Squaric acid derivatives as VLA-4 integrin antagonists

Article abstract of DOI:10.1016/S0960-894X(02)00075-6

SAR studies aimed at improving the rate of clearance by the incorporation of a 3,4-diamino-3-cyclobutene-1,2-dione group as an amino acid isostere in a series of VLA-4 integrin antagonists are described.

Full text of DOI:10.1016/S0960-894X(02)00075-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1051–1054
Squaric Acid Derivatives as VLA-4 Integrin Antagonists
John R. Porter,* Sarah C. Archibald, Kirstie Childs, David Critchley, John C. Head,
Janeen M. Linsley, Ted A. H. Parton, Martyn K. Robinson, Anthony Shock,
Richard J. Taylor, Graham J. Warrellow, Rikki P. Alexander and Barry Langham
Celltech R&D Ltd, 216 Bath Road, Slough SL1 4EN, UK
Received 10 December 2001; accepted 28 January 2002
Abstract—SAR studies aimed at improving the rate of clearance by the incorporation of a 3,4-diamino-3-cyclobutene-1,2-dione group
as an amino acid isostere in a series of VLA-4 integrin 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, including T-lymphocytes
and eosinophils, but not neutrophils. 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
in the bile of only minor amounts of metabolites com-
5
pared to the parent compounds.
Postulating that the a-amido group, although appar-
ently essential for potency, was responsible for the rapid
clearance in this series, we investigated a number of meth-
ods for its replacement. The 3,4-diamino-3-cyclobutene-
1,2-dione group has been recognised as an isostere for a-
1
6
sites of inflammation. Blocking such an interaction
amino acids as well as other functional groups such as
7
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
cyanoguanidines. We now wish to report that the repla-
cement of the a-amido carbonyl moiety in compounds
such as 2 with 3-cyclobutene 1,2-dione analogues gave
compounds such as 3 that are potent VLA-4 antagonists
and have an improved pharmacokinetic profile.
2
from early stage clinical trials are promising. Thus,
small molecule VLA-4 receptor antagonists represent an
attractive target for the treatment of various chronic
inflammatory diseases such as asthma, multiple sclerosis
3
and rheumatoid arthritis.
We have recently described the discovery of the thio-
4
proline CT5219, 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. Unfortunately both 1
and 2 displayed rapid clearance in a number of species,
including the rat, thereby preventing further develop-
ment. The rapid biliary clearance was shown to be the
result of an active transport mechanism by the presence
The compounds were readily prepared as outlined in
Scheme 1. The lithiated 3,5-dichloropyridine 4 was
*
5
Corresponding author. Tel.: +44-1753-534655; fax: +44-1753-
36632; e-mail: john.porter@celltechgroup.com
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00075-6

1052
J. R. Porter et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1051–1054
quenched with CO to generate the carboxylic acid that
2
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
was converted to the acid chloride 5 by treatment with
thionyl chloride. Coupling of 5 to the a-N-Boc pro-
tected ethyl ester of 4-aminophenylalanine in the pre-
sence of base gave the amine 6 after removal of the Boc
group with TFA. Heating with a solution of 3,4-diiso-
propoxy-3-cyclobuten-1,2-dione in ethanol gave the key
intermediate 7 that was further functionalised with a
variety of amines. Aqueous LiOH hydrolysis of the
ethyl ester gave the target acid 8.
8
liver (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,
This method was adapted for parallel synthesis as
shown in Scheme 2. a-N-FMOC protected 4-nitrophe-
nylalanine 9 was coupled to Wang resin and the nitro
9
10
ligand binding and a cell-based, adhesion assay.
group reduced with SnCl in DMF to give the resin
2
The initial preparation of a library of 23 compounds,
based on the scaffold 12, with a diverse range of amines
identified the methoxyethylamine analogue 13 as a pro-
mising lead with good potency in both the protein and
cell-based assays. Compound 13 also had a moderate
rate of clearance as measured by the IPRL assay, that
translated to a clearance of 5 mL/min/kg in the rat.
bound 4-aminophenylalanine 10. Addition of the acid
chloride 5 followed by FMOC deprotection gave the
amine 11, heating with a DMF solution of 3,4-dime-
thoxy-3-cyclobuten-1,2-dione gave the key intermediate
12. Derivatisation with a range of amines was followed
by cleavage from the resin and HPLC purification to
give the target acids 8.
