Triazole derivatives: A series of Darapladib analogues as orally active Lp-PLA2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.03.062

This Letter reports our efforts towards the optimization of our previously identified series of imidazole and triazole derivatives that lead to the discovery of a series of orally active Lp-PLA₂ inhibitors in C57 mice. These inhibitors are characterized by the presence of a diamine side chain in the molecules, such as 2c, 2f, and 4a. The introduction of the terminal-end amine succeeded in maintaining the in vitro activities at sub-nanomolar levels. The vivo activities could be greatly affected by variations in the two amines via modulating the metabolic stability and lipophilicity of the compounds.

Full text of DOI:10.1016/j.bmcl.2013.03.062

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2897–2901
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Triazole derivatives: A series of Darapladib analogues as orally active
Lp-PLA2 inhibitors
⇑
⇑
Kai Wang , Wenwei Xu , Wei Zhang, Mingguang Mo, Yiping Wang , Jianhua Shen
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:
This Letter reports our efforts towards the optimization of our previously identified series of imidazole
and triazole derivatives that lead to the discovery of a series of orally active Lp-PLA2 inhibitors in C57
mice. These inhibitors are characterized by the presence of a diamine side chain in the molecules, such
as 2c, 2f, and 4a. The introduction of the terminal-end amine succeeded in maintaining the in vitro activ-
ities at sub-nanomolar levels. The vivo activities could be greatly affected by variations in the two amines
via modulating the metabolic stability and lipophilicity of the compounds.
Received 28 January 2013
Revised 12 March 2013
Accepted 18 March 2013
Available online 26 March 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Atherosclerosis
Lp-PLA2 inhibitor
Imidazole
Triazole
Clinical studies have convincingly shown that a substantial
residual risk persists in cardiovascular patients despite aggressive
treatment with a variety of different therapeutic agents in combi-
nation, including lipid lowering drugs, hypotensors and antiplat-
elets. The development of new drugs with novel pharmacological
mechanisms to complement the existing pipelines and treat the
residual cardiovascular risks are therefore urgently required.1 In
recent years, a deeper understanding of the role of inflammation
in the pathogenesis of atherosclerosis has been developed and re-
sulted in the identification of a significant number of novel inflam-
matory mediators.2 Of these new mediators, lipoprotein-associated
phospholipase A2 (Lp-PLA2) has been identified as a crucial enzyme
in atherosclerosis, and is generally believed to catalyze the hydro-
lysis of oxidatively modified phospholipids at the sn-2 position
within low density lipoproteins (LDLs).3 The hydrolysis products,
including non-esterified fatty acids (NEFA) and lysophosphatidyl-
choline (LysoPC), are well-known pro-inflammatory factors that
are involved in every stage of the atherogenesis process.4 The accu-
mulation of evidence from both animal and epidemiologic studies
has demonstrated that Lp-PLA2 is an independent predictor of car-
diovascular risk.5 Accordingly, Lp-PLA2 has come to be regarded as
a promising new target for atherosclerosis, with this understand-
ing being boosted by the phase III development of Darapladib,6
In addition to this pyrimidone class of Lp-PLA2 inhibitors and its
analogues, several other Lp-PLA2 inhibitors have also been dis-
closed, including carbonyloxime,7 amides of xanthurenic acid,8
and, most recently, a series of carbamates.9 None of these three
classes, however, has been reported to be efficacious in vivo. To en-
rich the family of Lp-PLA2 inhibitors, we have attempted to explore
a series of Darapladib analogues by replacing the amide group of
Darapladib with an imidazole or a triazole ring (Fig. 1).10 The
resulting compounds, exemplified by compounds 1a and 1b (Ta-
ble 1), exhibited unsatisfactory levels of in vitro activity. In-house
structure–activity relationship (SAR) studies revealed that modifi-
cations to the 4-triflurobiphenyl and 4-flurobenzyl moieties had
little effect on potency enhancement. Consequently, we attempted
O
N
O
N
N
N
S
S
O
N
F
F
X
R
N
N
NEt2
which is
scientists.
a pyrimidone Lp-PLA2 inhibitor developed by GSK
⇑
Corresponding authors. Tel.: +86 21 50806733 (Y.W.); tel.: +86 21 20231969;
fax: +86 21 20231971 (J.S.).
CF3
Darapladib
CF3
X = CH, N
E-mail addresses: ypwang@mail.shcnc.ac.cn (Y. Wang), jhshen@mail.shcnc.ac.cn
(J. Shen).
These authors equally contributed to this work.
