Discovery of a potent, orally bioavailable pyrimidine VLA-4 antagonist effective in a sheep asthma model

Article abstract of DOI:10.1016/j.bmcl.2011.01.075

A series of N-(pyrimidin-4-yl)-phenylalanine VLA-4 antagonists is described. Optimization of substituents at the 2 and 5 positions of the pyrimidine ring gave 14, a very potent VLA-4 inhibitor which is orally active in a sheep asthma model.

Full text of DOI:10.1016/j.bmcl.2011.01.075

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1741–1743
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a potent, orally bioavailable pyrimidine VLA-4 antagonist
effective in a sheep asthma model
⇑
Christopher M. Semko , Linda Chen, Darren B. Dressen, Mark L. Dreyer, Whitney Dunn, Francine S. Farouz,
Stephen B. Freedman, Elizabeth J. Holsztynska, Michael Jefferies, Andrei W. Konradi, Anna Liao,
Judevin Lugar, Linda Mutter, Michael A. Pleiss, Kevin P. Quinn, Thomas Thompson,
Eugene D. Thorsett, Christopher Vandevert, Ying-Zi Xu, Ted A. Yednock
Elan Pharmaceuticals, Inc., 800 Gateway Blvd., S. San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-(pyrimidin-4-yl)-phenylalanine VLA-4 antagonists is described. Optimization of substitu-
ents at the 2 and 5 positions of the pyrimidine ring gave 14, a very potent VLA-4 inhibitor which is orally
active in a sheep asthma model.
Received 17 December 2010
Revised 13 January 2011
Accepted 18 January 2011
Available online 22 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
VLA-4
Integrin
Antagonist
Pyrimidine
VLA-4 (a4b1 integrin) is a cell surface receptor expressed on
O
N
many leukocytes, including T- and B-lymphocytes, eosinophils,
basophils, and mast cells.1 It is well established that VLA-4 plays
a key role in the recruitment of leukocytes from the bloodstream
to inflamed or injured tissues via a process known as cell adhe-
sion.2 This process is mediated by the binding of VLA-4 to the vas-
cular cell adhesion molecule-1 (VCAM-1), which is expressed on
activated endothelial cells. VLA-4 also binds to the extracellular
matrix protein fibronectin (FN). Inhibiting VLA-4/VCAM-1 and
VLA-4/FN binding offers therapeutic opportunity for the treatment
of inflammatory and autoimmune pathologies such as asthma,
multiple sclerosis (MS), rheumatoid arthritis and Crohn’s disease.3
This approach has been validated with the approval of the human-
ized anti-a4 antibody natalizumab for the treatment of relapsing–
remitting MS4 and Crohn’s disease.5 Since an antibody requires
intravenous administration and has the potential of immunogenic-
ity, an orally efficacious small molecule would serve as a valuable
alternative therapeutic approach.
N
N
O
S
N
O
O
O
OH
N
H
O
S
1
Figure 1. Peptidic VLA-4 antagonist.
of peptidic acids demonstrated poor bioavailability resulting from
low permeability and rapid biliary clearance. An ester prodrug ap-
proach was adopted to improve physical chemical characteristics
of this class of compounds to impart oral efficacy.7 In this Letter
we describe our efforts to identify an orally efficacious small mol-
ecule antagonist of VLA-4 not dependent on a prodrug strategy.
Because all attempts to replace the C-terminal carboxylate with
other functionality or a carboxylate isostere gave compounds of
greatly reduced potency, attention was directed at the central car-
boxamide.6 It was hypothesized that the carboxamide was a con-
tributor to the poor pharmacokinetic profile in the dipeptide
We have previously reported the discovery of dipeptide 1
(Fig. 1) as a potent antagonist of VLA-4. This compound demon-
strated efficacy in a sheep asthma model by reducing early and late
phase bronchoconstriction and carbachol induced airway hyperre-
sponsiveness when dosed via inhalation.6 Unfortunately, this class
series so an appropriate isostere was desired. Benzamide
(Fig. 2) had been identified as a relatively potent antagonist as
measured in an 4b1 positive Jurkat cell-recombinant soluble
VCAM-1 FACS assay.6 Efforts in the dipeptide series had
2
a
⇑
Corresponding author.
