2050 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Brief Articles
preparative thin-layer chromatography (PTLC) on 20 × 20 cm
silica gel GF uniplates (Analtech 0.25-1.0 mm thickness), by
silica gel column (E. Merck 230-400 mesh), and/or by reverse-
phase high-performance liquid chromatography (HPLC) using
Whatman Partisil 10 ODS (20.250 mm) using the same
gradient described above. Final deprotected products were
lyophilized from H2O and often retained as a partial solvate.
Melting points were taken on a Thomas-Hoover capillary
melting point apparatus and are uncorrected. IR spectra were
obtained on a Perkin-Elmer model 21 spectrophotometer, while
UV spectra were recorded on Perkin-Elmer 402 spectropho-
tometer. Proton NMR spectra were obtained on Varian VXR-
300 spectrometers. Coupling constants (J ) are reported in
hertz.
Mass spectra were recorded on a Kratos Ms-50 TA instru-
ment. The FAB (fast atom bombardment) ionization was
obtained with a FAB saddle field source operated with xenon
atoms at 7 kV. Elemental analyses were performed by Guelph
Chemical Laboratories Ltd., Ontario, Canada and are reported
in percent. Amino acid analyses were carried out on a Varian
5500 analyzer by BioChem ImmunoSystems after hydrolysis
with 6 M HCl, containing 0.1% phenol, at 110 °C for 20 h.
Gen er a l P r oced u r e for th e P r ep a r a tion of An ilid e 3.
Cou p lin g of r-N-CBZ-L-Am in o Acid s w ith 4-F lu or o-1,2-
p h en ylen ed ia m in e. To a mixture of R-N-CBZ-L-amino acid
(11 mmol), 4-fluoro-1,2-phenylenediamine (0.52 mmol), and
TEA (1.5 mL; 11 mmol) in CH2Cl2 (50 mL) was added EDC‚
HCl at 0 °C. After the mixture was stirred at this temperature
for 2 h and then at room temperature overnight, 5% HCl (25
mL) was added, and the mixture was washed with brine, 5%
sodium bicarbonate, and brine and dried over Na2SO4. Evapo-
ration of the solvent and crystallization of the residue from
CH2Cl2/hexanes afforded pure 3.
night at room temperature. Insolubles were removed, and the
solution was washed successively with 5% HCl (10 mL) and
brine (10 mL). The chloroform layer was dried over anhydrous
MgSO4 and chromatographed on silica gel.
N-BOC-Glycin yl-glycin e Ben zyl Ester 9: yield (75%); Rf
1
) 0.30 (EtOAc/hexanes 1:1); H NMR (CDCl3) δ 1.45 (s, 9H),
3.84 (d, 2H, J ) 5.77), 4.09 (d, 2H, J ) 5.83), 5.19 (s, 2H), 5.20
(bs, 1H), 6.80 (bs, 1H), 7.41 (s, 5H).
N-BOC-L-Tyr osin yl-th ioglycin yl-glycin e Ben zyl Ester
7: yield (62%); Rf ) 0.35 (EtOAc/hexanes 2:1); 1H NMR
(CDCl3) δ 1.43 (s, 9H), 2.98 (m, 2H), 4.30-4.56 (m, 5H), 5.19
(s, 2H), 5.38 (d, 1H), 6.70 (d, 2H, J ) 5.4), 6.92 (d, 2H, J )
5.5), 7.35 (m, 6H), 8.91 (bs, 1H).
N-BOC-L-Tyr osin yl-glycin yl-glycin e Ben zyl Ester 12:
1
yield (65%); Rf ) 0.15 (EtOAc/hexanes 2:1); H NMR (CDCl3)
δ 1.38 (s, 9H), 2.90 (m, 2H), 4.01 (t, 2H), 4.32 (t, 2H), 4.40 (m,
1H), 5.20 (s, 2H), 6.77 (d, 2H, J ) 5.3), 7.01(d, 2H, J ) 5.5),
7.38 (s, 5H).