Figure 1. Replacement of the amide of Darapladib with an imidazole or a triazole.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.062

2898
K. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2897–2901
Table 1
Inhibitory activities of the synthesized compounds in rabbit and human plasma assays, as well as the IC50 values of selected compounds in recombinant human Lp-PLA2 assay
O
N
S
N
N
F
X
R
N
F3C
% Inhibition in rabbit plasma
Compoundc
X
-R
% Inhibition in human plasma
10 nM
rhLp-PLA2 IC50 (nM)
100 nM
10 nM
NTa
NT
NT
NT
N
N
1a
1b
2a
2b
CH
N
88
85
74
75
10
75
78
76
76
21
N
5
N
COOH
O
H
N
N
13
N
2c
2d
2e
N
N
N
98
91
97
72
36
77
94
91
95
0.91
NT
NEt2
N
N
N
0.14
NMe2
N
N
2f
N
N
97
97
75
76
94
92
0.67
0.23
N
N
2g
N
2h
2i
N
N
98
97
73
35
93
82
0.55
NT
NMe2
H
N
NEt2
N
H
N
2j
N
N
N
N
98
97
98
97
32
33
55
41
85
79
82
82
NT
NT
NT
NT
O
H
N
N
N
2k
2l
H
N
H
N
2m
N(i-Pr)2
Et
N
2n
2o
2p
2q
3a
N
97
95
98
99
89
75
33
54
46
54
92
81
89
86
81
NT
NMe2
NEt2
i-Pr
N
N
NT
N
1.60
NT
N
NMe2
NMe2
N
N
N
CH
1.72
NEt2
N
N
3b
CH
92
54
84
NT
N
N
N
4a
4b
N
N
96
96
76
71
92
94
0.24
0.81
NEt2
NMe2
NMe2
NEt2
4c
5a
N
N
96
95
71
74
93
93
0.89
0.46

K. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2897–2901
2899
Table 1 (continued)
Compoundc
X
-R
% Inhibition in rabbit plasma
% Inhibition in human plasma
rhLp-PLA2 IC50 (nM)
100 nM
10 nM
73
10 nM
91
NEt2
N
5b
5c
N
N
96
97
0.96
0.86
75
93
5d
N
96
99
76
82
92
95
0.95
N
Darapladib
0.10b
a
Not tested.
Reported IC50 = 0.25 nM.
b
c
Compounds 1a and b are published compounds in Ref. 10. Compounds 2a–5d are new compounds.
to increase the potency by making further modifications of the R
region, namely the side chain of the molecule. Herein, we describe
our recent work towards the optimization of the side chain that led
to the identification of orally active Lp-PLA2 inhibitors in C57 mice.
Compounds 2a–q were prepared according to a slightly modi-
fied version of our previously published procedure,10 which started
with the condensation reaction of 1-bromo-4-isothiocyanoatom-
ethylbenzene with 2-hydroxyacetohydrazide under basic condi-
tion, to afford intermediate 7 (Scheme 1). Subsequent oxidative
desulfurization followed by Suzuki coupling gave alcohol 9, which
was converted to the corresponding azide 10 in the presence of
diphenylphosphoryl azide (DPPA). The azide 10 was then con-
verted to amine 11 using catalytic hydrogenation condition. The
enamine reaction of 11 with ethyl 2-oxocyclopentanecarboxylate
provided intermediate 12, followed by a ring closing reaction in
the presence of trimethylsilyl isothiocyanate to give thiouracil
13, which was subsequently heated in a formaldehyde solution
at reflux to yield 14. The alkylation of 14 with 4-fluorobenzyl bro-
mide provided the key intermediate 15. Treatment of this interme-
diate with SOCl2 provided the corresponding chloride 16, which
was reacted with a variety of different amines to give the desired
target compounds 2a–q. Intermediate 15 was also oxidized to
aldehyde 18 which was subsequently treated with CH3MgBr to af-
ford 19, followed by chlorination and substitution reactions to af-
ford compounds 4a–c. The formaldehyde 18 was converted to
propylaldehyde 20 via a two steps procedure involving Wittig
reaction and catalytic hydrogenation. The aldehyde 20 was then
converted to compounds 5b and 5c using reductive amination
chemistry. The synthesis of 5a began with the hydrazinolysis of
d-valerolactone to give 21, which was subjected to the same proce-
dures used for the preparation of 9 to give intermediate 23. The
application of successive Swern oxidation, reductive amination
and hydroxymethylation reactions provided alcohol 26, which
was subsequently converted to 5a according to the routine proce-
dures described above. The preparation of the imidazole deriva-
tives 3a and 3b was performed in a similar manner to that of 5a,
except for the construction of intermediate 2711 and the oxidation
of alcohol 28.
