Arylsulfonamide pyrimidines as VLA-4 antagonists

Article abstract of DOI:10.1016/j.bmcl.2013.03.010

A series of (S)-2-(2-(diethylamino)-5-(N-alkyl-N-sulfonamido)pyrimidin-4- ylamino)-3-(4-(carbamoyloxy)phenyl)propanoic acid is discovered as orally available VLA-4 antagonists. Representative compounds 11b and 11p showed efficacy in multiple in vivo anima

Full text of DOI:10.1016/j.bmcl.2013.03.010

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3070–3074
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Arylsulfonamide pyrimidines as VLA-4 antagonists
a
b
a
c
d
Ying-zi Xu a, , Andrei W. Konradi , Frederique Bard , Michael Dappen , Lilibeth Dofiles , Mark Dreyer ,
Ian Gallager b, Caroline Garrido b, Mike Krimm c, Zhenmei Liao c, Elizabeth Messersmith c, Linda Mutter d,
Michael A. Pleiss a, Bhushan Samant d, Christopher M. Semko a, Jennifer Smith a, Frank Stappenbeck a,
Brian Stupi a, Chris Vandervert b, Brent Welch b, Brian Wipke c, Ted Yednock b
⇑
a Department of Chemical Sciences, Elan Pharmaceuticals, Inc., 180 Oyster Point Blvd., South San Francisco, CA 94080, USA
b Department of Biology, Elan Pharmaceuticals, Inc., 180 Oyster Point Blvd., South San Francisco, CA 94080, USA
c Department of Pharmacology, Elan Pharmaceuticals, Inc., 180 Oyster Point Blvd., South San Francisco, CA 94080, USA
d Department of DMPK, Elan Pharmaceuticals, Inc., 180 Oyster Point Blvd., South 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 (S)-2-(2-(diethylamino)-5-(N-alkyl-N-sulfonamido)pyrimidin-4-ylamino)-3-(4-(carbamoyl-
oxy)phenyl)propanoic acid is discovered as orally available VLA-4 antagonists. Representative com-
pounds 11b and 11p showed efficacy in multiple in vivo animal models. The in vitro selectivity of 11p
is also described.
Received 11 December 2012
Revised 21 February 2013
Accepted 4 March 2013
Available online 15 March 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
a4b1
VLA-4
Antagonist
Orally available
CIA model
EAE model
Very late antigen 4 (VLA-4,
a
4b1 integrin) is an adhesion mole-
illustrated in Figure 1. In all four conformations, the proline residue
was in a gamma-turn conformation, with a Psi angle near zero.
Given the observed proline gamma turn conformation, we
hypothesized a 5-substituted pyrimidine might be an effective
replacement for the amide bond in 1. Applying the methods illus-
trated in Scheme 1, we converted the previously reported interme-
diate 4 into 3, which proved to be a very potent VLA-4 inhibitor,
albeit still with suboptimal oral availability.
cule, which is expressed on T-cells, B-cells, eosionophils, basophils
and mast cells.1 VLA-4 binds to vascular cell adhesion molecule 1
(VCAM-1), which is transiently expressed on endothelial cells in
response to cytokines, and to fibronectin (FN), which is present
in the extracellular matrix.2 Interactions of VLA-4 with these li-
gands mediate the adhesion, extravascularization, and survival of
lymphocytes, in certain beneficial or pathological states of inflam-
mation. The humanized monoclonal antibody Natalizumab blocks
the binding of VLA-4 to VCAM-1, and is an effective intravenous
therapy for multiple sclerosis (MS).3,4 Towards oral therapies for
MS and other inflammatory diseases, considerable research has
been devoted to the discovery of orally available small molecules,
which block the function of VLA-4.5–12 Dipeptide derivatives
including 1 (Fig. 1) are very potent VLA-4 inhibitor as demon-
strated by FN cell adhesion assay13 but with poor oral availabilities,
which have been reported by several research groups.14 During the
course of our VLA-4 inhibitor discovery program, we obtained the
X-ray crystal structure of the dicyclohexylammonium salt of the
moderately potent VLA-4 inhibitor Ts-Pro-Phe-OH 2. The unit cell
contained four very similar conformations of 2, one of which is
N
O
N
O
N
O
O
O
S
N
OH
N
H
O
S
1
2
FN Cell Adhesion IC50 =2nM
Figure 1. Very potent dipeptide derivative VLA-4 antagonist (1); Ts-Pro-Phe-OH (2)
dicyclohexylammonium salt X-ray crystal structure (dicyclohexylammonium ion
omitted for clarity).
