Discovery and evaluation of N-(triazin-1,3,5-yl) phenylalanine derivatives as VLA-4 integrin antagonists.

Article abstract of DOI:10.1016/S0960-894X(02)00237-8

SAR studies aimed at improving the rate of clearance of a series of VLA-4 integrin antagonists by the introduction of a 1,3,5-triazine as an amide isostere are described.

Full text of DOI:10.1016/S0960-894X(02)00237-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1591–1594
Discovery and Evaluation of N-(triazin-1,3,5-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; revised 9 April 2002; accepted 9 April 2002
Abstract—SAR studies aimed at improving the rate of clearance of a series of VLA-4 integrin antagonists by the introduction of a
1,3,5-triazine as an amide isostere 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, including T-lymphocytes
and eosinophils, but not neutrophils. The binding of
VLA-4 to ligands such as vascular cell adhesion mole-
cule-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.
Anti-a4 antibodies have shown efficacy in animal mod-
els of a number of inflammatory diseases and the results
from early stage clinical trials are promising.2 Thus,
small molecule VLA-4 receptor antagonists represent an
attractive target for the treatment of various chronic
inflammatory diseases such as asthma, multiple sclerosis
and rheumatoid arthritis.3
gave a series of potent compounds. Disappointingly,
these compounds displayed rapid clearance in a number
of species thereby hindering further development. That
this rapid clearance was a result of an active transport
mechanism was shown by the presence in the bile of
only trace amounts of metabolites compared to the
parent compounds.5
We have recently described the discovery of the thio-
proline CT5219,4 1, a small molecule VLA-4 antagonist
that showed excellent efficacy in inhibiting antigen-
induced airway responses in the allergic sheep model.
The potency of 1 could be further enhanced by the
replacement of the 2,6-dichlorobenzyl ether with a
3,5-dichloropyridylamide group, 2. Depeptidisation by
replacement of the thioproline group with a benzamide
or pyridyl amide as in, for example, compound 3 also
We postulated that the a-amido group, although
apparently essential for potency, was responsible for the
rapid clearance in this series. In an attempt to move
away from the lead acyl derivatives but retain the features
believed necessary for inhibitory activity, we envisaged
incorporating the carbonyl of the a-aminocarbonyl
*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)00237-8

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J. R. Porter et al. /Bioorg. Med. Chem. Lett. 12 (2002) 1591–1594
group into a heterocycle, to obtain N-(triazin-1,3,5-yl)-
phenylalanine derivatives such as 4. In this way, we
retained a degree of structural and electrostatic similar-
ity but also ensured that the a-amino group remained
non-basic and also possessed a hydrogen atom. These
features were felt to be necessary for potent VLA-4
antagonists based on a number of earlier structure–
activity studies which included attempts to replace the
a-aminocarbonyl group with isosteres. These included
CH2NH, –CH2O–, –CH¼CH– and N-methylation of
the a-aminocarbonyl. All such substitutions lead to a
significant reduction in activity.
give the ester 8. Aqueous LiOH hydrolysis of this ester
afforded the target compound 4.
Table 1 shows some of these compounds and compares
their activities against VLA-4 and their rate of clearance
with the amides 2 and 3. Although the potencies of the
N-heteroaryl derivatives 4, 9 and 10 are much reduced
compared to the N-acyl derivatives 2 or 3, there is a
marked improvement (reduction) in their rate of bilary
clearance. We initially focused on optimising the
N-triazin-1,3,5-yl derivative 4, because of the combina-
tion of reasonable potency, low rate of clearance, pro-
mising DMPK and ease of synthesis of analogues of this
type of compound. The triazine 4 displayed a clearance
of 3 mL/min/kg, a t1/2 of 1.6 h and 9% bioavailability in
the rat (10 mg/kg po and iv).
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
liver6 (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 binding7 and a cell-based, adhesion8 assay.
Further derivatisation of the triazines was achieved by
condensation of the appropriate 1,3-dialkoxy-5-chloro
triazine with the amino ester 7 in a similar fashion to
that shown in Scheme 1. Alternatively, condensation of
1,3-dichloro-5-methoxy triazine with the amino ester 7
followed by displacement of the remaining chloro atom
with either amines or alcohols gave the differentially sub-
stituted triazines 11 as shown in Scheme 2. In both routes
the esters were hydrolysed under aqueous conditions to
generate the target acids.
We opted, in the first instance, to retain the dichloro-
pyridyl amide derivatised analogues of phenylalanine
for the core of our structures. Incorporation of this
group had given consistently the most potent VLA-4
antagonists in other series. The compounds were readily
prepared as outlined in Scheme 1. The lithiated 3,5-
dichloropyridine 5 was quenched with CO2 to generate
the carboxylic acid that was converted to the acid chlor-
ide 6 by treatment with thionyl chloride. Coupling of 6
to the a-N-Boc protected ethyl ester of 4-aminophen-
ylalanine in the presence of base gave, after removal of
the Boc group, the amine 7. The amino group of this
intermediate 7 could then be reacted with a range of
reactive, commercially available heterocycles as shown,
for example with 1,3-dimethoxy-5-chloro triazine to
The resulting data for some of the prepared analogues is
given in Table 2. It is evident that a wide range of
alkoxy and amino groups are tolerated with regard to
potency and it was possible to improve potency 3–4 fold
Table 1. Potency and rate of clearance for lead compounds
Compd
R
VLA-4 protein7
IC50 (nM)
VLA-4 cell8
IC50 (nM)
IPRL
k (hꢀ1
)
2
0.6
0.9
8
0.5
0.5
3.7
3
4
3.9
1.1
900
9
80
1700
2500
2.1
1.0
Scheme 1. (i) LDA, THF, ꢀ78 ꢁC, 30 min then CO2; (ii) SOCl2, DCM;
(iii) a-N-Boc 4-aminophenylalanine ethyl ester, N-methyl morpholine,
DCM; (iv) 3 M HCl in EtOAc; (v) 1,3-dimethoxy-5-chloro triazine,
DIPEA (1 equiv), DCM; (vi) LiOH, THF, H2O.
10
260