Encouraged by these results, we set about making fur-
ther libraries based upon this scaffold initially using
primary unbranched amines, a representative selection
of which are shown in Table 1. Although most of these
compounds had a relatively flat SAR in the protein-
based assay there were noticeable trends in the cell-
based assay. The methyl analogue, 14, displays poor
potency but as the length of the carbon chain increases
an increase in potency is observed that reaches a max-
imum at propyl 3, and butyl 16, before appearing to
drop off again with pentyl 17. The clearance rate con-
stant of all of these compounds is reasonably low but
À1
appears to reach a minimum of 0.1 h with the propyl
analogue 3. This translated to a clearance of 0.1 mL/
min/kg in the rat. Interestingly the more lipophilic tri-
fluoromethyl analogue 19 is cleared at a faster rate. The
rate of clearance is not dependent on the stereo-
chemistry of the a-centre is shown by comparison of the
ꢀ
Scheme 1. (i) LDA, THF, À78 C, 30 min then CO
2 2
; (ii) SOCl , DCM;
(
iii) a-N-Boc 4-aminophenylalanine ethyl ester, N-methyl morpholine,
DCM; (iv) 3 M HCl in EtOAc; (v) 3,4-diisopropoxy-3-cyclobuten-1,2-
dione, DIPEA, EtOH; (vi) R NH, EtOH; (vii) LiOH, THF, H O.
Table 1. Potency and rate of clearance for secondary unbranched
amines
1
R
2
2
Compd
R
VLA-4
protein
IC50, nM) (IC50, nM) k h
VLA-4
10
cell
IPRL
9
À1
(
1
1
1
3
1
17
18
19
20
3
4
5
2-Methoxyethyl
Methyl
Ethyl
1.2
0.9
0.6
1.5
0.4
1.0
1
75
990
425
120
130
225
120
90
1.8
0.9
2.0
0.1
1.3
0.9
0.4
1.4
1.1
n-Propyl
n-Butyl
n-Pentyl
Allyl
6
Scheme 2. (i) Paramax Wang resin, 2,6-dichlorobenzoyl chloride,
pyridine, DMF; (ii) 20% acetic anhydride, DMF; (iii) SnCl (H O)
DMF; (iv) 5, DIPEA, DCM; (v) 20% piperidine, DMF; (vi) 3,4-
2
2
2
,
2-Trifluoromethylethyl
3-Trifluoromethylpropyl
nd
1
ꢀ
dimethoxy-3-cyclobuten-1,2-dione, DMF, 70 C; (vii)
DCM; (viii) 60% TFA, DCM; (ix) HPLC purification.
R
1
R
2
NH,
370

J. R. Porter et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1051–1054
1053
propyl analogue 3 with its distomer 3a (VLA-4 protein
IC 102 nM; VLA-4 cell IC50 >10,000 nM; IPRL k 0.1
of these tertiary amine derivatives display very high
rates of clearance.
5
0
À1
h
).
This correlation between the degree of amine substitu-
tion, potency and rate of clearance extended to replace-
ments for the dichloropyridyl amide. We prepared a
series of these analogues as either the n-propylamino or
diethylamino substituted squarates, a representative
sample of which are shown in Table 4. The 2,6-di-
methoxyphenyl substituent has been used in a number of
phenylalanine-based VLA4 antagonist analogues, but
the squarate analogues 43 and 44 display less potency than
their dichloropyridylamide counterparts 32 and 3, respec-
tively. However the relative potencies (tertiary amine being
more potent than secondary) and clearance rates (second-
ary amine being cleared more slowly than tertiary) remain
the same regardless of the 4-phenylalanine substituent for
all of the examples prepared. Obviously the squarate
amino substituent has a profound effect upon clearance,
and although we were unable, at this stage, to rule out
plasma protein binding, we speculated that conforma-
tional differences may have some influence.