⇑
Corresponding author. Tel.: +1 6508570520.
E-mail address: ying_zi_xu@yahoo.com (Y.-z. Xu).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.010

Y.-z. Xu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3070–3074
3071
OH
OH
N
O
N
O
N
c
Cl
a,b
b
a
N
N
O
O
O
N
N
O
N
H
O
H2N
O
H2N
NO2
O
N
H
O
O
NO2
O
5
8
7
4
O
N
O
R1
O
N
N
N
O
R1
O
N
S
N
N
O
O
c,d,e
N
N
N
N
OH
O
d,e,f,g
N
H
N
N
O
OH
O
N
H
N
H
N
O
O
O
S
N
H
NH2
O
O
R2
O
NO2
O
Ar
O
3
6
9a: R1
= dimethylamine
11a-f, j-r
9b: R1 = pyrolidine
9c: R1 = azetidine
Scheme 1. Reagents and conditions: (a) 2,4-chloro-5-nitropyrimidine, iPrN(Et)2,
CH2Cl2, 0 °C; (b) Pd/C, H2, NaHCO3, EtOH; (c) TsCl, pyridine; (d) Me2SO4, K2CO3, DMF
(e) HCO2H.
Scheme 2. Reagents: (a) 2,4-dichloro-5-nitropyrimidine, iPrN(Et)2, THF; (b) Et2NH,
iPrOH; (c) R1COCl, Et3N, CH2Cl2; (d) Pd/C, H2, EtOH; (e) ArSO2Cl, pyridine; (f)
Me2SO4 or EtI or propargyl chloride, K2CO3, DMF; (g) HCO2H
Previously, we reported that similar pyrimidines, with Et2N
groups attached on their 2-positions, are orally available VLA-4
inhibitors.9 Applying the methods illustrated in Scheme 2, we pre-
pared 11a (Fig. 2), which has in vitro potency comparable to 3, but
15% oral availability in rat.
O
N
O
N
S
N
OH
N
H
O
N
O
O
N
N
N
O
O
N
S
N
3
OH
N
H
FN Cell Adhesion IC50 = 2 nM
O
O
O
O
N
11a
N
O
FN Cell Adhesion IC50 =3 nM
F = 15%
N
S
N
OH
N
H
O
N
O
O
Given the success of 11a, we applied the methods illustrated in
Schemes 2 and Scheme 3 to prepare a panel of pyrimidines, struc-
tures and data for which are illustrated in Table 1. All the products
were determined to be very potent in our FN cell adhesion assay.
However, there were strong substituent effects on systemic expo-
sure as measured by cassette dosing.15 In general, the best expo-
sures were obtained for 4-fluorophenyl sulfonamides, with
unbranched N-substituents, and carbamates derived from
pyrrolidine. Compound 11p, which combines an N-propargyl
4-fluorophenyl sulfonamide with a pyrrolidine carbamate, achieves
very impressive exposure, as measured by either cassette dosing or
a 12-h individual PK experiment.
Compounds 11b and 11p also were tested in FN cell adhesion
assays including serum from humans, mice, or rats (Table 2). The
IC50s determined in the different sera suggest very strong binding
of these compounds to proteins in human and rat sera, but much
weaker binding to proteins in mouse serum.
