Design of novel and potent cPLA2α inhibitors containing an α-methyl-2-ketothiazole as a metabolically stable serine trap

Article abstract of DOI:10.1016/j.bmcl.2011.03.005

We report the design of novel, potent cPLA₂α inhibitors that possess an α-methyl-2-ketothiazole that acts as a serine-reactive moiety. We describe the optimization of the series for potency and metabolic stability towards ketone reduction. This

Full text of DOI:10.1016/j.bmcl.2011.03.005

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3128–3133
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design of novel and potent cPLA2a inhibitors containing
an -methyl-2-ketothiazole as a metabolically stable serine trap
a
Antonio Mete a, , Glen Andrews b, Mike Bernstein a, Stephen Connolly a, Paul Hartopp a, Clive G. Jackson b,
Richard Lewis a, Iain Martin c, , David Murray b, Rob Riley c, David H. Robinson a, Gill M. Smith b,
Edward Wells b, W. John Withnall a
⇑
a Department of Medicinal Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire LE11 5RH, United Kingdom
b Department of Discovery BioScience, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire LE11 5RH, United Kingdom
c Department of DMPK, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire LE11 5RH, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the design of novel, potent cPLA2a inhibitors that possess an a-methyl-2-ketothiazole that acts
Received 26 January 2011
Revised 1 March 2011
Accepted 2 March 2011
Available online 6 March 2011
as a serine-reactive moiety. We describe the optimization of the series for potency and metabolic stability
towards ketone reduction. This was achieved by attenuating the reactivity of the ketone using a combi-
nation of electronic and steric effects.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Cytosolic phospholipase A2
Enzyme inhibitor
Serine trap
Keto-thiazole
Metabolic stability
Cytosolic phospholipase A2
a
is an intracellular enzyme (cPLA2
a
,
have also observed that these molecules are highly metabolized
and in particular the ketone, which is electrophilic, undergoes
rapid reduction to the inactive hydroxyl derivative, rendering these
compounds unsuitable as drug candidates or even as tool
group IVA phospholipase) which generates free arachidonic
acid by selectively cleaving the sn-2 position of arachidonyl-
glycophospholipids.1 Further metabolism of arachidonic acid leads
to a variety of inflammatory mediators such as leukotrienes,
prostaglandins, thromboxanes and acetylation of the lyso glycero-
phosphatidyl choline generates platelet activating factor (PAF).2
O
cPLA2a is therefore considered as playing a key role in a number
O
O
O
O
N
N
of inflammatory conditions and as such as a potential target for
O
the treatment of a number of diseases such as rheumatoid arthritis,
S
S
S
asthma and osteoarthritis.3 Inhibitors of cPLA2
a are therefore still
1
OH
of great interest as potential new therapeutic agents.4
We have previously described a series of potent cPLA2
a
inhibi-
O
tors containing a 1,3-dioxypropan-2-one group as a serine-reactive
moiety,5 typified by compound 1 (Fig. 1). We have demonstrated
by NMR studies, analogous to those performed on the trifluorom-
R1
X
X
R2
ethylketone-arachidonyl (TFMA) cPLA2a
inhibitor6 that the ketone
2
in compound 1 interacts with a serine residue on the enzyme to
form a complex containing an ionized hemiketal.7 However, we
O
⇑
R3
Corresponding author. Tel.: +44 01509 644251.
3
E-mail address: Antonio.Mete@Astrazeneca.com (A. Mete).
Present address: DMPK, Merck Research Laboratories, 33 Avenue Louis Pasteur,
Boston, MA, United States.
Figure 1. Novel series of cPLA2a inhibitors.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.005

