Phosphopeptides, Inhibitors, and Prodrugs
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1231
(s, 9H, Boc), 2.75, 3.05 (dd, 2H, CH2â), 6.6, 6.85 (dd, 4H, H-Ar),
6.5 (s, 1H, NH), 9.15, (s, 1H, OH).
HOAT as described for the preparation of Boc-(R-Me)Tyr-Asn-
NH2. The crude product after evaporation of solvent was
purified by the semipreparative HPLC to give 29 mg of product
(yield, 3.6%). 1H NMR (DMSO-d6): δ 1.25 (s, 12H, RMe + Boc),
2.3 (s, 6H, 4 × CH3CO), 2.5-3 (m, 6H, 3 × CH2â), 3.1 (t, 4H
+ 2 × CH2S), 4.05-4.2 (m, 6H, 2CHR + 2 × CH2O), 6.85-7.3
(m, 13H, 8 × H-Ar + NH Tyr + 2 × CONH2), 7.8 (d, 1H, NH
Asn), 8.25 (s, 1H, + NH RMeTyr).
Boc-(r-Me)Tyr -Asn -NH2. Boc-(R-Me)Tyr-OH (2.5 g, 8.46
mmol) and HCl‚Asn-NH2 (1.70 g, 10.15 mmol) were dissolved
in 60 mL of DMF. The solution was adjusted to pH 9 by the
DIEA (about 5 mL). The HOAt (1.61 g, 11.85 mmol) and the
HATU (4.5 g, 11.85 mmol) were then added, and the mixture
was stirred at room temperature for 3 days before evaporation
to dryness. The residue was purified by chromatography with
CH2Cl2/MeOH/H2O/AcOH (7/3/0.6/0.3)/AcOEt, 1:1, as eluent.
An amount of 2.96 g of white solid was obtained with a yield
p Tyr (MeSATE 2)-(r-Me)p Tyr (MeSATE)2-Asn -NH2. This
product was obtained by the deprotection of Boc as described
for the preparation of (R-Me)pTyr(MeSATE)2-Asn-NH2 with
1
1
of 86%. H NMR (DMSO-d6): δ 1.1 (s, 3H, RMe), 1.35 (s, 9H,
85% yield. H NMR (DMSO-d6): δ 1.25 (s, 3H, RMe), 2.25 (s,
Boc), 2.3 (m, 2H, CH2âAsn), 2.75, 2.90 (dd, 2H, CH2âTyr), 4.5
(m, 1H, ChaAsn), 6.55, 6.85 (dd, 4H, H-Ar), 6.8-7.1 (m, 4H,
2CONH2), 7.3 (s, 1H, NHTyr), 8.0 (d, 1H, NHAsn), 9.15, (s,
1H, OH).
12H, 4 × CH3CO), 2.3-2.6 (m, 14H, 3 × CH2â + 4 × CH2S),
3.9-4.15 (m, 8H, 4 × CH2O), 4.2 (m, 2H, CHR), 7 (m, 8H,
H-Ar Tyr), 7.15 (d, 4H, 2 × CONH2), 7.3 (s, 1H, NH RMe Tyr),
+
8 (s, 3H, NH3 RMeTyr), 8.6 (d, 1H, NH Asn).
Boc-(r-Me)p Tyr (MeSATE)2-Asn -NH2. Boc-(RMe)Tyr-Asn-
NH2 (1.20 g, 2.93 mmol) was phosphorylated with bis(S-acetyl-
2-thioethyl)N,N-die´thylphosphoramidite as described.18 The
crude product was purified by chromatography with CH2Cl2/
MeOH/AcOH (100/5/1) as eluent. An amount of 2.34 g of white
solid was obtained with 69% yield. 1H NMR (DMSO-d6): δ 1.15
(s, 3H, RCH3), 1.41 (s, 9H, Boc), 2.3 (s, 6H, 2 × CH3CO), 2.4-
2.6 (m, 2H, CH2âAsn), 2.85, 3.1 (dd, 2H, CH2âTyr), 3.15 (t,
4H, 2 × CH2S), 4.15 (q, 4H, 2 × CH2O), 4.3 (m, 1H, CHRAsn),
6.8, 7.1 (ss, 4H, 2 × CONH2), 7.1 (q, 4H, H-Ar Tyr), 7.25 (s,
1H, NH-Tyr), 8 (d, 1H, NH Asn).
