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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