Design and synthesis of imidazole and triazole derivatives as Lp-PLA 2 inhibitors and the unexpected discovery of highly potent quaternary ammonium salts

Article abstract of DOI:10.1016/j.bmcl.2013.01.029

New Lp-PLA₂ inhibitors were synthesized by the bioisosteric replacement of the amide group of Darapladib with an imidazole or a triazole. Unfortunately, the inhibitory activities of these derivatives were lower than that of Darapladib. But interestingly, a series of quaternary ammonium salts that were isolated as by-products during this synthetic work were found with high potency. Of these by-products, compound 22c showed a similar profile to Darapladib both in vitro and in vivo.

Full text of DOI:10.1016/j.bmcl.2013.01.029

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1187–1192
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of imidazole and triazole derivatives as Lp-PLA2
inhibitors and the unexpected discovery of highly potent quaternary
ammonium salts
⇑
⇑
Kai Wang, Wenwei Xu, Yang Liu, Wei Zhang, Wenyi Wang, Jianhua Shen , Yiping Wang
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
New Lp-PLA2 inhibitors were synthesized by the bioisosteric replacement of the amide group of Darapla-
dib with an imidazole or a triazole. Unfortunately, the inhibitory activities of these derivatives were
lower than that of Darapladib. But interestingly, a series of quaternary ammonium salts that were iso-
lated as by-products during this synthetic work were found with high potency. Of these by-products,
compound 22c showed a similar profile to Darapladib both in vitro and in vivo.
Received 3 September 2012
Revised 7 November 2012
Accepted 8 January 2013
Available online 16 January 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Bioisosteric replacement
Imidazole
Triazole
Lp-PLA2 inhibitor
Quaternary ammonium salt
With modern progress in understanding the role of inflamma-
tion in the pathogenesis of atherosclerosis,1 researchers have fo-
cused significant research efforts on the development of drugs
that target the inflammatory pathways. Of the large number of po-
tential therapeutic targets, lipoprotein-associated phospholipase
A2 (Lp-PLA2) has been regarded as a particularly promising one
over the past decade.2 The enzyme of Lp-PLA2, also known as plas-
ma platelet-activating factor acetylhydrolase (PAF-AH), is a 50 kDa,
calcium-independent lipase that is secreted by inflammatory cells
and binds mainly to low-density lipoprotein (LDL).3 Lp-PLA2 is
responsible for the hydrolysis of oxidized phospholipids within
LDL into two pro-inflammatory mediators, including lysophospha-
tidylcholine and oxidized non-esterified fatty acids.4 Both products
have been shown to elicit a series of inflammatory-immune re-
sponses within the arterial endarterium, ultimately leading to the
initiation and progression of the atherosclerotic plaque.5 The inhi-
bition of Lp-PLA2 activity is therefore expected to slow the hydro-
lysis process, reduce the content of the afore mentioned pro-
inflammatory factors in circulation and stabilize the vulnerable
plaque, as indicated by many animal studies.6 Several epidemio-
logical studies7 have also presented a positive correlation between
the elevated level of Lp-PLA2 and the increased risk of adverse
cardiovascular events. Lp-PLA2 inhibitors as new therapeutic
agents have great potential to further reduce the clinical event
rates that cannot be entirely eliminated by current pharmaceuti-
cals (e.g., lipid-lowering and antiplatelet agents).
Several different classes of selective and reversible Lp-PLA2
inhibitors have been discovered in industrial and academic
groups.8 Some of these inhibitors have progressed into clinical
studies, such as SB-435495,8a Rilapladib8b and Darapladib,8c which
were all developed by GSK. Darapladib (Fig. 1) is now undergoing
phase III trials to determine its clinical efficacy in reducing the inci-
dence of death, myocardial infarction or ischemic stroke.
As the first drug in its class, Darapladib does not necessarily
represent the perfect drug candidate in terms of its physiochemical
properties, such as high logP value and low oral bioavailability.
Further optimization of Darapladib would be desirable and several
different research groups are already heavily involved in optimiza-
tion programs. A survey of available patents and general literatures
revealed that modifications to the pyrimidone moiety, the hydro-
phobic amino-chain and the biphenyl groups in Darapladib have
been thoroughly investigated, whereas any exploration of the cen-
tral amide has not yet been attempted. Bioisosteric replacement is
a commonly used and effective strategy in drug design. We envis-
aged that the replacement of the amide group in Darapladib with a
heterocyclic bioisostere would represent an interesting strategy for
the development of new Darapladib analogues (Fig. 2). Imidazole
and triazole were selected to evaluate this idea, mainly because
they frequently appeared in approved drugs and have been used
⇑
Corresponding authors. Tel.: +86 21 20231969; fax: +86 21 20231971 (J.S.);
Tel.: +86 21 50806733 (Y.W.).
E-mail addresses: jhshen@mail.shcnc.ac.cn (J. Shen), ypwang@mail.shcnc.ac.cn
(Y. Wang).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.029

