Discovery of AZD2716: A Novel Secreted Phospholipase A2 (sPLA2) Inhibitor for the Treatment of Coronary Artery Disease

Article abstract of DOI:10.1021/acsmedchemlett.6b00188

Expedited structure-based optimization of the initial fragment hit 1 led to the design of (R)-7 (AZD2716) a novel, potent secreted phospholipase A₂ (sPLA₂) inhibitor with excellent preclinical pharmacokinetic properties across specie

Full text of DOI:10.1021/acsmedchemlett.6b00188

Subscriber access provided by Northern Illinois University
Letter
Discovery of AZD2716: a novel secreted phospholipase A2
(sPLA2) inhibitor for the treatment of coronary artery disease
Fabrizio Giordanetto, Daniel Pettersen, Ingemar Starke, Peter Nordberg, Mikael
Dahlstrom, Laurent Knerr, Nidhal Selmi, Birgitta Rosengren, Lars-Olof Larsson,
Jenny Sandmark, Marie Castaldo, Niek Dekker, Ulla Karlsson, and Eva Hurt-Camejo
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00188 • Publication Date (Web): 09 Aug 2016
Downloaded from http://pubs.acs.org on August 10, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Medicinal Chemistry Letters is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Discovery of AZD2716: a novel secreted phospholipase A2
(sPLA2) inhibitor for the treatment of coronary artery disease
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fabrizio Giordanettoa*,†, Daniel Pettersena*, Ingemar Starkea, Peter Nordberga, Mikael Dahlströma, Lauꢀ
rent Knerra, Nidhal Selmia, Birgitta Rosengrenb, LarsꢀOlof Larssonc, Jenny Sandmarkd, Marie Castaldoe,
Niek Dekkere, Ulla Karlssonf, Eva HurtꢀCamejob
Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit
Departments of aMedicinal Chemistry, bBioscience, cDMPK.
Discovery Sciences
Sections of dStructure & Biophysics, eReagents and Assay Development and fScreening Sciences and Sample Management.
Astrazeneca, Mölndal, Pepparedsleden 1, SEꢀ431 83, Mölndal, Sweden
KEYWORDS. secreted phospholipase A2; sPLA2; inhibitor; fragment-based drug discovery, fragment screening, atheroscle-
rosis, coronary artery disease
ABSTRACT: Expedited structureꢀbased optimisation of the initial fragment hit 1 led to the design of (R)ꢀ7 (AZD2716) a novel,
potent secreted phospholipase A2 (sPLA2) inhibitor with excellent preclinical pharmacokinetic properties across species, clear in
vivo efficacy and minimized safety risk. Based on accumulated profiling data, (R)ꢀ7 was selected as a clinical candidate for the
treatment of coronary artery disease.
Secreted phospholipase A2 (sPLA2) are enzymes that hydroꢀ
lyze the acyl ester at the snꢀ2 position of snꢀ3 glycerophosphoꢀ
lipids1, a process characterised by complex interfacial kinetics
of substrateꢀenzyme binding and catalysis2. Eleven sPLA2
enzymes (group IbꢀXIIb) have so far been identified in mamꢀ
mals3ꢀ5, several of which have been detected in human atheroꢀ
sclerotic lesions6. Among these, group IIa, V, and X sPLA2
isoforms are present in human carotid atherosclerotic lesions
and have been associated with disease progression. They have
been implicated in several proatherogenic actions in the arteriꢀ
al wall.7ꢀ9 Due to their hydrolytic action on lipoprotein phosꢀ
pholipids, sPLA2s promote lipid accumulation, induce signifiꢀ
cant lipoprotein remodeling, macrophage activation and foam
cell formation.10ꢀ11 Furthermore, as the rateꢀlimiting step in
eicosanoid production, sPLA2ꢀmediated release of arachidonic
acid from the snꢀ2 position of phospholipids renders them
highly proꢀinflammatory enzymes.10ꢀ11 In addition, epidemioꢀ
logical data has shown that increased levels of sPLA2 protein
and sPLA2 activity are independently associated with risk of
cardiovascular events and prevalence of atherosclerosis.11
Owing to the pivotal role of sPLA2s in regulating lipoprotein
function and inflammatory mechanisms, two crucial compoꢀ
nents of atherogenesis, sPLA2 inhibitors12 could be useful for
the treatment of atherosclerosis. Interestingly, the archetypal
sPLA2 inhibitor varespladib methyl (Figure 1), was evaluated
in short duration clinical trials for the treatment of rheumatoid
studies to more properly assess the relevance of their lipoproꢀ
teinꢀmodifying effects15ꢀ17on cardiovascular disease, alongside
their antiꢀinflammatory properties.
