Discovery of a Novel Series of Imidazo[1,2-a]pyrimidine Derivatives as Potent and Orally Bioavailable Lipoprotein-Associated Phospholipase A2 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01024

Inhibition of lipoprotein-associated phospholipase A₂ (Lp-PLA₂) has been suggested to be a promising therapeutic strategy for several inflammation-associated diseases, including atherosclerosis, Alzheimer's disease, and diabetic macu

Full text of DOI:10.1021/acs.jmedchem.5b01024

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Article
Discovery of a Novel Series of Imidazo[1,2-a]pyrimidine Derivatives as Potent
and Orally Bioavailable Lipoprotein-Associated Phospholipase A2 Inhibitors
Xinde Chen, Wenwei Xu, Kai Wang, Mingguang Mo, Wei Zhang, Lili
Du, Xiaojing Yuan, Yechun Xu, Yi-Ping Wang, and Jianhua Shen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01024 • Publication Date (Web): 19 Oct 2015
Downloaded from http://pubs.acs.org on October 22, 2015
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Discovery of a Novel Series of Imidazo[1,2-a]pyrimidine Derivatives as
Potent and Orally Bioavailable Lipoprotein-Associated Phospholipase
A Inhibitors
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†
†
Xinde Chen, Wenwei Xu, Kai Wang, Mingguang Mo, Wei Zhang, Lili Du, Xiaojing
Yuan, Yechun Xu, Yiping Wang,* Jianhua Shen*
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China
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ABSTRACT
Inhibition of lipoproteinꢀassociated phospholipase A (LpꢀPLA ) has been suggested to be
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a promising therapeutic strategy for several inflammationꢀassociated diseases, including
atherosclerosis, Alzheimer’s disease, and diabetic macular edema. Herein, we report the
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discovery of
a
novel series of LpꢀPLA2 inhibitors constructed on an
imidazo[1,2ꢀa]pyrimidine scaffold through a conformational restriction strategy.
Structureꢀactivity relationship (SAR) analysis resulted in identification of several
compounds with high potency in vitro and good metabolic stability in liver S9 fractions.
Compounds 7c and 14b selected for further exploration in vivo demonstrated excellent
pharmacokinetic profiles and exhibited significant inhibitory efficacy in SD rats upon
oral dosing.
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INTRODUCTION
Lipoproteinꢀassociated phospholipase A2 (LpꢀPLA2) previously known as
plateletꢀactivating factor acetylhydrolase (PAFꢀAH), is a member of the phospholipase A2
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superfamily. LpꢀPLA is produced mainly by macrophages, monocytes, Tꢀlymphocytes,
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–4
and mast cells.
In human plasma, LpꢀPLA circulates mainly with lowꢀdensity
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5
lipoprotein and can hydrolyze oxidized modified phosphatidylcholine to generate
6
oxidized free fatty acids and lysophosphatidylcholine (lysoꢀPC), and both products have
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, 8
been shown to induce inflammatory responses. LysoꢀPC, for example, has been found
to be involved in leukocyte activation, induction of apoptosis, and mediation of
9
, 10
endothelial dysfunction.
Consistent with these results, LpꢀPLA has been considered to
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11–13
be a promising therapeutic target for atherosclerosis.
Very recently, it was reported
that inhibition of LpꢀPLA might protect against the damage to the bloodꢀbrain barrier
2
caused by hyperglycemia and hypercholesterolemia, suggesting that inhibition of
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4
LpꢀPLA could be used for treating neurodegenerative diseases. In addition, inhibition
2
of LpꢀPLA was recently reported to have beneficial effects against diabetic macular
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1
5, 16
edema (DME),
breakdown of the bloodꢀretinal barrier.
To date, a number of LpꢀPLA2 inhibitors have been reported, including
a severe complication whose pathogenesis is directly related to the
1
7, 18
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20, 21
22, 23
24
azetidinones, oximes,
amides of xanthurenic acid,
carbamates, and
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5–39
pyrimidones.
Among them, several compounds have progressed into clinical trials
4
0
(
Figure 1), and darapladib has been the most rapidly developed. Although darapladib
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was ineffective in two phaseꢀIII trials focusing on coronary heart diseases,
another
phaseꢀII trial revealed that oral administration of darapladib for 3 months modestly
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reduced macular edema and enhanced vision acuity in centerꢀinvolved DME patients.