electrostatic interactions with the protein. Only a few analogues
(2a–2d) were prepared initially to quickly evaluate the potential
of these compounds. Pleasingly, this limited panel of compounds
provided some exciting results. Based on the data shown in Table 1,
it was clear that the introduction of O-containing groups provided
no increase in activity (2a, 2b). In contrast, the introduction of N-
containing groups was greatly favored, with compound 2c display-
ing a level of activity comparable to that of Darapladib in rabbit and
human plasma assays. The pyridine-containing compound 2d was
less potent than the diethylamine derivative 2c, suggesting that
an sp3-N was more efficacious than an sp2-N. Consequently, the
type of ethylenediamine side chain became the focus of our study
and several different analogues were synthesized containing varia-
tions to the terminal amine. As expected, the frequently used
amines (2e–g) displayed similar levels of inhibitory activity in
rabbit and human plasma assays and increased potency against
rhLp-PLA2 in comparison with 2c. Furthermore, a one-carbon atom
extension between the two nitrogen atoms afforded compound 2h,
which also possessed good binding affinity. In general, the introduc-
tion of the amine to the terminal-end of the molecule has led to
significant improvements in the inhibitory activity, likely due to
the occurrence of an electrostatic interaction between the proton-
ated nitrogen atom and the protein. The introduction of the amine
was also favored in the imidazole series, although the resulting
compounds (3a, 3b) were less potent than the corresponding
triazoles. With this in mind, the decision was taken to continue
the optimization work primarily with the triazole series.
We then proceeded to explore the nature of the amine at the
internal-end of the side chain via the incorporation of a variety of
different substituents. In comparison with the methyl-substituted
derivatives, the corresponding un-substituted secondary amines
showed reduced levels of inhibition towards the enzyme (2i–m).
Although the replacement of the methyl group with an ethyl group
was well tolerated (2n), replacement with an isopropyl group sig-
nificantly weakened the inhibitory activity (2o). The inclusion of
the N atom within a ring system led to a significant loss in the activ-
ity (2p, 2q), demonstrating the importance of the flexibility of the
side chain to the high binding affinity. The results from previous
study10 implied a possible preference for the introduction of further
steric bulk alpha to the heterocycle. With this in mind, several com-
pounds were prepared bearing a branched methyl group to deter-
mine its effect on the potency (4a–c). Unfortunately, however, the
additional methyl group did not provide any further enhancement
to the inhibitory activities of the compounds. Finally, we assessed
the potential of replacing the N atom with an sp3-C atom. The result-
ing butylamine derivative 5a provided a similar level of inhibition to
that of the diamine analogue 2c, indicating that the internal-end N
of the side chain was not important to the observed in vitro activity
and could therefore be replaced without loss in potency.
The inhibitory activities of the compounds against the enzyme
of Lp-PLA2 were evaluated in rabbit and human plasmas according
to the reference method12 and expressed as the percentage inhibi-
tion of the enzyme activity (Table 1). The activities observed in
plasma factored in non-specific binding effects and were more
instructive for the SAR studies. The most promising compounds
were also evaluated their IC50 values using recombinant human
Lp-PLA2 (rhLp-PLA2).