E-mail address: semko@sbcglobal.net (C.M. Semko).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.075

1742
C. M. Semko et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1741–1743
Table 1
O
N
O
N
FACS potency and systemic exposure in Sprague–Dawley rat
O
O
Compd
R
FACS IC50 (nM)
AUCa
(lg h/mL)
n.t.b
0.2
0.6
0.2
0.6
0.6
0.3
<0.1
<0.1
n.t.
<0.1
0.3
0.6
n.t.
<0.1
O
N
N
OH
OH
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
Phenyl
o-Tolyl
300
25
90
N
N
H
H
O
O
2-Trifluoromethylphenyl
2-Methoxyphenyl
2-Fluorophenyl
2,6-Difluorophenyl
2,3-Dichlorophenyl
2,4,6-Trimethylphenyl
3-Nitrophenyl
3-Pyridyl
4-Pyridyl
Thiophen-2-yl
3-Methylthiophen-2-yl
1H-Pyrrol-2-yl
16
18
27
77
100
180
130
85
16
5
2
3
FACS IC50 = 130 nm
FACS IC50 = 190 nm
Figure 2. Benzamide and quinazoline VLA-4 antagonists.
demonstrated the importance of the amide carbonyl and N–H for
potency. These results suggested N-arylated heterocycles could
mimic the key structural and electronic features of the carboxam-
ide. The near equivalent potency of quinazoline 3 with benzamide
2 confirmed replacement of the carboxamide could be achieved
with an appropriately polarized heterocycle.8
Having success in replacing the carboxamide, we now focused
to improve the potency of 3 by preparing variants of the quinazo-
line heterocycle including 5-arylated pyrimidines prepared accord-
75
<1
3,5-Dimethylisoxazol-4-yl
a
AUC = area under curve.
Not tested.
b
ing to Scheme 1. Reacting suitably protected L-tyrosine 4 with
O
N
dimethylcarbamyl chloride followed by deprotection gave amino
ester 5, which was reacted with 4-chloro-5-iodopyrimidine to give
6. This iodopyrimidine underwent Suzuki couplings with a variety
of aryl boronic acids and subsequent treatment with formic acid
gave desired acids 7a–7o. Potency data and systemic exposure as
determined from area under curve (AUC) quantitation for the
5-arylated pyrimidines is shown in Table 1.9
N
O
N
N
OH
N
H
O
8
FACS IC50 = 8 nm
Compound 7o was determined to be the most potent, but this
compound exhibited poor exposure following oral dosing.
Efforts in the quinazoline series had revealed that 2-amino
Figure 3. Quinazoline VLA-4 Antagonist.
substitution increased potency with compound
8 containing
N-methyl-N-cyclohexyl being optimal (Fig. 3).
O
N
Cl
Cl
This substitution was incorporated into 7b via Scheme 2. The
reaction of 2,4-dichloro-5-iodo-pyrimidine 9 with 1 equiv of 5 pro-
ceeded with complete regioselectivity to give 10. Intermediate 10
was then treated with N-methyl-N-cyclohexylamine to give the
iodopyrimidine which following Suzuki coupling with o-tolylbo-
ronic acid and ester deprotection gave 11a.
O
N
N
N
N
b,c,d
a
OtBu
Cl
N
H
I
I
O
9
10
Although the expected increase in potency was not seen, 11a
did demonstrate a good increase in exposure (Table 2). Since 2,4-
diamino pyrimidines are known to be relatively basic (pKa >7),10
the AUC improvement may in part be explained by the zwitterionic
nature of these compounds in the GI tract which facilitates oral
absorption. A formal pharmacokinetic study of 11a determined a
bioavailability of 26% in the Sprague–Dawley (SD) rat with mini-
R1 R2
N
O
N
O
N
N
OH
N
H
O
11a-k
Scheme 2. Reagents and conditions: (a) 5, DIEA, IPA, 40 °C; (b) R1R2NH, IPA, 120 °C;
(c) o-tolylboronic acid, Pd(PPh3)4, 2 M Na2CO3, DME, reflux; (d) formic acid.
