Stabilized Analogs of Thymopentin. 1
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2395
1a (isomer A): FAB-MS m/e 663 (M + H+). Anal. Calcd for
C31H50N8O8‚3CF3CO2H: C, H, N, F.
Compound 1a (isomer B) was prepared in 21% yield in a
similar manner from 19a -B and 20: FAB-MS m/e 663 (M +
H). Anal. Calcd for C31H50N8O8‚3.25CF3COOH‚H2O: C, H,
N, F.
NCHCO); 13C-NMR (D2O) 212.88 (ketone). Anal. Calcd for
C27H49N7O8‚2.2CF3COOH‚H2O: C, H, N, F.
N-Acetyl-Ar g-P r o-Asp -Va l(k )P h e (1j-A). Resin-bound
N-BOC pentapeptide (28b-A, from preparation of 1b-A, 994
mg) was treated with 40% TFA/10% anisole in MeCl2 (5 and
30 min) to remove the BOC group and the resin washed with
MeCl2 and 2-PrOH, followed by drying in vacuo. The dried
resin (994 mg, as TFA salt) was neutralized with 5% diiso-
propylethylamine in MeCl2 and thrice washed with MeCl2. The
resin was then treated with 2 mL of Ac2O and 0.25 mL of
pyridine in 8 mL of MeCl2 for 2 h. The resin was washed with
MeCl2 and dried to leave 960 mg of N-acetyl resin-bound
product (29j). The material was treated with dry HF-anisole
(9:1) for 1.5 h at 0 °C to cause cleavage from the resin and
removal of blocking groups. The mixture was evaporated in
vacuo and the peptide extracted into 10% CH3CN containing
0.5% TFA. Lyophilization afforded 231 mg of crude material,
which was purified by preparative HPLC (14-20% CH3CN
gradient containing 0.1% TFA to afford 62 mg (98% pure) and
75 mg (90% pure): FAB-MS m/e 674 (M + H). Anal. Calcd
for C32H47N7O9‚1.25CF3COOH‚H2O: C, H, N, F.
N-Acetyl-Ar g-Nle-Asp -Va l(k )P h e (1k -A). In a manner
similar to that described for 1j, the resin-bound intermediate
28c (from preparation of 1c-A) was treated with TFA in anisole
to remove the BOC group and the free amine was acetylated
with Ac2O. The resulting resin-bound blocked peptide (29k )
was treated with HF-anisole to provide crude 1k -A, which
was purified by HPLC: FAB-MS m/e 690 (M + H). Anal. Calcd
for C33H51N7O9‚CF3COOH‚H2O: C, H, N, F.
Tr is(b en zyloxyca r b on yl)-L-a r gin yl-N-m et h yln or leu -
cin e ter t-Bu tyl Ester (31). A mixture of 2.45 g (10 mmol) of
N-methylnorleucine, 1.5 mL of concentrated H2SO4, and 40
mL of dioxane was stirred for 15 min. Liquid isobutylene (30
mL) was added slowly to the mixture using a Dewar condensor
with dry ice. The mixture was stirred for 5 h, poured into ice-
cold 1 N NaOH (200 mL), and extracted with Et2O (3 × 100
mL). The combined Et2O extracts were washed with saturated
NaHCO3 (100 mL) and brine (2 × 100 mL), dried over MgSO4,
and evaporated to leave 1.05 g (52%) of N-Me-norleucine tert-
butyl ester (30). A solution of NR,Nδ,Nω-tris(benzyloxycarbo-
nyl)-L-arginine (2.88 g, 5.0 mmol) in MeCl2 (50 mL) was cooled
to -10 °C, and Et3N (0.68 mL, 5.0 mmol) and isobutyl
chloroformate (0.78 mL, 6.0 mmol) were added successively.
After the reaction mixture was stirred for 20 min, a solution
of 1.05 g of 30 in MeCl2 (20 mL) was added, followed by Et3N
(0.68 mL, 5.0 mmol). The reaction mixture was allowed to
come to room temperature and stirred for 4 h. The material
was evaporated to an oil and subjected to flash chromatogra-
phy using a step gradient of 10, 20, and 30% ethyl acetate in
hexane (500 mL each), yielding 2.56 g (33.7%) of 31: MS m/e
760 (M + H+); 1H-NMR (CDCl3) δ 0.90 (t, 3H, CH3), 1.01-
1.95 (m, 10H, CH2); 1.39 (s, 9H, t-Bu), 2.83 (s, 3H, NCH3), 4.00
(m, 2H, CH2N), 4.62 (m, 1H, Arg-CH-N), 5.08 (s, 2H, CH2O),
5.12 (s, 2H, CH2O), 5.20 (s, 2H, CH2O), 5.58 (d, 1H, NleCH-
N), 7.23-7.45 (m, 15H, Ar), 9.33 (s, 2H, NH). Anal. Calcd
for C41H53N5O9: C, H, N.