Our optimization approach involved the introduction of an O- or
N-containing functional group into the side chain with the expecta-
tion that the group would form meaningful hydrogen-bonding or

2900
K. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2897–2901
O
EtO
HN
HS
N
N
N
N
R
N
N
O
N
N
N
9
R= -CH2OH
OH
H2NHN
a
d
N
c
f
b
OH
OH
NCS
N
10 R= -CH2N3
N
e
Br
11 R= -CH2NH2
6
Br
Br
Ar
7
12
8
Ar
O
N
O
O
N
O
O
N
HN
HN
N
N
N
S
S
S
S
N
N
S
N
N
N
N
N
F
F
F
N
N
N
N
N
h
g
N
N
N
N
N
j
i
k
OH
NR1R2
OH
Cl
Ar
13
Ar
15
Ar
Ar
14
Ar
17 NR1R2 = -NCH3COOEt
16
l
m
2a
2b-2q
O
O
N
O
N
O
O
N
N
N
N
N
S
N
S
S
S
N
S
N
N
N
N
N
N
N
F
F
F
F
F
N
N
N
N
N
N
N
N
N
p
o, e
j, k
n
NR1R2
O
OH
NR1R2
O
Ar
Ar
Ar
Ar
Ar
20
19
18
4a-4c
5b, 5c
HS
N
N
N
HO
N
N
N
O
23
24
25
O
R= -(CH2)4OH
N
N
N
b, c
a
q
r
O
H2NHN
(CH2)4OH
R= -(CH2)3CHO
R= -(CH2)4NEt2
(CH2)4OH
R
(CH2)4NEt2
p
d, e, f, g, i
h
5a
21
Br
Ar
Ar
26
22
HS
N
N
N
N
N
N
HO
HO
Ar:
NH2
N
N
N
N
NR1R2
OH
OH
O
O
p
d, e, f, g, i
s
b, c
h
m
3a, 3b
CF3
Br
Br
Ar
Ar
Ar
Ar
27
28
30
31
29
Scheme 1. 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, 3–5 h; (g) (CH3)3SiNCS, DMF, 140 °C, 4 h; (h) formaldehyde (37 wt % sol. in water), reflux, 8 h for 14, 25 or 36 h for 29; (i)
4-fluorobenzyl bromide, DBU, KI (cat.), CH3CN, rt, 5 h; (j) SOCl2, CH2Cl2, DMF (cat.), 0 °C, 1 h then NaHCO3 solution; (k) HNR1R2, DBU, KI (cat.), CH3CN, reflux, 1 h; (l) NaOH,
H2O–EtOH, rt, 2 h, then 6 N HCl; (m) MnO2, dioxane, 70 °C, 3 h; (n) CH3MgBr, THF, 0 °C, 2 h; (o) Ph3P = CHCHO, THF, rt, 12 h; (p) HNR1R2, NaBH(OAc)3, CH2Cl2, rt, 2 h; (q)
H2NNH2–H2O, EtOH, reflux, 2 h; (r) (COCl)2, DMSO, Et3N, CH2Cl2, À60 °C; (s) concd HCl, 0 °C, CH3CN, then 1,3-dihydroxyacetone, propionic acid, KSCN, 70 °C, 2 h.
Furthermore, the analogues containing one carbon less in their side
chains (i.e., the propylamine derivatives 5b–d) exhibited a similar
level of inhibition to that of 5a in rabbit and human plasma assays.
Together with the data from compounds 2h and 4c, these results
reveal that side chains consisting of three to five atom-tethered
amines all can be well accommodated by the active site of the en-
zyme in this particular region.
Based on the in vitro SAR studies, the most promising com-
pounds were subjected to preliminary evaluation in our C57 mouse
assay. The testing compounds were formulated in tartrate salts for

K. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2897–2901
2901
Figure 2. Relative serum Lp-PLA2 activities in C57 mice after a single dose (50 mg/kg, po, n = 5).
oral administration at 50 mg/kg.13 As depicted in Figure 2, the
Supplementary data
piperidine derivative 2f displayed the most prolonged level of inhi-
bition with activity extending over a 12-h period. The diethylamine
analogues 2c and 4a showed enhanced levels of inhibition whereas
the efficacy lasted over a shorter period of time compared with 2f,
suggesting that cyclic amine might possess better metabolic stabil-
ity than acyclic one. In contrast, the propylamine derivative 5b was
found to be a much weaker inhibitor in vivo, whereas the butyl-
amine derivative 5a was nearly inefficacious. The attenuation of
in vivo activities might be attributed to the poor absorption prop-
erties of 5a and 5b which were caused by the significantly elevated
logP values as a consequence of removing the internal-end N atom
(8.56, 9.36 and 8.94 for 2c, 5a and b, respectively, calculated using
ChemBioDraw Ultra 11.0). It has been shown in the SAR studies
that high binding affinities were well regulated by the presence
of the terminal-end amine. Herein, the studies in mice revealed
that variations in the two amines were able to change the meta-
bolic stability and lipophilicity of the analogues, leading to quite
different in vivo profiles. These findings indicated the possibility
of further improving the in vivo performances of our compounds
by rational modifications of the side chain, although they are still
incomparable to Darapladib at present.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
03.062.
