S.S. Schweiker et al. / European Journal of Medicinal Chemistry 84 (2014) 584e594
591
were deprotonated and histidines were protonated); Amber FF99
atoms types were assigned; charges were added using Gastei-
gereHückel method, and the side chain amines and side chain
bumps were fixed. The protomol threshold value was set to 0.10 Å
and bloat was set to 2 Å. All the other parameters were set to the
default value.
The initial conformations of compounds 5, 7, 9e13 and 19 were
optimized in SYBYL-X 2.0 [61]. Hydrogen's were added and Gas-
teigereHückel charges were assigned to the atoms. The structures
were minimized using the Conjugate Gradient method with the
Amber7FF94 force field to reach a final energy convergence
gradient value of 0.05 kcal/mol. The minimized structures offered
reasonable starting conformations for docking. The Surflex-Dock
program [62,63] interfaced with SYBYL-X 2.0, was used to dock
compounds 5, 7, 9e13 and 19 to each of the GP binding domains,
AMP (allosteric), purine and indole (new allosteric). An overlay of
compounds 5, 7, 9e13 and 19 docked in each of the AMP, purine and
indole sites indicated that none of 5, 7, 9e13 and 19 were sitting
outside the binding site, thus eliminating false positive results for
the Cscore.
4.20e4.39 (m, 1H,
CH), 1.66e1.79 (m, 1H,
(m, 2H, CH2).
a
CH), 3.05e3.20 (m, 2H, εCH2), 1.79e1.90 (m, 1H,
b
b
CH), 1.40e1.50 (m, 2H, CH2), 1.30e1.40
d
g
8.3.3. N-[(benzyloxy)carbonyl]leucine 27 [68]
Using Leu-OH 26 (38 mmol). A clear oil (8.022 g, 79%). ESI-MS m/
z 288 (MþNaþ), 266 (MþHþ). 1H NMR (400 MHz, CDCl3)
d: 7.33 (m,
5H, ArH), 5.10 (s, 2H, CH2Ar), 4.30e4.40 (m, 1H,
aCH), 1.60e1.80 (m,
2H,
bCH2), 1.46e1.60 (m, 1H,
gCH), 0.90 (s, 6H, 1ꢃ
g
CH3, CH3).
d
8.4. Typical procedure for coupling morpholine with amino acid
HOBt (959 mg, 7.10 mmol, 0.85 equiv) and EDC.HCl (1.6 g,
8.36 mmol,1.0 equiv) were dissolved in DCM (anhydrous, 50 mL). Z-
Phe-OH 23 (2.5 g, 8.36 mmol, 1.0 equiv) was added and the mixture
stirred for 4 h under an atmosphere of nitrogen. Morpholine (1.09 g,
12.54 mmol,1.5 equiv) and DIPEA (2.7 g, 20.9 mmol, 2.5 equiv) were
added and the reaction stirred for 20 h. The reaction mixture was
washed with water (50 mL). The organic layer was extracted with
brine (50 mL), dried (MgSO4, anhydrous) and concentrated in vacuo.
The residue was purification of the residue by silica column chro-
matography (ethyl acetate:hexane).
8.2. Computational studies
8.4.1. Benzyl-1-morpholino-1-oxo-3-phenylpropan-2-ylcarbamate
14 [69]
8.2.1. Ligand efficiency (LE)
Ligand efficiency [57] was calculated for a temperature of 300 K
using the following equation.
Purification (ethyl acetate:hexane, 55:45). A beige gum (2.35 g,
79.5%). ESI-MS m/z 391 (MþNaþ), 369 (MþHþ). 1H NMR (400 MHz,
LE ¼ Dg ¼ ðDGÞ=N
CDCl3)
d
: 7.13e7.30 (m, 10H, AreH), 6.20 (d, 1H, J ¼ 9 Hz, NH), 5.20
(q, 2H, J ¼ 9,11.5 Hz, CH2Ar), 4.92 (dd,1H, J ¼ 5, 9 Hz, H2), 3.50e3.55
(m, 2H, 2ꢃ H60), 3.29e3.45 (m, 3H, H50, 2ꢃ H20), 3.19e3.29 (m, 1H,
H50), 2.87e3.05 (m, 3H, H3, 2ꢃ H30), 2.77e2.87 (m, 1H, H3).
where
D
G ¼ ꢁRTln Kd and N is the number of non-hydrogen atoms.
The units used for LE were units kcal/mol per non-hydrogen atom.
Following the practice of substitution of pKd with pIC50, LE can be
expressed as LE ¼ (1.37/HA) ꢃ pIC50 [57]. pIC50 values were
calculated from the IC50 values [64]. The LE values were comparable
as the assay conditions were the same for all tested compounds.
8.4.2. Benzyl, N-(benzyloxy)-1-morpholino-1-oxohexan-2-
ylcarbanmate 16
Using Z-Lys(Z)-OH 25 (2.928 g, 7.07 mmol, 1.0 equiv) and puri-
fication with ethyl acetate:hexane, 80:20. A clear oil (1.337 g, 39%).
