A. Mollica et al. / Fitoterapia 98 (2014) 91–97
95
layer was dried over Na2SO4, filtered and dried under vacuum.
The crude residue was treated with Et2O to give the acylated
products (3), (6), and (14).
Product (5) was obtained in 56% of yield following the
general DKP preparation procedure. Rf = 0.76 (EtOAc/EtOH/
AcOH/H2O 7:1:1:1). 1H NMR (DMSO-d6) δ: 2.85 (2H, m, βCH2–
Tyr); 3.84 (2H, m, βCH2–Ser); 3.96 (2H, m, αCH Tyr); 4.85 (1H,
m, αCH Ser); 6.65 and 6.90 (4H, dd, aromatics); 7.83 and 7.95
(2H, br, NH–Tyr and NH–Ser); 8.10 (1H, t, OH Ser); 9.18 (1H, s,
OH–Tyr).
2.2.5. Compound 1 (Boc–Tyr–Gly–OMe)
The title Boc protected dipeptide was prepared following
the general coupling procedure described above starting from
Boc–Tyr–OH (0.986 g, 3.50 mmol). Usual work up gave a crude
residue which was purified by treatment with Et2O/petroleum
ether, to afford pure Boc–Tyr–Gly–OMe (0.860 g, 3.18 mmol,
76.6% yield) as oil. 1H NMR (CDCl3) δ: 1.34 (9H, s, Boc); 3.00
(2H, m, βCH2–Tyr); 3.73 (3H, s, OMe); 3.95 (2H, m, CH2–Gly);
4.34 (1H, m, αCH–Tyr); 5.10 (1H, br, NH–Tyr); 6.48 (1H, t, NH–
Gly); 6.71–7.04 (4H, dd, aromatics). Rf = 0.6 (CH2Cl2/EtOAc
1:1).
2.2.10. Compound 6 (c[Tyr(O-Dal)–Ser])
DKP (5) (0.05 g, 0.2 mmol) was alkylated in DMF (5 mL),
K2CO3 (138 mg) and 3,3-dimethylallyl-bromide (0.03 mL) by
following the general O-alkylation procedure described above.
After the work-up the crude residue was purified by silica gel
chromatography, using CHCl3/MeOH 95:5 as eluent to give the
pure product (6) in 35% of yield. Rf = 0.45 (CHCl3/MeOH 95:5).
1H NMR (DMSO-d6) δ: 1.66 and 1.71 (6H 2d, CH3 Dal); 2.90
(4H, m, βCH2 Tyr and CH2 Gly); 3.62 and 3.90 (2H, m, αCH Tyr
and αCH–Ser); 4.45 (2H, d, O-CH2 Dal); 4.95 (1H, t, OH Ser);
5.39 (1H, t,_CH\CH2\O); 6.81 and 7.05 (4H, 2d, aromatics);
7.87 and 7.96 (2H, br, NH Tyr and NH Gly).
2.2.6. Compound 2 (c[Tyr–Gly])
Dipeptide (1) (0.434 g, 1.23 mmol) was treated with a
mixture of TFA/CH2Cl2 1:1 (5 mL) following the general
procedure described above to give the Nα-deprotected dipeptide
(quantitative yield) which was used in the next step without
further purification.
2.2.11. Compound 7 (Boc–Ala–Gly–OMe)
The title N-Boc protected dipeptide was prepared following
the general coupling procedure described above starting from
Boc–Ala–OH (0.497 g, 2.63 mmol). Usual work-up gave an oily
residue which was used for the next step without further
purification (quantitative yield). Rf = 0.38 (CH2Cl2/EtOAc 8:2).
1H NMR (CDCl3) δ: 1.37 (3H, d, CH3Ala); 1.44 (9H, s, Boc); 3.75
(3H, s, OCH3); 4.03 (2H, m, CH2Gly); 4.22 (1H, m, CH–Ala); 4.99
(1H, br, NH–Boc); 7.05 (1H, br, NH–Gly).
Product 2 was obtained in 43% of yield following the general
DKP preparation procedure.
Rf
=
0.70 (EtOAc/EtOH/AcOH/H2O 7:1:1:1). 1H NMR
(DMSO-d6) δ: 2.85 (2H, m, βCH2–Tyr); 3.25 (2H, m, CH2–
Gly); 3.94 (1H, m, αCH–Tyr); 6.62–6.85 (4H, dd, aromatics);
7.84 and 8.08 (2H, br, NH–Tyr and NH–Gly); 9.28 (1H, s, OH–
Tyr).