R
O
O
O
H2N
O
O
N
H2N
O
1
R=Me, varespladib methyl
R=H, varespladib
Figure 1. sPLA2 inhibitor varespladib methyl, its active meꢀ
tabolite varespladib and initial fragment hit 1.
Given the competitive landscape, medicinal chemistry
precedents and available structural information for sPLA2
enzymes, we opted for a structureꢀbased fragment approach to
maximize the chances of novelty and developability.18 Analyꢀ
sis of potency data in combination with the available ligandꢀ
bound sPLA2 crystal structures19 indicated that primary amides
are extremely effective sPLA2 warheads, as they establish
three hydrogen bonds with sPLA2 and one coꢀordination bond
with the catalytic calcium ion. We assembled a selection of
primary aromatic carboxamideꢀcontaining fragments (heavy
atom count ≥ 10 and ≤18) by mining in house biochemical and
biophysical assay data against the sPLA2ꢀIIa and sPLA2ꢀX
isoforms. The selection was based on activity against sPLA2ꢀ
IIa, which is the most widely expressed isoform in humans20,
but also on inspection of crystal structures and chemical evoꢀ
lution potential.
arthritis and acute coronary syndrome with negative results.13ꢀ
14
We thus set out to identify novel sPLA2 inhibitors that could
be used in longer term coronary artery diseaseꢀbased clinical
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 7
Table 1. Initial profile for the fragment hit 1.
activity is the result of various sPLA2 isoforms and we were
1
2
3
4
5
6
7
8
interested in identifying a broad spectrum sPLA2 inhibitor, we
also monitored inhibition of sPLA2ꢀV and sPLA2ꢀX, given
their potential role in lipoprotein modulation.16 Lastly, to
avoid the need for a prodrug strategy (eg varespladib methyl)
we carefully evaluated compound lipophilicity against passive
permeability, solubility and metabolic stability, prior to verifyꢀ
ing the pharmacokinetic (PK) and pharmacodynamic (PD)
profile in vivo.
Plasma
sPLA2ꢀIIa
entry
Fu (%) LE/LLEb
ICu,50
IC50 (ꢁM)
(ꢁM)a
Varespladib
0.028
24
0.008
0.9
12.5
1.8
0.37/7.2
0.39/2.2
1
aCalculated as Plasma sPLA2 IC50 (ꢁM) × compound’s unꢀ
bound fraction in human plasma (Fu) /100. Fu = 100ꢀhuman proꢀ
tein binding(%). bLigand efficiency, LE (kcal/mol/HAC), calcuꢀ
lated as
ꢀRTln(sPLA2ꢀIIa IC50) / Heavy Atom Count. Ligand Lipophilicity
efficiency (LLE) calculated as pIC50 (sPLA2ꢀIIa) – logD.
The crystal structure of 1 bound to sPLA2ꢀX confirmed the
binding mode of of the primary amide, with hydrogenꢀbonding
to sPLA2ꢀX’s G28, H46 and D47 (corresponding to sPLA2ꢀIIa
G29, H47 and D48) and coordination to the calcium ion obꢀ
served, as shown in Figure 2. Additionally, the 4ꢀbenzyl subꢀ
stituent was located in a lipophilic pocket consisting of resiꢀ
dues I2, L5, A6, V9, P17, I18 and M21 (corresponding to L2,
F5, H6, I9, A17, A18 and G22 in sPLA2ꢀIIa, Figure 2). While
the two sPLA2 isoforms are identical in the amide coordinating
residues, the lipophilic pocket that accommodates the benzyl
group is slightly smaller in sPLA2ꢀIIa. However, superposiꢀ
tion of the sPLA2ꢀIIa and sPLA2ꢀX crystal structures suggestꢀ
ed that the benzyl group of 1 could fit in the slightly smaller
sPLA2ꢀIIa pocket.
