Molecular Pharmaceutics
Article
Scheme 1. Release of S-Allyl-GSH Following CP11 Ring-Opening
In this study, we gauged the chemical stability of CP11 at
different values of pH and the enzymatic stability in simulated
fluids and human plasma. The cell membrane permeation
characteristics of CP11 were measured using PAMPA-BBB, as
model for CNS permeability, while the anti-inflammatory activity
was assayed in vitro on LPS-activated U937 cells.
CH), 76.46 (OMe), 82.04 (OBut), 85.10 (Boc), 118.05 (S-CH2-
CH-CH2), 133.89 (S-CH2-CH-CH2), 170, 173, 174, 186, and
197 (5 × CO).
Boc-Gly(OCH2O)-Glu[Cys(Allyl)-OBut]-OMe (9). The dipep-
tide 7 (4.37 g, 9.48 mmol) was dissolved with TFA (9.43 mL,
123.24 mmol) and dry CH2Cl2 (19.55 mL) for 4 h at 0 °C. After
evaporation, the residue was treated with Et2O to afford the
corresponding pure dipeptide 8 (4.5 g, quantitative yield).
A suspension of Boc-Gly(OCH2O)-p-nitro-phenyl-carbonate
4 (3.33 g, 9.01 mmol), HOBt (1.22 g, 9.01 mmol), and NMM
(0.99 mL, 9.01 mmol) in dry DMF (210 mL) was added to a
solution of 8 (4.27 g, 9.01 mmol) and NMM (0.99 mL, 9.01
mmol) in DMF (210 mL); the flask was left for 2 h at 0 °C under
nitrogen atmosphere and 19 h at 20 °C. After evaporation, the
residue was washed with CH2Cl2/5% citric acid (×2), H2O (×1),
NaHCO3 (×2), H2O (×1), and NaCl (×1) and then evaporated.
Compound 9 (2.4 g) was obtained after chromatographic
purification using CHCl3:MeOH (97:3). Yield: 45%. Rf = 0.40,
CHCl3:MeOH (97:3). 1H NMR (CDCl3) δ: 1.42 (9H, s, OBut),
1.46 (9H, s, Boc), 1.96−2.24 (2H, m, Glu β-CH2), 2.29−2.38
(2H, m, Glu γ-CH2), 2.75−2.98 (2H, m, Cys β-CH2), 3.11−3.15
(2H, m, Cys S-CH2-CH-CH2), 3.73 (3H, s, OMe), 3.89 (2H, d,
Gly α-CH2), 4.36−4.41 (1H, m, Glu α-CH), 4.60−4.65 (1H, m,
Cys α-CH), 5.00 (1H, d, Gly NH), 5.09−5.12 (2H, m, Cys S-
CH2-CH-CH2), 5.64−5.76 (1H, m, Cys S-CH2-CH-CH2), 5.79
(2H, s, OCH2O), 6.02 (1H, d, Glu NH), 6.41 (1H, d, Cys NH).
13C NMR (CDCl3) δ: 28.04 (Glu β-CH2), 28.19 (OBut), 28.52
(Boc), 32.21 (Glu γ-CH2), 33.13 (Cys β-CH2), 35.33 (S-CH2),
42.53 (Gly α-CH2), 52.39 (Cys α-CH), 52.92 (Glu α-CH), 53.71
(OMe), 76.83 (OCH2O), 80.57 (OBut), 83.09 (Boc), 118.12 (S-
CH2-CH-CH2), 133.88 (S-CH2-CH-CH2), 154.41, 169.34,
169.88, 170.11, 171.66, and 172.12 (6 × CO).
MATERIALS AND METHODS
■
Chemistry. H-Cys(Allyl)-OH and Boc-Glu-OMe were
purchased from Bachem. The synthesis of Boc-Gly(OCH2O)-
p-nitro-phenyl-carbonate was performed as described in the
literature.14 All the organic layers were dried on Na2SO4.
Chromatography was executed on silica gel (Merck 60, 70−230
mesh). 1H and 13C NMR spectra were registered in ppm (δ) by
Varian VXR-300 spectrometer with Me4Si as internal standard.
Mass analysis was carried out using an LCQ ion trap mass
spectrometer (Thermo Finnigan, San Jose, CA, USA) equipped
with an ESI source. The injector temperature was 300 °C, and the
spray voltage was 4.25 kV. N2 was used as sheath and auxiliary
gas.