Cou p lin g of Th ioa cyla tin g Rea gen ts w ith Am in o Acid
or P ep tid es. F or m a tion of Th iop ep tid es 6 a n d 10. Free
amino acid benzyl ester (1 mmol) was dissolved in dry DMF
(0.5 mL) at 0 °C under a flow of argon. To this solution,
thioacylating agent (1.1 mmol) was added in two portions over
a period of 10 min. The resulting mixture was stirred at this
temperature for 2 h and then at room temperature overnight.
Insolubles were removed by filtration, and the solvent was
evaporated under reduced pressure. The remaining residue
was dissolved in EtOAc (15 mL) and washed successively with
5% NaHCO3 (20 mL), brine (20 mL), citric acid (20 mL), and
brine (20 mL). The organic layer was dried over anhydrous
MgSO4, evaporated, and chromatographed on silica gel.
N-BOC-Th ioglycin yl-glycin e Ben zyl Ester 6: yield (70%);
Rf ) 0.45(EtOAc/hexanes 1:1); 1H NMR (CDCl3) δ 1.48 (s, 9H),
4.22 (d, 2H, J ) 5.80), 4.46 (d, 2H, J ) 5.00), 5.22 (bs, 1H),
5.25 (s, 2H), 7.40 (s, 5H), 8.62 (bs, 1H).
r-N-CBZ-L-Ala n in e-2-a m in o-5-flu or oa n ilid e (3a ): yield
(94%); Rf ) 0.50 (EtOAc/hexanes 1:1); 1H NMR (CDCl3) δ 1.42
(d, 3H, J ) 6.6), 3.5 (bs, 2H), 4.39 (m, 1H), 5.02 (s, 2H), 5.52
(d, 1H, J ) 4.5), 6.40 (m, 2H), 7.00 (m, 1H), 7.30 (m, 5H), 7.82
N-BOC-O-ben zyl-L-th iotyr osin yl-glycin yl-glycin e Ben -
1
(s, 1H), LRMS(FAB) m/z ) 332 (MH+); HRMS calcd for C17H19
-
zyl Ester 10: yield (61%); Rf ) 0.3 (EtOAc/hexanes 2:1); H
FN3O3 (MH+) 332.1415, found 332.1403.
NMR (CDCl3) δ 1.42 (s, 9H), 3.10 (m, 2H), 4.01 (m, 2H), 4.30
(m, 2H), 4.60 (m, 1H), 5.20 (s, 2H), 5.25 (s, 2H), 6.90 (d, 2H, J
) 5.4), 7.10 (d, 2H, J ) 5.4), 7.25-7.45 (m, 10H), 8.30 (bs,-
1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of Th ioa n il-
id es 4. Under a flow of argon, phosphorus pentasulfide (1.70
mmol) was mixed with sodium carbonate (1.70 mmol) in dry
THF (100 mL). The mixture was stirred for 1 h at room
temperature and then cooled to 0 °C. To this clear solution
was added anilide 3 (1.80 mmol), and the reaction was kept
at this temperature overnight. To work up, an aqueous solution
of sodium tribasic phosphate was added (12%, 7 mL) followed
by EtOAc (20 mL) and heptane (20 mL). The organic layer
was separated and washed with brine (2 × 30 mL). Solvent
was then evaporated, and the residue was purified on silica
gel. This gave pure 4 as a pale yellow solid.
Gen er a l P r oced u r e for Rem ova l of P r otectin g Gr ou p s
of 7, 10, a n d 12 a n d HP LC An a lysis. The fully protected
peptides (1 mmol) were deblocked with 90% HF (5 mL) in the
presence of anisole, ethylmethyl sulfide, and thioanisole (1:
1:1 v/v, 0.8 mL) in an ice bath for 1 h. The excess HF was
removed by evaporation in vacuo at 0 °C, the residue was
treated with 10% AcOH (3 mL), washed with ether (35 mL),
and then dissolved in water (20 mL). The resulting solution
was then lyophilized and purified by HPLC to give the
expected product. The HPLC analysis revealed that com-
pounds 8 and 11 were >99% pure. The mobile phase was 10-
50% MeOH/NH4OAc 0.01 M, pH 6.0; pressure was 2000 psi;
and flow rate was 1 mL/min. For 8: tR ) 26.59 min; for 11: tR
) 28.80 min.
