The identification of clinical candidate SB-480848: A potent inhibitor of lipoprotein-associated phospholipase A2

Article abstract of DOI:10.1016/S0960-894X(03)00058-1

Modification of the pyrimidone 5-substituent in clinical candidate SB-435495 has given a series of inhibitors of recombinant lipoprotein-associated phospholipase A₂ with sub-nanomolar potency. Cyclopentyl fused derivative 21, SB-480848, showed an enhanced in vitro and in vivo profile versus SB-435495 and has been selected for progression to man.

Full text of DOI:10.1016/S0960-894X(03)00058-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1067–1070
The Identification of Clinical Candidate SB-480848: A Potent
Inhibitor of Lipoprotein-Associated Phospholipase A2
Josie A. Blackie, Jackie C. Bloomer, Murray J. B. Brown, Hung-Yuan Cheng,
Beverley Hammond, Deirdre M. B. Hickey, RobertJ. Ife, Colin A. Leach, V. Ann Lewis,
Colin H. Macphee, Kevin J. Milliner, Kitty E. Moores, Ivan L. Pinto, Stephen A. Smith,*
Ian G. Stansfield, Steven J. Stanway, Maxine A. Taylor and Colin J. Theobald
Medicines Research Centre, GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, UK
Received 15 November 2002; revised 9 January 2003; accepted 10 January 2003
Abstract—Modification of the pyrimidone 5-substituent in clinical candidate SB-435495 has given a series of inhibitors of recom-
binant lipoprotein-associated phospholipase A2 with sub-nanomolar potency. Cyclopentyl fused derivative 21, SB-480848, showed
an enhanced in vitro and in vivo profile versus SB-435495 and has been selected for progression to man.
# 2003 Elsevier Science Ltd. All rights reserved.
Atherosclerosis bears many hallmarks of a chronic
inflammatory disease.1 For this reason we have targeted
an enzyme, lipoprotein associated phospholipase A2
(Lp-PLA2), that is able to generate inflammatory pro-
ducts, lysophosphatidylcholine (lyso-PtdCho) and oxi-
dised fatty acids, on hydrolysis of oxidised low density
lipoprotein (LDL). Both of these hydrolysis products
have been implicated in atherosclerosis.2,3 Additionally,
the increased levels of lyso-PtdCho in oxidised LDL can
be completely accounted for by Lp-PLA2.3 Further-
more, a recent study has shown a strong, positive
correlation between levels of lipoprotein associated
phospholipase A2 (Lp-PLA2) and coronary events in
asymptomatic, hypercholesterolemic men and has sug-
gested that Lp-PLA2 is a new, independentmarker of
end, we decided to modify the pyrimidone 5-substituent
and now report the beneficial effects of this study.
Compounds 2–7, 10, 11 and 14 were synthesised using
methods similar to those previously described.6 Halo
derivatives 8 and 9 were prepared from 1,5-unsub-
stituted pyrimidones by reaction with the corresponding
N-halosuccinimide and subsequent introduction of the
1-substituent as before.6 Oxidation of 11 with meta-
chloroperbenzoic acid gave 12. Compound 13 was pre-
pared via known methods.7 Hydroxyethyl derivative 14
was converted into amine 15 via methanesulfonation,
azide displacementand reduciton (H 2, Pd/C). Com-
pounds 18–23 were prepared as exemplified for 21 in
Scheme 1.
4
coronary heartdisease risk. As a result of these data,
we soughtinhibiotrs of Lp-PLA
this lipase in atherosclerosis.
to assess the role of
2
We have previously reported the identification of com-
pound 1, SB-435495, a very potent inhibitor of Lp-
PLA2 with a suitable profile for evaluation in man.5 In
view of the exceptional potency of this compound, we
speculated that good activity would still be achievable in
simplified (lower molecular weight) analogues. To this
Compounds were evaluated using recombinant human
Lp-PLA2 (rhLp-PLA2).8 Non-specific binding effects in
plasma were assessed by evaluating compounds against the
enzyme in both whole human and Watanabe hereditable
*Corresponding author. Tel.: +44-1438-763492; fax: +44-1438-
763620; e-mail: stephen_1_smith@gsk.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00058-1

1068
J.A.Blackie et al./ Bioorg.Med.Chem.Lett.13 (2003) 1067–1070
Scheme 1. Reagents: (i) (a) thiourea, NaOEt, EtOH; (b) 4-F-PhCH2Cl, K2CO3, DMF; (ii) (a) tBuO2CCH2I, iPr2NEt, CH2Cl2; (b) TFA, CH2Cl2;
(iii) EDC, HOBT, DMF.