A 0.3 mg/kg oral dose of compound 11b was determined to in-
hibit the early and late phases of airway hyper-responsiveness and
subsequent carbachol sensitivity in the Abraham sheep model of
asthma16 (data not shown). Compounds 11b and 11p were tested
in a mouse model of acute pulmonary cellular influx.17 Female
Black/6 mice were sensitized on day 1 by intraperitoneal injection
(i.p.) of ova/alum followed by a booster injection on day 14. Mice
were challenged on day 28 and 29 with aerosolized ova and alum.
Mice were euthanized and bronchoalveolar lavage samples were
11a
FN Cell Adhesion IC50 = 2 nM
F = 15%
Figure 2. 5-Substituted pyrimidine as a proline replacement.
O
N
O
N
N
N
O
O
N
N
N
N
a
O
O
N
H
N
H
NO2
O
HN
O
10
9a
O
N
N
O
N
N
b,c
OH
N
O
S
H
N
O
Ar
O
11g-i
Scheme 3. Reagents: (a) acetone, AcOH, PtO2, H2, EtOH; (b) ArSO2Cl, pyridine; (c)
HCO2H.

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Y.-z. Xu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3070–3074
Table 1
In vitro potencies and exposures/pharmacokinetics in SD rat for compounds 11a–11r
Ar
R1
R2
FN Cell adhesiona IC50
M)
Cassette AUC (
mL)
l
g h/ AUC iv (
lg h/
iv t1/ AUC po (
l
g h/
C12 pob
mL)
(
lg/
Fc
(%)
(
l
mL)
mL)
2
11a
11b
11c
11d
11e
11f
p-Tolyl
4-Fluorophenyl
4-Chlorophenyl NMe2
4-Fluorophenyl NMe2
4-Chlorophenyl NMe2
NMe2
NMe2
Methyl
Methyl
Methyl
Ethyl
Ethyl
ethyl
0.003
0.002
0.003
0.001
0.007
0.004
41
27
32
32
17
17
1.6
4.1
7.1
3.3
4.2
3.6
6
20
23
14
5.1
4.2
0.10
15
74
73
44
33
24
(6h)
1.78
1.64
1.9
1.25
0.55
0.12
0.13
0.06
0.012
0.008
3,4-
NMe2
Difluorophenyl
4-Fluorophenyl
4-Chorophenyl
3,4-
11g
11h
11i
NMe2
NMe2
NMe2
i-Propyl
i-Propyl
i-Propyl
0.005
0.004
0.007
0.51
0.43
0.12
Difluorophenyl
p-Tolyl
4-Chlorophenyl Pyrrolidine Methyl
2,4-
Difluorophenyl
11j
11k
11l
Pyrrolidine Methyl
0.006
0.019
0.008
20.54
13.2
14.8
173
153
90
6.1
10.2
11.3
74
51
16
0.45
0.33
0.1
43
33
17
pyrrolidine Methyl
Pyrrolidine Ethyl
11m 4-Fluorophenyl
0.001
0.017
0.009
12.8
9.7
4.8
111
110
83
5.9
5.7
11.1
51
34
28
0.18
0.1
0.05
46
31
34
11n
11o
4-Chlorophenyl Pyrrolidine Ethyl
2,4-
Pyrrolidine Ethyl
Difluorophenyl
4-Fluorophenyl
2,4-
11p
11q
Pyrrolidine Propargyl 0.008
Pyrrolidine Propargyl 0.013
90.3
23.8
156
126
10.4
9.2
88
42
1.31
0.12
56
33
Difluorophenyl
4-Fluorophenyl
11r
Azetidine
Ethyl
0.002
0.9
34
6.9
6
0.01
16
a
b
c
Fibronectin mediated cell adhesion assay.
Compound level at 12 h post po dosing.
F = bioavailability.
Table 2
1 h prior and 12 h post challenge. The typical range of inhibition
achieved by PS/2 was 60–80%. As illustrated in Table 3, 11b and
11p had minimum effective po doses of 10 and 3 mg/kg,
respectively.