1H NMR (400 MHz, CDCl3)
d: 7.30 (s, 10H, ArH), 5.8e6.0 (brs, 2H, 2-
8.2.2. Ligand-efficiency-dependent lipophilicity (LELP)
Ligand-Efficiency-dependent Lipophilicity [65] was calculated
for compounds using the definition of LELP ¼ the ratio of log P and
ligand efficiency (LE).
NH, 6-NH), 4.95e5.13 (m, 4H, 2-NHC(O)OCH2, 6-NHC(O)OCH2),
4.5e4.59 (m, 1H, H2), 3.64e3.68 (m, 5H, 2ꢃ H20, H50, 2ꢃ H60),
3.56e3.64 (m, 3H, H50, 2ꢃ H30), 3.02e3.20 (m, 2H, 2ꢃ H6),
1.58e1.72 (m, 1H, H3), 1.40e1.58 (m, 2H, H5, H3), 1.22e1.40 (m, 2H,
H4, H5), 1.14e1.22 (m, 1H, H4). 13C NMR (75 MHz, CDCl3)
d: 170.3,
8.3. Typical procedure for N-protection (Cbz) of amino acid
156.3, 156.0, 156.5, 156.3, 128.3 (3C), 127.9 (2C), 127.9 (2C), 127.8
(3C), 67.2, 67.0, 66.8 (2C), 49.2, 46.4, 42.8, 40.8, 33.0, 29.1, 22.4. ESI-
MS m/z 506 (MþNaþ), 484 (MþHþ). HRMS m/z 484.2426 (calcd for
A solution of Phe-OH 22 (5.0 g, 30 mmol, 1.0 equiv) was dis-
solved in NaOH (2 M, 50 mL) and stirred at 0 ꢄC under an atmo-
sphere of nitrogen. Benzyl chloroformate (4.3 mL, 30 mmol,
1.0 equiv) was dissolved in toluene (5 mL) and NaOH (4 M, 5 mL)
was added. The reaction was stirred vigorously at rt for 30 min.
Water (50 mL) was added and the mixture extracted with diethyl
ether (2 ꢃ 50 mL). The combined organic layers were cooled to 0 ꢄC
and acidified to pH 1 with HCl (conc). A white solid formed which
was isolated by filtration. The solid was dissolved in DCM (200 mL).
The DCM layer was extracted with brine (50 mL), dried (MgSO4,
anhydrous) and concentrated in vacuo.
C26H33N3O6 þ 1H, 484.2448).
8.4.3. Benzyl 4-methyl-1-morpholino-1-oxopentan-2-ylcarbamate
18 [70]
Using Z-Leu-OH 27 (2.5 g, 9.43 mmol, 1.0 equiv) and purification
with ethyl acetate:hexane, 55:45. A colourless oil (3.133 g, 99.5%).
1H NMR (400 MHz, CDCl3)
d: 7.22 (m, 5H, ArH), 5.98 (d, 1H, NH),
5.00 (s, 2H, CH2Ar), 4.55e4.65 (m,1H,1ꢃ H2), 3.42e3.62 (m, 6H, 2ꢃ
H50, 2ꢃ H20, 1ꢃ H60), 3.32e3.42 (m, 2H, 2ꢃ H30), 1.58e1.70 (m, 1H,
1ꢃ H3), 1.41e1.52 (m, 1H, 1ꢃ H3), 1.30e1.39 (m, 1H, 1ꢃ H4), 0.90 (d,
3H, J ¼ 6.5 Hz, 3ꢃ H5), 0.85 (d, 3H, J ¼ 6.5 Hz, 4ꢁCH3). 13C NMR
8.3.1. N-[(benzyloxy)carbonyl]phenylalanine 23
A white solid (5.482 g, 60%). mp 135ꢁ136 ꢄCþ, (Lit [66]. mp
(100 MHz, CDCl3) d: 170.2, 155.9, 135.7, 129.8 (2C), 128.4, 127.2 (2C),
130ꢁ132 ꢄC). ESI-MS m/z 322 (MþNaþ), 300 (MþH ).
67.1, 67.0, 66.9, 49.0, 46.3, 42.7, 24.9, 23.6 (2C), 23.2. ESI-MS m/z 357
(MþNaþ), 335 (MþHþ).
8.3.2. N,N-[(benzyloxy)carbonyl]lysine 25 [67]
Using benzyl chloroformate (60 mmol) and purification by silica
column chromatography (ethyl acetate:hexane, 40:60). A clear oil
(8.9 g, 72%). ESI-MS m/z 437 (MþNaþ), 415 (MþHþ). 1H NMR
8.4.4. tert-Butyl 4-((benzyloxy) carbonyl)-1-morpholino-1-
oxobutan-2-ylcarbamate 29
Using Boc-Glu(OBzl)-OH 28 (2.0 g, 5.92 mmol, 1.0 equiv) and
purification with ethyl acetate:hexane, 50:50. A clear oil (1.213 g,
(400 MHz, CDCl3)
d
: 7.6 (brs,1H, Wh/z ¼ 58 Hz, OH), 7.30 (m,10H, 2ꢃ
C6H5), 6.35 (brs, 1H, Wh/z ¼ 19 Hz, NH), 5.00e5.20 (m, 4H, 2ꢃ CH2),
50%). 1H NMR (300 MHz, CD3OD)
d: 7.15e7.35 (m, 5H, ArH), 5.59