ꢁ
M. Ilic et al. / European Journal of Medicinal Chemistry 50 (2012) 255e263
261
3
134.13 (C-30, C-50), 133.88 (C-8a), 129.04 (d, JCeF ¼ 8.0 Hz, C-200, C-
600), 119.05 (CN), 115.81 (C-8), 115.71 (C-20, C-60), 114.82 (d,
2JCeF ¼ 21.0 Hz, C-200, C-600), 103.07 (C-40), 102.19 (C-7), 102.19 (C-7),
97.73 (C-5), 70.93 (CH2O), 54.16 (C-3), 46.56 (PheCH2), 38.17
H), 4.04 (d, J ¼ 9.9 Hz, 1H, CH2O), 4.12 (q, J ¼ 7.2 Hz, 2H, CH2eCH3,),
4.13 (d, J ¼ 9.9 Hz, 1H, CH2O), 4.21 (s, 2H, NeCH2eCO), 4.52 (s, 2H,
PheCH2), 5.84 (dd, J ¼ 8.6, 2.8 Hz,1H, AreH7), 5.95 (d, J ¼ 2.7 Hz,1H,
AreH5), 6.50 (d, J ¼ 8.6 Hz, 1H, AreH8), 7.18 (d, J ¼ 8.9 Hz, 2H,
0
0
00
(NeCH3), 20.97 (2-CH3); IR (KBr,
n
, cmꢃ1): 3423, 2221, 1604, 1508,
1219, 1036; HPLC: 95.4%, tr 13.5 min; HRMS (ESI) m/z calcd for
C25H25FN3O2 [M þ H]þ 418.1931, found 418.1942. Anal. Calcd. for
C25H24FN3O2: C, H, N.
AreH2 , AreH6 ), 7.20 (overlapped, 1H, PheH6 ), 7.34e7.44 (m, 2H,
0
0
0
0
0
0
PheH2 , PheH5 ), 7.80 (d, J ¼ 8.8 Hz, 2H, AreH3 , AreH5 ), 6.97 (br s,
2H, amidino-H), 9.15 (br s, 2H, amidino-H); 13C NMR (100 MHz,
DMSO-d6):
d ppm: 171.25 (COO), 164.57 (C(]NH)NH2), 162.81 (C-
1
2
10), 149.41 (dd, JCeF ¼ 244.7 Hz, JCeF ¼ 12.3 Hz, C-300), 148.12 (dd,
2
5.1.7. Ethyl 2-((2-((4-cyanophenoxy)methyl)-2,4-dimethyl-3,4-
dihydro-2H-1,4-benzoxazin-6-yl)(4-fluorobenzyl)amino)acetate
(7a)
Synthesized according to the A typical procedure for preparation
of N-alkylated compounds (5.1.2). The brown oil was purified by
column chromatography using petroleum ether/ethyl acetate
(2.5:1) as eluant to give yellow oil, yield 1.45 g (60%); 1H NMR
1JCeF ¼ 243.9 Hz, JCeF ¼ 12.4 Hz, C-400), 142.54 (C-8a), 137.97
3
(pseudo t, JCeF ¼ 4.0 Hz, C-100), 135.25 (C-6), 134.61 (C-4a), 130.13
3
4
(C-30, C-50), 123.28 (dd, JCeF ¼ 6.2 Hz, JCeF ¼ 3.1 Hz, C-600), 123.29
3
4
(dd, JCeF ¼ 6.2 Hz, JCeF ¼ 3.1 Hz, C-600), 119.85 (C-40), 117.26 (d,
2JCeF ¼ 16.9 Hz, C-500), 115.84 (C-8), 115.62 (d, JCeF ¼ 17.6 Hz, C-200),
2
114.97 (C-20, C-60), 102.56 (C-7), 97.72 (C-5), 73.55 (C-2), 71.01
(CH2O), 60.26 (CH2eCH3), 54.91 (PheCH2), 54.03(C-3), 53.65
(NeCH2eCO), 38.05 (NeCH3), 20.96 (2-CH3), 14.14 (CH2eCH3); IR
(300 MHz, CDCl3):
d
ppm 1.28 (t, J ¼ 7.1 Hz, 3H, CH2eCH3), 1.46 (s,
3H, 2-CH3), 2.78 (s, 3H, NeCH3), 3.02 (d, J ¼ 11.5 Hz, 1H, 3-H), 3.25
(d, J ¼ 11.5 Hz, 1H, 3-H), 3.90 (d, J ¼ 9.0 Hz, 1H, CH2eCH3), 4.01 (s,
2H, NeCH2eCO), 4.10 (d, J ¼ 9.0 Hz, 1H, CH2eCH3), 4.20 (d,
J ¼ 7.1 Hz, 1H, CH2O), 4.24 (d, J ¼ 7.1 Hz, 1H, CH2O), 4.56 (s, 2H,
PheCH2), 6.00e6.15 (m, 2H, AreH8, AreH7), 6.67 (d, J ¼ 9.1 Hz, 1H,
(KBr, n
, cmꢃ1): 3417, 1672, 1515, 1266, 1186, 1140, 1039; HPLC:
100.0%, tr 17.8 min; HRMS (ESI) m/z calcd for C29H33F2N4O4
[M þ H]þ 539.2470, found 539.2481. Anal. Calcd. for
C29H32F2N4O4 ꢂ1/4CF3COOH ꢂ 5/4H2O: C, H, N.