IC50s of 11b and 11p in FN cell adhesion assays with and without sera from human,
rat, and mouse
a4b1-FN IC50 (lM)
0.3%
BSA
100% Human
serum
100% Rat
serum
100% Mouse
serum
The robust efficacy of 11p in the mouse model of pulmonary
cellular influx prompted us to test this compound in a rat collagen
induced arthritis (CIA) model.18,19 Female Lewis rats with develop-
ing type II collagen arthritis were treated prophylactically by oral
gavage, bid, with 3, 10 and 30 mg/kg 11p for 21 days. In a separate
arm of the study, similar animals were given methotrexate (MTX)
0.05 mg/kg po, qd alone or in combination with 11p. Efficacy eval-
uation was based on ankle caliper measurements, expressed as
area under the curve (AUC), terminal hind paw weights and
11b 0.002
11p 0.009
0.045
4.6
0.28
1.9
0.006
0.036
collected on day 30, 48 h post first challenge. The a4 antibody, PS/2,
was administered iv at 10 mg/kg, 2 h prior to the first challenge.
Compounds 11b and 11p were administered twice daily on days
28 and 29 by oral gavage, at a constant dose volume of 5 ml/kg,
Table 3
Efficacy of 11b and11p in a mouse model of acute pulmonary cellular influx
11b
11p
10 mg/kg
3 mg/kg
3 mg/kg
1 mg/kg
a
Eos influx% inhibition wrt PS/2b
80**
45**
5
3
74*
41*
0
0
a
Eos influx% inhibition wrt vehicle group
a
With respect to.
b
PS/2 is an a4 antibody, it was used as positive control in the study. The typical range of inhibition achieved was 60–80%.
p <0.01.
p <0.05.
**
*
Table 4
Efficacy of 11p in the rat CIA model
11p
11p + MTX 0.05 mg/kg
MTX
30 mg/kg
10 mg/kg
3 mg/kg
30 mg/kg
10 mg/kg
3 mg/kg
0.05 mg/kg
AUC paw diameter
Final paw weight
Body weight
Ankle histology
Knee histology
37**
23*
15
31*
26*
14
25
15
0
41**
37**
26*
24*
11
21
24
25*
13
17
27*
23*
46*
28*
17
26**
37**
26*
40**
24*
39**
31**
46**
21*
17
31*
**
*
p <0.01.
p <0.05.

Y.-z. Xu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3070–3074
3073
In conclusion, we have identified a group of arylsulfonamide
pyrimidines, which are potent inhibitors of VLA-4. These com-
pounds retain the potency of the similar dipeptide inhibitors, but
display much greater bioavailability, and achieve efficacy in animal
models of asthma, rheumatoid arthritis and multiple sclerosis
when dosed orally.
References and notes
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W. K. Bioorg. Med. Chem. Lett. 2008, 18, 1688.
Figure 3. EAE experiment of 11p with Tysabri as positive control.
6. Gong, Y.; Barbay, J. K.; Kimball, E. S.; Santulli, R. J.; Fisher, M. C.; Dyatkin, A.
B.; Miskowski, T. A.; Hornby, P. J.; He, W. Bioorg. Med. Chem. Lett. 2008, 18,
1331.
7. Kamenecka, T. M.; Park, Y.-J.; Lin, L. S.; de, L. S.; McCauley, E. D.; Van, R. G.;
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9. Semko, C. M.; Chen, L.; Dressen, D. B.; Dreyer, M. L.; Dunn, W.; Farouz, F. S.;
Freedman, S. B.; Holsztynska, E. J.; Jefferies, M.; Konradi, A. W.; Liao, A.; Lugar,
J.; Mutter, L.; Pleiss, M. A.; Quinn, K. P.; Thompson, T.; Thorsett, E. D.;
Vandevert, C.; Xu, Y.-Z.; Yednock, T. A. Bioorg. Med. Chem. Lett. 2011, 21, 1741.