A. Mete et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3128–3133
3129
compounds for use in in vivo models of inflammation. Our efforts
in the area were therefore directed to improving the metabolic
stability of these compounds and render them more drug-like.
Our approach was to explore a number of modifications to the ke-
tone in 1, designed to maintain its ability to interact with the cat-
alytic serine residue in the enzyme,8 while making it resistant to
reduction.
After a review of the options, we were attracted to some keto-
heterocycles, as these have been used in a variety of amidase,
protease and lipase inhibitors.9 Furthermore, when incorporated
into some human neutrophil elastase (HNE) inhibitors, they have
been reported to possess good in vivo pharmacokinetic (PK)
properties.10
The initial design strategy was to use compound 1 as a template
in which to incorporate various keto-heterocycles. In the 1,3-
dioxypropan-2-one series of cPLA2 inhibitors,5 we established that
structural modifications close to the ketone were allowed only at
the benzoic acid end of the molecule. Introduction of keto-
heterocycles into this part of the molecule was therefore proposed
O
OH
O
O
a
OEt
R
R
S
S
5
4
b
c
O
O
O
N
H
R1
R
R
R
X
S
S
S
6
2
R2
d
OH
OH
CN
NH
O
O
e
R
OEt
S
8
7
f
OH
O
N
O
g
N
O
and the structures 2 and 3 were explored as potential cPLA2a
inhibitors (Fig. 1). The synthetic routes used to prepare such het-
erocyclic derivatives are outlined in Scheme 1.
X
R
X
R
R3
S
S
R3
9
3
The ester 5 was readily synthesized and proved a versatile
intermediate. This ester reacted with a variety of lithiated hetero-
cycles to afford the ketones 2 directly.11 Interestingly, very little
double addition to the ester was observed. Some of the bicyclic
derivatives 3 such as the benzothiazoles, were also amenable to
synthesis by this approach. Benzoxazole and benzimidazole deriv-
atives were synthesized from ester 5 by first converting it to the
R = Phenylpentyl
Scheme 1. Reagents and conditions: (a) ethyl bromoacetate, Cs2CO3, DMF, rt, 88%;
(b) 2-lithiated heterocycle, THF, À78 °C to rt, 50–80%; (c) dibal, THF, À78 °C to rt,
83%; (d) acetonecyanohydrin, TEA, rt, 60%; (e) HCl, EtOH, rt, >95%; (f) 1,2-
diaminobenzene or 2-aminophenol, MeCN, reflux, 45–70%; (g) Dess–Martin
reagent, rt, 50–70%.
Table 1
cPLA2
a inhibition and metabolic stability of keto-heterocycles
O
O
R
X
a
a
d
Compd
R
X
S
cPLA2 IC50
0.08
(l
M)
HL60 cells IC50
(lM)
% loss ketoneb
Clint rat hepsc
Khyd
O
OH
1
1.5
nt
nt
nt
O
N
N
N
10
11
S
S
1
>10
nt
74
62
nt
nt
<0.1
nt
S
>10
HN
12
S
>10
nt
nt
nt
<0.1
N
Me
N
13
14
S
S
>10
1
nt
nt
nt
nt
nt
nt
N
Me
N
N
30
0.48
S
HN
15
S
2.7
3.7
nt
nt
nt
OH
O
(continued on next page)