J. R. Porter et al. /Bioorg. Med. Chem. Lett. 12 (2002) 1591–1594
1593
Table 3. Potency and rate of clearance for phenylalanine substituents
Compd
R
VLA-4 protein7
IC50 (nM)
VLA-4 cell8
IC50 (nM)
IPRL
k (hꢀ1
)
23
50
4600
0.6
24
25
26
30
4000
nd
0.7
2.2
nd
1500
5500
Scheme 2. (i) 1,3-Dichloro-5-methoxy triazine, DIPEA (2 equiv),
DCM; (ii) RXH, NaH, THF or RNH2, DIPEA, DCM; (iii) LiOH,
H2O, dioxan, H+
.
nd
Table 2. Potency and rate of clearance for triazine compounds
preparation of analogues where this group was replaced
with structurally similar moieties (Table 3). In all cases
potency was lost when compared to the parent 4,
although the rates of clearance remained similar, sug-
gesting that this feature is not key for recognition by the
active transporter. The amide in this series appears to be
crucial as replacing it with an ether, for example com-
pound 25, effectively abolishes potency. It is also inter-
esting to note the effect of methylating the amide,
compound 26, also gives significantly less potency. Pos-
sibly the combination of conformational twist induced
by the two ortho substituents and the presence of the
relatively non-basic pyridyl nitrogen is optimal for these
compounds.
Compd R1
XR2
VLA-4 protein7 VLA-4 cell8 IPRL
IC50 (nM)
IC50 (nM) k (hꢀ1
)
13
14
15
16
17
18
19
nPr
Me
Me
M
Me
Me
Me
OnPr
OnPr
OPh
50
45
30
16
70
25
7
740
300
320
590
1620
740
320
0.2
2.3
0.6
1.1
nd
e
NM
e
2
NEt2
NHnPr
2.4
1.8
This work, and additional studies employing parallel
synthesis, failed to identify any analogues with poten-
cies less than 100 nMin the cell-based assay. Potencies
of this order were considered appropriate to show effi-
cacy in animal models and consequently this series was
not developed further. However, we had demonstrated
that it was possible to identify compounds that had
reasonable potencies in blocking the adhesion of VLA-4
to VCAM-1, yet were not subject to rapid clearance
in the rat. Our efforts in optimising the pyrimidinyl
and pyridine series are described in the following
communication.
20
21
22
Me
Me
Me
14
25
6
510
1000
240
nd
2.1
2.1
(with respect to activity in the cell-based assay) in the
best examples (14, 15, 19 and 22). Notably all analogues
exhibited the sought-after, low rates of clearance, with
some compounds displaying exceptionally low values
(for example, 13 and 15). In general the more lipophilic
the substituents the better the potency and the lower the
rate of clearance though this may be a result of the
enhanced plasma protein binding of these compounds.
Interestingly the incorporation of polar functionality,
for example the carboxylic acid 22, also has a profound
effect on potency whilst retaining a reasonable rate of
clearance suggesting that the SAR for both properties in
this region of the molecule is quite complex.
Acknowledgements
This paper is dedicated to the memory of Alex Zomaya.
References and Notes
The advantage of the dichloropyridyl amide derivatised
analogues of phenylalanine was demonstrated by the
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7. VLA4 (from HL60 lysate) was immobilised on a plate with
a non-blocking anti-b1 antibody (TS2/16). The test com-
pounds were titrated into a solution of two-domain VCAM-
Fc-Ig in a separate plate and added to the wells. The assay was
carried out in TBS, 1% BSA, 1 mMMnCl 2, 0.1% Tween.
After incubation for 2 h at room temperature the plates were
washed and residual VCAMvisualised with peroxidase
coupled anti-human Fc.
8. A Jurkat cell line expressing VLA4 was incubated at 37 ꢁC
for 30 min with human two-domain VCAM-1-Fc immobilised
on a plate with anti-human Fc in the presence of the test
compounds. The plates were washed and residual cells were
stained with Rose Bengal.