2406 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15
DeGraw et al.
a mixture of four compounds. Purification was achieved via
preparative HPLC with a gradient of 10-30% acetonitrile-
H2O-0.1% TFA, followed by rechromatography with an iso-
cratic elution by 17% acetonitrile-0.1% TFA and finally a
gradient elution with 12-25% acetonitrile-0.1% TFA. Evapo-
ration and lyophilization gave 23 mg of 3d , isomer A: FAB-
MS m/ e 677 (M + H+); 1H-NMR (D2O) δ 0.86 (d, 6H), 1.37 (m,
2H), 1.62-2.00 (m, 9H), 2.55 (m, 1H), 2.70 (m, 3H), 2.83 (m,
2H), 3.04 (m, 5H), 3.16 (m, 1H), 3.21 (m, 2H), 4.56 (m, 3H),
5.03 (m, 1H), 7.26-7.42 (m, 5H).
Ar g-Lys-Asp (k )-Ala -P h e (3e). Peptide 3e was similarly
prepared from the aspartyl ketomethylene alanine dipeptide
unit (17b) and the blocked Arg-Lys dipeptide (18) proceeding
through the resin-bound pentapeptide (22e). From 1.13 g of
18 there was obtained 396 mg of crude 3e. Preparative HPLC
using a 0-20% acetonitrile-H2O-0.1% TFA gradient afforded
64 mg of 3e (isomer A) and 79 mg of 3e (isomer B).
δ 212.11 (ketone). Anal. Calcd for C31H50N8O9‚2.4CF3COOH‚
3H2O: C, H (0.6), N, F.
Ar g-P r o-Asp (k )Va l-P h eNH2 (3g). Compound 17a (668
mg, 1.62 mmol) and N-hydroxysuccinimide (260 mg, 1.70
mmol) were dissolved in MeCl2 and cooled to 0 °C (ice bath).
DCC (351 mg, 1.70 mmol) was added, and the reaction was
stirred at 0 °C for 2 h and then refrigerated overnight. The
precipitated DCU was removed and the filtrate evaporated to
give the succinimide ester. The BOC group was removed from
BOC-Phe-methylbenzhydrylamine resin (2.5 g, 1.3 mmol at 0.5
mequiv/g) using the standard protocol, and the resin was
neutralized and washed. The succinimide ester was dissolved
in MeCl2 (10 mL) and added to the resin along with a catalytic
amount of HOBt (5 mg). The reaction was allowed to proceed
7 days with periodic monitoring using the Kaiser test. The
resin was then capped using acetic anhydride (1 mL) and
pyridine (0.2 mL) in MeCl2 (10 mL). The BOC was removed
as usual, leaving the resin as the TFA salt. Meanwhile, Z3-
L-Arg-L-Pro (20, 1.05 g, 1.56 mmol) and HOBt (250 mg, 1.64
mmol) were dissolved in MeCl2 and cooled to 0 °C, after which
DCC (350 mg, 1.70 mmol) was added. The resin was neutral-
ized (2×) with 5% diisopropylethylamine and washed with
MeCl2 (3×) just before the activated dipeptide was added. The
vessel was then shaken overnight. The reaction was deter-
mined to be complete (negative Kaiser test), and the resin was
washed and dried to give 2.81 g. HF removal of peptide from
resin yielded 352 mg of crude peptide. The product was
partially purified by preparative HPLC using a 10-25%
gradient of acetonitrile in water containing 0.1% TFA. Further
preparative HPLC purification was performed in an isocratic
system using 22% acetonitrile in water containing 0.1% TFA.
Fractions containing isomer A were pooled, evaporated of
acetonitrile, frozen, and lyophilized, yield 42 mg (98% pure)
of 3g (isomer A). Isomer B appeared unstable, giving rise to
multiple peaks in the analytical HPLC.
Isom er A: FABS-MS m/ e 635 (M + H+); 1H-NMR (D2O) δ
1.06 (d, 3H), 1.44 (m, 2H), 1.58-1.76 (m, 4H), 1.82 (m, 2H),
1.93 (m, 2H), 2.59 (dd, 1H), 2.70 (dd, 1H), 2.76 (dd, 1H), 2.81
(m, 2H), 2.91 (dd, 1H), 2.99 (t, 2H), 3.04 (dd, 1H), 3.21 (t, 2H),
3.23 (dd, 1H), 4.07 (t, 1H), 4.37 (t, 1H), 4.54 (dd, 1H), 4.64
(dd, 1H), 7.28-7.42 (m, 5H); 13C-NMR (D2O) δ 208.85 (ketone).