References and notes
1. Koenig, W. J. Am. Coll. Cardiol. 2008, 51, 1642.
2. Björkbacka, H.; Fredrikson, G. N.; Nilsson, J. Atherosclerosis 2013, 227, 9.
3. Zalewski, A.; Macphee, C. H. Arterioscler. Thromb. Vasc. Biol. 2005, 25, 923.
4. Wilensky, R. L.; Macphee, C. H. Curr. Opin. Lipidol. 2009, 20, 415.
5. Anderson, J. L. Am. J. Cardiol. 2008, 101, S23.
6. Blackie, J. A.; Bloomer, J. C.; Brown, M. J.; Cheng, H. Y.; Hammond, B.; Hickey, D.
M.; Ife, R. J.; Leach, C. A.; Lewis, V. A.; Macphee, C. H.; Milliner, K. J.; Moores, K.
E.; Pinto, I. L.; Smith, S. A.; Stansfield, I. G.; Stanway, S. J.; Taylor, M. A.;
Theobald, C. J. Bioorg. Med. Chem. Lett. 2003, 13, 1067.
7. Jeong, H. J.; Park, Y. D.; Park, H. Y.; Jeong, I. Y.; Jeong, T. S.; Lee, W. S. Bioorg. Med.
Chem. Lett. 2006, 16, 5576.
8. Lin, E. C. K.; Hu, Y.; Amantea, C. M.; Pham, L. M.; Cajica, J.; Okerberg, E.;
Brown, H. E.; Fraser, A.; Du, L.; Kohno, Y. Bioorg. Med. Chem. Lett. 2012, 22,
868.
9. Nagano, J. M. G.; Hsu, K. L.; Whitby, L. R.; Niphakis, M. J.; Speers, A. E.;
Brown, S. J.; Spicer, T.; Vega, V. F.; Ferguson, J.; Hodder, P.; Srinivasan, P.;
Gonzalez, T. D.; Rosen, H.; Bahnson, B. J.; Cravatt, B. F. Bioorg. Med. Chem.
Lett. 2013, 23, 839.
In summary, we have designed and synthesized a series of oral-
ly bioactive Lp-PLA2 inhibitors in C57 mice by making precise mod-
ifications to the side chain of our previously discovered triazole
scaffold. The most promising compounds, including compound
2c, 2f, and 4a featured a diamine side chain, in which the termi-
nal-end amine provided binding interaction with the protein as
the key factor for high affinity. The analogues that were similarly
potent in vitro gave very different in vivo performances, probably
due to the differences in their metabolic or absorption properties
that could be modulated by the two amines. The research findings
disclosed in this study should prove of great value in further design
of improved Lp-PLA2 inhibitors.
10. Wang, K.; Xu, W. W.; Liu, Y.; Zhang, W.; Wang, W. Y.; Shen, J. H.; Wang, Y. P.
Bioorg. Med. Chem. Lett. 2013, 23, 1187.
11. Maligres, P. E.; Waters, M. S.; Weissman, S. A.; McWilliams, J. C.; Lewis, S.;
Cowen, J.; Reamer, R. A.; Volante, R. P.; Reider, P. J.; Askin, D. J. Heterocycl. Chem.
2003, 40, 229.
12. Activities in rabbit, human plasma and recombinant human Lp-PLA2 (rhLp-
PLA2) assays were all assessed using [3H]PAF as the substrate. Briefly, DMSO
solutions of the compounds were added to assay buffer containing 50 mmol/L
HEPES (pH 7.4), 150 mmol/L NaCl, 150
PLA2) in a total volume of 200 L. Following 5 min of preincubation at 37 °C,
10
L [3H]PAF (1uCi) was added and incubated for 10 min at 37 °C. After
the incubation, 600 L of CHCl3/MeOH (2:1) was added and the resulting
mixture was mixed thoroughly. After a period of centrifugation at 12000g
for 15 min at 4 °C, 200 of the supernatant was collected and mixed
with 200 of CHCl3. An 80 portion of the supernatant was then
lL dd H2O and 10 lL plasma (or rhLp-
l
l
l
lL
lL
lL
collected and its radioactivity was measured in a liquid scintillation counter.
The inhibition rate was determined by the following equation:Inhibitory
rate (%) = 1 À (CPMcompound À CPMblank)/(CPMpositive À CPMblank)⁄100%The blank
sample contained no plasma or test compound in the assay buffer and the
positive sample contained no test compound.
Acknowledgments
This work was financially supported by Grants from the Na-
tional Science & Technology Major Project ‘Key New Drug Creation
and Manufacturing Program’ (Grant Nos. 2012ZX09301001-001
and 2012ZX09103101-008).
13. A group of five C57 mice that had been fasted for 16 h were administrated with
the test compound (50 mg/kg po) and blood samples were drawn before or 1,
3, 6, 12, and 24 h after administration to measure the serum Lp-PLA2 activity
according to the method described in Ref. 10.