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5.24. 4-[1-[2,5-Dichloro-4-[N0-(2-methylphenyl)ureido]phen-
ylacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid
(20b)
(2H, m), 3.58–3.98 (5H, m), 4.10–4.69 (2H, m), 5.32–5.55 (1H,
m), 7.01–7.06 (3H, m), 7.34–7.44 (2H, m), 7.63–7.65 (1H, m),
7.85–7.98 (4H, m). Anal. Calcd for C27H23BrClF2N3O5ꢀ0.5H2O: C,
51.32; H, 3.83; N, 6.65. Found: C, 51.29; H, 3.93; N, 6.36.
Yield 70% (two steps) Colorless powder. IR (KBr) 3340, 1604,
1511, 1251, 1166, 750 cmꢂ1
;
1H NMR (DMSO-d6) d 2.27 (3H, s),
5.30. 4-[1-[4-[N0-(2-bromophenyl)ureido]-2,5-dichloro-
phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic
acid (20h)
2.30–2.35 (2H, m), 3.63–4.00 (5H, m), 4.12–4.70 (2H, m), 5.33–
5.55 (1H, m), 6.94–7.20 (6H, m), 7.45–7.49 (1H, m), 7.76–7.81
(1H, m), 7.86–7.91 (2H, m), 8.26–8.31 (2H, m); MS (ESI), m/z 574
(M++1). Anal. Calcd for C28H26Cl2FN3O5ꢀ1.5H2O: C, 55.92; H, 4.86;
N, 6.99. Found: C, 56.23; H, 4.78; N, 6.59.
Yield 49% (two steps). Colorless solid. IR (KBr) 3328, 1604, 1513,
1434, 1251, 1166, 750 cmꢂ1 1H NMR (DMSO-d6) d 2.25–2.33 (2H,
;
m), 3.63–4.00 (m, 5 H), 4.10–4.72 (2H, m), 5.42–5.55 (1H, m), 7.01–
7.07 (4H, m), 7.35 (1H, t, J = 7.5 Hz), 7.47–7.50 (1H, m), 7.63 (1H, d,
J = 7.3 Hz), 7.86–7.94 (4H, m), 8.19–8.21 (1H, m); MS (ESI), m/z 638
(M++1). Anal. Calcd for C27H23BrCl2FN3O5ꢀ0.25H2O: C, 50.37; H,
3.68; N, 6.53. Found: C, 50.57; H, 4.06; N, 5.99.
5.25. 4-[1-[5-chloro-4-[N0-(2-methylphenyl)ureido]-2-methyl-
phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic
acid (20c)
Yield 70% (two steps). Colorless powder. IR (ATR) 3342, 1604, 1533,
1251, 1166, 755 cmꢂ1; 1H NMR (DMSO-d6) d 2.17 (3H, s), 2.25 (3H, s),
2.29–2.32 (2H, m), 3.68–3.78 (2H, m), 3.80–3.99 (3H, m), 4.10-–4.43
(2H, m), 5.32–5.53 (1H, m), 6.98 (1H, t, J = 7.5 Hz), 7.03–7.07 (2H,
m), 7.12–7.22 (3H, m), 7.78–7.80 (1H, m), 7.86–7.91 (3H, m); MS
(FAB), m/z 554 (M++1). Anal. Calcd for C29H29ClFN3O5ꢀ1.25H2O: C,
60.42; H, 5.51; N, 7.29. Found: C, 60.19, H, 5.38; N, 7.04.
5.31. 4-[1-[4-[N0-(2-bromophenyl)ureido]-5-chloro-2-methyl-
phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic
acid (20i)
Yield 27% (two steps). Colorless powder. IR (ATR) 3324, 1581,
1529, 1434, 1025, 748 cmꢂ1 1H NMR (DMSO-d6) d 2.19 (3H, s),
;
2.25–2.32 (2H, m), 3.59–3.74 (2H, m), 3.85–4.00 (3H, m), 4.10–
4.40 (2H, m), 5.32–5.54 (1H, m), 6.98–7.07 (3H, m), 7.20–7.24
(1H, m), 7.32–7.35 (1H, m), 7.62 (1H, d, J = 8.0 Hz), 7.84–7.91
(3H, m), 7.96 (1H, d, J = 6.8 Hz); MS (FAB), m/z 618 (M++1). Anal.
Calcd for C28H26BrClFN3O5ꢀ1.25H2O: C, 52.43; H, 4.48; N, 6.55.
Found: C, 52.42, H, 4.34; N, 6.11.
5.26. 4-[1-[3-Chloro-4-[N0-(2-chlorophenyl)ureido]-6-fluoropheny-
lacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid (20d)
Yield 43% (two steps). Colorless powder. IR (KBr) 3322, 1529,
1249, 1168, 750, 644 cmꢂ1 1H NMR (DMSO-d6) d 2.26–2.33 (2H,
;
m), 3.68–3.99 (5H, m), 4.10–4.69 (2H, m), 5.41–5.55 (1H, m),
7.04–7.10 (3H, m), 7.30–7.34 (1H, m), 7.39–7.49 (2H, m), 7.85–
7.91 (2H, m), 7.96–7.99 (1H, m), 8.05–8.07 (1H, m); MS (ESI), m/z
578 (M++1), 576 (M+ꢂ1). Anal. Calcd for C27H23Cl2F2N3O5ꢀ1H2O:
C, 54.37; H, 4.22; N, 7.05. Found: C, 54.41; H, 4.13; N, 6.80.
