compound also possessed
excellent inhibitory potency, it was cleared quickly in both human and rat liver S9
fractions. Assuming that the sulfur atom can be oxidized to sulfoxide and sulphone,
sulphone 13p was then prepared in an effort to block this hypothetical metabolic pathway,
however, 13p also showed relatively high intrinsic clearance in both human and rat liver
S9 fractions.
0
1
2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
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8
9
0
1
2
3
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5
6
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On the basis of its excellent inhibitory potency against rhLpꢀPLA , low intrinsic
2
clearance, and favorable Cacoꢀ2 permeability, compound 13h was selected for further
optimization by investigating the left hand side of the structure. Similar to that described
in our previously reported imidazo[1,2ꢀa]pyrimidine derivatives, most of the structural
modifications of the linker and the terminal benzene ring resulted in different degrees of
reduction in potency (details in Supporting Information), therefore we quickly focused
our attention on the exploration of the central benzene ring (Table 3). To our delight,
compounds with the central benzene ring as benzene (14a) or monofluoroꢀbenzene (14b)
both displayed similar potency, metabolic stability, and Cacoꢀ2 permeability as compared
with 13h. The cyanoꢀsubstituted analogue 14c, which was somewhat unstable in rat liver
S9 fractions, showed excellent inhibitory potency and the best permeability among these
pyrimidone derivatives. The chlorineꢀsubstituted analogue 14d and methylꢀsubstituted
analogue 14e both showed a decrease in potency, indicating that a bulkier
electronꢀwithdrawing substituent or electronꢀdonating substituent in the central benzene
ring was detrimental for the inhibitory potency. These SAR results were similar to those
10
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12
13
14
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19
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21
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23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
9
described for our previously reported imidazo[1,2ꢀa]pyrimidine series.
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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
Table 3. SAR, Metabolic Stability, and Caco-2 Permeability of Analogues 14a–e
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
%
inhibition against
rhLpꢀPLA2
liver S9 stability
(mL/min/kg)
Cacoꢀ2 permeability
compd
IC50 （nM）
A to B
100 nM
10 nM
human
rat
efflux ratio
ꢀ6
(
10 cm/s)
13h
100
100
100
100
91
89
81
90
92
49
44
1.6
1.9
2.7
2.2
4.09
3.89
5.55
19.7
25.40
13.3
1.39
2.05
2.01
6.54
0.77
1.09
0.73
1.03
1
4a
4b
1
15.10
94.8
14c
1
4d
1
4e
84
On the basis of the results outlined in Tables 2 and 3, morpholine analogues 13h, and
14a–c were selected to further evaluate their potency against LpꢀPLA in human plasma.
2
As shown in Table 4, these selected compounds demonstrated similar potency in human
plasma when compared with their potency against rhLpꢀPLA . For example, at a
2
concentration of 10 nM, compound 13h displayed similar inhibition of 89% and 95%
against rhLpꢀPLA and LpꢀPLA in human plasma, respectively. These results illustrated
2
2
that nonspecific binding had a negligible effect on the binding affinity between these
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pyrimidone analogues and the LpꢀPLA enzyme in human plasma. The inhibitory
2
potency against LpꢀPLA in rat plasma of these selected compounds was also evaluated
2
before an in vivo assessment was conducted. As shown in Table 4, these morpholine
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14
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28
29
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analogues displayed somewhat different potency against LpꢀPLA in rat plasma. In
2
particular, difluorineꢀsubstituted analogue 13h (39% at 10 nM) had relatively lower
potency in rat plasma when compared with analogues 14a–c (14a, 72% at 10 nM; 14b,
6
0% at 10 nM; 14c, 94% at 10 nM). Among these morpholine analogues,
cyanoꢀsubstituted analogue 14c displayed the best potency in rat plasma, and its
inhibitory potency in rat plasma was further confirmed by determination of IC value as
5
0
1
.0 nM. Driven by the different SAR results of the substituents on the central benzene
2
ring in rat plasma, several compounds with different R groups (13a, 13d, 13f, 13g) were
2
then selected to further investigate the SAR of R in rat plasma (Table 4). Unlike the
2
substituents on the central benzene ring, the SAR of R in rat plasma was similar to that
observed for rhLpꢀPLA , though the potencies of these compounds in rat plasma were
2
relatively lower when compared with their potencies against rhLpꢀPLA2.
