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S.D. Dattoli et al. / European Journal of Medicinal Chemistry 73 (2014) 225e232
2.19 (m, 4H, CH3 þ PyrrolidineH-4), 2.74 (m, 1H, AspH
(m, 2H, PyrrolidineH-3 þ AspH ), 3.36 (m, 2H, PyrrolidineH-
5< PyrrolidineH-2>), 3.41e3.50 (m, 2H, PyrrolidineH-
b
), 2.90e2.99
J ¼ 7.6 Hz, 1H, ArH), 7.02e7.14 (m, 4H, ArH), 7.15e7.26 (m, 3H, ArH),
7.35 (t, J ¼ 8.4 Hz, 2H, ArH), 7.58 (s, 1H, NH), 7.79 (d, J ¼ 8.4 Hz, 1H,
ArH), 7.81 (br.t, 1H, NH>), 7.85 (br.t, 1H, NH<), 8.40 (d, J ¼ 7.6 Hz, 1H,
AspNH<), 8.55 (d, J ¼ 7.6 Hz, 1H, AspNH>), 8.62 (s, 1H, NH>), 8.64 (s,
1H, NH<). Elem. Anal. for C31H31Cl2N5O6, calcd C 58.13, H 4.88, N
10.93, found C 57.00, H 4.81, N 10.15; ESI-MS m/z 640.1 (M þ H)þ,
calcd 640.2.
b
þ
2> þ PyrrolidineH-5<), 3.54 (d, J ¼ 5.2 Hz, 2H, ArCH2CON), 3.51e
3.60 (m, 4H, PyrrolidineH-2< þ PyrrolidineH-5>), 4.78 (m, 1H,
AspH
a
), 5.07 (br.s, 2H, CH2Ph), 6.96 (t, J ¼ 7.6 Hz, 1H, ArH), 7.00e
7.08 (m, 3H, ArH), 7.09e7.18 (m, 5H, ArH), 7.24e7.32 (m, 4H,
ArH þ NH), 7.35 (d, J ¼ 8.8 Hz, 1H, AspNH), 7.56 (s, 1H, NH), 7.60 (d,
6. 1H NMR (DMSO-d6, 400 MHz)
d (ppm): (two sets of reso-
J ¼ 8.0 Hz, 1H, ArH); 13C NMR (CDCl3)
d
(ppm): 18.3, 28.3, 36.7, 36.4,
nances: ꢃmajor conformer; ꢅminor conformer) 1.95 (m, 2H, Pyr-
40.6, 42.4, 44.1, 47.7, 48.8, 48.9, 49.2, 118.4, 123.0, 126.4, 127.0, 128.0,
128.4,129.2,129.3,130.4,132.7,136.4,137.7,139.1,153.1,169.0,170.3,
172.0, 172.2. Elem. Anal. for C32H34N4O7, calcd C 65.52, H 5.84, N
9.55, found C 63.88, H 6.00, N 9.64; ESI-MS m/z 587.2 (M þ H)þ,
calcd 587.2.
rolidineH-4<), 2.05 (m, 2H, PyrrolidineH-4>), 2.29 (s, 3H, CH3Ph),
2.56 (m, 1H, AspH
3.25 (m, 1H, Prod<), 3.47 (m, 1H, PyrrolidineH-2>), 3.60 (m, 2H,
PyrrolidineH-5< PyrrolidineH-5>), 3.69e3.79 (m, 2H,
Proa> þ Proa<), 3.99 (m, 1H, Prod>), 4.11 (m, 1H, Proa<), 4.27 (br.s,
2H, CH2Ph), 5.09 (m, 1H, AspH ), 6.81 (br.t, 1H, ArH), 7.01e7.14 (m,
b), 2.92e2.98 (m, 2H, PyrrolidineH-3 þ AspH
b),
þ
3. 1H NMR (8:2 DMSO-d6/H2O, 400 MHz)
d
(ppm): 1.89 (m, 1H,
a
PyrrolidineH-4cis), 1.92 (m, 1H, PyrrolidineH-4trans), 2.00 (m, 1H,
PyrrolidineH-4cis), 2.06 (m, 1H, PyrrolidineH-4trans), 2.22 (s, 3H,
CH3), 2.39 (m, 1H, AspHb), 2.72 (m, 1H, AspHb), 2.91 (m, 1H, Pyr-
4H, ArH), 7.15e7.26 (m, 3H, ArH), 7.27e7.35 (m, 2H, ArH), 7.52 (s,
1H, NH), 7.70 (d, J ¼ 8.4 Hz, 1H, ArH), 7.75 (br.t, 1H, NH>), 7.81 (br.t,
1H, NH<), 8.20 (br.d, 1H, AspNH<), 8.25 (br.d, 1H, AspNH>), 8.51 (s,
rolidineH-3trans), 2.96 (m, 1H, PyrrolidineH-3cis), 3.21 (m, 1H, Pyr-
rolidineH-5cis), 3.28 (m, 1H, PyrrolidineH-2trans), 3.39e3.45 (m, 3H,
PyrrolidineH-5trans þ NCH2Ar), 3.45 (m, 1H, PyrrolidineH-5cis), 3.54
(m, 1H, PyrrolidineH-2cis), 3.56 (m, 1H, PyrrolidineH-2trans), 3.63
(m, 1H, PyrrolidineH-5trans), 3.75 (t, J ¼ 8.8 Hz, 1H, PyrrolidineH-
1H, NH); 13C NMR (CDCl3)
d (ppm): 18.2, 28.4, 29.5, 39.4, 42.7, 48.7,
49.0, 51.2, 118.6, 121.8, 124.1, 127.0, 128.6, 129.5, 131.0, 131.4, 132.8,
133.8, 136.6, 136.8, 138.0, 153.4, 167.4, 171.2, 172.0, 173.1. Elem. Anal.