OH
O
N
O
mal biliary excretion (<3%) when dosed orally at 10 mg/kg. The
favorable pharmacokinetic properties of this compound prompted
a sheep asthma study in collaboration with Dr. William Abraham.11
This study determined that 11a prevented both the early and late
phase response and carbachol induced hyperresponsiveness with a
minimum effective oral dose of 10 mg/kg.
a,b
c
OtBu
OtBu
ZHN
H2N
O
O
4
5
O
N
O
N
The efficacy demonstrated by 11a inspired the preparation of a
family of compounds which introduced a variety of amines at the
2-position of the pyrimidine to identify the amino substitution that
retained the potency of 11a and maximized exposure. Several
compounds not only profiled with greater exposure but also higher
potency including 11i (Table 2).
O
O
N
N
N
N
d,e
OH
OtBu
N
N
H
R
O
H
I
O
6
7a-o
Having discovered that simple diethylamino substitution at C-2
could impart good exposure as shown with 11i, the analog of 7o
incorporating this moiety was prepared according Scheme 3. Treat-
Scheme 1. Reagents and conditions: (a) ClCONMe2, Et3N, DMAP, CH2Cl2; (b) Pd/C,
H2, EtOH; (c) 4-chloro-5-iodopyrimidine, DIEA, THF, reflux; (d) ArB(OH)2, Pd(PPh3)4,
2 M Na2CO3, DME, reflux; (e) formic acid.

C. M. Semko et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1741–1743
1743
Table 2
Compound 14 retained the potency of 7o but exhibited a vastly
improved systemic exposure. Furthermore, this compound was
found to be efficacious in the Abraham sheep model of asthma
when dosed orally at 1 mg/kg while no effect was noted at
0.3 mg/kg. The pharmacokinetic profile of compound 14 in three
species is summarized in Table 3. Notable is the acceptable oral
availability and low clearance across species.
In conclusion, we have identified a class of pyrimidinyl VLA-4
antagonists that retain the potency of the peptidic series. Optimi-
zation of this class identified a bioavailable VLA-4 antagonist
which achieved robust efficacy in a sheep asthma model when
dosed as the parent carboxylic acid.
FACS potency and systemic exposure in Sprague–Dawley rat
Compd
R1
R2
FACS IC50
(nM)
AUC
g h/mL)
(l
11a
11b
11c
11d
11e
11f
11g
11h
11i
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Ethyl
Cyclohexyl
Methyl
Isobutyl
Cyclopropyl
Cyclobutyl
(1-Methyl) piperidin-4-yl
4-Chloro phenyl
Methyl
20
29
7
0.9
0.4
0.3
0.4
1.8
<0.1
5.7
0.7
1.6
1.9
0.5
20
10
68
107
5.3
5.7
4.9
32
Ethyl
Ethyl
Piperidin-1-yl
Ethyl
Propyl
11j
11k
Acknowledgment
We thank Professor John G. Topliss for his insightful
suggestions.
O
N
N
O
N
Cl
N
I
O
O
O
N
N
N
N
a
c
b
OtBu
OtBu
References and notes
N
N
H
H
I
O
O
O
1. Jackson, D. Y. Curr. Pharm. Des. 2002, 8, 1229.
2. Cox, D.; Brennan, M.; Moran, N. Nat. Rev. Drug Disc. 2010, 9, 804.
3. Tilley, J. W. Expert Opin. Ther. Pat. 2008, 18, 841.
4. Sheremata, W. A.; Minagar, A.; Alexander, J. S.; Vollmer, T. CNS Drugs 2005, 19,
909.
5. Sandborn, W. J.; Colombel, J. F.; Enns, R.; Feagan, B. G.; Hanauer, S. B.;
Lawranace, I. C.; Panaccione, R.; Sanders, M.; Schreiber, S.; Targan, S.; van
Deventer, S.; Goldblum, R.; Despain, D.; Hogge, G. S.; Rutgeerts, P. N. Eng. J. Med.