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Two other compounds, rilapladib (once also in phaseꢀII trial for atherosclerosis ) and
GSK2647544 (structure not disclosed), which are in phaseꢀII and phaseꢀI trials,
respectively, are in development for the treatment of Alzheimer’s disease.
`
Figure 1. Chemical structures of LpꢀPLA inhibitors in clinical trials.
2
Previously, our research team reported a series of darapladib analogs with high
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potency in vitro.
However, high oral efficacy through further structural modifications
was hard to obtain because of low plasma exposure. Likewise, darapladib as the initial
compound also shows relatively low oral plasma exposure because of low oral
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bioavailability.
We postulated that the limited oral plasma exposure of darapladib and
its analogs should be mainly due to their large molecular sizes and high lipophilicity
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7
(darapladib: molecular weight (MW), 666.78; cLogP, 8.33). Therefore, we sought to
develop a new class of LpꢀPLA inhibitors with lower MW and cLogP to obtain better
2
pharmacokinetic properties, so that sufficient plasma concentrations of these compounds
could be achieved to effectively inhibit LpꢀPLA enzyme in plasma after oral
2
administration.
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In our ongoing exploration of novel molecular scaffolds of LpꢀPLA inhibitors, we
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recently undertook rational drug design on compound 1 (Figure 2A), which was newly
developed by GlaxoSmithKline (GSK) scientists, and reported to have high potency in
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vitro. Moreover, compound 1 provided a smaller molecular size as well as lower
lipophilicity (MW, 502.88; cLogP, 4.48) compared with darapladib, and our preliminary
pharmacokinetic experiments revealed that compound 1 demonstrated greater plasma
exposure than darapladib when dosed orally to SD rats (Table 1). Nevertheless, besides
these virtues, compound 1 showed limited elimination halfꢀlife in vivo (t1/2 = 2.08 h).
Through further evaluation of metabolic stability in rat liver S9 fractions, we confirmed
that the high clearance of compound 1 in vivo was mainly due to its poor metabolic
stability (CL = 74.4 mL/min/kg). Structural restriction has been reported to contribute
int
4
8–51
to
greater metabolic stability,
and
bicyclic structures,
such
as
imidazo[1,2ꢀa]pyrimidine, have been commonly used as a versatile building block in
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medicinal chemistry,
so we were tempted to use a structural restriction strategy to
replace the pyrimidone group in compound 1 with a new imidazo[1,2ꢀa]pyrimidine
scaffold (Figure 2A). Based on this design, the firstꢀprepared compound (7a)
demonstrated better metabolic stability in human and rat liver S9 fractions as compared
with compound 1, and displayed good potency against recombinant human LpꢀPLA2
(
rhLpꢀPLA ). Encouraged by these results, we undertook systematic structureꢀactivity
2
relationship (SAR) explorations of imidazo[1,2ꢀa]pyrimidine derivatives.
Herein, we describe the discovery of imidazo[1,2ꢀa]pyrimidine derivatives as a novel
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class of LpꢀPLA inhibitors. These compounds are potent in vitro, metabolically stable,
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and exhibit excellent pharmacokinetic profiles and high inhibitory efficacy in vivo. The
synthesis and pharmacological evaluation are reported.
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Figure 2. (A) Design of imidazo[1,2ꢀa]pyrimidine derivatives as LpꢀPLA inhibitors. (B)
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Systematic SAR explorations of imidazo[1,2ꢀa]pyrimidine derivatives.
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Table 1. Pharmacokinetic parameters of compound 1 and darapladib after oral
a
administration to SD rats
compd
AUC0ꢀ∞ (µgꢁh/mL)
t1/2 (h)
2.08
Cmax (µg/mL)
0.78
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b
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c
darapladib
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0.15
a
b
c
n = 5 animals/group; administered at 25 mg/kg; administered at 50 mg/kg. Data are the
mean.
CHEMISTRY
The synthesis of imidazo[1,2ꢀa]pyrimidine derivatives 7a-e is summarized in Scheme 1.