3130
A. Mete et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3128–3133
Table 1 (continued)
a
a
d
Compd
R
X
S
cPLA2 IC50
>10
(l
M)
HL60 cells IC50
(lM)
% loss ketoneb
Clint rat hepsc
Khyd
N
O
HN
16
17
>10
nt
nt
nt
OH
O
N
N
O
S
S
0.14
0.08
3.6
1.6
86
35
28
5
O
S
OH
18
100
nt
OH
O
N
N
S
S
19
20
S
S
>10
0.3
nt
30
44
nt
nt
OMe
1.1
11
0.61
nt
OH
O
N
N
N
S
S
S
21
22
S
S
S
0.5
0.5
54
nt
nt
nt
nt
OH
OH
O
O
4.3
3
nt
23
24
0.08
0.5
44
<0.1
OH
O
N
N
O
S
2
6
40
21
nt
nt
S
S
OH
O
25
0.28
1.3
31
<0.1
OH
a
Values are means of at least two experiments. nt = not tested.
Loss of parent ketone by reduction in rat liver S9 fraction.
b
c
Intrinsic clearance in rat hepatocytes: l
L/min/10À6 cells.
d
Khyd: Equilibrium constant for hydration of the ketone in DMSO/water.
imidate 8 by standard methods. This reacted with a variety of 1,2-
diaminobenzenes or 2-amino-phenols to afford the hydroxy inter-
mediates 9 which were oxidized to the required keto derivatives
3.16 A number of these hydroxy-heterocyclic derivatives 9, and
appearance of the hydroxy metabolite.13 The intrinsic clearance
in rat hepatocytes was subsequently measured for some key com-
pounds.14 Firstly, a range of unsubstituted mono- and bi-cyclic het-
erocyclic derivatives, 10–14 were evaluated. All had disappointing
levels of activity compared to compound 1. The effect of incorpo-
rating an acidic substituent onto the heterocycle, to mimic the acid
found in 1, was then explored. Encouragingly, addition of the acid
group afforded compounds 15–20 with interesting levels of po-
tency, and analogues 17 and 18 possessing similar potency to that
seen in compound 1. However, the intrinsic clearance of both com-
pounds in rat hepatocytes was high (35 and 28, respectively) and
this appeared to be due to ketone reduction, as both compounds
were extensively reduced in the rat liver S9 assay. Compound 17
was assessed in a rat in vivo PK study which confirmed its poor
metabolic profile, exhibiting very high clearance (Cl >200) and very
short plasma half-life (T½ <0.2 h). Although we had designed a new
the equivalent hydroxy derivatives of 2, were tested for cPLA2
a
activity throughout the course of this work and in all cases exhib-
ited no inhibition of the enzyme. Table 1 summarizes the initial set
of compounds made.
The key properties tabulated include cPLA2a potencies in a cell-
free enzyme assay and a cellular assay and metabolic stability to
ketone reduction. The enzyme assay measured inhibition of cleav-
age of a soluble fluorogenic substrate12 by recombinant enzyme as
previously described.5a The cell assay measured inhibition of [3H]-
arachidonate release following A23187 challenge of DMSO differ-
entiated HL60 cells.5a Ketone stability to metabolic reduction was
initially assessed by an in vitro assay using rat liver S9 fraction
which measured the disappearance of the parent ketone and
series of potent cPLA2a inhibitors, we had not made any progress