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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
Table 4. Inhibitory Activity against Lp-PLA in Human and Rat Plasma
2
% inhibition in plasma
compd
structure
human
0 nM
rat
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
1 nM
74
100 nM
98
10 nM
39
1
3h
95
92
85
92
89
90
76
1
4a
61
28
67
68
56
0
100
100
100
84
72
60
94
30
28
12
1
4b
1
4c
1
3a
1
3d
92
1
3f
65
1
3g
68
94
0
54
98
4
darapladib
59
84
Pharmacokinetic Studies in Vivo. Compound 14c, although somewhat unstable in
rat liver S9 fractions, displayed the most promising inhibitory potency in vitro, and the
best permeability in the Cacoꢀ2 assay, was selected to investigate its pharmacokinetic
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properties in male SD rats. As shown in Table 5, after intravenous administration at a
dose of 5 mg/kg, 14c displayed a moderate clearance of 18.6 mL/min/kg, and a relatively
large volume of distribution (9.1 L/kg). At an oral dose of 50 mg/kg, 14c achieved good
oral plasma exposure, with a peak plasma concentration (Cmax) of 1879 ng/mL, an AUC
value of 19.9 µgꢁh/mL, and a decent oral bioavailability of 45%. These PK values were
all much higher than those of darapladib (Cmax, 115 ng/mL; AUC, 1.3 µgꢁh/mL; F, 11%).
10
11
12
13
14
15
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17
18
19
20
21
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23
24
25
26
27
28
29
30
31
32
33
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35
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39
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a
Table 5. In Vivo Pharmacokinetic Parameters in SD Rats of 14c and Darapladib
Pharmacokinetics (iv)
Pharmacokinetics (po)
compd
AUC0ꢀ24h
Cl
AUC0ꢀ24h
(µgꢁh/mL)
19.9
Cmax
t1/2 (h)
V (L/kg)
Tmax (h)
(ng/mL)
F (%)
ss
(µgꢁh/mL)
(ml/min/kg)
b
14c
4.4
5.7
3.3
18.6
9.1
1879
115
4.6
4.2
45
11
c
darapladib
1.2
121.0
34.4
1.3
a
b
n = 5 animals/group, data are the mean values. Dosed iv: administered at 5 mg/kg,
vehicle was 5% DMSO/5% Tweenꢀ80 in saline; dosed po: administered at 50 mg/kg,
c
vehicle was 0.5% carboxymethylcellulose sodium. Dosed iv: administered at 10 mg/kg
in tartrate salt form, vehicle was water; dosed po: administered at 50 mg/kg in tartrate salt
form, vehicle was water.
Assessment of the Inhibitory Activity of Lp-PLA in Vivo. In view of the
2
promising inhibitory potency in vitro and decent pharmacokinetic profiles in vivo, it came
as no surprise that compound 14c demonstrated robust inhibitory potency against
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
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3
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5
5
5
6
LpꢀPLA in male SD rats after oral administration (Figure 3). And it was worth
2
mentioning that 14c could still efficiently inhibit LpꢀPLA activity even at a relatively
2
low dose of 3 mg/kg, and this activity was relatively better than that of darapladib, which
was administered at a single oral dose of 25 mg/kg in the tartrate salt form.
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 3. Relative activity of LpꢀPLA in the serum of SD rats after a single oral dose (n
2
= 5). Error bars represent the SD. 14c was formulated in 0.25% carboxymethylcellulose
sodium, darapladib in the tartrate salt form was dissolved in water.
Effects of 14c on Retinal Thickening in STZ-induced Diabetic Rats. DME is
characterized by retinal thickening, which is induced by the accumulation of fluid in the
retinal layers, and it was reported that hyperglycemia would induce retinal thickening in
40ꢀ42
STZꢀinduced diabetic rats.