A
nucleophilic
substitution
reaction
between
benzyl
mercaptan
and
4ꢀchloropyrimidinꢀ2ꢀamine in the presence of sodium hydride yielded intermediate 2, and
5
5
subsequent cyclization of 2 provided intermediate 3. Direct arylation of 3 with
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ꢀbromopyrimidine using palladium(II) acetate as a catalyst gave intermediate 4, which
was oxidized using m-CPBA to provide 5 as a mixture of the sulphone and sulfoxide. A
nucleophilic substitution reaction between 5 and hydroxyl or thiol derivatives 6a-e (6a-e:
see Supporting Information for synthesis details) using sodium hydride as a base afforded
compounds 7a-e.
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a
Scheme 1. Synthesis of analogs 7a-e
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a
Reagents and conditions: (a) 4ꢀchloropyrimidinꢀ2ꢀamine, sodium hydride, THF, rt; (b)
2ꢀbromoacetaldehyde diethyl acetal, 48% HBr aqueous solution, 80 °C, 6 h, then rt,
NaHCO , 2, 70 °C, 2 h; (c) 5ꢀbromopyrimidine, Pd(OAc) , KOAc, DMA, 140 °C; (d)
3
2
m-CPBA, DCM, rt; (e) sodium hydride, THF, rt.
Compounds 10a-d were synthesized starting from benzyl alcohol 6c and
ꢀchloropyrimidinꢀ2ꢀamine using a method similar to that described for intermediate 4
4
(
Scheme 2). Compound 10e was obtained by an alternative route: bromination of 9 gave
intermediate 11, and subsequent Suzuki coupling of 11 and
1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)boronic acid using bis(triphenylphosphine)palladium(II)
(
dichloride as a catalyst gave the desired compound 10e. The synthesis of 10f was
commenced by a cyclization reaction between 8 and 12 in ethanol.
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a
Scheme 2. Synthesis of compounds 10a-f
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a
Reagents and conditions: (a) 4ꢀchloropyrimidinꢀ2ꢀamine, sodium hydride, THF, rt; (b)
2
ꢀbromoacetaldehyde diethyl acetal, 48% HBr aqueous solution, 80 °C, 6 h, then rt,
1
NaHCO , 8, 70 °C, 2 h; (c) R Br, Pd(OAc) , KOAc, DMA, 140 °C; (d) NBS, THF, rt; (e)
3
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(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)boronic acid, PdCl (PPh ) , Na CO , dioxane, H O,
2
3 2
2
3
2
microwave, 150 °C, 30 min; (f) ethanol, H O, reflux.
2
As shown in Scheme 3, the biaryl analogs 14aꢀm were synthesized in a manner
similar to that described for compounds 7aꢀe starting from 5 and the corresponding
benzyl alcohol 13aꢀm (13aꢀm: see Supporting Information for synthesis details).
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Scheme 3. Synthesis of biaryl analogs 14aꢀm
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Reagents and conditions: (a) sodium hydride, THF, rt.
RESULTS AND DISCUSSION
SAR in vitro. Compounds were first evaluated for their inhibitory activity against
rhLpꢀPLA at two concentrations (100 nM and 10 nM) in vitro, and rhLpꢀPLA activity
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was measured using 2ꢀthioꢀPAF as the substrate. IC values against rhLpꢀPLA for those
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compounds with an acceptable inhibitory activity (>30% at 10 nM) were tested to
ascertain their inhibitory potency precisely.
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SAR analyses mainly focused on the influences of three regions on rhLpꢀPLA2
inhibitory activity (Figure 2B): the linker between the imidazo[1,2ꢀa]pyrimidine moiety
and diphenyl ether moiety; substituents at the 3ꢀposition of imidazo[1,2ꢀa]pyrimidine
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1
(
R ); diphenyl ether moiety.
Effects of the linker were first explored (Table 2). Compound 7b with the oxygen
atom directly connected to the diphenyl ether moiety showed a complete loss of potency,
and elongation of the linker to three carbon atoms (7d) was also not tolerated. Compound
7c with a single methylene spacer demonstrated comparable potency with the initial hit
compound 7a. Replacement of the oxygen atom with a sulfur atom (7e) reduced potency
markedly. These results suggested that rhLpꢀPLA inhibitory activity was affected
2
considerably by the length of the linker and linking atom.