A. Mete et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3128–3133
3131
O
The Khyd was initially measured for a small group of keto-
heterocyclic derivatives 10, 12, 14 and 17 to see how it varied
and is summarized in Table 1. Figure 3 shows the hydration of
compound 14 as a typical example and how the amount of hydrate
present increases dependent on the mole fraction of water in the
sample. In order to determine the ratio of ketone to hydrate it
HO OH
Het
H2O
O
O
Het
R
R
DMSO
S
S
Figure 2. Assessing ketone reactivity by K hydration.
was most convenient to integrate the methylene singlet at
15
5.78 ppm (ketone) and 4.34 ppm (hydrate), to define the Khyd
.
These data were used to rank ketone electrophilicity and to prior-
itize the choice of keto-heterocycles to optimize.
Mol fraction water
Ketone
4
Hydrate
The data show that the least reactive ketones towards hydration
are in the monocyclic imidazole and thiazole compounds 10 and
12. Interestingly, the addition of a fused phenyl ring onto 10, to
give the benzothiazole 14, led to a more reactive ketone. The benz-
oxazole derivative 17 has an even more electrophilic ketone, pre-
sumably a result of a combination of the fused phenyl ring and
an electron-withdrawing acid substituent. The chosen strategy,
based on these data, was to attach an acid onto the thiazole 10
using an alkyl chain to try and increase potency by optimizing
the interaction of the acid with the enzyme. Benzo-fusion was
not used as this had led to increased ketone electrophilicity in
14. The chain length connecting the acid to the thiazole ring was
varied in 20, 21 and 23, and the linking chain was rigidified with
a double bond in 22. The position of attachment to the thiazole ring
was also explored in 24, as were other interactive groups such as
hydroxyl 25. It was encouraging to see that activity was found in
these compounds 20–25. Both the chain length of the acid and
its position on the ring influenced activity, indicating a specific
interaction with the enzyme. This led to compound 23 which com-
bined excellent potency with a measured Khyd which was very
much lower than that of 17. However, rather disappointingly the
liver S9 assay showed that the ketone in 23 was still being reduced.
This compound was also assessed in a rat in vivo PK study, and
although the clearance had been reduced compared to 18, (71 vs
>200) the plasma half-life was still very short (T½ ꢀ0.2 h). It was
concluded that clearance needed to be reduced even further in this
series.
0.37
3
0.28
2
0.16
0.00
1
7.2
6.6
6.0
5.4
f1 (ppm)
4.8
4.2
3.6
Figure 3. Detail of the NMR spectrum of compound 14 and its hydrate.
over the 1,3-dioxypropan-2-one series in terms of improved PK
properties.
At this stage of the program the design strategy was re-assessed
and it was decided to optimize potency in a keto-heterocyclic
series in which the ketone had low electrophilicity and hence
potentially better metabolic stability. To this end a method was
needed to assess the electrophilicity of the ketone in various
keto-heterocycles. It had been observed that the ketone in 1 was
reactive towards water and readily formed a hydrate. A convenient
NMR method was developed for measuring the equilibrium con-
stant for this hydration process (Khyd) for the various ketone con-
taining compounds (Fig. 2).15
Analogues 26–29, in which the phenylpentylthio group was re-
placed with other groups, were briefly investigated, to see if these
would influence metabolism (Table 2). More polar groups such as
Table 2
cPLA2
a inhibition and metabolic stability of keto-thiazoles
O
O
N
R5 R6
R4
S
X
OH
O
a
a
Compd
R5
R6
R4-X-
cPLA2 IC50
(lM)
HL60 cells IC50
(
lM)
% loss of ketoneb
Clint rat hepsc
26
27
28
29
30
31
32
33
34
35
36
37
38
H
H
H
H
Me
Me
Et
Isopropyl
Phenyl
Me
Me
Me (S)
Me (R)
H
H
H
H
H
Me
H
H
H
H
H
H
H
Ph(CH2)5–O
PhCH2S
DecylSO2
0.24
3
1.2
2
17
nt
33
13
0
nt
nt
nt
nt
nt
nt
nt
nt
20
nt
41
nt
nt
nt
nt
nt
nt
10
nt
nt
8
0.3
0.04
0.2
6.7
7.9
4.4
3.6
0.7
1.5
>1
0.2
0.23
0.7
>10
nt
5.6
3.2
2
Decyl–S
Ph(CH2)5–S
Ph(CH2)5–S
4-Chlorobenzyl–S
Ph(CH2)5–S
Ph(CH2)5–S
Ph(CH2)5–O
4-Cl-benzyl–S
Ph(CH2)5–S
Ph(CH2)5–S
1.5
nt
nt
0.2
a
b
c
Values are means of at least two experiments. nt = not tested.
Loss of parent ketone by reduction in rat liver S9 fraction.
Intrinsic clearance in rat hepatocytes l
L/min/10À6 cells.