0
0
AreH5), 6.96 (d, J ¼ 8.9 Hz, 2H, AreH2 , AreH6 ), 6.99e7.07 (m, 2H,
5.1.10. 2-((2-((4-Carbamimidoylphenoxy)methyl)-2,4-dimethyl-
3,4-dihydro-2H-1,4-benzoxazin-7-yl)(4-fluorobenzyl)amino)-2-
oxoacetic acid (9d)
Synthesized according to the A typical procedure for alkaline
hydrolysis of alkyl esters (5.1.5). White solid was obtained, yield
124 mg (40%); mp 174e177 ꢁC; 1H NMR (400 MHz, DMSO-d6):
0
0
Ph), 7.24e7.36 (m, 2H, Ph), 7.58 (d, J ¼ 8.9 Hz, 2H, AreH3 , AreH5 );
IR (KBr, n
, cmꢃ1): 3421, 2220, 1654, 1507, 1253, 1019; HPLC: 100.0%,
tr 22.4 min; HRMS (ESI) m/z calcd for C29H31FN3O4 [M þ H]þ
504.2299, found 504.2292. Anal. Calcd. for C29H30FN3O4: C, H, N.
5.1.8. Ethyl 2-((2-((4-cyanophenoxy)methyl)-2,4-dimethyl-3,4-
dihydro-2H-1,4-benzoxazin-7-yl)(3,5-difluorobenzyl)amino)-2-
oxoacetate (7p)
Synthesized according to the A typical procedure for preparation
of N-acylated compounds (5.1.3). The brown oil was purified by
column chromatography using petroleum ether/ethyl acetate
(1.5:1) as eluant to give yellow oil, yield 1.41 g (55%) of a yellow oil;
d
ppm 1.29 (s, 3H, 2-CH3), 2.76 (s, 3H, NeCH3), 2.91 (d, J ¼ 11.8 Hz,
1H, 3-H), 3.09 (d, J ¼ 11.7 Hz, 1H, 3-H), 3.88 (d, J ¼ 10.1 Hz, 1H,
CH2O), 3.92 (d, J ¼ 10.1 Hz, 1H, CH2O), 4.74 (d, J ¼ 15.3 Hz, 1H,
PheCH2), 4.83 (d, J ¼ 15.3 Hz, 1H, PheCH2), 6.58 (d, J ¼ 2.1 Hz, 1H,
AreH8), 6.61 (d, J ¼ 8.7 Hz, 1H, AreH5), 6.66 (dd, J ¼ 8.6 Hz,
0
0
J ¼ 2.1 Hz, 1H, AreH6), 7.00 (d, J ¼ 8.8 Hz, 2H, AreH2 , AreH6 ), 7.13
(t, J ¼ 8.8 Hz, 2H, Ph),07.22 (dd, J ¼ 8.5 Hz, J ¼ 7.7 Hz, 2H, Ph), 7.77 (d,
0
1H NMR (400 MHz, DMSO-d6):
d
ppm 0.85 (t, J ¼ 7.1 Hz, 3H,
J ¼ 8.8 Hz, 2H, AreH3 , AreH5 ), 9.02 (br s, 2H, amidino-H), 9.92 (br
CH2eCH3), 1.36 (s, 3H, 2-CH3), 2.85 (s, 3H, NeCH3), 3.08 (d,
J ¼ 11.1 Hz, 1H, 3-H), 3.29 (d, J ¼ 11.0 Hz, 1H, 3-H), 3.96 (q, J ¼ 7.1 Hz,
2H, CH2eCH3), 4.10 (d, J ¼ 7.1 Hz, 1H, CH2O), 4.14 (d, J ¼ 7.0 Hz, 1H,
CH2O), 4.88 (s, 2H, PheCH2), 6.57e6.62 (m, 2H, AreH6, AreH8), 6.68
s, 2H, amidino-H); 13C NMR (100 MHz, DMSO-d6):
d ppm: 167.88
(COeCOOH), 164.33 (COeCOOH), 162.34, 162.28 (C-10, C(]NH)
NH2), 161.14 (d, 1JCeF ¼ 242.4 Hz, C-100), 140.90 (C-4a, C-8a), 134.07
4
(d, JCeF ¼ 2.2 Hz, C-400), 133.96 (C-7), 129.74 (C-30, C-50), 129.34 (d,
00
00
(d, J ¼ 9.2 Hz, 1H, AreH5), 6.90 (d, J ¼ 6.2 Hz, 2H, PheH2 , PheH6 ),
3JCeF ¼ 8.1 Hz, C-200, C-600), 120.08 (C-40), 119.24 (C-6), 115.04 (d,
2JCeF ¼ 21.3 Hz, C-300, C-500), 114.88 (C-20, C-60), 114.15 (C-8), 112.05
(C-5), 74.90 (C-2), 71.40 (CH2O), 53.43 (C-3), 49.21 (PheCH2), 38.14
0
0
7.12 (d, J ¼ 8.9 Hz, AreH2 , AreH6 ), 7.16 (tt, J ¼ 9.4 Hz, J ¼ 2.4 Hz, 1H,
00
0
0