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Table 2. SAR of the linker
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%
inhibition against
compd
X
n
rhLpꢀPLA
2
IC （nM）
50
100 nM
10 nM
7
a
O
O
O
O
S
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1
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11.1
7b
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10.6
7d
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7e
1
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98
78
88
3.4
a
darapladib
92
0.7
a
Reported IC = 0.25 nM.
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On the basis of the high inhibitory activity, 7c was selected for further optimization.
1
Our next objective was to investigate the influences of R on potency, and representative
compounds are shown in Table 3. Among the modifications, we found that removal of the
pyrimidine ring resulted in a loss of potency (9). Replacement of the pyrimidine ring with
a pꢀmethoxyꢀphenyl ring was not well tolerated (10a), whereas the pyridine derivatives
1
0b and 10c restored potency to a certain degree. These SARs suggested that an aromatic
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ring and hydrogenꢀbond acceptor(s) were required at this moiety in the molecule. This
hypothesis was further confirmed by the 2ꢀmethoxyꢀpyrimidine compound 10d and
1
ꢀmethylꢀpyrazole compound 10e: both compounds were more potent than the
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pꢀmethoxyꢀphenyl compound 10a. Loss of potency after relocation of the pyrimidine ring
to the 2ꢀposition (10f) suggested that substitution at the 3ꢀposition was favored for
potency.
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Table 3. Influences of R on the inhibitory activity of rhLp-PLA
2
%
inhibition against
1
2
compd
R
R
rhLpꢀPLA
2
IC （nM）
50
100 nM
89
10 nM
7
c
H
H
H
H
H
H
H
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9
H
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1
0a
0b
0c
0d
0e
0f
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76
19.3
16.0
13.4
16.9
1
78
1
79
1
78
1
H
12
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6
Finally, we explored the effects of the diphenyl ether moiety. As shown in Table 4,
we first investigated the influences of the central benzene ring. Introduction of a methyl
substituent ortho to the diphenyl ether linkage resulted in a decrease in potency (14a). In
contrast, introduction of a small, electronꢀwithdrawing fluorine group (14b) or cyano
group (14c) increased the activity as compared to 7c. Encouraged by the results of 14b
and 14c, we then turned our attention to the introduction of electronꢀwithdrawing groups
in the central benzene ring. However, compounds with a bulkier substituent such as a
chloro group (14d) or trifluoromethyl group (14e) were less potent. In addition, further
incorporation of a fluorine group in the fluorineꢀsubstituted derivative 14b maintained
inhibitory potency (14f). However, replacement of the central benzene ring with a
pyridine ring or fiveꢀmembered heteroaromatic ring provided a lower cLogP value was
not tolerated (data not shown). These SARs led us to speculate that slight electron
deficiency and hydrophobicity of the central aromatic ring was beneficial for the binding
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
to the LpꢀPLA enzyme. Next, keeping the diꢀfluorine benzene ring constant, we
2
examined the effects of the terminal benzene ring. Representative compounds are shown
in Table 4. A benzene ring without substituents (14g) was less active than compound 14f.
Replacement of the chlorine atom with a fluorine atom (14h) or removal of the chlorine
atom (14i) resulted in compounds with slightly decreased potency. Replacement of the
trifluoromethyl group with a chlorine atom (14j) also resulted in a slight decrease in
potency, and migration of the trifluoromethyl group to the para position (14k) was poorly
tolerated. As with the central benzene ring, alteration of the terminal benzene ring to the
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pyridine ring also resulted in lowꢀactivity compounds (14l and 14m). Thus, we
speculated that the terminal benzene ring played a key role in hydrophobic interactions
with LpꢀPLA enzyme, and LpꢀPLA inhibitory activity was obviously affected by the
2
2
10
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12
13
14
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substituents on the terminal benzene ring.
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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. Effects of the diphenyl ether on the inhibitory activity of rhLp-PLA2
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
compd
rhLpꢀPLA
2
IC （nM）
50
1
00 nM
10 nM
55
7c
89
10.6
1
4a
4b
4c
4d
4e
4f
4g
4h
70
15
1
95
78
3.7
2.9
1
97
68
1
74
11
1
3
F
1
100
47
82
2.3
O
F
1
1
1
96
66
47
57
29
8
6.5
10.9
6.3
1
4i
91
4j
94
1
4k
4l
14m
79
1
57
21
To further gain insight into the binding interactions of these
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imidazo[1,2ꢀa]pyrimidine derivatives with LpꢀPLA enzyme, docking studies were then
2
performed to investigate the docking pose of compound 14b into LpꢀPLA (Figure 3).