Anal. Calcd for C29H46N8O8‚3CF3COOH‚0.5H2O: C, H, N, F.
Isom er B: FABS-MS m/ e 635 (M + H+); 1H-NMR (D2O) δ
0.90 (d, 3H), 1.43 (m, 2H), 1.63 (m, 2H), 1.69 (m, 2H), 1.79 (m,
2H), 1.92 (m, 2H), 2.61 (dd, 1H), 2.73-2.87 (m, 3H), 2.91-
3.20 (m, 4H), 3.20 (t, 2H), 3.25 (dd, 1H), 4.05 (t, 1H), 4.36 (t,
1H), 4.64 (m, 2H), 7.25-7.40 (m, 5H); 13C-NMR (D2O) δ 208.95
(ketone).
Ar g-Lys-Asp (k )Va l-Tyr (3f). Compound 17a (566 mg, 1.38
mmol), HOBt (217 mg, 1.41 mmol), BOP (617 mg, 1.40 mmol),
and N-methylmorpholine (467 µL, 4.27 mmol) were combined
in MeCl2 (10 mL) and stirred at room temperature for 30 min.
Meanwhile, the BOC group was removed from BOC-L-Tyr-
(OCBZ)-O-resin (2.59 g, 1.50 mmol at 0.58 mequiv/g) with 40%
TFA/10% anisole in the usual manner, and the resin was
washed with MeCl2 (3×), neutralized with 10% diisopropyl-
ethylamine in MeCl2 (2×), and washed with MeCl2 (3×). The
activated ketomethylene was added to the resin and shaken
for 7 days. The untreated amines were capped using acetic
anhydride (1.0 mL) and pyridine (0.1 mL) in MeCl2 (9 mL).
The BOC group was removed from the Asp portion as usual,
leaving the resin-bound material as the TFA salt. Meanwhile,
Z3-L-Arg-Nꢀ-Z-L-Lys (18, 1.26 g, 1.50 mmol) and HOBt (230 mg,
1.50 mmol) were dissolved in MeCl2 and cooled to 0 °C, after
which DCC (340 mg, 1.65 mmol) was added. The resin was
neutralized (2×) with 5% diisopropylethylamine and washed
with MeCl2 (3×) just before the activated dipeptide was added.
The vessel was then shaken overnight. The reaction was
determined to be complete (negative Kaiser test), and the resin
was washed and dried to give 3.43 g of peptide-resin. HF
removal of peptide from resin yielded 588 mg of crude peptide.
The products were separated and partially purified by pre-
parative HPLC using a 0-17% gradient of acetonitrile in water
containing 0.1% TFA. Isomer A was further purified by
preparative HPLC using a 3-12% gradient of acetonitrile in
water containing 0.1% TFA. Fractions were pooled, evapo-
rated of acetonitrile, frozen, and lyophilized; yield, 92 mg (99%
pure) of 3f (isomer A). Isomer B was further purified by
preparative HPLC using a 7-17% gradient of acetonitrile in
water containing 0.1% TFA to yield 31 mg (98% pure) of 3f
(isomer B).
1
Isom er A: FAB-MS m/ e 631 (M + H+); H-NMR (D2O) δ
0.43 (d, 3H), 0.58 (d, 3H), 1.45 (m, 1H), 1.68 (m, 2H), 1.91 (m,
3H), 2.00 (m, 2H), 2.33 (m, 2H), 2.72 (m, 1H), 2.79-2.91 (m,
4H), 3.19 (t, 2H), 3.00 (dd, 1H), 3.57 (m, 1H), 3.72 (m, 1H),
4.36 (t, 3H), 4.48 (dd, 1H), 4.59 (dd, 1H), 4.63 (t, 1H), 7.22-
7.34 (m, 5H); 13C-NMR (D2O) δ 210.94 (ketone). Anal. Calcd
for C30H45N8O7‚1.8CF3COOH‚2.5H2O: C, H, N, F.