2005, 353, 1912.
12
10
O
O
N
N
N
O
N
N
N
N
OtBu
OH
N
N
H
H
O
6. Huryn, D. M.; Konradi, A. W.; Ashwell, S.; Freedman, S. B.; Lombardo, L. J.;
Pleiss, M. A.; Thorsett, E. D.; Yednock, T.; Kennedy, J. D. Curr. Top. Med. Chem.
2004, 4, 1473.
7. Huryn, D. M.; Ashwell, S.; Baudy, R.; Dressen, D. B.; Gallaway, W.; Grant, F. S.;
Konradi, A.; Ley, R. W.; Petusky, S.; Pleiss, M. A.; Sarantakis, D.; Semko, C. M.;
Sherman, M. M.; Tio, C.; Zhang, L. Bioorg. Med. Chem. Lett. 2004, 14,
1651.
8. For similar approaches see: (a) Doherty, G. A.; Kamenecka, T.; McCauley, E.;
Van Riper, G.; Mumford, R. A.; Tong, S.; Hagmann, W. K. Bioorg. Med. Chem. Lett.
2002, 12, 729; (b) Porter, J. R.; Archibald, S. C.; Brown, J. A.; Childs, K.; Critchley,
D.; Head, J. C.; Hutchinson, B.; Parton, T. A. H.; Robinson, M. K.; Shock, A.;
Warrellow, J.; Zomaya, A. Bioorg. Med. Chem. Lett. 2002, 12, 1591; (c) Porter, J.
R.; Archibald, S. C.; Brown, J. A.; Childs, K.; Critchley, D.; Head, J. C.; Hutchinson,
B.; Parton, T. A. H.; Robinson, M. K.; Shock, A.; Warrellow, J.; Zomaya, A. Bioorg.
Med. Chem. Lett. 2002, 12, 1595.
9. Systemic exposure was determined as follows: Three fasted male Sprague–
Dawley rats were administered test compound mixtures at a total dose of
10 mg/5 mL/kg orally. Individual test compounds typically varied from 1.1 to
2.0 mg/kg. Blood samples were collected at 4, 8, and 12 h post dose by
intracardiac puncture. Separated acidified plasma was extracted with ethyl
acetate, evaporated to dryness and reconstituted in the chromatographic
mobile phase and subjected to LC/MS/MS analysis on a PE-Sciex API-365 triple
quadurpole mass spectrometer. Quantitation was performed with PE-Sciex
software (MacQuan v1.6).
N
N
O
O
13
14
FACS IC50 < 1 nm
AUC = 1.8 µg.h/mL (SD rat)
Scheme 3. Reagents and conditions: (a) Et2NH, IPA, 120 °C; (b) 3,5-dimethyl-4-
isoxazoleboronic acid, Pd(PPh3)4, K3PO4, DMF, 100 °C; (c) formic acid.
Table 3
Pharmacokinetic data for compound 14
Species
F%
CL (mL/h/kg)
T1/2 (h)
Vd (mL/kg)
SD rata
19
59
16
170
404
808
1.4
0.9
1.1
346
524
1291
Beagle dogb
Cynomolgus monkeyc
a
b
c
Male rats dosed at 10 mg/kg iv and po.
Male dogs dosed at 3 mg/kg iv and 10 mg/kg po.
Male monkeys dosed at 3 mg/kg iv and 10 mg/kg po.
10. Brown, D. J. Chemical Science, Part 2B In Comprehensive Heterocyclic Chemistry;
Boulton, A. J., McKillop, A., Eds.; Pergamon Press: Oxford, 1984; Vol. 3, pp 57–
155.
11. Abraham, W. M.; Delehunt, J. C.; Yerger, L.; Marchette, B. Am. Rev. Respir. Dis.
1983, 128, 839.
ing 10 with diethylamine gave iodopyrimidine 12 which under-
went Suzuki coupling with 3,5-dimethyl-4-isoxazoleboronic acid
to give ester 13. Treatment of 13 with formic acid gave acid 14.