2
Key interactions between this compound and LpꢀPLA are hydrogen bonds and several
2
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hydrophobic interactions. Q352 formed two hydrogen bonds with 14b. The pyrimidine
1
ring in position R formed an edgeꢀtoꢀface πꢀπ stacking interaction with W298. In the
case of imidazo[1,2ꢀa]pyrimidine ring, the pyrimidine moiety formed hydrophobic
interactions with H351 and L153. The central and terminal benzene rings are participated
in a number of hydrophobic interactions, most notably to F110, G154, Y160, and F357.
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0
1
2
3
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9
0
1
2
3
4
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6
7
8
9
0
Figure 3. Docking pose of compound 14b showing key interactions with LpꢀPLA2
binding site. Carbon, oxygen, nitrogen, fluorine and chlorine atoms are colored cyan, red,
blue, limon, and green, respectively. Compound 14b is represented as a ballꢀandꢀstick
model. The protein is shown as light grey cartoons. (A) The binding pocket is shown by
molecular surface. (B) Interactions between 14b and LpꢀPLA . Hydrogen bonds are
2
depicted as dotted lines. Key residues at the binding site are shown as sticks.
To account for any nonꢀspecific binding effects in plasma, selected compounds with
relatively good activity against rhLpꢀPLA were further evaluated for their potency
2
against LpꢀPLA in human plasma. As shown in Table 5, potency against LpꢀPLA in
2
2
human plasma correlated well with potency against rhLpꢀPLA . For example, compounds
2
1
4c and 14f act as the most potent compounds against rhLpꢀPLA , displayed rather high
2
potency against LpꢀPLA in human plasma, and potency against rhLpꢀPLA of 14c and
2
2
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1
4f (14c, 68% at 10 nM; 14f, 82% at 10 nM) were similar to their potency against
LpꢀPLA in human plasma (14c, 83% at 10 nM; 14f, 87% at 10 nM). These findings
2
suggested that nonꢀspecific binding had a negligible effect on the binding affinity
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59
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between these imidazo[1,2ꢀa]pyrimidine derivatives and LpꢀPLA enzyme in human
2
plasma. Before we evaluated efficacy in vivo, we also assessed the activity of selected
compounds against LpꢀPLA in rat and mouse plasma (Table 5). Surprisingly, potency in
2
the plasma of rats and mice differed from the potency observed in human plasma for
these imidazo[1,2ꢀa]pyrimidine derivatives. For instance, compound 14f displayed a high
activity against LpꢀPLA in human plasma (87% at 10 nM), but showed a relatively low
2
activity in rat plasma (26% at 100 nM). In addition, in mouse plasma, all of these
imidazo[1,2ꢀa]pyrimidine derivatives showed quite low potency against the LpꢀPLA2
enzyme. Despite this species difference, most of these imidazo[1,2ꢀa]pyrimidine
derivatives provided ＞50% inhibition of LpꢀPLA in rat plasma at 100 nM, which were
2
better than that observed for compound 1.
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1
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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. Inhibitory activity against Lp-PLA in human plasma, rat plasma, and
2
mouse plasma
%
inhibition in plasma
rat
0
1
2
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8
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8
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0
1
2
3
4
5
6
7
8
9
0
compd
human
mouse
100 nM
100 nM
87
10 nM
56
100 nM
49
10 nM
1 µM
53
56
28
13
0
7a
2
7c
91
58
76
10
13
11
14
10
1
0e
4b
4c
4f
85
42
64
1
84
60
70
1
94
83
68
1
96
87
26
8
1
96
86
48
91
99
18
96
darapladib
100
93
98
84
Metabolic stability tests in liver S9 fractions. Compounds with relatively good activity
against rhLpꢀPLA were also selected to evaluate their metabolic stability in human and
2
rat liver S9 fractions (Table 6). Compound 7c with a single methylene spacer displayed
much better metabolic stability compared with the hit compound 7a. Replacement of the
pyrimidine ring with 1ꢀmethylꢀpyrazole (10e) also resulted in good metabolic stability.