r-N-CBZ-L-Ala n in e-2-a m in o-5-flu or oth ioa n ilid e (4a ):
1
yield (65%); Rf ) 0.60 (EtOAc/hexanes 1:1); H NMR (CDCl3)
δ 1.54 (d, 3H, J ) 6.4), 4.01 (bs, 2H), 4.65 (m, 1H), 5.05 (S,
2H), 5.65 (d, 1H, J ) 6.8), 6.46 (m, 2H), 7.05 (m, 1H), 7.33 (m,
5H), 9.56 (bs, 1H); LRMS(FAB) m/z ) 348 (MH+); HRMS calcd
for C17H19FN3O2S (MH+) 348.1182, found 348.1200.
L-Tyr osin yl-th ioglycin yl-glycin e 8: yield (63%); Rf ) 0.35
(EtOH/NH4OH/H2O 8:1:1); UV λmax (50% aqueous ethanol) 267
nm; amino acid analysis Gly (1.92), Tyr (1.00); 1H NMR
(DMSO) δ 2.50 (m, 1H), 2.86 (m, 1H), 3.38 (m, 1H), 4.11 (d,
2H, J ) 2.8), 4.34 (s, 2H), 6.67 (d, 2H, J ) 8.36), 7.01 (d, 2H,
J ) 8.52), 8.40 (bs, 1H); 13C NMR (DMSO) δ 39.94, 48.75,
49.86, 55.66, 115.56, 127.19, 130.66, 156.49, 169.71, 172.40,
197.59; LRMS(FAB) m/z ) 312 (MH+); HRMS calcd for
Gen er a l P r oced u r e for th e P r ep a r a tion of Ben zim i-
d a zolon e 5. A solution of phosgene in toluene (1.7 mmol) was
added to thioanilide 4 (1.8 mmol) in dry THF. The reaction
mixture was stirred for 4 h at 25 °C. Insolubles were removed
by filtration, and the solvent was evaporated under reduced
pressure. The residue was chromatographed on silica gel. This
gave pure 5 as a colorless solid.
C
13H18N3O4S(MH+) 312.1018, found 312.0998.
1-(r-N-CBZ-L-Th ion oa la n in yl)-6-flu or oben zim id a zolo-
n e (5a ): yield (70%); Rf ) 0.40 (EtOAc/hexanes 1:1); 1H NMR
(CDCl3) δ 1.58 (d, 3H, J ) 6.8), 5.10 (S, 2H), 5.92 (d, 1H, J )
7.2), 6.35 (m, 2H), 6.58 (m, 1H), 7.40 (m, 5H), 8.61 (m, 1H),
10.00 (s, 1H), LRMS (FAB) m/z ) 374 (MH+); HRMS calcd for
L-Th iotyr osin yl-glycin yl-glycin e 11: yield (57%); Rf )
0.40 (EtOH/NH4OH/H2O 8:1:1); UV λmax(50% aqueous ethanol)
1
268 nm; amino acid analysis Gly (2.00), Tyr (0.96); H NMR
(DMSO) δ 2.84 (m, 1H), 2.99 (m, 1H), 3.80 (m, 2H), 4.11-4.33
(m, 3H), 6.69 (d, 2H, J ) 8.52), 7.05 (d, 2H, J ) 8.30), 8.41 (t,
1H, J ) 5.55), 9.32 (bs, 1H); 13CNMR (DMSO) δ 40.59, 41.00,
48.14, 59.73, 115.57, 125.70, 130.77, 156.79, 167.22, 171.28,
200.52; LRMS (FAB) m/z ) 312 (MH+); HRMS calcd for
C
18H17FN3O3S (MH+) 374.0975, found 374.0986.
Cou p lin g w ith th e Ad ja cen t Am in o Acid . Free amino
or thioamino acid benzyl ester (2 mmol) was dissolved in CHCl3
(10 mL) together with BOC amino acid (2.2 mmol). After the
addition of EEDQ (2.3 mmol), the solution was stirred over-
C
13H18N3O4S (MH+) 312.1018, found 312.1033.