for C31H31Cl2N5O6, calcd C 58.13, H 4.88, N 10.93, found C 57.60, H
4.79, N 11.13; ESI-MS m/z 640.1 (M þ H)þ, calcd 640.2.
2
cis), 4.19 (s, 2H, PhCH2COcis), 4.21 (s, 2H, PhCH2COtrans), 4.75 (m,1H,
AspHacis), 4.81 (m, 1H, AspHatrans), 6.93 (t, J ¼ 7.2 Hz, 1H, ArH4),
4.3. Scintillation proximity-binding assay (SPA)
0
0
7.09e7.18 (m, 4H, ArH3,5 þ ArH3 ,5 ), 7.16e7.28 (m, 5H, PhCH2CON),
0
0
7.39 (d, J ¼ 8.4 Hz, 2H, ArH2 ,6 ), 7.81 (d, J ¼ 8.0 Hz, 1H, ArH6), 7.89 (s,
1H, NHa), 8.43 (br.t, 1H, NHctrans), 8.46 (br.t, 1H, NHccis), 8.54 (br.d,
1H, AspNHtrans), 8.56 (br.d, 1H, AspNHcis), 8.98 (s, 1H, NHb); 13C
We developed a SPA assay to detect competitive binding of
drugs to soluble 125I-human FN (Mw approximately 440 kDa)
bound to an antibody-captured integrin complex [46,47]. The
assay uses microspheres coated with an anti-rabbit IgG antibody
capable of binding the complex a4 integrin anti-a4 antibody. The
radioligand binds to the a4b1 integrin and the close proximity of
the isotope to the scintillant incorporated in the beads allows the
radiation energy to transfer to the scintillant where it can be
detected as counts per min (cpm). The procedure is described in
detail in S.M.
NMR (DMSO-d6) d (ppm): 18.3, 28.3, 29.9, 39.4, 40.2, 42.4, 44.1, 45.8,
46.0, 47.4, 48.8, 48.9, 49.2, 118.4, 121.5, 126.4, 126.6, 127.8, 128.4,
129.3, 130.4, 132.7, 133.0, 136.4, 137.7, 139.1, 153.1, 168.8, 170.3, 171.7,
172.0. Elem. Anal. for C32H35N5O6, calcd C 65.63, H 6.02, N 11.96,
found C 66.20, H 6.18, N 11.70; ESI-MS m/z 586.2 (M þ H)þ, calcd
586.3.
4. 1H NMR (8:2 DMSO-d6/H2O, 400 MHz)
PyrrolidineH-4cis), 1.95e2.05 (m,
4trans þ PyrrolidineH-4cis), 2.15 (m, 1H, PyrrolidineH-4trans), 2.18 (s,
3H, CH3), 2.57 (m, 1H, AspH ), 2.94 (m, 1H, AspH ), 2.97 (m, 1H,
d
(ppm): 1.85 (m, 1H,
2H, PyrrolidineH-
4.4. Adhesion assays
b
b
PyrrolidineH-3trans), 3.22 (dd, J ¼ 6.0,10.0 Hz,1H, PyrrolidineH-5cis),
Jurkat cell adhesion assays were done as described [46,47].
