RPR203494 a pyrimidine analogue of the p38 inhibitor RPR200765A with an improved in vitro potency

Article abstract of DOI:10.1016/S0960-894X(01)00034-8

Following the discovery of RPR200765, a series of pyrimidine analogues have been prepared as backups. Amongst them, RPR203494 was identified with a better in vitro profile than RPR200765A.

Full text of DOI:10.1016/S0960-894X(01)00034-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 693±696
RPR203494 a Pyrimidine Analogue of the p38 Inhibitor
RPR200765A with an Improved In Vitro Potency
Alan J. Collis, Martyn L. Foster, Frank Halley,* Christopher Maslen, Iain M. McLay,
Kenneth M. Page, E. Jane Redford, John E. Souness and Nicola E. Wilsher
Aventis Pharma, Dagenham Research Centre, Rainham Road South, Dagenham, Essex, RM10 7XS, UK
Received 1 August 2000; accepted 10 January 2001
AbstractÐFollowing the discovery ofRPR200765, a series ofpyrimidine analogues have been prepared as backups. Amongst
them, RPR203494 was identi®ed with a better in vitro pro®le than RPR200765A. # 2001 Elsevier Science Ltd. All rights reserved.
Results and Discussion
Introduction
p38 is a member ofthe intracellular afmily ofmitogen
activated protein kinase (MAPK), implicated in a
phosphorylation cascade leading to the release ofIL-1 b
and TNF-a from leukocytes.1 IL-1b and TNF-a have
been shown to be two important cytokines associated
with the initiation and progression ofrheumatoid
arthritis (RA).2 A p38 inhibitor would inhibit the
release ofthese cytokines and hence be bene®cial in the
treatment ofRA. Two p38 inhibitors, VX-745 and SB
242235 are currently in clinical phase II and I, respec-
tively, for rheumatoid arthritis and the disease-modify-
It has been shown recently that the pyridine ring ofthe
pyridinylimidazoles class ofp38 inhibitors could be
responsible for the inhibition of the cytochrome P450
enzymes and that replacement ofthe pyridine with a
substituted
pyrimidine
would
be
bene®cial.5
RPR200765A did indeed show some inhibition of
CYP1A2 (IC50=4.2 mM) or CYP2C9 (IC50=12 mM),4
and the synthesis ofpyrimidine analogues was under-
taken. The criteria followed for the selection of the
amines (R1R2NH) used in the synthesis ofthe ®nal
aminopyrimidines (chemistry section, Scheme 1) was
that the ®nal compounds should ful®l the Lipinski rule
ing activity ofSB 242235 in an animal model has been
3
6
the subject ofa recent publication.
Following the
of5 but with a modi®ed molecular weight of600. The
discovery ofRPR200765A, a 4-phenyl-5-pyridyl-imi-
dazol-1,3-dioxanyl-morpholinyl methanone4 a series
of2-aminopyrimidines was prepared. RPR203494, a 2-
cyclopropylamino-pyrimidine analogue was found to
be more potent than RPR200765A in vitro (see
Table 1) and had minimal inhibition ofhuman CYP
isozymes:
compounds were also found to have acceptable polar
surface area7 (PSA) with most ofthe compounds having
a PSA<140 A2. All the compounds were screened in a
functional assay of lipopolysaccharide (LPS) induced
TNF-a release on human monocytes (EC50) and were
evaluated in parallel for their in vitro metabolic stability
in rat hepatic microsomes. The remaining active and
metabolically stable compounds were further evaluated
in a combinatorial-pharmacokinetic (combi-PK) study
in the rat to establish oral bioavailability. The results
are summarised in Table 1. Only the compounds reach-
ing 20% bioavailability were considered for in vivo
evaluation, followed by single PK study to con®rm
absolute bioavailability and to test inhibition ofhuman
CYP enzymes. Finally the most interesting compounds
were evaluated on p38 kinase (see Table 1 for IC50).
These results showed that di-substitution ofthe amine
was detrimental for activity (entry 2) and cyclic secondary
*Corresponding author. Tel.: +33-0-15571-3614; fax: +33-0-15571-
8014; e-mail: frank.halley@aventis.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00034-8