9
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The triaging process identified compound 1 (LE: 0.39)
originating from a legacy fragment/HTS campaign as the most
promising fragment lead . As the translatability to a clinical
setting was of special importance, we also triaged the actives
for sPLA2 activity inhibition in human plasma (the modeꢀofꢀ
action biomarker to be used in clinical trials), as measured
using a previously established protocol.21
Compound 1 inhibited sPLA2ꢀIIa (IC50: 24 ꢁM) and human
plasma sPLA2 activity (ICu,50: 0.9 ꢁM) in a concentration
dependent manner (Table 1). Going forward, as plasma sPLA2
Table 2. sPLA2 potency and ligand efficiencies for compounds 2 – 6.a
R
O
H2N
sPLA2ꢀIIa
sPLA2ꢀV
sPLA2ꢀX
Plasma
Entry
R
LE/LLEc
IC50 (ꢁM)
0.028
IC50 (ꢁM)
0.12
IC50 (ꢁM)
0.041
ICu,50 (ꢁM)b
0.008
varespladib
1
0.37/7.2
0.39/2.2
H
24
NAd
2.2
0.9
*
HO
2
3
0.91
>10
NAc
NAd
0.33/4.9
0.38/5.9
*
O
O
0.07
1.4
1.1
0.04
HO
*
*
HO
4
5
6
0.012
0.11
0.19
0.36
4.1
0.28
5.4
0.007
0.03
NDe
0.4/6.4
0.34/5
O
O
HO
*
HO
3.7
3.7
0.34/5.4
O
*
O
aResults are mean of at least two experiments. Experimental errors within 20% of value. bCalculated as Plasma sPLA2 IC50 (ꢁM) ×
compound’s unbound fraction in human plasma (Fu) /100. Fu = 100ꢀhuman protein binding(%) cLigand efficiency, LE (kcal/mol/HAC),
calculated as ꢀRTln(sPLA2ꢀIIa IC50) / Heavy Atom Count, Ligand Lipophilicity efficiency (LLE) calculated as pIC50 (sPLA2ꢀIIa) – logD.
dNot active at maximum tested concentration (25 ꢁM). eNot determined.
ACS Paragon Plus Environment

Page 3 of 7
ACS Medicinal Chemistry Letters
gation at the carboxylic acid position by introduction of methꢀ
1
2
3
4
5
6
7
8
ylene units (3 – 5) had a parabolic effect on potency, with the
3ꢀphenylpropionic acid side chain yielding the most potent and
ligand efficient derivative (4, Table 2). Replacement of the
benzylic methylene by an ether oxygen or further elongating
the hydrocarbon chain were not tolerated (cf. 5 and 6, Table
2), hinting at a specific conformational requirement for the
carboxylic acidꢀcontaining side chain. The coꢀcrystal structure
of sPLA2ꢀIIa and 4 confirmed the previously hypothesised
ligandꢀmediated calcium chelation, as displayed in Figure 3.
9
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Superposition of the crystal structure of 1 bound
to sPLA2ꢀX (cyan) and a crystal structure of sPLA2ꢀIIa
(grey). Residues that differ between the two isoforms are
labelled in cyan and grey, respectively. The calcium ion is
depicted as a purple sphere and hydrogen bonds are disꢀ
played as dashed lines.
We therefore devised a chemical exploration strategy startꢀ
ing from the binding mode of 1. Here, special effort was
placed upon establishing a second coordination bond to the
catalytic calcium ion. The reasoning was twoꢀfold: a) to inꢀ
crease affinity and functional inhibition of the enzyme as a
result of a bidentate calcium chelate and additional van der
Waals contact with the enzyme, b) to allow a more balanced
lipophilicity profile of the final compounds as the additional
calcium interacting moiety was anticipated to be a carboxylic
acid. The ortho position of the benzamide ring was identified
as a favourable substitution vector to deploy such a strategy.
Based on iterative molecular modelling and careful consideraꢀ
tion of theoretical affinity gain and ligand efficiencypredicꢀ
tion, we synthesised compounds 2 – 6, according to Scheme 1
(for compound 4 refer to Scheme 2). Key steps involved the
formation of the boronic acid by ortho lithiation22 of the 4ꢀ
benzylbenzonitrile (step b) followed by a SuzukiꢀMiyaura
coupling and a controlled hydrolysis (step d) to generate both
the amide and carboxylic acid functions (Scheme 1).
Figure 3. Cocrystal structure of 4 bound to sPLA2ꢀIIa. The
calcium ion is depicted as a purple sphere, and relevant
hydrogen bonds are displayed as dashed lines.