Compound 1a -A was also obtained by hydrogenation of 0.90
g of 18 (isomer A) over 0.80 g of Pd in HOAc containing 0.1 N
HCl for 24 h. Filtration of catalyst followed by evaporation of
solvent and washing with Et2O afforded 0.36 g (56%) of 1a as
the HCl salt, equal to 1a above by HPLC. Anal. Calcd for
C31H50N8O8‚3HCl‚2.5H2O: C, H, N, Cl. Other pseudopen-
tapeptides in this series (1b-h ) were obtained by the resin
method as described above for 1a . The reactants and analyti-
cal data are recorded below for these compounds which were
obtained in 17-22% yields.
Ar g-P r o-Asp -Va l(k )P h e. 1b-A from 19a -A and 21: FAB-
MS m/e 632 (M + H); partial NMR (D2O) δ 0.76 (d, 3H, CH3),
0.88 (d, 3H, CH3), 4.37 (m, 2H, NCHCO), 4.50 (dd, 1H,
NCHCO), 4.67 (dd, 1H, NHCHCO), 7.30 (m, 5H, C6H5); 13C-
NMR (D2O) 210.04 (ketone). Anal. Calcd for C30H45N7O8‚
2CF3COOH‚H2O: C, H (0.6), N, F (0.8).
1b-B from 19a -B and 21: FAB-MS m/e 632 (M + H); partial
NMR (D2O) δ 0.74 (d, 3H, CH3), 0.81 (d, 3H, CH3), 4.23 (d,
1H, NCHCO), 4.34 (t, 1H, NCHCO), 4.46 (dd, 1H, NCHCO),
4.63 (dd, 1H, NCHCO), 7.26 (m, 5H, C6H5); 13C-NMR (D2O)
210.68 (ketone). Anal. Calcd for C30H45N7O8‚2CF3COOH‚
H2O: C, H, N, F (0.5).
Ar g-Nle-Asp -Va l(k )P h e. 1c-A from 19a -A and 22; FAB-
MS m/e 648 (M + H); partial NMR (D2O) δ 0.74 (d, 3H, CH3),
0.83 (t, 3H, CH3), 0.86 (d, 3H, CH3), 4.01 (t, 1H, NCHCO), 4.29
(t, 1H, NCHCO), 4.35 (d, 1H, NCHCO), 4.70 (dd, 1H, NCHCO),
7.28 (m, 5H, C6H5); 13C-NMR (D2O) 210.00 (ketone). Anal.
Calcd for C31H49N7O8‚2.3CF3COOH: C, H, N, F.
1c-B from 19a -B and 22: FAB-MS m/e 648 (M + H); partial
NMR (D2O) δ 0.76 (d, 3H, CH3), 0.82 (t, 3H, CH3), 0.83 (d,
3H, CH3), 4.01 (t, 1H, NCHCO), 4.23 (d, 1H, NCHCO), 4.29
(t, 1H, NCHCO), 4.70 (dd, 1H, NCHCO), 7.28 (m, 5H, C6H5);
13C-NMR (D2O) 210.59 (ketone). Anal. Calcd for C31H49N7O8‚
2.25CF3COOH: C, H, N, F.
Ar g-Leu -Asp -Va l(k )P h e. 1d -A from 19a -A and 23; FAB-
MS m/e 648 (M + H); partial NMR (D2O) δ 0.74 (d, 3H, CH3),
0.87 (d, 6H, CH3), 0.90 (d, 3H, CH3), 4.10 (t, 1H, NCHCO),
4.36 (m, 1H, NCHCO), 4.39 (m, 1H, NCHCO), 4.70 (m, 1H,
NCHCO), 7.29 (m, 5H, C6H5); 13C-NMR (D2O) 211.00 (ketone).
Anal. Calcd for C31H49N7O8‚1.75CF3COOH: C, H, N, F.