hyperlipidaemic (WHHL) rabbitplasma ata single
concentration of inhibitor.8 High potency in rabbit and
human plasma, alongside little interaction with a bat-
tery of cytochrome P450 enzymes5 and reasonable levels
of permeability5 were normally required before com-
pounds were evaluated in vivo in WHHL rabbits,8 and
for selected inhibitors, in the rat and dog.9
Compounds bearing a number of other non-polar sub-
stituents typically proved highly potent inhibitors with
only the 5-methoxy derivative 10 less active (cf. 8, 9 and
11 with 10). Of the more polar derivatives prepared
(compounds 12–17) none matched the potency of the
5-ethyl derivative 6 and, in contrast to the less polar
analogues (3–10), none proved sufficiently permeable
across artificial membranes.5
Although removal of the entire 5-substituent in 1 resul-
ted in a marked reduction in potency (Table 1, cf. 1 and
2), sub-nanomolar inhibition could be regained on the
introduction of a simple methyl, or more preferably
ethyl, group (see compounds 1–6). Increasing the chain
length further (see 7 vs 6) was not advantageous either
in terms of potency or CYP450 interaction (see below).
A simple methyl substituent when placed at the 6-posi-
tion gave a compound of lower potency (Table 2, cf. 18
and 4). However in contrast to our original work with
compounds bearing
a
5-heterocyclylmethyl sub-
stituent,11 the introduction of a 6-methyl group into the
5-methyl series was not detrimental (see 19 and 4), likely
Table 1. 5-Substituent modification
No.a
Z
R5
% Inhibition in plasma
b
IC50 nM
Human 10 nM
Rabbit100 nM
Permeability cm/h
1
2
3
4
5
6
7
8
CF3
Cl
Cl
CF3
Cl
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CH2(1-Me-pyrazol-4-yl)
0.06
50
5
87
1
10
33
48
69
18
12
29
NT
22
NT
18
18
95
10
41
63
0.12
0.10
43
47
60
NT
45
NT
56
29
NT
33
0.017
NTc
0.10
0.08
H
Me
Me
Et1
Et0.4
Pr
Cl
Br
1
24
48
0.8
3
0.13
0.11
0.09
0.025
0.03
0.006
0.002
0.003
NT
0.003
0.005
9
1
10
11
12
13
14
15
16
17
OCH3
SCH3
SOCH3
19
0.8
0.8
1.5
2
38
6
1
CH2OH
CH2CH2OH
CH2CH2NH2
CH2CH2NHAc
CH2CH2NHSO2CH3
NT
17
NT
NT
aAll new compounds gave satisfactory analytical/spectral data.10
bSee ref 5. Permeability >0.01 cm/h considered acceptable.
cNot tested.

J.A.Blackie et al./ Bioorg.Med.Chem.Lett.13 (2003) 1067–1070
1069
Table 2. Pyrimidone-5,6-substitution
Table 3. Interaction of selected compounds with CYP450 2D6 and
3A4
No.a R5 R6
Z
Inhibition in plasma
No.
R5
R6
Z
Lp-PLA2
IC50 nM
CYP450
2D6
IC50 (mM)a
3A4
IC50 nM Human Rabbit Permeability
cm/hb
10 nM 100 nM
1
3
4
5
6
7
8
9
11
18
19
20
21
22
23
b
Me
Me
EtH
Et
Pr
Cl
Br
H
H
H
Cl
H
H
H
H
H
Me
Me
CF3
Cl
CF3
0.06
5
1
37
15
39
10
13
17
4
Me
H
H CF3
Me CF3
1
3
33
6
39
36
63
39
71
67
79
NT
NT
0.08
0.05
0.03
0.14
0.10
NT
18
19
20
21
22
23
Me Me CF3
0.5
0.4
0.25
0.25
0.1
1
14
7
(CH2)3
(CH2)3
(CH2)4
(CH2)4
Cl
CF3
Cl
CF
3
CF3
0.4
0.8
3
1
0.8
3
0.5
0.4
0.25
0.25
0.1
24
22
83
56
39
47
24
25
26
6
11
5
60
NTc
NT
CF3
CF3
CF3
CF3
CF3
Cl
CF3
Cl
CF3
29
39
15
22
22
18
27
5
CF3
0.06
SMe
H
Me
aAll new compounds gave satisfactory analytical/spectral data.10
bSee ref 5. Permeability >0.01 cm/h considered acceptable.
cNot tested.