The carbonyl oxygen atom of the amide group of 4 provided
the first coordination bond to calcium, analogously to 1. The
carboxylate moiety established the second coordination bond
to calcium (d: 2.4 Å) and a hydrogen bond to the backbone
amide group of G31, as shown in Figure 3, while the additionꢀ
al phenyl ring made significant van der Waals contacts with
the side chains of L2, G29 and V30. The 4ꢀbenzylbenzamide
componentof 4 displayed a similar interaction pattern as in 1,
except for the benzamide ring which was rotated by ca. 50
degrees. This rotation is induced by the introduction of the
substituent at the 2ꢀposition and, in the case of sPLA2ꢀX, this
comes with a penalty. This is exemplified by 2 and 3, where
the affinity gain is very limited, despite the addition of more
lipophilic interactions (Table 2). In type IIa, on the other hand,
this conformational lock is further stabilized by a edgeꢀtoꢀface
pi interaction with F5, which is not available in sPLA2ꢀX,
This is reflected by the steep improvement in affinity when
adding the substituents. For example, 3 despite having a linker
too short to form the additional calcium interaction, still gains
more than 300ꢀfold in affinity. This is further improved by the
propanoic acid side chain of 4 where the second calcium coorꢀ
dination bond is properly established leading to a 2000 fold
increase in potency compared to 1. The active site of sPLA2ꢀ
IIa is smaller, F5 (L5 in sPLA2ꢀX) affects the benzamide
moiety and I9 (V9 in sPLA2ꢀX) is located close to the hinge
between the two benzyl groups of 1 (d:3.7Å), thereby
slightly altering the angle in which the 4ꢀbenzyl enters the
pocket and potentially introducing some strain in the fragꢀ
ment, where the larger pocket of sPLA2ꢀX offers a less
restrained binding mode
Scheme 1. General synthesis of compounds 2, 3, 5, 6a
Br
A
N
N
N
O
OH
B
X
c
OR
OH
a
b
80%
35%
57-88%
Br
N
H2
N
O
O
O
X
OR
X
OH
d
2:X= -
3:X= -CH2-
4:X= -CH2CH2-
5:X= -CH2CH2CH2-
6:X= -OCH2-
45-88%
For 4, see Scheme 2
a Conditions: (a) benzylzinc bromide, Pd(Ph3)4, THF, 60 °C.
(b) 1. nBuLi, tetramethylpiperidine 2. B(OiPr)3, THF ꢀ78 °C.
(c) Compound 2, 3, 5: 3ꢀbromophenyl carboxylic acid A,
PdCl2(dppf), Cs2CO3, DMF, 60ꢀ90 °C. Compound 6:
Pd(P(Ph3)4, Cs2CO3, DMF 90 (d) Compound 2, 3, 5: nꢀ
propanol:H2O (10:1), KOH (10 eq), 80ꢀ100 °C, or THF,
H2SO4. Compound 6: KOH (10 eq), MeOH/H2O, microwawe
130 °C, 20 min.
The high ligand efficiency and potency of 4, coupled with
its marked plasma sPLA2 inhibition ability (ICu,50 7 nM) trigꢀ
gered a broad characterisation campaign to identify potential
shortcomings.
Introduction of a 3ꢀbenzoic acid moiety at the 2ꢀposition of
4ꢀbenzylbenzamide 1, albeit notoptimal, confirmed the potenꢀ
tial for growth at that position. (2, Table 2). Progressive elonꢀ
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 7
As summarised in Table 3, compound 4 proved to be soluꢀ
tures (careful monitoring of the reaction to avoid over hydrolꢀ
ysis to the corresponding diꢀacid is needed).
Scheme 2. General synthesis of sPLA2 inhibitors 4, 7-9a
1
2
3
4
5
6
7
8
ble, highly permeable and metabolically stable;characteristics
which translated well in vivo with high bioavailability and low
systemic clearance recorded in rat and dog (Table 3). This
provided a significant improvement over varespladib, which
required a methyl ester prodrug approach (ie. varespladib
methyl) to afford moderate oral absorption (F: 40 – 55%) in
the same species. Compound 4 did not show any significant
inhibition of cytochrome P450 enzymes or ion channel activity
relevant to cardiac function. Nevertheless, 4 inhibited the
uptake of pivastatin in HEK293 cells transfected with the
human organic anionic transporter polypeptide 1B1
(OATP1B1) at an estimated IC50 of 2.2 µM, as shown in Table
3. The OATP1B1 transporter is necessary for statin’s hypoꢀ
cholesterolemic action as it mediates their access to the liver
compartment where they can then inhibit the function of
HMGꢀCoA reductase. 23ꢀ25
X=Br or I
a
b-d
e
95%
46-76%
75-90%
B
C
Compound 4,7:
9
f
g
h
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4: R=H
7:R=Me
8:R=Et
9:R=cPr
19-82%
58-84%
77%
~60%
i
Compound 8-9:
B
(step b, Scheme 1)
Table 3. Profilea of compound 4.
a Conditions: (a) appropriate malonate, Cs2CO3, DMF, 70 °C,
2h. (b) NaOH (4 eq), H2O:MeOH 3:1 80 °C 2h. (c) HOAc (3 M),
reflux. (d) HCl, MeOH 60 °C, 2.5 h. (e) (Bpin)2 (1.3 eq), KOAc
(2.5 eq), PdCl2(dppf) (6.5mol%), dioxane, 90 °C. (f) BnZnBr (1.5
eq, 0.5 M in THF), Pd(PPh3)4 (5 mol%), THF, 60 °C, 2h. (g)
Compound 4: 3ꢀ(3ꢀmethoxyꢀ3ꢀoxopropyl)phenylboronic acid
(commercial), Pd(Ph3)4. Compound 7: PdCl2(dbpf) (5 mol%), C.