To preliminary determine if these pyrimidone derivatives
could display potential therapeutic effects for DME, compound 14c was evaluated for its
effects on retinal thickening in STZꢀinduced diabetic SD rats as a pathological model of
DME with a relatively high oral dose of 25 mg/kg/day. As shown in Figure 4, diabetic
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retinals were significantly thicker than control retinals, and a significant inhibitory effect
was observed on retinal thickening in diabetic rats treated with 14c after oral dosing for 4
weeks. Additionally, the inhibitory efficacy of 14c on retinal thickening was achieved
without significantly affect the metabolism of blood glucose and serum lipids (details in
Supporting Information). Although the inhibitory efficacy on retinal thickening of 14c
was not differentiated from that of darapaldib in this animal model at a dose of 25
mg/kg/day, these preliminary positive results warrant comprehensive investigation of
these pyrimidone derivatives in more animal studies including doseꢀresponse experiment
to further understand their potential as therapeutic agents for DME.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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35
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38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Effect of compound 14c on retinal thickening in STZꢀinduced diabetic rats (n =
5
, 25 mg/kg, po, 4 weeks). ###, p<0.001 vs normal control. **, p<0.01 vs diabetic control.
Error bars represent SEM. Oneꢀway ANOVA statistical analysis
was used. 14c was formulated in 0.25% carboxymethylcellulose sodium, darapladib in
the tartrate salt form was dissolved in water.
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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
CONCLUSION
We have described the discovery of a series of pyrimidone derivatives as potent LpꢀPLA2
inhibitors. Our preliminary SAR explorations indicated that the 2ꢀposition of the
pyrimidone moiety could tolerate a range of substitutions and thus suggested that
inhibitory potency and pharmacokinetic properties might be modulated through structural
modifications at this position. Our chemical efforts at this position first led to the
identification of morpholine analogue 13h, further structural modifications of the left
hand side of the structure resulted in the identification of several morpholine derivatives
with promising inhibitory potency and pharmacokinetic properties in vitro.
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
Compound 14c selected for in vivo evaluation displayed decent pharmacokinetic
profiles, and demonstrated robust inhibitory potency against LpꢀPLA in male SD rats
2
after oral administration. Furthermore, 14c significantly inhibited retinal thickening in
STZꢀinduced diabetic SD rats as a model of DME after oral dosing for 4 weeks. Our
preliminary results warrant further in vivo experiments with 14c or other pyrimidone
derivatives to fully understand their potential as therapeutic agents for DME.
EXPERIMENTAL SECTION
43
In Vitro Assay to Measure the Inhibitory Activity of Lp-PLA . Activities against
2
rhLpꢀPLA , human plasma, rat plasma, and mouse plasma LpꢀPLA were measured using
2
2
2
ꢀthioꢀPAF as the substrate. Briefly, 10 ꢂL of the rhLpꢀPLA enzyme (or plasma) and 10
2
ꢂL of a DMSO solution of the compound were added to 0.1 mol/L TrisꢀHCl (pH 7.2)
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containing 1 mmol/L EGTA, 50 ꢂmol/L 2ꢀthioꢀPAF, and 10 ꢂL of 2 mmol/L 5,5′ꢀdithiobis
(2ꢀnitrobenzoic acid) in a total volume of 200 ꢂL. The assay was carried out using a plate
reader to obtain absorbance values at 414 nm every minute for 10 min. Percent inhibition
was determined using the following equation:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
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ꢃꢄꢅꢆꢇꢈꢉꢊꢈꢋꢌꢍ ꢀ− ꢃꢄꢅꢆꢎꢏꢐꢌꢑ
inhibitionꢀꢁ%ꢂ = 1 −
× 100%ꢀ
ꢃꢄꢅꢆꢊꢈꢒꢓꢔꢓꢕꢖ ꢀ− ꢃꢄꢅꢆꢎꢏꢐꢌꢑ
Vmax: slope of absorbance values for 10 min, calculated by MolecularDevice,
SpectraMax M2e. The blank sample contained no rhLpꢀPLA enzyme (or plasma) or test
2
compound in assay buffer. The positive sample contained no test compound.
This study has been approved and supervised by Institutional Animal Care and Use
Committee (IACUC), Shanghai Institute of Materia Medica, Chinese Academy of
Sciences (IACUC Approval Number: SIMMꢀ2014−08ꢀWYPꢀ18) and the Ethics
Committee of Shanghai Xuhui Central Hospital.