(CH2)3
(CH2)3
(CH2)4
(CH2)4
due to the absence of any significant conformational
effect in this series. Following this result, and in view of
the good activity of compound 6, we prepared cyclic
analogues 20–23. All of these compounds proved very
potent inhibitors of rhLp-PLA2 with the tri-
fluoromethylbiphenyl derivatives again more potent
than their 4-chloro analogues.5 Furthermore, whilst
compounds of Table 2 passed the permeability guide-
lines, cyclopentyl fused derivatives 20 and 21 proved
particularly permeable.
9
10
aSee ref 5, target IC50 ꢀ10 mM.
bCH2(1-Me-pyrazol-4-yl).
In contrast to our previous series,5 the majority of
compounds passed our criteria for CYP450 interaction
(target IC50 ꢀ10 mM) although the potent cyclohexyl
fused pyrimidones 22 and 23 proved important excep-
tions to this finding (results against key isozymes 2D6
and 3A4 are shown for compounds of interest in Table
3).
With these data in hand, the most potent compounds in
whole plasma (6, 19 and 21) were progressed to the
WHHL rabbit for evaluation and comparison with
compound 1. In this model, compounds 6 and 19 gave
very similar profiles to 1 (data not shown), whilst
cyclopentyl fused derivative 21 proved considerably
more effective, showing prolonged inhibition of plasma
Lp-PLA2 over 24 h (Fig. 1) and a good correlation of
pharmacodynamic and pharmacokinetic effects (Fig. 2).
As a consequence of these findings, 21 was chosen for a
more detailed evaluation.
Figure 1. Inhibition of plasma Lp-PLA2 in the WHHL rabbit @ 10
mg/kg (n=2) po.
Additional in vivo studies with 21 indicated an oral
bioavailability of 11Æ2% in the fed rat. This was a
marked improvementover compound 1 for which fur-
ther studies had shown that rat bioavailability fell from
13Æ1% in the fasted state to 2Æ1% in the fed animal.
The fed:fasted ratio for compound 21 was 1:1.9Æ0.4,
again a considerable advance over 1 and consistent with
an oral bioavailability of ꢁ21% in the fasted rat.9 The
oral bioavailability of 21 was 28Æ4% in the dog (cf.
24Æ7% for 1).9 Furthermore excellent inhibition of Lp-
PLA2 within the atherosclerotic plaque was achieved for
21, wit h 95Æ1% inhibition observed 2 h after an oral
dose of 30 mg/kg to the WHHL rabbit.12
Mechanistic studies using steady state and transient
kinetics indicated compound 21 to be a freely reversible,
non-covalently bound, inhibitor of rhLp-PLA2 with a Ki
of 110 pM and an off-rate of 27 min.5 Potent inhibition
of the enzyme in whole human plasma was confirmed
(IC50=5Æ2 nM). Furthermore, the presence of com-
pound 21 during the copper catalysed oxidation of
human LDL prevented the production of lyso-PtdCho
(IC50=4Æ3 nM) and subsequentmonocyte chemotaxis
(IC50=4Æ1 nM).5

1070
J.A.Blackie et al./ Bioorg.Med.Chem.Lett.13 (2003) 1067–1070
K. E.; Krishna, M.; Wilkinson, F. E.; Rumley, A.; Lowe,
G. D. O. N.Engl.J.Med. 2000, 343, 1148.
5. Blackie, J. A.; Bloomer, J. C.; Brown, M. J. B.; Cheng, H.-
Y.; Elliott, R. L.; Hammond, B.; Hickey, D. M. B.; 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.; Whittaker, C. M.
Bioorg.Med.Chem.Lett. 2002, 12, 2603, and references therein.
6. Bloomer, J. C.; Boyd, H. F.; Hickey, D. M. B.; Ife, R. J.;
Leach, C. A.; Macphee, C. H.; Milliner, K. J.; Pinto, I. L.;
Rawlings, D. A.; Smith, S. A.; Stansfield, I. G.; Stanway, S. J.;
Taylor, M. A.; Theobald, C. J.; Whittaker, C. M. Bioorg.
Med.Chem.Lett. 2001, 11, 1925.
7. Ghosh, C.; Schmidt, D. G.; Pal, B. C. J.Org.Chem. 1984,
49, 5257.