10. Rehman, A.; Soni, A.; Naik, K.; Nair, S.; Palle, V. P.; Dastidar, S.; Ray, A.; Alam, M.
S.; Salman, M.; Cliffe, I. A.; Sattigeri, V. Bioorg. Med. Chem. Lett. 2010, 20, 5514.
11. Saku, O.; Ohta, K.; Arai, E.; Nomoto, Y.; Miura, H.; Nakamura, H.; Fuse, E.;
Nakasato, Y. Bioorg. Med. Chem. Lett. 2008, 18, 1053.
Table 5
Activity of 11p in adhesion assays involving different integrins
Assays
IC50 (lM)
a
a
a
a
a
4b1-FN
0.009
7.24
>30
0.5
4b7-MadCAM
Lb2-ICAM
vb1-OPN
5b1-FN
>30
histopathologic evaluation of ankles and knees. Results were ex-
pressed using only animals that had definite clinical and histopath-
ologic evidence of disease induction that resulted in connective
tissue damage. As illustrated in Table 4, 11p had significant effects
on inflammation, cartilage destruction, and bone resorbtion. Rats
treated with 11p at 30 mg/kg or 10 mg/kg alone and in combina-
tion with MTX had significant inhibition of clinical arthritis as as-
sessed by AUC for ankle swelling. Rats given 11p at 3 mg/kg had
25% non-significant inhibition of AUC and the effect was similar
to the groups treated with combination of MTX or MTX alone. A
very similar trend was observed with final paw weights. Significant
effect was observed with higher doses but not 3 mg/kg 11p alone.
Animals treated with 11p alone had slight but non-significant
improvement of body weight gain at 10 and 30 mg/kg.
The effects on histopathology indicate 11p has disease modify-
ing anti rheumatic drug (DMARD) activity at all doses, alone or in
combination with MTX. The lack of a dose–response for the
DMARD effects of 11p suggest even 3 mg/kg achieves a maximal
effect. Compound 11p at all doses also had stronger DMARD activ-
ity than Methotrexate at 0.05 mpk.
12. Muro, F.; Iimura, S.; Yoneda, Y.; Chiba, J.; Watanabe, T.; Setoguchi, M.;
Takayama, G.; Yokoyama, M.; Takashi, T.; Nakayama, A.; Machinaga, N. Bioorg.
Med. Chem. 2009, 17, 1232.
13.
a
4b1-Dependent adhesion: Jurkat TM adhesion to human plasma fibronectin:
96 wells plate (Costar 3590) is coated with fibronectin (FN) (Calbiochem cat #
341635) at 1.25 g/well in PBS++ (PBS + 1 mM CaCl2 + 1 mM MgCl2) overnight
l
at 40 °C. Then the plate is blocked with assay buffer (Hepes 20 mM, NaCl
140 mM, CaCl2 1 mM, MgCl2 1 mM and 0.3% BSA) for 30 min at rt and washed
3 times with assay buffer. Calcein labeled Jurkat TM cells (Molecular probes cat
#
C-1430, according to manufacturer procedure), at 105 cells/well (final
volume: 100 l /well) in either assay buffer + 10 g/ml TS2/16 (activating
anti-a4 antibody, Elan) or 100% human or rat serum (Seracare), are pre-
incubated in a 96 wells Flexi plate with 5 g/ml anti-a5 (Pharmingen cat #
555614) and with a dose response of compound, for 30 min on wet ice. 21/6
(anti- 4, Elan) at 10 g/ml is used as a positive control to inhibit the 4b1
l
l
l
a
l
a
dependent binding. Cells are then transferred to the FN coated plate and
binding is allowed for 35 min at 37 °C, 5% CO2 (incubator). Plate is washed 3
times with assay buffer and read for fluorescence (Applied Biosystems,
CytoFluor series 4000).
14. 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.