1188
K. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1187–1192
O
O
O
N
O
O
O
N
CF3
S
N
O-
P
O
O
F
O
N+
N
Darapladib (GSK, phase III)
C16-PAF
Figure 1. The structure of Darapladib and C16-PAF (a substrate of Lp-PLA2).
O
O
N
N
S
N
S
N
N
O
F
F
X
N
N
O
N
Et2N
Ar
Ar
Et2N
N
I
S
3
2
N
F
O
O
N
X
1
R1
N
N
N
R2
S
N
S
O
N
F
F
X
N
N
X = CH, N
NEt2
Ar
Darapladib
NEt2
Ar
II
Figure 2. Design of imidazole/triazole derivatives as Lp-PLA2 inhibitors.
as amide bioisosteres to form H-bonds with proteins through their
N-3 sites in the same way as the O-atom of the original amide.
Herein, we describe our work on the preliminary structure–activity
relationship (SAR) studies of the imidazole and triazole derivatives.
Furthermore, we describe our discovery of a series of quaternary
ammonium salts as highly potent Lp-PLA2 inhibitors.
The synthesis of the imidazole derivatives together with part of
the triazole derivatives is outlined in Scheme 1. Using the method
initially reported by Thompson,9 alcohol 1 was converted to the
azide 2 by reaction with diphenylphosphoryl azide (DPPA). A sub-
sequent Staudinger reaction provided the corresponding amine 3,
which underwent a condensation reaction with ethyl 2-oxocyclop-
entanecarboxylate in the presence of tetraethyl orthosilicate10 to
give 4, which was then treated with trimethylsilyl isothiocyanate
to produce 5. The subsequent treatment of 5 with 4-fluorobenzyl
bromide in boiling acetone gave rise to 6. The intermediates repre-
sented by structure 1 were prepared according to procedures pre-
viously published in the literature and have therefore not been
illustrated here.
material 1-bromo-4-isothiocyanoatomethylbenzene was con-
verted to 7 via a condensation reaction with 2-hydroxyacetohyd-
razide, followed by oxidative desulfurization11 and Suzuki
coupling to form 9. Azidation of 9 with DPPA, followed by catalytic
hydrogenation of the resulting azide gave amine 11, which was
subsequently transformed into 13 under the same conditions as
described for the conversion of intermediate 5 mentioned above.
Using DBU as the base instead of K2CO3, compound 6h was ob-
tained in excellent yield via the reaction of 13 with 4-fluorobenzyl
bromide in acetonitrile at ambient temperature. This process was
also applied for the preparation of the key intermediate 15 from
14, which was acquired itself from the hydroxymethylation of 13
in boiling formaldehyde solution. Compound 15 was then oxidized
to 16 using MnO2, followed by reaction with methylmagnesium
bromide to afford compound 17. The synthesis of the target com-
pounds 18a–h was completed by the reductive amination reac-
tions of compound 16 with the corresponding amines HNRaRb. In
a separate experiment, the azidation of 15 and subsequent cata-
lytic hydrogenation led to the formation of 20, which was ulti-
mately converted to compounds 21a and 21b by treatment with
the corresponding acylating agents. Compared with the synthetic
Compounds 6h, 15, 17, 18a–h and 21a–b were synthesized
according to a well established procedure (Scheme 2). The starting
O
O
O
N
N3
NH2
HN
OH
EtO
HN
S
N
N
d
N
N
b
e
c
S
N
a
X
X
X
N
F
N
N
N
N
N
X
R1
R2
X
R1
R2
X
R1
R2
N
N
N
2
3
R1
R2
1
R1
R2
R1
R2
6a-6s except for 6h
4
5
Scheme 1. Reagents and conditions: (a) DPPA, DBU, THF, reflux, 2–3 h; (b) Ph3P, THF–H2O, rt, 3 h; (c) ethyl 2-oxocyclopentanecarboxylate, Si(OEt)4, EtOH, reflux, 3 h; (d)
(CH3)3SiNCS, DMF, 140 °C , 3–4 h; (e) 4-fluorobenzyl bromide, K2CO3, acetone, reflux, 0.5–1 h.

K. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1187–1192
1189
O
HS
N
N
NH2
N3
N
N
EtO
HN
O
N
N
N
N
N
OH
N
H2NHN
a
N
N
c
b
d
N
OH
OH
e
f
NCS
N
N
N
OH
Ar
N
Br
Ar
Ar
N
11
10
9
8
Br
Br
7
Ar
12
6h
O
N
O
N
O
O
N
N
HN
HN
k
h
j
S
h
S
N
S
17
g
S
N
N
N
F
N
F
N
N
N
N
N
N
N
N
i
N
O
OH
Ar
OH
Ar
Ar
16
Ar
15
13
14
l
d
O
O
N
O
N
Ar:
N
N
N
e
ClCOCH3
ClSO2CH3
S
N
S
S
21a
21b
N
N
N
F
F
F
N
N
N
N
N
N
Ra
Rb
N
N3
CF3
20
NH2
19
Ar
18a-18h
Ar
Ar
Scheme 2. Reagents and conditions: (a) EtOH, reflux, 2 h then K2CO3, H2O, reflux, 1 h; (b) H2O2 (30 wt % sol in water), AcOH, CH2Cl2, reflux, 1 h; (c) 4-
trifluoromethylphenylboronic acid, Cs2CO3, Pd(Ph3P)4, dioxane, reflux, 18 h; (d) DPPA, DBU, THF, reflux, 3 h; (e) H2, 10%Pd/C, 1 atm, EtOH, rt, 12 h; (f) ethyl 2-
oxocyclopentanecarboxylate, Si(OEt)4, EtOH, reflux, 5 h; (g) (CH3)3SiNCS, DMF, 140 °C , 4 h; (h) 4-fluorobenzyl bromide, DBU, KI (cat.), CH3CN, rt, 5 h; (i) formaldehyde (37
wt% sol in water), reflux, 8 h; (j) MnO2, dioxane, 70 °C , 3 h; (k) CH3MgBr, THF, 0 °C , 2 h; (l) HNRaRb, NaBH(OAc)3, CH2Cl2, rt, 2 h.
Table 1
Inhibitory activity of imidazole/triazole derivatives: study of the linkage site of the biphenyl group
O
N
S
N
N
F
X
N
R1
R2
Compound
R1
R2
X
% Inhibition in rabbit plasma
10
20
34
lM
1
l
M
100 nM
NTa
CF3
CF3
6a
6b
–CH2CH2NEt2
–Et
CH
N
16
21
NT
6c
6d
–CH2CH2NEt2
–Et
n-C10H21
n-C10H21
CH
N
6
27
NT
NT
CF3
6e
6f
–Et
N
40
88
NT
42
n-C12H25
–H
–H
CH
NT
17
CF3
CF3
Cl
6g
6h
6i
CH
N
97
98
50
3
80
95
2
–H
–H
–H
–H
67
CH
CH
CH
6j
NT
NT
CH3
6k
28
a
Not tested. Usually, if inhibitory ratio was less than 50%, the compound would not be tested at lower concentration.
route shown in Scheme 1, this procedure was characterized by its
high-efficiency in producing the derivatives that were commonly
generated in the last step. Furthermore, over 9 steps in the prepa-
ration of the key intermediate 15, only compound 10 required
purification by column chromatography. All of the other interme-
diates could be purified either by recrystallization in moderate to
high yields or used directly in the subsequent step without isola-
tion. The synthetic route was therefore amenable to the prepara-