Compounds with different substituents in the central benzene ring demonstrated different
metabolic stability. The cyanoꢀsubstituted derivative 14c showed relatively poor
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metabolic stability, whereas the fluorineꢀsubstituted derivatives 14b and 14f were quite
stable in human and rat liver S9 fractions.
Table 6. Metabolic stability in liver S9 fractions of selected compounds
liver S9 stability (t , min)
10
11
12
13
14
15
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17
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19
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44
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47
48
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50
51
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53
54
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56
57
58
59
60
1
/2
compd
human
rat
7
a
42
110
348
7
c
116
180
331
106
212
33
a
10e
4b
4c
NC
169
40
1
1
14f
1
196
51
a
NC = cannot be calculated. The halfꢀlife was too long to be calculated in this system.
Pharmacokinetic evaluation. For assessment of the pharmacokinetic properties of these
imidazo[1,2ꢀa]pyrimidine derivatives in vivo, compounds 7c, 14b, and 14c were selected
for evaluation in male SD rats through oral administration. The concentrationꢀtime curves
are shown in Figure 4 (with the data of compound 1 and darapladib included for
comparison), and the key pharmacokinetic parameters are summarized in Table 7.
Pharmacokinetic studies showed that the in vivo halfꢀlives of these compounds correlated
well with the metabolic stability in rat liver S9 fractions. With good metabolic stability in
rat liver S9 fractions, in vivo halfꢀlives of 7c and 14b were 9.44 h and 13.34 h,
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respectively, much longer than that of compound 1 (2.08 h). Compound 14c was less
stable in rat liver S9 fractions, and displayed a shorter in vivo halfꢀlife (3.24 h).
Furthermore, 7c, 14b, and 14c achieved a maximum concentration (Cmax) of 3.52, 2.60
and 1.40 µg/mL, respectively, whereas the Cmax of compound 1 was 0.78 µg/mL.
Consequently, AUC values for 7c (88.05 µgꢁh/mL), 14b (78.14 µgꢁh/mL), and 14c (16.13
µgꢁh/mL) were higher than that for compound 1 (3.38 µgꢁh/mL). Taken together, these
data suggested that imidazo[1,2ꢀa]pyrimidine derivatives possess promising
pharmacokinetic profiles in vivo.
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
Table 7. Pharmacokinetic parameters of selected compounds after oral
a
administration to SD rats
compd
AUC0ꢀ∞ (µgꢁh/mL)
88.05
t1/2 (h)
9.44
Cmax (µg/mL)
3.52
7c
1
4b
78.14
13.34
3.24
2.60
14c
16.13
1.40
a
n = 5 animals/group, administered at 25 mg/kg. Data are the mean.
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Figure 4. Concentrationꢀtime curves of selected compounds after oral administration to
SD rats (n=5). Compounds 7c, 14b, 14c and 1 were administrated at 25 mg/kg,
darapladib was administered at 50 mg/kg.
Assessment of the inhibitory activity of Lp-PLA in vivo. Based on their very
2
promising profiles, compounds 7c and 14b were selected for evaluating inhibitory
activity in male SD rats through oral administration at a single dose of 25 mg/kg, with the
data for compound 1 and darapladib included for comparison (Figure 5). The relatively
low inhibitory activity of compound 1 in vivo could be due to its modest pharmacokinetic
profile and relatively low potency against LpꢀPLA in rat plasma in vitro. With much
2
better pharmacokinetic properties and slightly greater potency against LpꢀPLA in rat
2
plasma in vitro, 7c and 14b exhibited much better inhibitory activity in vivo when
compared with compound 1. Both compounds produced ≈75% inhibition of LpꢀPLA2
activity after 5 h and could effectively inhibit LpꢀPLA activity over 24 h.
2
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Figure 5. Relative activity of LpꢀPLA in the serum of SD rats after a single oral dose of
2
25 mg/kg (n = 5).
CONCLUSION
Among reported LpꢀPLA inhibitors, darapladib has been the most rapidly developed
2
compound. However, darapladib demonstrated low oral bioavailability due to its
physicochemical properties, such as high values of MW (666.78) and cLogP (8.33). In an
effort to find potent and orally bioavailable LpꢀPLA inhibitors, we focused on the
2
development of compounds with lower values of MW and cLogP.