(Sharjah, United Arab Emirates) 2004, 4, 1473.
Compound 11p also was tested at 30 and 300 mg/kg po in gui-
nea pig EAE.20 In this model of EAE animals were immunized and
only animals with established disease (clinical score21 >0) were
randomized into treatment group at 16–18 days post immuniza-
tion. Compound 11p was dosed orally bid for 12 days as Natal-
izumab (Tysabri) was dosed every other day. As illustrated in
Figure 3, Natalizumab and high dose 11p were able to reverse par-
alytic symptoms of EAE in guinea pigs as measured by clinical
score. Mean clinical scores in the Natalizumab treated group were
reduced from 1.3 before treatment to an average score of 0.5 by
treatment day 12. 300 mg/kg 11p administration reduced the
mean clinical score by half from 1.3 to 0.7 over the treatment per-
iod. In guinea pigs treated with either vehicle or low dose 11p, clin-
ical scores increased steadily over the treatment period. Vehicle
treated animals experienced an increase in mean clinical score
from 1.2 to 1.6, while 30 mg/kg 11p treated animal’s scores like-
wise increased from 1.2 to 1.9 over the same period.
15. Systemic exposure was determined as follow: Three fasted male Sprague–Dawley
rats were adminstered 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 etheyl 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).
16. Abraham, W. M.; Gill, A.; Ahmed, A.; Sielczak, M. W.; Lauredo, I. T.;
Botinnikova, Y.; Lin, K. C.; Pepinsky, B.; Leone, D. R.; Lobb, R. R.; Adams, S. P.
Am. J. Respir. Crit. Care Med. 2000, 162, 603.
17. Kung, T. T.; Jones, H.; Adams, G. K., 3rd; Umland, S. P.; Kreutner, W.; Egan,
R. W.; Chapman, R. W.; Watnick, A. S. Int. Arch. Allergy Immunol. 1994, 105,
83.
18. Trentham, D. E.; Townes, A. S.; Kang, A. H. J. Exp. Med. 1977, 146, 857.
19. Bendele, A.; McComb, J.; Gould, T.; McAbee, T.; Sennello, G.; Chlipala, E.; Guy,
M. Toxicol. Pathol. 1999, 27, 134.
20. Yednock, T. A.; Cannon, C.; Fritz, L. C.; Sanchez-Madrid, F.; Steinman, L.; Karin,
N. Nature (London) 1992, 356, 63.
21. Clinical scoreing criteria for guinea pig EAE: (0) No abnormality; (1) Abnormal
gait. Pig may ‘wobble’ or lean to one side as it walks. Partial paralysis of one leg
may also be present; (2) Bi-lateral hind limb weakness. Animal is unable to
support its hind limbs consistently. Inability to right itself when placed on its
back and held in hand; (3) Complete hind limb paralysis. Animal is unable to
move hind limbs, motility limited to fore limbs only; (4) Moribund, that is, the
animal is unable to right itself for locomotion, or complete paralysis of all
limbs.
As illustrated in Table 5, the activities of 11p on several inte-
grins were determined. Compound 11p showed no activity on
LFA122 or
a
5b1.23 Compound 11p was much weaker in assays
involving binding of
a a4b7 to
vb1 to osteopontin (OPN),24 or
MadCAM.25

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Y.-z. Xu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3070–3074
22.
23.
a
Lb2-Dependent adhesion: 8866 cell adhesion to ICAM1-FC, protocol similar as
24.
25.
a
Vb1-Dependent adhesion: 293/
OPN-HIS, protocol similar as described in Ref. 13.
4b7-DEPENDENT ADHESION: 8866 cell adhesion to MadCAM-FC, protocol
similar as described in Ref. 13.
aVb1 Transfected Cells Adhesion To Mouse-
described in Ref. 13.
a
5b1-Dependent adhesion: THP-1 cell adhesion to human plasma fibronectin,
a
protocol similar as described in Ref. 13.