Metabolic Stability Tests In human and Rat Liver S9 Fractions. The test compound
was dissolved in DMSO and diluted to the desired concentration with an aqueous
solution of 0.1% BSA. Liver S9 (0.33 mg/mL; pooled human liver S9 purchased from
Celsis In Vitro Technologies; Wistar rat liver S9 purchased from Research Institute for
Liver Diseases), test compounds (0.1 µM), MgCl (5.0 mM), BSA (0.005%), and
2
NADPH (1.0 mM) in Tris buffer (0.1 M, pH 7.4) were incubated in a 96ꢀwell plate at
3
7 °C. An aliquot was removed at each time point and the enzymatic reaction stopped by
protein precipitation in cold methanol. Halfꢀlives of the compounds in liver S9 fractions
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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
were calculated based on the firstꢀorder rate constants that were measured. Intrinsic
clearance was calculated using the following equation:
0
.693
mLꢀofꢀincubation
mgꢀofꢀliverꢀS9ꢀprotein
gꢀofꢀtissue
gꢀofꢀtissue
CLꢓꢌꢔ
=
×
ꢀ×
ꢀ×ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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8
9
0
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8
9
0
1
2
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5
6
7
8
9
0
ꢗꢘꢀꢙꢗꢚꢛꢜꢀtꢝ/ꢞ mgꢀofꢀliverꢀS9ꢀprotein
kgꢀofꢀweight
Scaling factors for the human liver (145 mg of S9 protein/g liver and 24.3 g of liver/kg
body weight) and rat liver (179 mg of S9 protein/g liver and 40 g of liver/kg body weight)
were employed in this calculation.
Caco-2 Permeability Assay. Cacoꢀ2 cells were obtained from the ATCC
(Cat#HTBꢀ37) and maintained in Dulbecco’s modified Eagle’s Medium containing 10%
fetal bovine serum, 1% glutamine, 1% nonessential amino acids, 100 ꢂg/mL streptomycin,
and 100 ꢂg/mL penicillin. Cacoꢀ2 cells were cultured at 37 °C in a 5% CO and 90%
2
relative humidity environment. Cacoꢀ2 cells were passaged every 7 days at a ratio of 1:10.
Cells were used between passages 30 and 40. After 21 days of culture, the integrity of the
cell monolayer was verified by measuring the transepithelial electrical resistance (TEER).
Drug transport from the apical side to the basolateral side (AꢀB) and from the basolateral
side to the apical side was measured simultaneously under the same condition.
Propranolol and Nadolol were used as the hypertonic and hypotonic control, respectively.
Digoxin was used as the positive control for Pgpꢀmediated drug efflux. In brief, the
method was as follows. After washing the monolayer with HBSS three times, the
compounds were diluted and added to the appropriate well (pH 6.8 for apical side and pH
7.4 for basolateral side). The plate was incubated at 37 °C for 95 min. Samples were
collected from the donor side at 5 and 95 min, and from the receiver side at 35 and 95
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min postꢀincubation. The concentration of samples was measured by LCꢀMS/MS. The
Papp was calculated from the following equation:
ꢃ ꢀ
ꢀ[ drug]ꢐꢇꢇꢖꢊꢔꢈꢢ
ꢟ
Pꢐꢊꢊ
=
×
10
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ꢠꢟ ꢀ× ꢡ ꢀ[ drug]ꢓꢌꢓꢔꢓꢐꢏꢀꢍꢈꢌꢈꢢ
Where V is the volume of the acceptor well, S is the surface area of the membrane, T is
A
A
the total transport time, [drug]acceptor is the drug level at the acceptor side, and [drug]initial
is the drug level at the donor side at T = 0.
donor
Animals. Male Sprague−Dawley (SD) rats were obtained from Shanghai SLAC
Laboratory Animal Co. Ltd. (Shanghai, China). Animal experiments were approved by
Animal Care and Use Committee, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, and Zhejiang Chinese Medicine University Animal Care
Committee.
Pharmacokinetic Studies. Test compounds were subjected to pharmacokinetic studies
on male SD rats with five animals in each group. Compound 14c was formulated for
intravenous (5 mg/kg) and oral (50 mg/kg) administrations in 5% DMSO, 5% Tweenꢀ80
in saline and 0.5% carboxymethylcellulose sodium, respectively. Darapladib was
formulated for intravenous (10 mg/kg) and oral (50 mg/kg) administrations in water.
Serial specimens were collected at predose, 5, 15 and 30 min and 1, 3, 5, 7, and 24 h
following intravenous administration and at predose, 0.5, 1, 2, 3, 5, 7, and 24 h following
oral administration. The concentration of compounds in the plasma samples was
determined with a liquid chromatography−mass spectrometry. Pharmacokinetic
parameters were calculated from the mean serum concentration by noncompartmental
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analyses using DAS software 2.1.1.