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A. J. Collis et al. / Bioorg. Med. Chem. Lett. 11 (2001) 693±696
Table 1. Molecular weight, PSA, EC50, in vitro rat metabolism and rat oral bioavailability ofthe pyrimidine analogues
d
Compounds
R1
R2
MW
PSA (A2)
EC50 (nM)
Metabolism (%)
F (%)
29
IC50
1
2
3
4
5
6
7
8
Methyl
Methyl
Cyclopropyl
Cyclohexyl
H
Methyl
H
482.5
496.5
508.5
550.6
536.6
526.5
540.5
511.5
539.6
553.6
512.5
526.6
540.6
468.5
508.5
522.6
521.5
510.6
558.6
564.6
572.6
572.6
559.6
588.6
576.6
544.6
545.6
545.6
574.6
127
38% (300 nM)
8% (300 nM)
60
24
26% (300 nM)
8.1
40.7
43.6
98.6
99
n/ra
3.5
12.0
29
18
18.2
n/ra
44
17.6
89.1
87.0
n/ra
94.5
68
92
81
71
25
112.8
127.6
126.4
116.4
169.4
169.3
153.7
131.7
131.8
149.9
149.7
137.9
142
125.3
125.5
151.6
125.3
126
125.6
122.1
122.1
139.4
137.8
125.7
127.2
141.6
141.7
139.3
9
H
-CH2-(CH2)3-CH2À
Carboxymethyl
Carboxyethyl
Aminoethyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>10,000
6% (300 nM)
>10,000
9
N,N-Dimethylaminoethyl
N,N-Dimethylaminopropyl
Hydroxyethyl
Hydroxypropyl
3-Methoxypropyl
H
570
27% (300 nM)
319
57
54
39% (300 nM)
110
69
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
2
15
7
5
Allyl
Cyclopropylmethyl
Cyanoethyl
Propyl
28% (300 nM)
124
12
Benzyl
24
2-Thienyl-methyl
(R)-a-Methyl-benzyl
(S)-a-Methyl-benzyl
2-Pyridyl-methyl
4-Methoxybenzyl
4-Fluorobenzyl
Phenyl
3-Pyridinyl
4-Pyridinyl
3-Methoxyphenyl
70
10
1.5
45
49
62
54
231
41
33
10
25
1
n/db
29
11
16
23.9
44.6
51
24.6
n/ra
54.1
n/cc
6
an/r=no result.
bn/d=not detected.
cn/c=not calculated due to very low level ofdetection. IC on p38 kinase.
50
amines were found to lead to inactive compounds as in
the case ofpiperidine (entry 5). The introductions of
polar groups such as an acid substituent (entries 6 and
7), a primary amine (entry 8) or a tertiary amine (entries
9 and 10) were also found to be detrimental for activity.
The potent compounds have nonpolar alkyl or aryl
residue, the most potent being the (S)-a-methyl-benzyl
substituent (entry 22, EC50=1.5 nM) which is 10 times
more potent than (R)-isomer 21 or the benzyl 19 as
previously found on a related series.8 Many compounds
had high metabolic turnover and were discarded from
further consideration although the whole cell activity
was high (as in entry 4 for example). The selection cri-
teria for the progression of the compounds were
obtained from previous experience, the cut o€ for
metabolism was determined to be 80% for this series
and only compounds having an acceptable potency
(EC50<100 nM) were considered. Eleven compounds
ful®lling these criteria were evaluated in a combi-PK
study in the rat (entries 3, 12, 13, 19, 22, 23, 24, 25, 26,
28 and 29). Four compounds were identi®ed with an
oral bioavailability suitable for further investigation
(F>20%, entries 3, 19, 23 and 26). The p38 kinase
activity measured for the selected compounds showed
good correlation with the EC50. The four compounds
were screened in vivo in a rat model ofSCW induced
arthritis, using RPR200765A as a benchmark. Com-
pound 19 was surprisingly found to be almost inactive
and compound 23 did not show superiority over
RPR200765A. Compound 26 was cytotoxic in several
whole cell assays and subsequently discarded. Com-
pound 3 was found to be potent both in the SCW model
(Fig. 1) and in the mouse LPS induced TNF-a release
assay (Fig. 2).
Compound 3 signi®cantly inhibited the reactivation of
ankle swelling at doses from 10 mg/kg/day to 30 mg/kg/
day. Compound 3 was then evaluated in a rat PK study
as a single compound following administration by the iv
and oral routes, the results are shown in Table 2.
The bioavailability ofRPR 203494 has been found to be
good at 48% but the terminal elimination T1/2 following
iv and oral administration was only just longer than 1 h.