Briefly, 96-well plates were coated at 4 ꢄC overnight with 2 mg/mL
of VCAM-1 and a saturation curve for the ligand was plotted to
establish the best signal-to-noise ratio. Non-specific hydrophobic
binding sites were blocked by incubation with a BSA (1%)/HBSS
(w/v) solution for 30 min at 37 ꢄC. SK-MEL-24 cell adhesion assays
were done as described [44]. The procedure is described in detail
in S.M.
3.39e3.48 (m, 4H, PyrrolidineH-3cis þ NCH2Ar þ PyrrolidineH-
2
trans), 3.49e3.55 (m, 3H, PyrrolidineH-5trans þ PyrrolidineH-
5cis þ PyrrolidineH-2trans), 3.58 (m, 1H, PyrrolidineH-5trans), 4.21 (d,
J ¼ 6.0 Hz, 2H, PhCH2CO), 4.34 (dd, J ¼ 4.8, 8.8 Hz, 1H, PyrrolidineH-
2cis), 4.43 (t, J ¼ 9.2 Hz, 1H, PyrrolidineH-2cis), 4.59 (m, 1H,
AspHacis), 4.63 (m, 1H, AspHatrans), 6.90 (t, J ¼ 7.2 Hz, 1H, ArH4),
00
0
0
7.10e7.19 (m, 4H, ArH3,5 þ ArH3 ,5 ), 7.13e7.24 (m, 5H, ArH ), 7.40 (d,
0
0
J ¼ 8.2 Hz, 2H, ArH2 ,6 ), 7.79 (d, J ¼ 8.0 Hz, 1H, ArH6), 7.90 (s, 1H,
NHa), 8.41 (br.t, 1H, NHctrans), 8.45 (br.t, 1H, NHccis), 8.50 (br.d, 1H,
AspNHtrans), 8.54 (br.d, 1H, AspNHcis), 8.91 (s, 1H, NHb); 13C NMR
4.5. Data analysis
(DMSO-d6, 400 MHz)
d
(ppm): 18.3, 28.3, 29.5, 29.9, 39.4, 42.4, 42.7,
All data are expressed as mean ꢀ S.E.M., for the number of ex-
periments indicated. SPA binding data and ligand concentratione
response curves were analyzed using GraphPad software (Graph-
Pad Software Inc., San Diego, CA, USA). IC50 values indicate the
molar ligand concentration required to inhibit the response by 50%
and were converted, in SPA experiments, to Ki values using the
method of Cheng and Prusoff [54].
44.1, 45.0, 47.7, 48.2, 49.2, 55.0, 118.4, 121.5, 126.4, 127.8, 128.4,
129.2, 129.3, 130.4, 132.7, 136.4, 137.7, 139.1, 153.1, 171.7, 172.0, 172.2,
172.3. Elem. Anal. for C32H35N5O6, calcd C 65.63, H 6.02, N 11.96,
found: C 66.94, H 6.19, N 11.72; ESI-MS m/z 586.1 (M þ H)þ, calcd
586.3.
5. 1H NMR (DMSO-d6, 400 MHz)
d (ppm): (two sets of reso-
nances: ꢃmajor conformer; ꢅminor conformer) 2.00e2.18 (m, 2H,
PyrrolidineH-4<), 2.20e2.38 (m, 2H, PyrrolidineH-4>), 2.39 (s, 3H,
4.6. In vitro metabolic stability
CH3Ph), 2.58 (m, 1H, AspH
b), 2.90 (m, 1H, PyrrolidineH-3>), 2.98
(m, 1H, AspH ), 3.08 (m, 1H, PyrrolidineH-3<), 3.35 (m, 1H, Prod<),
b
Enzymatic degradation studies of 1, 3, and 4 were carried out in
triplicate and repeated three times using mouse serum purchased
from SigmaeAldrich. Peptides were dissolved in Tris buffer pH 7.4
3.46 (dd, J ¼ 8.0, 12.0 Hz, 1H, Proa>), 3.59 (m, 2H, PyrrolidineH-
5< þ PyrrolidineH-5>), 3.70 (dd, J ¼ 8.0, 12.0 Hz, 1H, Proa>), 3.79
(dd, J ¼ 7.6, 9.2 Hz, 1H, Proa<), 4.00 (m, 1H, Prod>), 4.09 (m, 1H,
and 10
mL aliquots of a 10 mM peptide stock solution were added to
Proa<), 4.24 (d, J ¼ 5.6 Hz, 2H, CH2Ph), 5.12 (m, 1H, AspH
a
), 6.81 (t,
190
m
L of serum. Incubations were carried out at 37 ꢄC for 120 min.