Assay to Measure the Inhibitory Activity of Lp-PLA in Vivo. A group of five male
2
SD rats were fasted overnight, and administered test compounds by gavage. 14c were
formulated in 0.25% carboxymethylcellulose sodium, darapladib in the tartrate salt form
was dissolved in water. Blood samples were drawn predose as well as 1, 3, 5, 7 and 24 h
after administration to measure LpꢀPLA activity in serum. LpꢀPLA activity in the serum
0
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0
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0
2
2
of SD rats was measured according to the method described for measurement of LpꢀPLA2
inhibitory activity in vitro.
Effects of 14c on Retinal Thickening in STZ-Induced Diabetic Rats. Male SD rats
were induced to diabetic by intraperitoneal injection of streptozotocin (STZ, Sigma) in 10
mmol/L sodium citrate (PH 4.6) at 50 mg/kg after fasting for 16 h. Blood glucose was
measured from blood samples 1 week after injection of STZ. Rats with blood glucose
values >10 mmol/L were considered diabetic. Test compounds were subjected to studies
on STZꢀinduced diabetic SD rats with five animals in each group, and were administered
at an oral dose of 25 mg/kg/day for 4 weeks starting 1 week after injection of STZ.
Compound 14c were formulated in 0.25% carboxymethylcellulose sodium, darapladib in
the tartrate salt form was dissolved in water. To determine the retinal thickness, retinal
cryosections (8 µM) were prepared and stained with hematoxylin/eosin. Digitized images
of the retina were captured at 400× magnification (Eclipse 80i, Nikon). The retinal
thickness was defined from the internal limiting membrane to the external membrane,
and was measured using ImageꢀPro plus 6.0.
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Materials and Methods. All reagents were purchased from commercial suppliers and
used without further purification unless otherwise stated. Yields were not optimized.
1
13
Microwave reactions were performed in a Biotage Initiator. H NMR and C NMR
spectra were recorded on a Bruker AC400 or a Bruker AC500 NMR spectrometer using
tetramethylsilane as an internal reference. Lowꢀresolution mass spectra were determined
on an Agilent liquidꢀchromatography mass spectrometer system that consisted of an
Agilent 1260 infinity LC coupled to Agilent 6120 Quadrupole mass spectrometer
(electrospray positive ionization; ESI). Highꢀresolution mass spectra were conducted on a
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+
triple TOF 5600 MS/MS system (AB Sciex, Concord, Ontario, Canada) in the positive
ESI mode. The purity of test compounds was determined by HPLC (Agilent ChemStation,
Agilent Eclipse XDBꢀC18, 5 ꢂM, 4.6×150 mm, 30 °C, UV 240 nM, flow rate = 1.0
mL/min) with aqueous CH CN (50−90%) containing ammonium formate (10 mmol/L)
3
for 25 min. All the assayed compounds possess ≥95% purity. Column chromatography
was performed on silica gel (200−300 mesh), and preparative TLC was performed on
HSGF 254 (0.4−0.5 mm thickness; Yantai Jiangyou Company, Yantai, Shangdong,
China).
4-(4-Chloro-3-(trifluoromethyl)phenoxy)-3,5-difluorobenzaldehyde
solution of 4ꢀchloroꢀ3ꢀ(trifluoromethyl)phenol (5.00 g, 25.51
,4,5ꢀtrifluorobenzaldehyde (3.16 mL, 28.03 mmol), and K CO (4.58 g, 33.19 mmol) in
(4).
A
mmol),
3
2
3
DMF (50 mL) was heated at 120 °C for 2 h under nitrogen. Then, the solution was cooled
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down and poured into ice water (150 mL), and extracted with ethyl acetate (80 mL × 3).
The organic layers were combined, washed with brine, dried over Na SO , filtered, and
2
4
concentrated. The residue obtained was purified with column chromatography (petroleum
0
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ether/ethyl acetate = 20:1) to yield intermediate 4 as a yellow solid (8.13 g, 94%). H
NMR (400 MHz, chloroformꢀd) δ 9.95 (s, 1H), 7.65–7.56 (m, 2H), 7.46 (d, J = 8.8 Hz,
+
1
H), 7.32 (d, J = 3.0 Hz, 1H), 7.06 (dd, J = 8.8, 2.9 Hz, 1H). MS (ESI): m/z 337 [M+H] .