Cs2CO3, DMF, 90 °C, 1.5 h. (h) nꢀpropanol, H2O (10:1), KOH (10
eq), 80ꢀ100 °C. i) B, PdCl2(dppf), Cs2CO3, DMF, 90 °C.
Solubility (pH=7.4) (µM)
Papp (10ꢀ6 cm/s)
HEP Clint (ꢁL/min/10ꢀ6 cells)
98
40.1
5.2
hERG, Nav1.5, IKs, Kv4.3, Cav3.2,
Cav1.2 IC50 (µM)
>33.3
CYP450 IC50 (µM)
>20
2.2
OATP1B1 IC50 (µM)
Addition of a methyl group (7, Table 4) displayed an isoꢀ
formꢀspecific effect on potency: while it was neutral at sPLA2ꢀ
IIa, it enhanced inhibition of sPLA2ꢀV and reduced that of
sPLA2ꢀX. Overall, this yielded excellent plasma sPLA2 inhibiꢀ
tion (ICu,50: 0.1 nM). Remarkably, structural manipulation of
the carboxylic acid surroundings by an alphaꢀmethyl group
demonstrated the intended effect at a transporter level and 7
was devoid of OATP1B1 inhibition at the maximum tested
concentration (25 µM). Ethyl and cyclopropyl substitutions (8,
9) were not as efficient and had a deleterious effect on metaꢀ
bolic stability in human hepatocytes (cf. 8, 9 and 7, Table 4).
We therefore proceeded to separate and characterise the two
enantiomers of 7. In line with expectations, sPLA2 inhibition
was affected by stereochemistry and the R enantiomer proved
to be the most active, halfꢀmaximally inhibiting sPLA2ꢀIIa, ꢀV
and ꢀX at 10, 40 and 400 nM, respectively (Table 4). As a
result, (R)ꢀ7 was the most potent plasma sPLA2 inhibitor in
this study (ICu,50: 0.1 nM) and, based on its minimized risk for
drugꢀdrug interactions with statins, was progressed to further
profiling.
PK
Rat
Dog
Dose i.v./p.o. (µmol/kg)
CL (mL/min/kg)
Vss (L/kg)
2/4
1
1/2
0.3
0.26
82
0.22
81
F (%)
a Please see the supporting information for experimental details.
Considering that an eventual sPLA2 inhibitor for the treatꢀ
ment of coronary artery disease will need to be coꢀ
administered with a statin, as an established standard of care,
minimizing the risk for such drugꢀdrug interaction was reꢀ
quired. As OATP1B1 recognises anionic compounds, we
reasoned that modification of the molecular environment
around the carboxylic acid of 4 might alleviate its interaction
with OATP1B1. More specifically, substituting the carbon
atom alpha to the carboxylic acid was of special interest as a)
it was postulated to provide a steric impediment to OTAP1B1,
b) it seemed compatible with the binding pocket of the sPLA2ꢀ
IIa enzyme, and c) could enhance potency and/or selectivity
through conformational “freezing” of the carboxylic acid side
chain via gaucheꢀlike effects. Due to structureꢀbased conꢀ
straints and in order to minimize the impact on compound
lipophilicity, we targeted small substituents and synthesised
compounds 7 – 9, following Scheme 2.
When incubated with HepG2 cells, (R)ꢀ7 effectively inhibitꢀ
ed sPLA2 activity (IC50 <14 nM) and suppressed production of
sPLA2ꢀIIa (IC50 176 ± 28 nM) via a mechanism not yet eluciꢀ
dated.7 Importantly, (R)ꢀ7 demonstrated significant sPLA2
activity inhibition (IC50 56 ± 10 nM) in atherosclerotic plaque
homogenates, as obtained from carotid endarterectomy of
coronary artery disease patients (N = 7).26
According to scheme 2, the appropriate dimethyl malonate
was alkylated using 3ꢀbromobenzylbromide. The propionic
acid derivative was then obtained by hydrolysis, decarboxylaꢀ
tion and reꢀesterification to yield the methyl ester. Boronylaꢀ
tion was accomplished by standard protocols using (Bpin)2
and PdCl2(dppf) to yield the pinacol borane ester which could
be used in the subsequent SuzukiꢀMiyaura coupling using the
benzylated chloro benzonitrile. Finally, racemic 7 – 9 could be
obtained by hydrolysis using hydroxide in alcohol/water mixꢀ
In vivo PK analysis of (R)ꢀ7 showed consistent high bioaꢀ
vailability and low clearance across different animal species,
as summarised in Table 5.
ACS Paragon Plus Environment

Page 5 of 7
ACS Medicinal Chemistry Letters
Table 4. sPLA2 potency and optimisation parameters for compounds 4 – 9 .