1190
K. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1187–1192
Table 2
Inhibitory activity of imidazole/triazole derivatives: modification at the R2 position
O
N
N
S
N
F
X
N
F3C
R2
Compound
R2
X
% Inhibition in rabbit plasma
% Inhibition in human plasma
100 nM
10 nM
10 nM
1 nM
6l
–Me
n-Pr
i-Pr
c-Pr
–CH2NMe2
–CH2NEt2
N
N
N
N
CH
CH
79
88
91
87
87
88
10
25
34
34
13
10
73
88
92
93
75
75
11
65
71
68
54
52
6m
6n
6o
6p
6q
6r
-CH2N
CH
79
10
67
21
6s
-CH2N
CH
86
11
69
58
15
17
18a
18b
18c
18d
–CH2OH
–CH(OH)CH3
–CH2NMe2
–CH2NEt2
–CH2N(i-Pr)CH3
–CH2N(c-Pr)CH3
N
N
N
N
N
N
73
90
88
85
85
86
23
29
21
21
13
16
65
89
85
78
77
77
58
68
62
60
35
38
N
Et
18e
-CH2N
N
N
N
N
90
76
82
67
20
16
11
15
82
69
71
51
64
23
57
45
O
18f
-CH2N
NMe2
18g
18h
-CH2N
-CH2N
N
COOCH3
21a
21b
Darapladib
–CH2NHCOCH3
–CH2NHSO2Et
N
N
67
75
99
8
7
82
67
68
95
44
49
80
tion of selected compounds in multi-gram quantities, although 12
steps were usually required.
Then we proceeded to evaluate the potential for further replace-
ments at the R2 position. The results of this work have been sum-
marized in Table 2. For synthetic convenience, we started with
some of the alkyl substituted compounds 6l–o. The increase in ste-
ric hindrance that occurred in the change from methyl to isopropyl
was found to improve the activity, with a similar trend also being
observed in the comparison of 15 with 17. In spite of this trend, we
decided not to explore the growth of the alky chain any further be-
cause the compounds invariably had high logP values and were not
suitable for the formation of pharmaceutical salts. The inclusion of
compounds with these physicochemical properties would very
likely lead to poor oral absorbability. With a robust and well estab-
lished synthetic route in hand, we proceeded to focus on the ami-
nomethyl substituted derivatives (6p–21b). Amino groups of
different shapes, sizes and electronic properties were therefore ta-
ken into consideration. We found that both the imidazole and tri-
azole derivatives showed moderate potency in the rabbit plasma
assay. The compounds behaved as potent inhibitors at 100 nM
(80–90% inhibition) but suffered an abrupt drop in activity at
10 nM (10–20% inhibition). In the human plasma assay, the com-
pounds performed much better. Several compounds, such as 18a
and 18b showed significant levels of potency, with both inhibiting
more than 60% of the enzyme activity at 1 nM. Unfortunately, this
activity was still lower than the positive control Darapladib and
the gap was even more pronounced in the rabbit plasma assay
(only 21% inhibition at 10 nM vs 82% inhibition for Darapladib).
The most potent compound in the series was 18e, which was only
poorly active in comparison to the n-propyl substituted compound
The assay for evaluating the potency of the compounds against
the enzyme of Lp-PLA2 was built in rabbit and human plasma
according to the reference method.12 The percentage inhibition of
the enzyme activity at the tested concentration was specified as
being indicative of the potency. Two kinds of scaffolds (repre-
sented by structures I and II) could be derived from Darapladib
(Fig 2), which were distinguishable by the linking site of biphenyl
group on the heterocycle (R2 and R1). In the first step, we estab-
lished which scaffold represented the preferred conformation. For
the biphenyl group at R2 (6a and 6b in Table 1), only limited inhi-
bition (20–30%) of Lp-PLA2 activity could be achieved at 10 lM
concentration in rabbit plasma, regardless of whether the R1 group
was an alkyl or aminoalkyl substituent. The introduction of the
more flexible and lipophilic decyl and biphenylmethyl groups gave
no further increase in the activity (6c–e). In contrast, the introduc-
tion of a long aliphatic chain or biphenylmethyl group at the R1 po-
sition (6f–h) provided significant improvements in the inhibitory
ratio (98% of 6h vs 34% of 6b at 10 lM), even in the absence of
any substitution at the R2 position. On the basis of these results,
scaffold II (i.e. with a biphenyl group at the R1 position) was se-
lected for further modification. Furthermore, the effect of substitu-
tion at the para-position of the biphenyl ring was also briefly
evaluated (6i–k). The results indicated that trifluoromethyl was
more effective than any of the other substituents tested in main-
taining the potency, likely because of its strong electrophilic and
high lipophilic properties.13

K. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1187–1192
1191
Table 3
Inhibitory activity of quaternary ammonium derivatives
O
N
S
N
N
F
X
N
R1
R2
Compound
R2
X
% Inhibition in rabbit plasma
% Inhibition in human plasma
100 nM
10 nM
10 nM
1 nM
Ph-4-F
Br
N
22a
22b
CH
CH
99
97
97
93
97
85
N
88
79
Br
4-F-Ph
N
Ph-4-F
22c
22d
N
N
N
97
90
91
10
99
93
95
71
Br
Ph-4-F
N
N
Br
22e
93
99
91
82
97
95
77
80
Darapladib
6m. These results indicated that the amino N-atom did not effec-
tively contribute to the potency of the compounds. Furthermore,
when the N-atom was acylated (21a and 21b) or hindered (18c,
d, f, h), a reduction in the activity was observed. The significant
loss in activity encountered following the replacement of the
amide in Darapladib with a heterocycle might result from the asso-
ciated reduction of the rotational degrees of freedom in the mole-
cule. That was, the rotatable C–N bond in the amide was restricted
in a cycle, preventing the molecule from reaching the optimal con-
formation necessary for a high level of affinity.
Although we were disappointed that the target compounds
were not as potent as what we had expected, we were encouraged
by the results achieved when we screened three quaternary
ammonium byproducts 22a–c (Table 3) that were generated from
the over alkylation of intermediate 5 with an excess of 4-fluorob-
enzyl bromide during the preparation of 6p, 6r, and 18a, respec-
tively.14 All of these compounds showed similar or higher levels
of potency to Darapladib in both the rabbit and human plasma as-
says. At this stage we became interested in establishing whether
the dramatically elevated potency observed in these compounds
was derived from the introduction of the 4-flurophenyl group or
the ammonium ion. To figure out this point, we synthesized com-
pounds 22d and 22e and compared their activities with that of 22c.
Compound 22d showed reduced activity, whereas 22e maintained
a similar level of potency to 22c. These results confirmed that the
ammonium ion was responsible for the significant improvement
observed in the activity. Interestingly, both our most potent inhib-
itors and PAF (Fig. 1) contained quaternary amines and it was
envisaged that these quaternary amines might bind to a similar po-
sition in the enzyme. But the accurate binding mode needs to be
determined by the establishment of the crystal structure of Lp-
PLA2 with our compounds.
In view of their good in vitro activities, the quaternary ammo-
nium compounds were selected for evaluation in the Apo-E mouse
model. The mice were fasted for 16 h prior to being given a single
dose of 50 mg/kg of the test compounds through an intraperitoneal
injection and blood samples were then draw at different time
points to measure the plasma Lp-PLA2 activity.15 Of the com-
pounds tested, 22c gave the best in vivo performance. As shown
in Figure 3, compound 22c effectively inhibited the plasma Lp-
PLA2 activity over a prolonged period of 24 h and showed a similar
profile to that of Darapladib during the first 12 h after dosing.
In summary, we have designed and synthesized a series of imid-
azole and triazole derivatives as Lp-PLA2 inhibitors using a bioisos-
teric replacement strategy. Unfortunately, these compounds
provided only moderate levels of activity. Pleasingly, however, a
series of quaternary ammonium salts that were obtained as
byproducts showed comparable activity to Darapladib both
in vitro and in vivo, as exemplified by compound 22c. Compounds
of this particular type could be valuable for researching the mech-
anism of drug action and help in the future design of Lp-PLA2
inhibitors.
Acknowledgments
This work was financially supported by grants from the
National Science & Technology Major Project ‘Key New Drug Crea-
tion and Manufacturing Program’ (Nos. 2012ZX09301001-001 and
2012ZX09103101-008).
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
01.029.
Figure 3. The relative plasma Lp-PLA2 activity in the Apo-E mouse after a single
dose (n = 5).

1192
K. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1187–1192
11. Ivanova, N. V.; Sviridov, S. I.; Shorshnev, S. V.; Stepanov, A. E. Synthesis 2006, 1,
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l
l
l
l
l
a
determined by the following equation:
Inhibitory rate (%) = 1ꢀ(CPMcompoundꢀCPMblank)/(CPMpositiveꢀCPMblank)⁄100%
The blank sample contained no plasma or test compound in assay buffer and
the positive sample contained no test compound.
13. Topliss, J. G. J. Med. Chem. 1972, 15, 1006.
14. Prior to the development of the synthetic route depicted in Scheme 2, the
synthesis of 18a was performed according to the method shown in Scheme 1
via the reaction of the corresponding intermediate 5 with p-fluorobenzyl
bromide. Compound 22c was obtained in this procedure as the byproduct. For
more synthetic details, please see the Supplementary data.
15. The serum Lp-PLA2 activity of the Apo-E mouse was measured using 2-thio-
PAF as the substrate. Briefly, 10
HCl (pH 7.2) containing 1 mmol/L EGTA, 50
2 mmol/L 5,50-dithiobis (2-nitrobenzoic acid) in a total volume of 200
l
L of the plasma were added to 0.1 mol/L Tris–
mol/L 2-thio-PAF and 10 L of
L. The
l
l
l
assay was performed using a plate reader to obtain absorbance values at
414 nm every minute. The Lp-PLA2 activity was calculated from the change in
absorbance per minute.
O
O
O
S
O-
O
P
O
N+
O
2-thio PAF
10. Zhao, Y. H.; Zhao, J. F.; Zhou, Y. Y.; Lei, Z.; Li, L.; Zhang, H. B. New J. Chem. 2005,
29, 769.