Ack n ow led gm en t. This work was sponsored by
J apan Tobacco Co., Ltd. We thank Dr. D. Thomas for
mass spectrometric analyses and Mr. G. Detre for
certain NMR analyses.
Refer en ces
(1) DeGraw, J . I.; Almquist, R. G.; Hiebert, C. K.; Colwell, W. T.;
Crase, J .; Hayano, T.; J udd, A. K.; Dousman, L.; Smith, R. L.;
Waud, W. R.; Uchida, I. Stabilized analogs of thymopentin. 1.
4,5-Ketomethylene pseudopeptides. J . Med. Chem. 1997, 40,
2386-2397.
(2) Tishio, T. P.; Patrick, J . E.; Weintraub, H. S.; Chasin, M.;
Goldstein, G. Short in vitro half-life of thymopentin pentapeptide
in human plasma. Int. J . Pept. Protein Res. 1983, 14, 479-484.
(3) Sisto, A.; Mariotti, S.; Groggia, A.; Marcozzi, G.; Villa, L.;
Neucioni, L.; Silvestri, S.; Verdini, A.; Pessi, A. Biologically active
retro-inverso analogs of thypentin. Pept.: Struct., Biol. Proc. Am.
Pept. Symp., 11th 1989, 772-773.
(4) Garcia-Lopez, M. T.; Gonzalez-Muniz, R.; Harto, J . R. Synthesis
of ketomethylene dipeptides containing basic amino-acid analogs
at C-terminus. Tetrahedron 1988, 44, 5131-5138.
Isom er A: FABS-MS m/ e 679 (M + H+); 1H-NMR (D2O) δ
0.81 (d, 3H), 0.82 (d, 3H), 1.42 (m, 2H), 1.52-1.82 (m, 7H),
1.89 (q, 2H), 2.49 (m, 1H), 2.60 (dd, 1H), 2.67 (dd, 1H), 2.78-
2.97 (m, 5H), 3.07 (dd, 1H), 3.17 (m, 2H), 4.03 (t, 1H), 4.34
(dd, 1H), 4.47 (dd, 1H), 4.54 (dd, 1H), 6.80 (m, 2H), 7.12 (m,
2H); 13C-NMR (D2O) 209.38 (ketone). Anal. Calcd for C31H50N8-
O9‚2.6CF3COOH‚3H2O: C, H, N, F.
(5) Garcia-Lopez, M. T.; Gonzalez-Muniz, R.; Harto, J . R. A simple
and versatile route to ketomethylene dipeptide analogs. Tetra-
hedron Lett. 1988, 29, 1577-1580.
(6) Umezawa, H.; Nakamura, T.; Fukatsu, S.; Aoyagi, T.; Tatsuta,
K. Synthesis of arphamenine-A and epi-arphamenine-A. J .
Antibiot. 1983, 36, 1787-1788.
(7) Aplin, R. T.; Christiansen, J .; Young, G. T. Amino-acids and
peptides. 48. Synthesis of bradykinyl-chloromethane. Int. J . Pept.
Protein Res. 1983, 21, 555-561.
Isom er B: FABS-MS m/ e 679 (M + H+); 1H-NMR (D2O) δ
0.56 (d, 3H), 0.64 (d, 3H), 0.79-0.92 (m, 1H), 1.41 (m, 2H),
1.53-1.71 (m, 5H), 1.78 (m, 2H), 1.90 (q, 2H), 2.52 (m, 1H),
2.62 (dd, 1H), 2.79 (dd, 1H), 2.81-2.87 (m, 2H), 2.89 (dd, 1H),
2.96 (t, 2H), 3.18 (m, 3H), 4.02 (t, 1H), 4.34 (t, 1H), 4.57 (dd,
1H), 4.66 (dd, 1H), 6.80 (m, 2H), 7.14 (m, 2H); 13C-NMR (D2O)
(8) Grieco, P. A.; Bahsas, A. Immonium ion based synthetic meth-
odology. A novel method for N-methylation of dipeptides and
amino acid derivatives via retro aza Diels-Alder reactions. J .
Org. Chem. 1987, 52, 5465-5749.
J M9508043