Boc-m AZ-p Tyr (MeSATE)2-(r-Me)p Tyr (MeSATE)2-Asn -
NH2. H3N+-pTyr(MeSATE)2-(RMe)pTyr(MeSATE)2-Asn-NH2
(1.85 g, 1.61 mmol) in DMF was adjusted to pH 9 by DIEA
addition. Boc-mAZ-ONp (0.69 g, 1.77 mmol) was added, and
the mixture was stirred at room temperature for 5 days. The
solvent was then evaporated and the residue was purified by
chromatography with CH2Cl2/MeOH/AcOH (100/5/1) as eluent
1
to give 1.88 g of product (yield, 91%). H NMR (DMSO-d6): δ
1.2 (s, 3H, RMe), 1.4 (s, 9H, Boc), 2.3 (s, 12H, 4 × CH3CO),
2.5-3 (m, 6H, 3 × CH2â), 3.1 (m, 8H, 4 × CH2S), 4-4.3 (m,
10H, 2CHR + 4 × CH2O), 4.8 (q, 2H, CH2φ), 6.85 (m, 4H, 2 ×
CONH2), 7-7.5 (m, 12H, H-Ar), 7.7 (d, 1H, NH Tyr), 7.9 (d,
1H, NH Asn), 8.3 (s, 1H, NH RMe Tyr), 9.3 (s, 1H, NH mAZ).
Boc-(r-Me)p Tyr (Bzl2)-Asn -NH2. Boc-(RMe)Tyr-Asn-NH2
(0.5 g, 1.22 mmol) was phosphorylated with dibenzyl N,N-
diethylphosphoramidite as described.51 The crude product was
purified by chromatography with CH2Cl2/MeOH/AcOH (100/
5/1) as eluent. An amount of 0.49 g of white solid was obtained
with 60% yield. 1H NMR (DMSO-d6): δ 1.15 (s, 3H, RCH3), 1.40
(s, 9H, Boc), 2.3-2.6 (m, 2H, CH2âAsn), 2.93, 3.1 (dd, 2H,
CH2âTyr), 4.25 (m, 1H, CHRAsn), 5.1 (d, 4H, CH2 Bzl), 7.05
(q, 4H, H-Ar Tyr), 7.25 (m, 11H, H-Ar Bzl and NH Tyr), 7.3
(m, 4H, 2 × CONH2), 8 (d, 1H, NH Asn).
m AZ-p T y r (Me S AT E )2-(r-Me )p T y r (Me S AT E )2-As n -
NH2 (P 10). The product was obtained by the deprotection of
Boc-mAZ-pTyr(MeSATE)2-(R-Me)pTyr(MeSATE)2-Asn-NH2 (1.87
g, 1.45 mmol) as described for the preparation of (R-Me)pTyr-
(MeSATE2)-Asn-NH2. The final product was purified by semi-
preparative HPLC to give 1.15 g of product (yield, 61%). It
has the same characteristics as those observed in the previous
synthesis (purity in HPLC, 98%).
m AZ-p Tyr (MeSATE)2-(r-Me)p Tyr -Asn -NH2 (P 11). The
deprotection of Boc-pTyr(MeSATE)2-(R-Me)pTyr-Asn-NH2 was
performed as described for the preparation of (R-Me)pTyr-
(MeSATE)2-Asn-NH2. The product obtained (22 mg, 0.023
mmol) was coupled with Boc-mAZ-ONp (18 mg, 0.046 mmol).
The crude product Boc-mAZ-pTyr(MeSATE)2-(R-Me)pTyr-Asn-
NH2, obtained after evaporation of the solvent, was submitted
to deprotection of the Boc group. The final product was purified
by semipreparative HPLC to give 6 mg of final product (yield,
26%). MS, m/z: 985.0 for 984.23 calculated. tR ) 11.0 min (0-
80% of solvent B in 30 min, purity 98%).
(r-Me)p Tyr (MeSATE)2-Asn -NH2. A cold solution of TFA/
CH2Cl2 (1/1, 15 mL) was added to Boc-(R-Me)pTyr(MeSATE)2-
Asn-NH2 (2.24 g, 3.23 mmol). The mixture was stirred at 0 °C
for 1 h. Heptane (50 mL) was added, and the solution was
evaporated to dryness. The residue was washed with ether to
give a white solid (2.48 g, quantitative yield). 1H NMR (DMSO-
d6): δ 1.15 (s, 3H, RCH3), 2.25 (s, 6H, 2 × CH3CO), 2.35-2.6
(m, 2H, CH2âAsn), 2.95, 3.2 (dd, 2H, CH2âTyr), 3.15 (t, 4H, 2
× CH2S), 4.1 (q, 4H, 2 × CH2O), 4.6 (m, 1H, CHRAsn), 7.1 (d,
+
4H, H-Ar Tyr), 7.3 (m, 4H, 2 × CONH2), 7.9 (s, 3H, NH3
-
Tyr), 8.5 (d, 1H, NH Asn).