PheH4 ), 7.76 (d, J ¼ 8.8 Hz, 2H, AreH3 , AreH5 ); 13C NMR
(100 MHz, DMSO-d6):
d
ppm 162.40, 161.86, 161.75 (COeCOO,
(NeCH3), 21.16 (2-CH3); IR (KBr, n
, cmꢃ1): 3366, 1608, 1511, 1489,
COeCOO, C-10), 162.30 (dd, 1JCeF ¼ 247 Hz, JCeF ¼ 13.2 Hz, C-300, C-
1265, 1183, 1041, 843; HPLC: 97.6%, tr 12.6 min; HRMS (ESI) m/z
calcd for C27H28FN4O5 [M þ H]þ 507.2044, found 507.2057. Anal.
Calcd. for C27H27FN4O5 ꢂ 3/4HCl: C, H, N.
3
3
500), 142.12 (C-8a), 141.01 (t, JCeF ¼ 9.0 Hz, C-100), 135.41 (C-4a),
134.16 (C-30, C-50), 128.70 (C-7), 120.27 (C-6), 119.01 (CN), 115.65 (C-
2
20, C-60), 114.48 (C-8), 111.85 (C-5), 110.89 (dd, JCeF ¼ 18.3 Hz,
4JCeF ¼ 6.6 Hz, C-200, C-600), 103.21 (C-40), 103.04 (t, JCeF ¼ 25.7 Hz,
5.2. Docking studies
2
C-400), 74.56 (C-2), 70.89 (CH2O), 61.17 (CH2eCH3), 53.27 (C-3), 50.19
(PheCH2), 38.06 (NeCH3), 20.59 (2-CH3), 13.33 (CH2eCH3); IR (KBr,
The binding mode for the ligands to both targets was studied by
CDOCKER, a docking tool based on the CHARMm force field, which
is incorporated into Discovery Studio 3.0 (Accelrys Software Inc.).
All ligands were docked in all possible stereoisomeric forms and
were ionized at pH ¼ 7.5 (protonated benzamidine moiety and
anionic carboxylate moiety). The crystal structure of thrombin (PDB
entry code: 1KTS) and GP IIb/IIIa (PDB entry code: 2VDM) was
extracted from the Brookhaven Protein Database. Ligands were
removed from the crystal structure prior to docking studies.
n
, cmꢃ1): 3504, 2225, 1741, 1666, 1509, 1204, 1033; HPLC: 100.0%, tr
19.08 min; HRMS (ESI) m/z calcd for C29H28F2N3O5 [M þ H]þ
536.1997, found 536.2006. Anal. Calcd. for C29H27F2N3O5: C, H, N.
5.1.9. Ethyl 2-((2-((4-carbamimidoylphenoxy)methyl)-2,4-
dimethyl-3,4-dihydro-2H-1,4-benzoxazin-6-yl)(3,4-difluorobenzyl)
amino)acetate (8i)
Synthesized according to the A typical procedure for preparation
of amidines from nitriles (5.1.4). The brown crude product was
purified by reverse phase column chromatography using gradient
of methanol/0.1% TFA (40e80%) as eluant to give white solid, yield
353 mg (39%); mp 159e162 ꢁC; 1H NMR (400 MHz, DMSO-d6):
ꢂ
Ligands were docked in a sphere with radius 15 A in the active site.
In CDOCKER, random ligand conformations are generated through
molecular dynamics, and a variable number of rigid-body rotations/
translations are applied to each conformation to generate the initial
ligand poses. The conformations are further refined by grid-based
simulated annealing in the receptor active site, which makes the
d
ppm 1.20 (t, J ¼ 7.1 Hz, 3H, CH2eCH3), 1.34 (s, 3H, 2-CH3), 2.72 (s,
3H, Ne-CH3), 3.00 (d, J ¼ 11.6 Hz,1H, 3-H), 3.23 (d, J ¼ 11.5 Hz,1H, 3-