N-(pyrimidin-4-yl) and N-(pyridin-2-yl) phenylalanine derivatives as VLA-4 integrin antagonists.

Article abstract of DOI:10.1016/S0960-894X(02)00241-X

The SAR studies to optimise both potency and rate of clearance in the rat for a series of pyrimidine and pyridine based VLA-4 antagonists are described.

Full text of DOI:10.1016/S0960-894X(02)00241-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1595–1598
N-(Pyrimidin-4-yl) and N-(Pyridin-2-yl) Phenylalanine Derivatives
as VLA-4 Integrin Antagonists
John R. Porter,* Sarah C. Archibald, Julien A. Brown, Kirstie Childs, David Critchley,
John C. Head, Brian Hutchinson, Ted A. H. Parton, Martyn K. Robinson,
Anthony Shock, Graham J. Warrellow and Alex Zomaya
Celltech R&D Ltd, 216 Bath Road, Slough SL1 4EN, UK
Received 28 February 2002; accepted 9 April 2002
Abstract—The SAR studies to optimise both potency and rate of clearance in the rat for a series of pyrimidine and pyridine based
VLA-4 antagonists are described. # 2002 Elsevier Science Ltd. All rights reserved.
The integrin very late antigen-4 (VLA-4) is a hetero-
dimeric (a4b1) adhesion molecule, expressed on the
surface of many leukocytes. The binding of VLA-4 to
ligands such as vascular cell adhesion molecule-1
(VCAM-1) expressed on endothelial cells is recognised
as a key step in the processes of adhesion, migration and
activation of inflammatory leukocytes at sites of
inflammation.1 Blocking such an interaction would be
expected to be of therapeutic benefit in a variety of
inflammatory and autoimmune diseases. Consequently
the design of VLA-4 antagonists is currently a major
target for the pharmaceutical industry.2
the N-(pyrimidin-4-yl) and N-(pyridin-2-yl) phenylala-
nine lead structures, 1 and 2. This communication out-
lines the SAR studies aimed at optimising the potency
and rate of clearance on this series of compounds.
We have recently reported on our initial discovery of
N-acyl derivatives of phenylalanine that afforded highly
potent antagonists of VLA-4 binding to VCAM-1.3
However, virtually all such analogues suffered from
rapid bilary clearance from the body by an as yet unde-
termined active transport mechanism. Postulating that
the central amide bond was playing a key role in the
recognition of these structures by the transporter, we
sought to replace this functionality with a suitable iso-
stere. The preceding communication4 describes our
efforts to incorporate the amide carbonyl group into a
heterocycle system to give N-(triazin-1,3,5-yl)-phenyl-
alanine derivatives. These compounds displayed much
reduced clearance, but initial analogues possessed only
modest potency as VLA-4 antagonists. We also described
Our initial structure–activity study focused on opti-
mising the N-pyrimidin-4-yl compound 1. Once again
we opted to retain the dichloropyridyl amide derivatised
analogues of phenylalanine for the core of our struc-
tures and to explore the effect of introducing sub-
stituents in the 2- and 6-positions of the pyrimidine.
Compounds in this series were prepared by condensa-
tion of the appropriately substituted pyrimidine with
the 3,5-dichloropyrid-4-ylcarboxamido substituted phe-
nylalanine ester 3, followed by aqueous LiOH hydro-
lysis of the ester to give the target acid 5 as shown in
Scheme 1. 4-Chloro or sulphonylalkyl substitutents on
the pyrimidine were found to be acceptable leaving
groups for this transformation, and where not commer-
cially available were prepared as shown in Scheme 2.
Additionally, where a 2- or a 6-sulphonyl functionality
was required this group could be introduced by mCPBA
oxidation of the ester 4 (where R=thioalkyl).
*Corresponding author. Tel.: +44-1753-534655; fax: +44-1753-
536632; e-mail: john.porter@celltechgroup.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00241-X