In this work, we chose compound 1 (MW, 502.88; cLogP, 4.48) from GSK as a
starting point. In view of the poor metabolic stability of compound 1, structural
modification using conformational restriction yielded imidazo[1,2ꢀa]pyrimidine as a
novel scaffold. SAR explorations resulted in identification of several
imidazo[1,2ꢀa]pyrimidine derivatives with good inhibitory activity in vitro and favorable
metabolic stability in liver S9 fractions. Several compounds that proceeded to
pharmacokinetic evaluation in vivo showed much better pharmacokinetic profiles
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compared with those observed for darapladib and compound 1. Compounds 7c and 14b
selected for efficacy assessment in vivo demonstrated robust inhibitory potency in male
SD rats at a single oral dose of 25 mg/kg, this activity was comparable with that for
darapladib and much better than that for compound 1.
10
11
12
13
14
15
16
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18
19
20
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22
23
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25
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29
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41
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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59
60
Taken together, results generated so far suggest that imidazo[1,2ꢀa]pyrimidine
derivatives with reasonable physicochemical properties, are potent, and orally
bioavailable LpꢀPLA inhibitors. In this way, they can serve as valuable probes to further
2
study the role of LpꢀPLA in vivo across various inflammationꢀassociated diseases. We
2
also observed a species difference across humans, rats and mice. Additional studies on
identification of compounds to overcome this species difference are underway.
EXPERIMENTAL SECTION
5
7
In vitro assay to measure the inhibitory activity of Lp-PLA2. Activities against
recombinant human LpꢀPLA (rhLpꢀPLA ), human plasma, rat plasma and mouse plasma
2
2
LpꢀPLA were measured using 2ꢀthioꢀPAF as the substrate. Briefly, 10 µL of the
2
rhLpꢀPLA enzyme (or plasma) and 10 µL of a DMSO solution of the compound were
2
added to 0.1 mol/L TrisꢀHCl (pH 7.2) containing 1 mmol/L EGTA, 50 µmol/L 2ꢀthioꢀPAF
and 10 µL of 2 mmol/L 5,5′ꢀdithiobis (2ꢀnitrobenzoic acid) in a total volume of 200 µL.
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:
Vmaxꢃꢄꢅꢆꢄꢇꢈꢉ ꢀ− Vmaxꢊꢋꢌꢈꢍ
Inhibitionꢀꢁ%ꢂ = 1 −
× 100%ꢀ
Vmaxꢆꢄꢎꢏꢐꢏꢑꢒ ꢀ− Vmaxꢊꢋꢌꢈꢍ
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4
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5
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5
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5
5
5
5
5
6
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: SIMMꢀ2014ꢀ08ꢀWYPꢀ18) and the Ethics
Committee of Shanghai Xuhui Central Hospital.
0
1
2
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0
1
2
3
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8
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0
1
2
3
4
5
6
7
8
9
0
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 NADPH (1.0 mM) in
2
Tris buffer (0.1 M, pH 7.4) were incubated in a 96ꢀwell plate at 37 °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 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
CLꢏꢈꢐ
=
×
ꢀ×
ꢀ×ꢀ
ꢓꢔꢀꢕꢓꢖꢗꢘꢀtꢙ/ꢚ mgꢀofꢀliverꢀS9ꢀprotein
kgꢀofꢀweight
Scaling factors for the human liver (145ꢀmg S9 protein/g liver and 24.3ꢀg liver/kg body
weight) and rat liver (179ꢀmg S9 protein/g liver and 40ꢀg liver/kg body weight) were
employed in this calculation.
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Page 27 of 58
Journal of Medicinal Chemistry
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Docking protocols. A crystal structure of LpꢀPLA (PDB ID: 3D59) was used as the
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receptor structure. The active site was defined according to the crystal structure of
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LpꢀPLA in complex with its substrate (PDB ID: 3D5E). Water molecules and ions
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were deleted. Receptor was prepared using Protein Preparation and Grid Preparation tools
in the Schrödinger Maestro interface. The 3D structures of substrate were generated with
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the Schrödinger program LigPrep. Automated docking was performed with Glide 5.5.
Standard Precision (SP) calculations were carried out with default settings. The
OPLSꢀ2005 force field was used for minimization and grid generation, while OPLSꢀ2001
force field was used for docking. The docked conformation of the compound with the
lowest energy was selected for study.
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.