H-Cys(Allyl)-OBut (6). A suspension of H-Cys-(Allyl)−OH
(4.16 g, 25.8 mmol) in AcOBut (375 mL) was slowly added to
HClO4 (7.74 mL) at 20 °C. The resulting solution was
maintained under stirring for 4 h, then neutralized with solid
NaHCO3, and extracted with AcOEt. After evaporation under
vacuum, the crude was chromatographed using CHCl3:MeOH
(97:3) to afford the tert-butyl ester 6 (4.5 g). Yield: 80%. Rf =
1
0.65, CHCl3:MeOH (97:3). H NMR (CDCl3) δ: 1.46 (9H, s,
OBut), 2.36 (2H, s, Cys NH2), 2.64−2.88 (2H, m, Cys β-CH2),
3.14−3.16 (2H, m, Cys S-CH2-CHCH2), 3.53 (1H, m, Cys α-
CH), 5.09−5.15 (2H, m, Cys S-CH2-CH-CH2), 5.75 (1H, m,
Cys S-CH2-CH-CH2). 13C NMR (CDCl3) δ: 28.24 (OBut),
35.35 (Cys β-CH2), 35.82 (S-CH2), 54.71 (Cys α-CH), 82.03
(OBut), 117.80 (S-CH2-CH-CH2), 134.19 (S-CH2-CH-CH2).
Boc-Glu[Cys(Allyl)-OBut]-OMe (7). Boc-Glu-OMe (2.55 g,
9.77 mmol) was treated with dry DMF (147 mL), isobutyl
chloroformate (1.28 mL, 9.77 mmol), and TEA (1.36 mL, 9.77
mmol) under stirring for 20 min at −20 °C. H-Cys(Allyl)-OBut
(4.26 g, 19.54 mmol) was added to the reaction flask and left for 3
h at 0 °C, then 19 h at 20 °C. After filtration, the solution was
extracted with CHCl3/KHSO4 1 N, NaHCO3ss, NaClss, and then
evaporated. The crude was purified with CHCl3 providing
Cyclo-{Gly(OCH2O)-Glu[Cys(Allyl)]-OMe} (CP11). The tri-
peptide 9 (2.26 g, 3.82 mmol) was treated with TFA (63 mL,
821.3 mmol) for 5 h at 20 °C. After evaporation, the treatment
with ether provided the fully deprotected tripeptide 10 (2.1 g)
(quantitative yield).
Compound 10 (1.8 g, 3.37 mmol) and NMM (3.92 mL, 35.7
mmol) in CH2Cl2 (800 mL) were added over 2 h to an ice-cold
mixture of bis(2-oxo-3-oxazolidinyl)phosphonic chloride (4.29
g, 16.85 mmol) and 4-(dimethylamino)pyridine (410 mg, 3.37
mmol) in CH2Cl2 (600 mL). The reaction mixture was left for 72
h at 20 °C. After evaporation, the residue was treated with
CH2Cl2 and washed with 5% citric acid (×2), H2O (×1),
NaHCO3ss (×2), H2O (×1), and NaClss (×1), and the organic
layers were evaporated. After RP-chromatographic purification
on silica gel with gradient scale of CH3CN:H2O (from 5:95 to
40:60) as eluant, the final product CP11 (300 mg) was achieved.
Yield: 20%. Rf = 0.7, CHCl3:MeOH (9:1). 1H NMR (CDCl3) δ:
1.93−2.21 (2H, m, Glu β-CH2), 2.27−2.35 (2H, m, Glu γ-CH2),
2.61−2.99 (2H, m, Cys β-CH2), 3.16−3.19 (2H, m, Cys S-CH2-
CH-CH2), 3.70 (3H, s, OMe), 3.92 (2H, d, Gly α-CH2), 4.40−
1
dipeptide 7 (4.5 g). Yield: 99%. Rf = 0.22, CHCl3. H NMR
(CDCl3) δ: 1.41 (9H, s, OBut), 1.45 (9H, s, Boc), 1.88−2.19
(2H, m, Glu β-CH2), 2.21−2.35 (2H, m, Glu γ-CH2), 2.75−2.97
(2H, m, Cys β-CH2), 3.11−3.14 (2H, m, Cys S-CH2-CH-CH2),
3.72 (3H, s, OMe), 4.32−4.36 (1H, m, Cys α-CH), 4.61−4.68
(1H, m, Glu α-CH), 5.08−5.14 (2H, m, Cys S-CH2-CH-CH2),
5.31 (1H, d, Cys NH), 5.67−5.81 (1H, m, Cys S-CH2-CH-CH2),
6.53 (1H, d, Glu NH). 13C NMR (CDCl3) δ: 28.20 (OBut),
28.53 (Boc), 32.39 (Glu β-CH2), 33.11 (Cys β-CH2), 35.36 (S-
CH2), 52.51 (Glu γ-CH2), 52.71 (Cys α-CH), 53.09 (Glu α-
B
dx.doi.org/10.1021/mp500431r | Mol. Pharmaceutics XXXX, XXX, XXX−XXX