(4-(4-Chloro-3-(trifluoromethyl)phenoxy)-3,5-difluorophenyl)methanol (5). To a
solution of 4 (4.00 g, 11.83 mmol) in ethanol (100 mL) was added NaBH (438 mg, 11.83
4
mmol) at 0 °C, and the reaction was stirred at rt for 0.5 h. After the reaction was complete,
the solvent was evaporated under reduced pressure, water was added to the residue, then
extracted with ethyl acetate (100 mL × 2), washed with brine, dried over Na SO , filtered,
2
4
and concentrated. The residue obtained was purified with column chromatography
(petroleum ether/ethyl acetate = 4:1) to yield intermediate 5 as a white solid (4.00 g,
1
99%). H NMR (400 MHz, chloroformꢀd) δ 7.42 (d, J = 8.8 Hz, 1H), 7.28 (d, J = 2.9 Hz,
1
H), 7.07 (d, J = 8.3 Hz, 2H), 7.02 (dd, J = 8.8, 2.8 Hz, 1H), 4.73 (d, J = 4.2 Hz, 2H). MS
+
(ESI): m/z 321 [M
−
H O+H] .
2
4
-((4-(4-Chloro-3-(trifluoromethyl)phenoxy)-3,5-difluorobenzyl)oxy)-6-methylp
yrimidin-2-amine (7a). To a solution of 5 (4.00 g, 11.76 mmol) in anhydrous THF (100
mL) was added sodium hydride (1.41 g, 35.25 mmol, 60% in mineral oil) at 0 °C, 6a
(1.52 g, 10.58 mmol) was added after the mixture was stirred at 0 °C for 0.5 h, and the
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mixture was stirred at rt overnight. Then, the reaction was quenched with NH Cl solution,
4
extracted with ethyl acetate (100 mL × 3), washed with brine, dried over NaSO , filtered,
4
and concentrated, the residue obtained was purified with column chromatography
10
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(
petroleum ether/ethyl acetate = 1:1) to yield intermediate 7a as a white solid (1.70 g,
1
32%). H NMR (400 MHz, chloroformꢀd) δ 7.42 (d, J = 8.8 Hz, 1H), 7.29 (d, J = 3.0 Hz,
1
3
H), 7.12–7.06 (m, 2H), 7.02 (dd, J = 8.8, 2.9 Hz, 1H), 6.05 (s, 1H), 5.32 (s, 2H), 2.29 (s,
+
H). MS (ESI): m/z 446 [M+H] .
4
-((4-(4-Chloro-3-(trifluoromethyl)phenoxy)-3,5-difluorobenzyl)oxy)-6-methoxy
pyrimidin-2-amine (7b). To a solution of 5 (2.00 g, 5.88 mmol) in anhydrous THF (80
mL) was added sodium hydride (0.70 g, 17.64 mmol, 60% in mineral oil) at 0 °C, 6b
(0.84 g, 5.29 mmol) was added after the mixture was stirred at 0 °C for 0.5 h, and the
mixture was stirred at 70 °C overnight. Then, the reaction was quenched with NH Cl
4
solution, extracted with ethyl acetate (80 mL × 3), washed with brine, dried over NaSO4,
filtered, and concentrated, the residue obtained was purified with column
chromatography (petroleum ether/ethyl acetate = 1:1) to yield intermediate 7b as a white
1
solid (0.70 g, 30%). H NMR (400 MHz, chloroformꢀd) δ 7.41 (d, J = 8.8 Hz, 1H), 7.30
(d, J = 2.9 Hz, 1H), 7.13–7.05 (m, 2H), 7.01 (dd, J = 8.8, 2.9 Hz, 1H), 5.56 (s, 1H), 5.29
+
(s, 2H), 3.86 (s, 3H). MS (ESI): m/z 462 [M+H] .
4
-((4-(4-Chloro-3-(trifluoromethyl)phenoxy)-3,5-difluorobenzyl)oxy)-6-methylp
yrimidin-2(1H)-one (8a). To a solution of 7a (250 mg, 0.56 mmol) in acetic acid (15 mL)
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