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A. Mete et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3128–3133
make the two enantiomers of compound 30 by the route outlined
in Scheme 2.
O
OH
a
O
OEt
R
S
R5 R6
It was gratifying to see that the activity resided in only one of
the enantiomers 38. The intrinsic clearance in rat hepatocytes of
this compound was also measured and it was found to be as low
as that seen for its close analogue 35.
We have demonstrated in compounds such as 35, 36 and 38
that a ketone-based serine trap can be made metabolically stable
to reduction, while maintaining good levels of enzyme inhibition.
This was achieved by attenuating the reactivity of the ketone using
a combination of electronic and steric effects. The balance of po-
tency and PK properties for these compounds make them suitable
for use in in vivo models of inflammation to help elucidate the role
S
R
R
S
S
4
5a
b
c
O
O
R5 R6
O
N
R1
O
OEt
X
R
R5
O
R2
30 - 36
5b
d
of cPLA2a in various disease states. The metabolic stability of these
O
N
derivatives to other metabolic pathways needs to be assessed and
in combination with further improvements in their potency and
physical properties, may lead to compounds which exhibit a much
more drug-like profile.
R1
R5
37 - 38
R
X
S
R2
Scheme 2. Reagents and conditions: (a) ethyl bromo-R5,R6-acetate, Cs2CO3, DMF, rt,
40–70%; (b) 2-lithiated thiazole, THF, À78 °C to rt, 30–70%; (c) ethyl 2-(S)-
hydroxypropionate or ethyl 2-(R)-hydroxypropionate, DEAD, PPh3, THF, 0 °C to rt,
60% (d) 2-lithiated thiazole, THF, À78 °C to rt, 55%.
References and notes
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Table 3
Rat in vivo PK properties of ketothiazole derivatives
Compd
Cl (mL/min/Kg)
Vss (L/Kg)
T ½ (h)
4. (a) McKew, J. C.; Foley, M. A.; Thakker, P.; Behnke, M. L.; Lovering, F. E.; Sum, F.-
W.; Tam, S.; Wu, K.; Shen, M. W. H.; Zhang, W.; Gonzalez, M.; Liu, S.;
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Ipek, M.; Wu, K.; Wooder, L.; Ramarao, M. K.; Murphy, E. A.; Goodwin, D. G.;
Albert, L.; Xu, X.; Donahue, F.; Ku, M. S.; Keith, J.; Nickerson-Nutter, C. L.;
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et al Bioorg. Med. Chem. Lett. 2006, 16, 2798.
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17
23
35
36
>200
71
10.5
10.5
nd
0.4
0.35
0.55
<0.2
0.2
1.25
1.15
Compounds dosed intra-venously to rats at 2 mg/Kg.
nd = not defined, due to extremely high Cl.
phenylpentyloxy 26 maintained reasonable potency, but benzyl-
thio 27 led to much lower potency. The more lipophilic decylsul-
phonyl 28 and decylthio groups 29 gave very potent compounds,
exhibiting some of the best enzyme and cell potencies observed
in this series (ꢀ200 nM). However, all these compounds were still
reduced by liver S9 and two that were assessed in rat hepatocytes,
26 and 28, also had high intrinsic clearances of 20 and 41, respec-
tively. Despite the lower ketone reactivity, the compounds still had
poor metabolic stability and were not suitable for in vivo studies.
Since the ketone reactivity to hydration had been reduced as much
as could be measured, other approaches were considered to further
stabilize these ketones to reduction.
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1994, 222, 110.
It was decided to evaluate the effect of steric bulk close to the
ketone on its stability. Compounds with a range of substituents
on the
The synthetic route to these
lined in Scheme 2.16 Interestingly, only compound 30, which bears
a single methyl group - to the ketone, retained good potency.
Incorporating two methyl groups or bulkier groups - to the ke-
tone led to a large drop in activity. Gratifyingly, the -methyl sub-
stituent had also improved the stability of the ketone to reduction
and in the rat liver S9 assay no loss of compound 30 was observed.
Some less lipophilic analogues, 35 and 36, were also made and they
a
-position of the ketone 30–36 were evaluated (Table 2).
a
-substituted keto-thiazoles is out-
a
a
a
maintained reasonable levels of potency against cPLA2a. These
13. Mandagere, A. K.; Thompson, T. N.; Hwang, K. K. J. Med. Chem. 2002, 45, 304.
14. Soars, M. G.; Burchell, B.; Riley, R. J. J. Pharmacol. Exp. Ther. 2002, 301, 382.
15. Approximately 8 mg of compound was divided between four NMR tubes and
dissolved in 0.45 ml dry DMSO-d6. To each tube was added either 0, 20, 40 or
were evaluated in rat in vivo PK studies and both had low in vivo
clearance (Cl ꢀ10) and improved plasma half-lives (T½ ꢀ1.2 h).
This represents a 10-fold improvement over the non-methylated
derivatives such as 23 (Table 3). These results encouraged us to
60 ll water (or deuterium oxide). The samples were mixed and equilibrated for

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3133
5 days at room temperature. The ratio of hydrate to ketone present in each
sample was determined by accurate integration of the proton NMR spectrum.
This ratio was plotted against the mol fraction of water (or deuterium oxide)
present and Khyd was then determined from the slope. Additional
measurements suggested that the isotope effect on the equilibrium constant
was very small and that values obtained from water or deuterium oxide
measurements could be used interchangeably.
16. Connolly, S.; Mete, A. WO Patent 2000034254; Chem. Abstr. 2000, 133, 30725.