1
2
3
4
5
6
7
Plasma
ICu,50 (nM) b (ꢁL/min/10ꢀ6 cells)c
OATP1B1
sPLA2ꢀIIa
IC50 (ꢁM)a
sPLA2ꢀV
sPLA2ꢀX
HEP Clint
Entry
R
IC50 (ꢁM)a
IC50 (ꢁM)a
IC50 (ꢁM)d
8
9
4
7
H
Me
0.012
0.011
0.021
0.018
0.038
0.010
0.36
0.07
0.07
0.25
1.2
0.28
0.75
0.43
0.58
3.8
7
5.2
9.6
25
2
1
NAe
NDf
NDf
NDf
NAe
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
Et
0.8
0.9
ND
0.1
9
CyPr
(S)ꢀMe
(R)ꢀMe
21
(S)ꢀ7
9.3
(R)ꢀ7
0.04
0.4
12
a Mean of at least two experiments. Experimental errors within 20% of value. bCalculated as Plasma sPLA2 IC50 (µM) × unbound fraction
in human plasma (Fu) /100. cIntrinsic clearance of test compounds after incubation with human hepatocytes. dInhibition of pivastatin uptake
to HEK293 cells transfected with human OATP1B1. eNot active at maximum tested concentration (25 ꢁM). fNot determined.
.
A 30 mg dose of (R)ꢀ7 was orally administered to cynomolꢀ
gus monkeys (N = 2), as shown in Figure 4. This generated a
concentrationꢀdependent inhibition of sPLA2 activity in plasꢀ
ma (ICu,80 13 ± 3 nM) that well reflected the time course of
(R)ꢀ7’s exposure profile (Figure 4).
Table 5. PK parameters of compound (R)-7
Cynoꢀ
Mouse
2/2
Rat
Dog
molgus
N i.v./p.o.
2/2
2/8
70
2/2
1/3
2/2
5/15.8
71.3
2.8
In summary, starting from the original fragment hit 1, two
design cycles based on structural information, ligand effiꢀ
ciency reasoning, physicochemical property control, medicꢀ
inal chemistry tactics and readily available experimental
data resulted in the discovery of (R)ꢀ7 (AZD2716), a novel,
potent sPLA2 inhibitor with excellent PK properties, in vivo
efficacy and minimized risk for drugꢀdrug interactions.
Based on the available results and the favourable toxicologꢀ
ical profile in rats, dogs and cynomolgus monkeys, (R)ꢀ7
was selected as a clinical candidate for the treatment of
coronary artery disease
Dose i.v./p.o. (µmol/kg) 10/50
Oral AUC0ꢀinf (µM×h)
Cl (mL/min/kg)
Vss (L/kg)
69.7
9.2
284.1
0.2
1
2.8
0.21
84
0.15
91
1.1
F (%)
76.3
81
Building on the observed PK parameters, and the in vitro
sPLA2 inhibition measured in cynomolgus monkey plasma
(ICu,50 1 nM), we resolved to evaluate the in vivo sPLA2 inꢀ
hibitory effect of (R)ꢀ7.
SUPPORTING INFORMATION
Synthesis details for 2 ꢀ 9, assay protocols and Xꢀray data are
enclosed in the supporting information. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
* Fabrizio Giordanetto, Phone, +1ꢀ212ꢀ4780ꢀ822; eꢀmail, fabꢀ
rizio.giordanetto@deshawresearch.com.
* Daniel Pettersen, Phone, +46 31 7065 663; eꢀmail,
daniel.pettersen@astrazeneca.com
Present Addresses
† D.E. Shaw Research, 120W 45th Street, New York, NY 10036,
USA
Notes
The authors declare no competing financial interest.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script. Project leader, lead author FG; Author DP; Medicinal
chemists DP, PN, MD, IS, LK, NS; Bioscientists EHꢀC, BR;
Enzyme assays UK; DMPK LꢀOL; Xꢀray JS, ND, MC.
Figure 4. Vehicleꢀcorrected plasma sPLA2 activity inhibiꢀ
tion, corresponding PKꢀPD relationship (a) and time course
(b) following oral administration of 30 mg (R)ꢀ7 to cynomolꢀ
gus monkeys (N=2).
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
14. Nicholls, S.J.; Kastelein, J. J.; Schwartz, G.G.; Bash,
ACKNOWLEDGMENT
1
2
3
4
5
6
7
8
D. Rosenson, R.S.; Cavender, M.A.; Brennan, D.M.;
Koenig, W.; Jukema, J. W.; Nambi, V.; Wright, R.S.;
Menon V.; Lincoff, A. M.; Nissen, S. E. Varespladib
and cardiovascular events in patients with an acute
coronary syndrome: the VISTA-16 randomized clinical
trial. JAMA 2014, 3, 252-262.