(r-Me)p Tyr -Asn -NH2. This product was obtained by treat-
ing Boc-(R-Me)pTyr(Bzl2)-Asn-NH2 (0.48 g, 0.72 mmol) with
TFA/CH2Cl2 (1/1) for 3 h at room temperature. The residue
obtained from evaporation was washed with ether and col-
lected by centrifugation (0.34 g, yield, 93%). 1H NMR (DMSO-
d6): δ 1.4(s, 3H, RCH3), 2.4 (m, 2H, CH2âAsn), 2.85, 3.1 (dd,
2H, CH2âTyr), 4.5 (m, 1H, CHRAsn), 7.05, 7.2 (dd, 4H, H-Ar
Tyr), 7.3 (m, 4H, 2 × CONH2), 8.0 (s, 3H, NH3+Tyr), 8.6 (d,
1H, NH Asn).
B o c -p T y r (Me S AT E 2 )-(r-Me )p T y r (Me S AT E )2 -As n -
NH2. Boc-pTyr(MeSATE)2-OH (2.11 g, 3.73 mmol) and (R-Me)-
pTyr(MeSATE)2-Asn-NH2 (2.40 g, 3.39 mmol) were coupled
with 1.1 equiv of HATU/HOAT as described for the preparation
of Boc-(R-Me)Tyr-Asn-NH2. The crude product after evapora-
tion was taken up in AcOEt, washed with 10% NaHCO3, 10%
citric acid, and brine and dried over Na2SO4. The residue
obtained after evaporation of solvent was purified by chroma-
tography with CH2Cl2/MeOH/AcOH (100/5/1) as eluent to give
2.55 g of product (yield 60%). 1H NMR (DMSO-d6): δ 1.2 (s,
12H, RMe + Boc), 2.3 (s, 12H, 4 × CH3CO), 2.5-3 (m, 14H, 3
× CH2â + 4 × CH2S), 4.1 (m, 10H, 2CHR + 4 × CH2O), 6.85
(m, 4H, 2 × CONH2), 7.05 (m, 8H, H-Ar), 7.3 (d, 2H, NH Tyr
+ NH RMe Tyr), 7.9 (d, 1H, NH Asn).
F m oc-Asp (tBu )-NH-(CH2)3-(1-n a p h th yl). Fmoc-Asp(tBu)-
OH (1.11 g, 2.7 mmol) and 3-(1-naphthyl)-1-propylamine21
(0.50 g, 2.7 mmol) were dissolved in 10 mL of DMF. BOP (1.2
g, 2.7 mmol) and then DIEA (0.95 mL, 5.4 mmol) were added,
and the solution was stirred at room temperature overnight.
DMF was then evaporated, and the residue was taken in ethyl
acetate. The solution was washed with 1 M KHSO4, saturated
NaHCO3, water, and brine before drying with anhydrous Na2-
SO4. After filtration, the filtrate was evaporated to dryness
and the residue was purified by chromatography with EtOAc/
c-hexane as eluent to give 1.3 g of white powder (yield, 83%).
1H NMR (CDCl3): δ 1.3 (s, 9H, tBu), 1.85 (m, 2H, 2-CH2 of
propyl), 2.55 and 2.85 (dd, 2H, CH2â of Asp), 3.0 (t, 2H, CH2
of naphthyl), 3.3 (m, 2H, CH2-N), 4.15 (t, 1H, CHR of Asp),
4.4 (m, 3H, 9-H and CH2 of Fmoc), 5.85 (bs, 1H, NH-propyl),
6.45 (bs, 1H, NH of Asp), 7.2-8.0 (m, 15H, H-Ar of Fmoc and
naphthyl).
F m oc-Asp -NH-(CH 2)3-(1-n a p h th yl). Fmoc-Asp(tBu)-NH-
(CH2)3-(1-naphthyl) (1.3 g, 2.25 mmol) was dissolved in 4 mL
of 50% TFA in CH2Cl2 with 1% anisol. The solution was stirred
for 15 min at 0 °C after 4 h at room temperature. The solvent
was then evaporated, and the residue was precipitated with a
solution of petroleum ether/diethyl ether (2/1). The precipitate
was collected by centrifugation and washed three times with
a mixture of petroleum ether/diethyl ether to give 1.1 g of white
powder (yield, 94%). 1H NMR (DMSO-d6): δ 1.70 (m, 2H,
Boc-p Tyr (MeSATE )2-(r-Me)p Tyr -Asn -NH 2. Boc-pTyr-
(MeSATE)2-OH (0.48 g, 0.85 mmol) and (R-Me)pTyr-Asn-NH2
(0.33 g, 0.85 mmol) were coupled with 1.1 equiv of HATU/