A. J. Collis et al. / Bioorg. Med. Chem. Lett. 11 (2001) 693±696
695
Figure 2. E€ect ofRPR203494 at 0.3, 1, 3, 10 and 30 mg/kg on
Figure 1. Dose±response relationship for RPR 203494 in the SCW-
induced model ofarthritis. This ®gure shows the area under the curve
for the change in medial ankle width following iv challenge with SCW
and oral administration ofcompound at 3, 10 and 30 mg/kg.
LPS (0.1 mg/kg) induced TNF alpha release in male Balb/c mice.
MeanÆSEM *p<0.05, **p<0.01, ANOVA with post hoc Dunnett's
test compared to vehicle-treated animals.
Table 2. Pharmacokinetic parameters and determination ofthe absolute oral bioavailability ofRPR203494 in the rat
Oral administration at 5 mg/kg iv administration at 1 mg/kg
Cp max
(ng/mL)
T max
(h)
AUC0-1
(h.ng/mL)
Terminal
T1/2 (h)
AUC0-1
(h.ng/mL)
ClT
(L/h/kg)
Vdss
(L/kg)
Terminal
T1/2 (h)
F%
48
620
0.25±0.5 h
1388
1.4
580
1.8
1.2
1.2
Biology
Compound 3 was ®nally screened using recombinant
human CYP enzymes and found to have no
inhibitory e€ect on CYP2D6, CYP1A2 and CYP2C9 up
to 50 mM.
Monocyte TNF-ꢀ release assay
Adherent human monocytes (100,000 cells/well) were
incubated with LPS (10 ng/mL) in the absence and pre-
sence ofcompound ofr 18 h. Individual experiments
were carried out in quadruplicate samples. TNFa was
measured by sandwich ELISA and EC50 values calcu-
lated for the activity of individual compounds. EC50
values shown from repeat experiments are means
ÆSEM (n=3).
Chemistry
The chemistry was adapted from the synthesis of RPR
200765A,9 starting from a 2-methythio-4-methylpyr-
imidine instead ofa 4-methylpyridine (Scheme 1).
Scheme 1. (i) DMFDMA, toluene, re¯ux (82%); (ii) ethyl 4-¯uorobenzoate, HMDSNa, THF, rt (99%); (iii) 48% aq HBr, DMSO, 65 ꢀC (86%); (iv)
glyoxaldimethylacetal, NH4OAc, AcOH, methyl-tert-butyl ether, rt (80%); (v) 2,2-dihydroxymorpholinylpropionamide, pTSA, DMF, 80 ꢀC (50%);
(vi) mCPBA, DCM, rt (80%); (vii) R1R2NH, N-methyl pyrrolidinone, 80 ꢀC (20±80%).

696
A. J. Collis et al. / Bioorg. Med. Chem. Lett. 11 (2001) 693±696
Kinase assay
(EC50=60 nM, IC50=9 nM on p38 kinase). RPR203494
moreover demonstrated a decreased inhibition ofhepa-
tic cytochrome P450 enzymes. Furthermore,
RPR203494 was found to have no inhibitory e€ect on
COX1 at 10 mM.
The p38 enzyme assay is carried out at room tempera-
ture for 1 h, using 40 ng/well of the mouse enzyme. The
substrate, 50 mg/mL ATF-2 is coated onto 96-well
plates, the assay is carried out in 25 mM Hepes bu€er,
pH 7.7 containing 25 mM magnesium chloride, 2 mM
dithiothreitol, 1 mM sodium orthovanadate and 100 mM
ATP. Phosphorylated ATF-2 is quantitated using
phospho-speci®c ATF-2 primary antibodies. IC50 values
shown from repeat experiments are meansÆSEM
(n=3).
Acknowledgements
The authors thank Adnan Alshaar, Mustafa Bhimani,
Brenda Burton and Stuart Parker for technical support.
Rat SCW model
References
Lewis rats received an i.a. injection of10 mg SCW 100P
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(5 mL/kg/day). Ankle width was measured daily from
day 21 to 25. * p<0.05 compare to vehicle, ANOVA
with post hoc Dunnett's test.
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Conclusion
A rapid investigation ofRPR200765A pyrimidine ana-
logues led to the identi®cation ofRPR203494 (com-
pound 3) which was shown to be more potent in vitro
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