Tomas Åkerud, Cristian Bodin, Kenth Hallberg, Thomas Olsson
and HansꢀGeorg Beisel are acknowledged for early lead generaꢀ
tion work and useful discussions. Åsa Månson and Fana
Hunegnaw are acknowledged for synthetic chemistry contribuꢀ
tions.
15. Ishimoto, Y.; Yamada, K.; Yamamoto, S.; Ono, T.; No-
toya, M.; Hanasaki, K. Group V and X Secretory
Phospholipase A(2)s-Induced Modification of High-
Density Lipoprotein Linked to the Reduction of Its An-
tiatherogenic Functions. Biochim. Biophys. Acta 2003,
1642, 129–138.
16. Hanasaki, K.; Yamada, K.; Yamamoto, S.; Ishimoto,
Y.; Saiga, A.; Ono, T.; Ikeda, M.; Notoya, M.; Kamitani,
S.; Arita, H. Potent Modification of Low Density Lipo-
protein by Group X Secretory Phospholipase A2 Is
Linked to Macrophage Foam Cell Formation. J. Biol.
Chem. 2002, 277, 29116–29124.
17. Beer, F. C. de; Connell, P. M.; Yu, J.; Beer, M. C. de;
Webb, N. R.; Westhuyzen, D. R. van der. HDL Modifi-
cation by Secretory Phospholipase A2 Promotes
Scavenger Receptor Class B Type I Interaction and
Accelerates HDL Catabolism. J. Lipid Res. 2000, 41,
1849–1857.
REFERENCES
1. Murakami, M.; Sato, H.; Miki, Y.; Yamamoto, K.;
Taketomi, Y. A new era of secreted phospholipase A₂.
J Lipid Res. 2015, 56, 1248-1261.
2. Singer, A. G.; Ghomashchi, F.; Le, C. C.; Bollinger, J.;
Bezzine, S.; Rouault, M.; Sadilek, M.; Nguyen, E.;
Lazdunski, M.; Lambeau, G.; Gelb M. H. Interfacial ki-
netic and binding properties of the complete set of
human and mouse groups I, II, V, X, and XII secreted
phospholipases A2. J. Biol. Chem. 2002, 277, 48535-
48549.
9
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3. Kudo, I.; Murakami, M. Phospholipase A2 enzymes. In
Prostaglandins and Other Lipid Mediators.; Elsevier:
2002, vol. 68-69, pp. 3–58.
4. Murakami, M.; Taketomi, Y.; Miki, Y.; Sato, H.; Hira-
bayashi, T.; Yamamoto, K. Recent progress in phos-
pholipase A2 research. In Cells to animals to humans.
In Progress in Lipid Research; 2011, pp. 152–192.
5. Kimura-Matsumoto, M.; Ishikawa, Y.; Komiyama, K.;
Tsuruta, T.; Murakami, M.; Masuda, S.; Akasaka, Y.;
Ito, K.; Ishiguro, S.; Ito, K.; Ishiguro, S.; Morita, H.;
Sato, S.; Morita, H.; Sato, S.; Ishii, T. Expression of
secretory phospholipase A2s in human atherosclero-
sis development. Atherosclerosis 2008, 196, 81-91.
6. Hurt-Camejo, E.; Camejo, G.; Peilot, H.; Öörni, K.; Ko-
vanen, P. Phospholipase A2 in Vascular Disease. Circ.
Res. 2001; 89, 298-304.
7. Rosenson, R. S.; Hurt-Camejo, E. Phospholipase A2
enzymes and the risk of therosclerosis. Eur. Heart. J.
2012, 33, 2899-2909.
8. Mallat, Z.; Lambeau, G.; Tedgui, A. Lipoprotein-
Associated and Secreted Phospholipases A2 in Cardi-
ovascular Disease. Circulation 2010, 122, 2183-2200.
9. Rosenson, R. S.; Gelb, M. H. Secretory phospholipase
A2: a multifaceted family of proatherogenic enzymes.
Curr Cardiol Rev. 2009, 11, 445-51.
10. Shridas, P.; Webb, N. R.; Diverse Functions of Secre-
tory Phospholipases A2. Advances in Vascular Medi-
cine 2014, 1-11.
11. Lind, L.; Simon, T.; Johansson, L.; Kotti, S.; Hansen,
T-; Machecourt, J.; Ninio, E.; Tedgui, A.; Danchin, N.;
Ahlström, H.; Mallat, Z. Circulating levels of secretory-
and lipoprotein-associated phospholipase A2 activities:
relation to atherosclerotic plaques and future all-cause
mortality. Eur Heart J. 2012, 33, 2946-2954.