5.32. 4-[1-[3-Chloro-4-[N0-(3-fluoro-2-methylphenyl)-
ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidiny-
lmethoxy]benzoic acid (20j)
Yield 53% (two steps). Light brown solid. IR (KBr) 1604, 1581,
5.27. 4-[1-[4-[N0-(2-chlorophenyl)ureido]-2,5-dichloroph-
enylacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid
(20e)
1527, 1240, 1220, 1167, 773 cmꢂ1 1H NMR (DMSO-d6) d 2.17
;
(3H, s), 2.21–2.34 (2H, m), 3.65–3.68 (2H, m), 3.78–4.28 (4H, m),
4.38–4.71 (1H, m), 5.31–5.52 (1H, m), 6.87 (1H, t, J = 8.5 Hz),
7.04–7.07 (2H, m), 7.16–7.20 (2H, m), 7.32–7.36 (1H, m), 7.65
(1H, d, J = 8.1 Hz), 7.87–7.90 (2H, m), 7.99–8.01 (1H, m), 8.69
(1H, s), 8.73 (1H, s), 12.60 (1H, broad s); MS (ESI), m/z 558
(M++1). Anal. Calcd for C28H26ClF2N3O5ꢀ1H2O: C, 58.39; H, 4.90;
N, 7.30. Found: C, 58.58; H, 4.78; N, 7.14.
Yield 46% (two steps). Colorless solid. IR (KBr) 3328, 1604, 1513,
1251, 1166, 750 cm-1 1H NMR (DMSO-d6) d 2.27–2.34 (2H, m),
;
3.67–4.01 (5H, m), 4.12–4.72 (2H, m), 5.33–5.55 (1H, m), 7.05–
7.10 (4H, m), 7.30 (1H, t, J = 7.8 Hz), 7.47–7.50 (2H, m), 7.86–7.91
(3H, m), 8.07 (1H, d, J = 8.6 Hz), 8.20–8.22 (1H, m); MS (ESI), m/z
594 (M++1). Anal. Calcd for C27H23Cl3FN3O5ꢀ1.25H2O: C, 52.53; H,
4.16; N, 6.81. Found: C, 52.78; H, 4.18; N, 6.31.
5.33. 4-[1-[3-Chloro-4-[N0-(4-fluoro-2-methylphenyl)-
ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidiny-
lmethoxy]benzoic acid (20k)
5.28. 4-[1-[5-Chloro-4-[N0-(2-chlorophenyl)ureido]-3-methy-
lphenylacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic
acid (20f)
Yield 76% (two steps). Colorless solid. IR (KBr) 1604, 1525, 1496,
1248, 1167, 773 cmꢂ1 1H NMR (DMSO-d6) d 2.18–2.32 (2H, m),
;
2.27 (3H, s), 3.47–3.97 (5H, m), 4.05–4.68 (2H, m), 5.31–5.52
(1H, m), 6.98 (1H, dt, J = 2,9, 8.6 Hz), 7.08–7.13 (3H, m), 7.14 (1H,
t, J = 8.3 Hz), 7.31–7.35 (1H, m), 7.68–7.72 (1H, m), 7.86–7.91
(2H, m), 7.99–8.03 (1H, m), 8.55–8.59 (2H, m), 12.61 (1H, broad
s); MS (ESI), m/z 558 (M++1). Anal. Calcd for C28H26ClF2N3O5ꢀ1H2O:
C, 58.39; H, 4.90; N, 7.30. Found: C, 58.38; H, 4.73; N, 7.10.
Yield 25% (two steps). Colorless powder. IR (ATR) 3338, 1583,
1531, 1438, 750 cm-1 1H NMR (DMSO-d6) d 2.18 (3H, s), 2.26–
;
2.33 (2H, m), 3.59–3.73 (2H, m), 3.84–3.99 (3H, m), 4.09–4.42
(2H, m), 5.32–5.54 (1H, m), 7.05–7.07 (3H, m), 7.20–7.24 (1H,
m), 7.30 (1H, t, J = 8.0 Hz), 7.48 (1H, d, J = 7.8 Hz), 7.86–7.91 (3H,
m), 8.10 (1H, d, J = 8.4 Hz); MS (FAB), m/z 574 (M++1). Anal. Calcd
for C28H26Cl2FN3O5ꢀ1.25H2O: C, 56.34; H, 4.81; N, 7.04. Found: C,
56.28, H, 4.71; N, 6.72.
5.34. VLA-4/VCAM-1 binding assay
A human VLA-4-expressing cell line, 4B4, was established at
Pharmacopeia Inc., by transfecting both the a4 gene and b1 gene
of VLA-4 into CHO-K1 cells. The 4B4 cells were maintained in
Ham0s F-12 medium (Sigma Corporation) supplemented with 10%
(v/v) fetal calf serum (REHATUIN Fetal Bovine Serum, Serologicals
Corporation), 100 U/ml penicillin (Invitrogen Corporation),
5.29. 4-[1-[4-[N0-(2-Bromophenyl)ureido]-3-chloro-6-
fluorophenylacetyl]-(4S)-fluoro-(2S)-
pyrrolidinylmethoxy]benzoic acid (20g)
Yield 38% (two steps). Colorless powder. IR (KBr) 3320, 1525,
1434, 1249, 1168, 750 cmꢂ1
;
1H NMR (DMSO-d6) d 2.26–2.33
100 lg/ml streptomycin (Invitrogen Corporation), and 2 mM L-glu-