2.2.7. Compound 3 (c[Tyr(O-Dal)–Gly])
2.2.12. Compound 8 (c[Ala–Gly])
DKP 2 (0.108 g, 0.5 mmol) was alkylated in DMF (5 mL),
K2CO3 (0.339 mg) and 3,3-dimethylallyl-bromide (0.3 mL) by
following the general O-alkylation procedure described above.
After the work-up the crude residue was treated with Et2O to
give the pure product 3 in 46% of yield. Rf = 0.2 (CHCl3/MeOH
95:5). 1H NMR (DMSO-d6) δ: 1.66 and 1.71 (6H, 2d, 2 × CH3
Dal); 2.65–3.02 (4H, m, βCH2 Tyr and CH2 Gly); 4.00 (2H, d, α-
CH Tyr); 4.45 (2H, d, O-CH2 Dal); 5.39 (1H, t,_CH\CH2\O);
6.81 and 7.15 (4H, 2d, aromatics); 7.87 and 8.11 (2H, br, NH Tyr
and NH Gly).
Dipeptide (7) (0.622 g, 2.39 mmol) was treated with a
mixture of TFA/CH2Cl2 1:1 (5 mL) following the general
procedure described above to give the N-deprotected dipeptide
(quantitative yield) which was used in the next step without
further purification.
Product (8) was obtained in 77% of yield following the
general DKP preparation procedure. Rf = 0.55 (EtOAc/EtOH/
AcOH/H2O 7:1:1:1).
2.2.13. Compound 9 (N,N′-diacetyl-c[Ala–Gly])
A mixture of DKP (8) (0.224 g, 1.75 mmol) in acetic
anhydride (10 mL) was stirred under reflux for 7 h. The solvent
was removed by azeotropic distillation with methanol and
toluene under reduced pressure. The residue was crystallized
from EtOAc/Et2O to yield (9) (91.5% yield) as a brown oil. 1H
NMR (CDCl3) δ: 1.50 (3H, d, CH3 Ala); 2.52–2.54 (6H, 2 × s,
CH3CO); 4.02 and 5.10 (2H, dd, CH2–Gly); 5.20 (1H, m, αCH–
Ala).
2.2.8. Compound 4 (Boc–Tyr–Ser–OMe)
The title Nα–Boc protected dipeptide was prepared following
the general coupling procedure described above starting from
Boc–Tyr–OH (0.497 g, 1.76 mmol). Usual work-up gave an oily
residue which was purified by silica gel column chromatography
(CH2Cl2/EtOAc from 8:2 to 100% EtOAc), to afford pure Boc–Tyr–
Ser–OMe (0.550 g) 78% of yield as oil. Rf = 0.6 (CH2Cl2: EtOAc
1:1). 1H NMR (CDCl3) δ: 1.40 (9H, s, Boc); 2.98 (2H, m, βCH2–
Tyr); 3,73 (3H, s, OCH3); 3.88 (2H, m, βCH2–Ser); 4.27 (1H, m,
αCH–Tyr); 4.57 (1H, m, αCH–Ser); 5.18 (1H, br, NH–Tyr); 6.77
and 7.05 (4H, dd, aromatics); 6.83 (1H, t, NH–Ser).
2.2.14. Compound 10 (c[Δz–Tyr(O-Ger)-N-acetyl-Ala])
Compound (9) (0.054 g, 0.25 mmol) was dissolved in
DMF, then (14) (0.164 g, 0.64 mmol) and tBuOK 0.5 M in
tBuOH (1.2 eq.) were added at 0 °C. The mixture was allowed
to warm to r.t. and stirred for 6 h. Then the reaction was
quenched with aqueous NH4Cl solution and extracted three
times with EtOAc. The combined organic layers were dried
on Na2SO4, filtered and evaporated.
2.2.9. Compound 5 (c[Tyr–Ser])
Boc–Tyr–Ser–OMe (4) (0.434 g, 1.23 mmol) was treated
with a mixture of TFA/CH2Cl2 1:1 (5 mL) following the general
procedure described above to give the Nα-deprotected dipeptide
(quantitative yield) which was used in the next step without
further purification.
The obtained crude was purified on silica gel chromatogra-
phy, using CH2Cl2/EtOAc 9:1 as eluent (73% yield). Rf = 0.65