12. For recent literature and references: Vasilakaki, S.;
Barbayianni, E.; Leonis, G.; Papadopoulos, M.G.;
Mavromoustakos, T.; Gelb, M.C.; Kokotos, G. Devel-
opment of a potent 2-oxoamide inhibitor of secreted
phospholipase A2 guided by molecular docking calcu-
lations. Bioorg. Med. Chem. 2016, 24, 1683-1695.
13. Bradley, J. D.; Dmitrienko, A. A.; Kivitz, A. J.; Gluck,
O. S.; Weaver, A.L.; Wiesenhutter, C.; Myers, S.L.;
Sides, G. D. A randomized, double-blinded, placebo-
controlled clinical trial of LY333013, a selective inhibi-
tor of group II secretory phospholipase A2, in the
treatment of rheumatoid arthritis. J. Rheumatol. 2005,
32, 417-23.
18. Erlanson, D. A. Introduction to fragment-based drug
discovery. Top Curr Chem. 2012, 317, 1-32.
19. Schevitz,
G.; Chirgadze, N. Y.; Clawson, D. K.; Dillard, R.
D.; Draheim, S. E.; Hartley, L.W.; Jones, N.
R.
W.; Bach,
N.
J.; Carlson,
D.
D.; Mihelich, E.D.; Olkowski, J. L.; Snyder, D.
W.; Sommers, C.; Wery, J.-P. Structure-based design
of the first potent and selective inhibitor of human non-
pancreatic secretory phospholipase A2. Nat. Struct.
Biol. 1995, 2, 458-465.
20. Lambeau, G.; Gelb, M. H. Biochemistry and physiolo-
gy of mammalian secreted phospholipase A2. Annu.
Rev. Biochem. 2008, 77, 495-520.
21. Pernas, P.; Masliah, J.; Olivier, J. –L.; Salvat, C.; Ryb-
kine, T.; Bereziat, G. Type II Phospholipase A2 recom-
binant overexpression enhance stimulated arachidonic
acid release. Biochem. Biophys. Res. Comm. 1991,
178, 1298-1305.
22. Kristensen, J.; Lysén, M.; Vedsö, P.; Begtrup, M. Syn-
thesis of ortho substituted arylboronic esters by in situ
trapping of unstable lithio intermediates. Org. Lett.
2001, 10, 1435-1437.
23. Hsiang, B.; Zhu, Y.; Wang, Z.; Wu, Y.; Sasseville, V.;
Yang, W. P.; Kirchgessner, T. G. A novel human he-
patic organic anion transporting polypeptide (OATP2).
Identification of a liver-specific human organic anion
transporting polypeptide and identification of rat and
human hydroxymethylglutaryl-CoA reductase inhibitor
transporters. J. Biol. Chem.1999, 274, 37161–37168.
24. Mizuno N, Niwa T, Yotsumoto Y and Sugiyama Y
(2003) Impact of drug transporter studies on drug dis-
covery and drug development. Pharmacol Rev 55:
425-461.
25. Shitara Y, Horie T and Sugiyama Y (2006) Transport-
ers as a determinant of drug clearance and tissue dis-
tribution. Eur J Pharmaceut Sci 27: 425-446.
26. Hurt-Camejo, E.; Andersen, S.; Standal, R.; Rosen-
gren, B.; Sartipy, P.; Stadberg, E.; Johansen, B. Local-
ization of Non Pancreatic Secretory Phospholipase A2
in Normal and Atherosclerotic Arteries: Activity of the
Isolated Enzyme on Low Density Lipoproteins. Arterio-
scler Thromb Vasc Biol. 1997, 17, 300-309.
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
1
2
For Table of Contents Use Only
3
4
5
6
7
8
9
Discovery of AZD2716: a novel secreted phospholipase A2
(sPLA2) inhibitor for the treatment of coronary artery disease
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
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fabrizio Giordanettoa*,†, Daniel Pettersena*, Ingemar Starkea, Peter Nordberga, Mikael Dahlströma, Lauꢀ
rent Knerra, Nidhal Selmia, Birgitta Rosengrenb, LarsꢀOlof Larssonc, Jenny Sandmarkd, Marie Castaldoe,
Niek Dekkere, Ulla Karlssonf, Eva HurtꢀCamejob
Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit
Departments of aMedicinal Chemistry, bBioscience, cDMPK.
Discovery Sciences
Sections of dStructure & Biophysics, eReagents and Assay Development and fScreening Sciences and Sample Management.
Astrazeneca, Mölndal, Pepparedsleden 1, SEꢀ431 83, Mölndal, Sweden
Table of Contents (TOC)
1) Fragment expansion
2) Focused optimization
1
AZD2716
Plasma sPLA2 ICu,50 = 0.9 uM
Plasma sPLA2 ICu,50 = 0.1 nM
7
ACS Paragon Plus Environment