Pharmacokinetic studies. Test compounds (0.5% carboxymethylcellulose sodium) were
subjected to pharmacokinetic studies on male SD rats with five animals in each group.
Test compounds were administered via the oral route by gavage at 25 mg/kg. Serial
specimens were collected preꢀdose as well as 1, 3, 5, 7, 24 and 48 h after administration
and quantified by liquid chromatographyꢀmass spectrometry. Pharmacokinetic parameters
were calculated from the mean serum concentration by nonꢀcompartmental analyses
using DAS software 2.1.1.
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Journal of Medicinal Chemistry
Page 28 of 58
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Assay to measure the inhibitory activity of Lp-PLA in vivo. A group of five male SD
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rats were fasted overnight, and administered test compounds by gavage at 25 mg/kg. Test
compounds were formulated in 0.5% carboxymethylcellulose sodium. Blood samples
were drawn preꢀdose as well as 1, 3, 5, 7 and 24 h after administration to measure
LpꢀPLA activity in serum. LpꢀPLA activity in the serum of SD rats was measured
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according to the method described for measurement of LpꢀPLA inhibitory activity in
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vitro.
Materials and methods. All reagents were purchased from commercial suppliers and
used without further purification unless otherwise stated. Yields were not optimized.
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Microwave reactions were performed in a Biotage Initiator. H NMR and C NMR
spectra were recorded on a VarianꢀMercucy Plusꢀ300 or a Bruker AC400 or a Bruker
AC500 NMR spectrometer using tetramethylsilane as an internal reference.
Lowꢀresolution mass spectra were determined on Agilent liquidꢀchromatography mass
spectrometer system that consisted of an Agilent 1260 infinity LC coupled to Agilent
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120 Quadrupole mass spectrometer (electrospray positive ionization; ESI).
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Highꢀresolution mass spectra were conducted on a 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,
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.6×150 mm, 30 °C, UV 240 nM, flow rate = 1.0 mL/min) with aqueous CH CN
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(50ꢀ90%) containing ammonium formate (10 mmol/L) for 25 min. All the assayed
compounds possess ≥95% purity. Column chromatography was performed on silica gel
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Journal of Medicinal Chemistry
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(
200ꢀ300 mesh), preparative TLC was performed on HSGF 254 (0.4ꢀ0.5 mm thickness;
Yantai Jiangyou Company, Yantai, Shangdong, China).
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4
-(benzylthio)pyrimidin-2-amine
(2).
To
a
suspension
of
4ꢀchloropyrimidinꢀ2ꢀamine (2.00 g, 15.44 mmol) and sodium hydride (1.85 g, 46.25
mmol, 60% in mineral oil) in THF (50 mL) at 0 °C，was added benzyl mercaptan (1.60
mL, 13.65 mmol) dropwise over a period of 0.5 h, the reaction mixture was stirred at rt
overnight. Then the reaction was quenched with NH Cl solution, diluted with ethyl
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acetate (50 mL). The organic layer was separated, washed with brine, part of the organic
solvent was evaporated under reduced pressure, the precipitates formed were collected by
1
filtration to give 2 as a white solid (1.80 g, 54%). H NMR (400 MHz, Chloroformꢀd) δ
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.85 (d, J = 5.5 Hz, 1H), 7.41 (d, J = 7.5 Hz, 2H), 7.32−7.18 (m, 3H), 6.50 (d, J = 5.5 Hz,
+
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H), 4.40 (s, 2H). MS (ESI): m/z 218 [M+H] .
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-(benzylthio)imidazo[1,2-a]pyrimidine (3). A solution of 2ꢀbromoacetaldehyde
diethyl acetal (7.80 mL, 66.00 mmol) and HBr (2.70 mL, 24.02 mmol, 48% aqueous
solution) in 95% ethanol (40 mL) was heated at 80 °C for 6 h. After the solution was
cooled down to rt, solid NaHCO (3.50 g, 41.66 mmol) was added in small portions,
3
follow by 2 (1.80 g, 8.29 mmol), the mixture was further stirred at 70 °C for 3 h. After the
reaction was complete, the solvent was evaporated under reduced pressure, DCM (50 mL)
was added to the residue, dried over MgSO , filtered, and concentrated. The residue was
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purified with column chromatography (DCM/MeOH = 20:1) to yield 3 (684 mg, 34%) as
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