8. Boyd, H. F.; Fell, S. C. M.; Flynn, S. T.; Hickey, D. M. B.;
Ife, R. J.; Leach, C. A.; Macphee, C. H.; Milliner, K. J.;
Moores, K. E.; Pinto, I. L.; Porter, R. A.; Rawlings, D. A.;
Smith, S. A.; Stansfield, I. G.; Tew, D. G.; Theobald, C. J.;
Whittaker, C. M. Bioorg.Med.Chem.Lett. 2000, 10, 2557.
9. Oral bioavailability was determined for the free base of 21
as described in ref 5, except that the iv infusion was in normal
saline containing 10% (w/v) Encapsin and 2% (v/v) DMSO
and the oral dose by gavage administration in 1% aq methyl-
cellulose (rat) or by loose filled gelatine capsule (dog). The
fed:fasted ratio in the rat was measured by oral gavage
administration (10 mg/kg in 1% Tween 80 and 1% aq
methylcellulose) to the conscious, cannulated fed or fasted
male rat. Serial blood samples were collected up to 14 h post
dose and analysed by LC/MS/MS. The fed:fasted ratio was
calculated as AUC (fed)/AUC (fasted).
Figure 2. Pharmacodynamic/pharmacokinetic correlation in the
WHHL rabbit: Compound 21 @ 10 mg/kg (n=2) po.
As a result of the excellent in vitro effects of 21 along-
side both an enhanced in vivo profile and shorter route
of synthesis versus SB-435495, compound 21,
SB-480848, was selected to replace SB-435495 for our
evaluation of the effects of Lp-PLA2 inhibition in man.
The encouraging initial human studies with SB-480848
will be published shortly.
10. Representative examples: compound 6 bitartrate salt 1H
NMR (DMSO-d6, rotamer mixture) d 0.96 (3H, m), 1.07 (6H,
m), 2.27 (2H, m), 2.59 (2H, m), 2.84 (2H,m), 3.37/3.50 (4H,
m), 4.26 (2H, s), 4.39/4.43 (2H, 2Âs), 4,64/4.72 (2H, 2Âs),
4,94/5.09 (2H, 2Âs), 7.11/7.14 (2H, 2Âm), 7.36–7.49 (5H, m),
7.63/7.72 (2H, 2Âd), 7.84 (4H, m); MS (APCI+) found
(M+1)=655; C35H38F4N4O2S requires 654. Compound 21
In conclusion, we have shown that highly potent inhib-
itors of Lp-PLA2 may be obtained on simplification of
the pyrimidone 5-substituent in compound 1. One of
these simplified inhibitors, compound 21, SB-480848,
demonstrated excellent in vitro and in vivo profiles and
has been chosen for progression to man. Our assessment
of Lp-PLA2 as a therapeutic target will undoubtedly be
enhanced by further studies with SB-480848.
1
free base H NMR (CDCl3, rotamer mixture) d 0.99 (6H, t),
2.10 (2H, m), 2.50 (4H, q), 2.58/2.62 (2H, 2Ât), 2.70/2.82 (2H,
2Ât), 2.86 (2H, t), 3.28/3.58 (2H, 2Ât), 4.45/4.52 (2H, 2Âs),
4.68/4.70 (2H, 2Âs), 4.93 (2H, s), 6.95 (2H, m), 7.31 (2H, d),
7.31/7.37 (2H, 2Âm), 7.48/7.52 (2H, d), 7.65 (2H, m), 7.72
(2H, m); MS (APCI+) found (M+1)=667; C36H38F4N4O2S
requires 666.
References and Notes
1. Libby, P.; Ridker, P. M.; Maseri, A. Circulation 2002, 105,
1135.
2. Suckling, K. E.; Macphee, C. H. Exp.Opin.Ther.Targets
2002, 6, 309.
3. Macphee, C. H. Curr.Opin.Pharmacology 2001, 1, 121,
and references therein.
4. Packard, C. J.; O’Reilly, D. St. J.; Caslake, M. J.; McMa-
hon, A. D.; Ford, I.; Cooney, J.; Macphee, C. H.; Suckling,
11. Boyd, H. F.; Flynn, S. F.; Hickey, D. M. B.; Ife, R. J.;
Jones, M.; Leach, C. A.; Macphee, C. H.; Milliner, K. J.;
Rawlings, D. A.; Slingsby, B. P.; Smith, S. A.; Stansfield, I. G.;
Tew, D. G.; Theobald, C. J. Bioorg.Med.Chem.Lett.
10, 395.
2000,
12. Determined as in ref 5, data from 15 month old female
WHHL rabbits. Compound 1 gave an inhibition of 74Æ9% in
sex and age (14 months) matched WHHL rabbits.