Ar g-Ala -Asp -Va l(k )P h e. 1e-A from 19a -A and 24; FAB-
MS m/e 606 (M + H); partial NMR (D2O) δ 0.75 (d, 3H,
CH3), 0.86 (d, 3H, CH3), 1.37 (d, 3H, CH3), 4.00 (t, 1H,
NCHCO), 4.36 (m, 2H, NCHCO), 4.67 (dd, 1H, NCHCO), 7.28
(m, 5H, C6H5); 13C-NMR (D2O) 211.16 (ketone). Anal. Calcd
for C28H43N7O8‚2CF3COOH‚2H2O: C, H, N, F.
Ar g-D-Lys-Asp -Va l(k )P h e. 1f-A from 19a -A and 25: FAB-
MS 663 (M + H); partial NMR (D2O) δ 0.76 (d, 3H, CH3), 0.86
(d, 3H, CH3), 4.00 (t, 1H, NCHCO), 4.23 (t, 1H, NCHCO), 4.36
(dd, 1H, NCHCO), 7.27 (m, 5H, C6H5); 13C-NMR (D2O) 211.29
(ketone). Anal. Calcd for C31H50N8O8‚2.85CF3COOH‚2H2O:
C (0.6), H, N, F (0.5).
Ar g-Nle-Asp -Ala (k )P h e. 1g-A and 1g-B from 19b and 22.
The crude pseudopentapeptide obtained (80%) by cleavage
from the resin was purified by preparative HPLC (24% CH3-
CN‚0.1% TFA) to afford 1g-A (21%) [FAB-MS m/e 620 (M +
H). Anal. Calcd for C29H45N7O8‚2CF3COOH‚H2O: C, H, N]
and 1g-B (19%) [FAB-MS m/e 620 (M + H). Anal. Calcd for
C29H45N7O8‚2CF3COOH‚2H2O: C, H, N].
Nr,Nδ,Nω-Tr is(ben zyloxyca r bon yl)-L-a r gin yl-N-m eth yl-
L-n or leu cin e (32). Compound 31 (1.14 g, 1.5 mmol) was
dissolved in a mixture of TFA (10 mL) and MeCl2 (10 mL) and
stirred for 40 min. The material was concentrated to an oil,
diluted in MeCl2 (50 mL), washed with H2O (4 × 50 mL) and
brine (50 mL), dried over MgSO4, and evaporated to give 940
mg (89%) of a white solid: MS m/e 552 (M - Z - H2O); 1H
NMR (CDCl3) δ 0.88 (t, 3H, CH3), 1.01-1.95 (m, 10H, CH2),
2.86 (s, 3H, NCH3), 3.83 (m, 2H, CH2N), 4.72 (m, 1H, ArgCH-
N), 5.0-5.3 (m, 6H, OCH2), 5.96 (d, 1H, Nle-CH-N), 7.23-7.45
(m, 15H, Ar), 8.2-9.4 (bs, 3H, NH). Anal. Calcd for
C37H45N5O9: C, H, N.
Ar g-Nle-Asp -Va l(k )Va l. 1h -A and 1h -B from 19c and 22.
The crude pseudopentapeptide (86%) was purified by HPLC
(16-30% CH3CN-0.1% TFA gradient elution) to give 1h -A
(18%) and 1h -B (17%). 1h -A: FAB-MS m/e 600 (M + H);
partial NMR (D2O) δ 0.77 (d, 3H, CH3), 0.83 (m, 3H, CH3),
0.89 (m, 9H, CH3), 4.01 (t, 1H, NCHCO), 4.30 (t, 1H, NCHCO),
4.44 (d, 1H, NCHCO); 13C-NMR (D2O) 211.81 (ketone). Anal.
Calcd for C27H49N7O8‚2CF3COOH: C, H, N, F. 1h -B: FAB-
MS m/e 600 (M + H); partial NMR (D2O) δ 0.90 (m, 15H, CH3),
4.04 (t, 1H, NCHCO), 4.32 (t, 1H, NCHCO), 4.33 (d, 1H,
Nr,Nδ,Nω-Tr is(ben zyloxyca r bon yl)-L-a r gin ylm eth yl-L-
n or leu cyl-L-a sp a r tic Acid â-Cycloh exyl-r-ter t-bu tyl Ester
(34). Compound 32 (910 mg, 1.30 mmol) in MeCl2 (10 mL)
and HOBT (213 mg, 1.40 mmol) in DMF (1 mL) were combined
and cooled to -10 °C. Dicyclohexylcarbodiimide (310 mg, 1.50
mmol) in MeCl2 (5 mL) was added dropwise over 5 min. The
reaction mixture was stirred for 20 min at -10 °C and then
at room temperature for 20 min. Aspartic acid â-cyclohexyl-