The next library of compounds was based around
branched primary amines and a representative selection
is shown in Table 2. This series displayed similar levels
of potency to the unbranched analogues with the iso-
propyl 21 and isobutyl 25 compounds being the most
active in the cell-based assay, though they have a clear-
ance rate constant higher than the essentially equipotent
propyl analogue 3. Increasing the steric bulk, for exam-
ple the t-butyl analogue 28, has a detrimental effect on
potency and the rate of clearance.
1
1
The preparation of a library of compounds based on
secondary amines shows that the amino hydrogen is not
required for potency (Table 3). Although the dimethyl
analogue 31 has poor activity in the cell based assay (as
may be expected from the result with the methyl com-
pound 14) the diethyl 32 and the di-n-butyl 34 analo-
gues display excellent levels of activity. This trend is
continued with the cyclic amines, notably the thiomor-
pholine 37 and the piperidine 38. The presence of a
basic centre, for example the piperazine 39, is less well
tolerated. However, despite their enhanced potency, all
Opportunities for probing the local structural environ-
ment around the squarate using NMR methods such as
Table 2. Potency and rate of clearance for secondary branched
amines
Table 4. Dichloropyridyl amide replacements
Compd
R
VLA-4 protein9 VLA-4 cell10 IPRL
À1
(
IC50, nM)
(IC50, nM)
k h
2
2
2
2
2
2
2
2
2
3
1
2
3
4
5
6
7
8
9
0
i-Propyl
c-Propyl
c-Butyl
c-Pentyl
i-Butyl
1.0
1.5
2.6
9
0.5
0.5
0.6
3.5
50
95
250
155
290
85
160
50
690
845
320
2.6
1.6
0.5
0.5
1.3
3.1
2.4
3.5
1.1
4.2
Compd
R1
R2
VLA-4
protein
IC50, nM)
VLA-4
10
cell
(IC50, nM)
IPRL
9
À1
(
k h
1-Methylpropyl
1-Methylbutyl
t-Butyl
41
NEt2
1.5
6.9
0.6
2300
2.7
0.6
3.9
Phenyl
Benzyl
3.7
4
2
3
NHPr
8200
180
Table 3. Potency and rate of clearance for tertiary amines
4
NEt
2
4
4
4
4
4
5
6
7
NHPr
2.1
1.2
6.7
25
710
1160
0.7
2.2
nd
3.9
nd
NEt
2
Compd
R1
R2
VLA-4 protein9 VLA-4 cell10 IPRL
À1
(
IC50, nM)
(IC50, nM) k h
NHPr
10,800
12,400
18,300
31
32
32
34
35
36
37
38
39
40
Methyl
Methyl
Ethyl
Methyl n-Propyl
n-Butyl n-Butyl
Morpholino
Thiomorpholino
Piperidine
Piperazine
Methyl
Ethyl
Ethyl
0.5
0.2
0.4
0.2
2.0
0.5
0.3
0.4
0.4
0.5
590
100
4.6
4.9
5.8
4.5
6.6
5.0
5.7
4.7
4.5
3.7
3.5
13
5.7
11
3.1
8.4
40
60
NEt
2
48
NHPr
67
N-Ethyl piperazine

1054
J. R. Porter et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1051–1054
Table 5. Comparison of NHCHa chemical shift temperature depen-
dence gradient against IPRL
also make this series more prone to the observed rapid
clearance.
À1
Compd
Gradient (ppb/K)
IPRL k h
As the n-propyl analogue, 3, appears to have the opti-
mum combination of reasonable potency and low rate
of clearance it was selected for further study in animal
models of inflammatory disease and optimisation for
improved oral bioavailability. The results of these stud-
ies will be reported at a later date.
4
4
4
4
4
4
3
3
3
1
1
3
4
4
1
2
5
6
3
2
2
À7.7
À4.4
À7.2
À4.2
À7.6
À4.5
À7.7
À4.3
À4.2
À4.4
À4.3
À6.3
À7.9
À4.7
2.7
0.6
2.2
nd
3.9
0.7
5.8
0.1
0.1
0.4
0.8
1.3
3.9
nd
a
8
6
6
7
8
Acknowledgements
The authors wish to acknowledge the assistance of Mr.
Lloyd King in analysing the IPRL samples.