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J. R. Porter et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1595–1598
Table 1. Potency and rate of clearance for 2- and/or 6-substituted
pyrimidines
Compd
R1
R2
VLA-4 protein6 VLA-4 cell7 IPRL
IC50 (nM)
IC50 (nM) k (hÀ1
)
1
6
7
8
9
10
11
12
13
14
15
16
17
Cl
nPrS
nPrSO2
nPrSO
PhCH2
PhO
MeSO2
PhCH2SO2
H
H
H
H
H
H
H
H
H
MeS
nPrSO2
MeSO2
nPrSO2
nPrSO2
80
60
1
1700
460
40
2.1
nd
4.4
4.3
6.4
3.1
4.0
5.2
nd
4.4
4.9
5.6
6.5
6
50
40
20
6
10
60
40
7
1100
520
670
50
3400
430
1000
140
140
Scheme 1. (i) Pyrimidine, DIPEA, DCM; (ii) LiOH, water, dioxan.
H
MeO
MeO
Me
4
2
Table 2. Potency and rate of clearance for carboxylic acid substituted
pyrimidines
Scheme 2. (i) NaSR (2 equiv), EtOH; (ii) NaSR (1 equiv), EtOH; (iii)
mCPBA, DCM.
In order to gain some understanding of the SAR for
clearance it was considered essential to have access to a
relatively high throughput screen that could provide this
data. The method chosen was the isolated perfused rat
liver5 (IPRL), whereby five compounds (including a
reference compound) could be dosed as a cassette. The
elimination of each compound from the perfusate is
expressed in terms of a rate constant, k, and normalised
to the reference compound. The higher the value of k
the more rapidly the compound was cleared. Com-
pounds were assayed for their ability to inhibit the
binding of VLA-4 to VCAM-1 in a protein-based,
ligand binding6 and a cell-based, adhesion7 assay.
Compd
R1
R2
VLA-4 protein6 VLA-4 cell7 IPRL
IC50 (nM)
IC50 (nM) k (hÀ1
)
18
19
20
21
5-CO2H
MeS
660
4
14
30
32,000
180
1500
520
1.3
0.9
3.1
5.9
6-CO2H nPrSO2
6-CO2H nHexSO2
6-CO2H iBuSO2
a clearance of 27 mL/min/kg, a t1/2 of 0.5 h and an oral
bioavailability of 4% after dosing in the rat (10 mg/kg
po and iv).
Initially we looked at a range of 6-substituted pyr-
imidines as shown in Table 1. Disappointingly, although
these compounds were more potent in the cell based
assay than the lead compound, 1, they all displayed
relatively rapid rates of clearance. However, the greatly
improved potency of the sulphonyl derivative 7 was
striking and prompted the synthesis of a number of
sulphonyl analogues. In general an alkylsulphonyl sub-
stituent afforded high potency in either the 2- or 6-pyr-
imidine position, and the larger alkylsulphonyl group
was more active than the methylsulphonyl analogue
(compare 7 with 11 and 15 with 16). Interestingly, the
sulphinyl analogue 8 is equipotent to the sulphonyl
equivalent 7. However, despite the variation in the phy-
sicochemical properties (logP, pKa) of these N-pyr-
imidinyl analogues, all such compounds displayed
relatively rapid clearance; for example compound 7 had
It had previously been observed in related and unrelated
series8 that incorporation of carboxylate functionality in
this region of the molecule could have a profound effect
upon the rate of clearance with only moderate loss in
potency. When we incorporated this modification into
the pyrimidine series, we observed a similar effect
(Table 2). Compound 18 had a clearance of 3 mL/min/
kg, and a t1/2 of 0.8 h after dosing in the rat (1 mg/kg iv).
The corresponding N-pyridin-2-yl analogues were also
investigated. Their synthesis by nucleophilic substitu-
tion was limited to analogues in which the pyridine
precursor bore a sufficiently powerful electron-with-
drawing group. Hence an alternative route based on
Rh-catalysed carbene insertion9 was developed; the

J. R. Porter et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1595–1598
Table 3. Potency and rate of clearance for pyridines
1597
Compd
R
VLA-4 protein6 VLA-4 cell7
IPRL
k (hÀ1
)
IC50 (nM)
IC50 (nM)
2
260
2400
1.0
24
25
26
27
28
29
30
31
50
410
1400
7
1300
53,000
nd
nd
0.2
nd
Scheme 3. (i) Isoamyl nitrite, CHCl3, AcOH, reflux; (ii) 2-amino-4-
thiopropylpyridine, Rh2(OAc)2, toluene, 80 ꢀC; (iii) mCPBA, DCM;
(iv) LiOH, water, dioxan.
synthesis of the 4-sulphonyl substituted analogue 29 is
illustrative (Scheme 3). Treatment of the 3,5-dichloro-
pyrid-4-ylcarboxamido substituted phenyl alanine ester
3 with isoamyl nitrite in chloroform at reflux for 1 h
gave the diazo compound 22 in good yield. Heating
with the appropriately substituted pyridine in the pre-
sence of catalytic rhodium (II) acetate in toluene affor-
ded the pyridyl compound 23. Oxidation (where the
pyridyl substituent was a thioalkyl group) was per-
formed with mCPBA, and aqueous hydrolysis with
LiOH gave the target acid 29. Although this method
affords relatively easy access to the desired pyridine
analogues it does destroy the chiral centre, all reported
data for these compounds (Table 3), is for the racemic
mixture.
80
4.6
2.1
5.2
nd
4
70
2
50
50
10
1600
90
0.2
The activity of 28 and 29 (Table 3) demonstrated again
the potency-enhancing effect of the alkylsulphonyl
group relative to the alkylthio or chloro substituents.
The alkylsulphinyl group of 27 is equipotent and cleared
at the same rate as the sulphonyl analogue 28. The
greatly reduced (though not abolished) potency of the
phenyl analogue 30 [compared to the corresponding
pyridinyl or pyrimidinyl analogues (29 and 7)] illustrates
the importance of having at least one electronegative
nitrogen atom in the ortho ring position. This atom
presumably mimics the oxygen of the carbonyl in the
original N-acyl lead structures. Although the potent
antagonist 28 displays a relatively good clearance value,
this study generally showed that alkylsulphonyl sub-
stituted pyridine analogues, like their pyrimidine coun-
terparts, suffer from high clearance rates. Again, it is
interesting to note that the combination of sulphonyl
and carboxylate functionality, 31, gives a compound
that has a respectable potency and a much slower rate
of clearance.
space to the acetyl or sulphonyl group in a potent series
of proline based antagonists.3 We have also observed
that the presence of carboxylate functionality can
improve the rate of clearance in the rat. The reason for
this is not clear, though it is thought unlikely to be a
result of plasma protein binding. Possibly the transpor-
ter cannot recognise the more polar molecule. However,
the increased polarity of these molecules suggests that they
will be poorly absorbed following oral administration.
Acknowledgements
This paper is dedicated to the memory of Alex Zomaya.
In conclusion, we have identified a novel series of VLA-
4 antagonists and have gained useful insights into their
SAR for potency and rate of clearance. In particular the
incorporation of sulphonyl or sulphinyl functionality
appears to enhance potency, possibly as these hydrogen
bond acceptor groups can access a similar region of
References and Notes
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