NOEs are limited as there are so few protons associated
with this moiety. The closest protons to the squarate
moiety are those attached to the nitrogen atoms on
either side. One technique that has been used success-
fully in the past is the study of the amide NH chemical
shift temperature dependencies in non-exchanging sol-
vents. The gradients obtained (in ppb/K) give an
indication of the extent of exposure that the NH proton
has to its surroundings. Values >À2.0 ppb/K suggest
hydrogen bonding, intermediate values <À3.0 ppb/K
but >À6.0 ppb/K suggest some degree of shielding
from the environment while values <À6.0 ppb/K indi-
cate almost total exposure. The gradients are always
negative, that is, there is an increase in magnetic shield-
ing (lower d) for the NH resonance as temperature
increases.
References and Notes
1
. Elices, M. J. In Cell Adhesion Molecules and Matrix Pro-
teins: Role in Health and Diseases; Mousa, S. A., Ed.; Springer
and R.G. Landes Co.: Berlin, Germany, 1998; p. 133.
2. Sorbera, L. A.; Martin, L.; Rabasseda, X. Drugs Future
2000, 9, 917.
1
2
3
. Porter, J. R. IDrugs 2000, 3, 788.
4. Archibald, S. C.; Head, J. C.; Gozzard, N.; Howat, D. W.;
Parton, A. H.; Porter, J. R.; Robinson, M. K.; Shock, A.;
Warrellow, G. J.; Abraham, W. M. Bioorg. Med. Chem. Lett.
000, 10, 997.
. Unpublished data.
. Kinney, W. A.; Lee, N. E.; Garrison, D. T.; Podlesny, E. J.,
2
5
6
Jr.; Simmonds, J. T.; Bramlett, D.; Notvest, R. R.; Kowal,
D. M.; Tasse, R. P. J. Med. Chem. 1992, 35, 4720.
7. Butera, J. A.; Antane, M. M.; Antane, S. A.; Argentieri,
T. M.; Freeden, C.; Graceffa, R. F.; Hirth, B. H.; Jenkins, D.;
Lennox, J. R.; Matelan, E.; Norton, N. W.; Quagliato, D.;
Sheldon, J. H.; Spinelli, W.; Warga, D.; Wojdan, A.; Woods,
M. J. Med. Chem. 2000, 43, 1187.
. Frink, E. J., Jr.; Kramer, T. H.; Banchy, S. M.; Brown,
B. R. Anesth. Analg. 1990, 71, 484.
. VLA-4 (from HL60 lysate) was immobilised on a plate with
The data are summarised in Table 5. In each case com-
pounds with a tertiary amine have a steeper temperature
gradient (typically À6.0 to À9.0 ppb/K than those with
a secondary amine (typically À4.0 to À5.0 ppb/K). This
effect appears to be independent of the nature of the
phenylalanine substituent. In the secondary amine series
the gradients for the two squarate NH protons are con-
sistently similar, in many cases their resonances being
isochronous across the temperature range. We postulate
that it may be the presence or absence of a hydrogen
atom on the amine nitrogen that influences the degree of
exposure of the NHCHa to solvent. In terms of potency
and clearance, in the secondary amine series where the
NHCHa is more shielded, it may be that the ligand’s
interaction with the protein is slightly weakened by this
apparent shielding effect, however this shielding may
also be responsible for the slower clearance. Conversely,
in the tertiary amine series the greater exposure of the
NHCHa may relate to the improved activity but may
8
9
a non-blocking anti-b1 antibody (TS2/16). The test com-
pounds were titrated into a solution of 2-domain VCAM-Fc-
Ig in a separate plate and added to the wells. The assay was
2
carried out in TBS, 1% BSA, 1 mM MnCl , 0.1% Tween.
After incubation for 2 h at room temperature the plates were
washed and residual VCAM visualised with peroxidase cou-
pled anti-human Fc.
1
3
0. A Jurkat cell line expressing VLA-4 was incubated at
ꢀ
7 C for 30 min with human 2-domain VCAM-1-FC immo-
bilised 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.
1. For examples see ref 3.
12. Wright, P. E.; Dyson, H. J.; Lerner, R. A. Biochem. 1988,
27, 7167.
1