Mouse Melanocortin Receptor Agonists
A R T I C L E S
mixture was stirred for 15 min after the evolution of gas had subsided,
at which time the reaction was incomplete according to TLC (petroleum
ether/ethyl acetate 1:1). Another portion of NaBH4 (50 mg in 1 mL of
H2O) was added, and after 30 min no starting material could be detected
by TLC. The reaction mixture was diluted with water (100 mL), and
the crude product was isolated by filtration. The white solid was washed
with water and petroleum ether, and dried in vacuo. Recrystallization
was achieved by dissolving the crude product in 96% EtOH, diluting
with water until near saturation, and cooling in a freezer for 2 h. The
white solid was isolated by filtration, washed with water and petroleum
ether, and lyophilized to give the title compound. Yield: 592 mg (61%).
General Procedure for DIBAL Redution of Fmoc Amino Esters
(5-10 mmol Scale).38,59 The Fmoc amino ester was dissolved in CH2Cl2
(50 mL) and cooled to -78 °C under Ar. Diisobutylaluminum hydride
(1 M solution in CH2Cl2, 2.5 equiv) was added slowly via a syringe.
The solution was stirred at -78 °C for 2 h and then quenched at -78
°C with a saturated solution of Rochelle’s salt (5 mL). Drying over
MgSO4 and evaporation gave a white solid. This material was purified
by column chromatography.
(S)-N-r-(Fluorenylmethyloxycarbonyl)-phenylalaninal (13a).57
This compound was flash chromatographed on silica using petroleum
ether/ethyl acetate 4:1. Yield: 49%. Rf (petroleum ether/ethyl acetate
1
1
Rf ) 0.22. H NMR (CDCl3, 250 MHz): δ 1.14-1.19 (app t, J ) 7,
2:1) ) 0.43. H NMR (CDCl3, 250 MHz): δ 2.98-3.0 (d, J ) 7.5,
2H), 1.42 (br s, 18H), 1.52 (br s, 2H), 1.9-2.3 (v br, 1H), 3.31-3.68
(m, 5H), 4.11-4.16 (t, J ) 6.5, 1H), 4.34-4.37 (d, J ) 6.3, 2H), 5.41-
5.44 (d, J ) 7.3, 1H), 7.19-7.35 (m, 4H), 7.51-7.54 (m, 2H), 7.67-
7.70 (d, J ) 7.3, 2H), 8.30 (br s, 1H), 11.40 (s, 1H). 13C NMR (CDCl3,
62.9 MHz): δ 26.6, 28.5, 28.7, 40.9, 47.7, 53.5, 65.4, 66.9, 83.6, 79.8,
121.4, 125.5, 127.5, 129.1, 141.7, 144.3, 153.7, 156.8, 157.1, 163.8.
ES-MS: mass calcd for C31H43N4O7 583.31 (MH+). Found 583.42.
(S)-N-r-(Fluorenylmethyloxycarbonyl)-N-E-(tert-butyloxycar-
bonyl)-N-γ-(tert-butyloxycarbonyl)-arginal (11). General guidelines
from previous procedures were used.33-35 Compound 10 (450 mg) was
dissolved in DCM (10 mL). Dess-Martin periodinane (393 mg, 2
equiv) was added, and the mixture was stirred for 30 min, at which
time the reaction was incomplete according to TLC (petroleum ether/
ethyl acetate 1:1). A further 100 mg of oxidant was added, and the
mixture was stirred for 2 h. The reaction was quenched by addition of
saturated aqueous NaHCO3 and 10% aqueous Na2S2O3 (5 mL of each).
After 15 min of vigorous stirring, the organic layer was washed with
brine and dried with MgSO4. Evaporation of the solvent gave an off-
2H), 4.03-4.09 (t, J ) 6.8, 1H), 4.22-4.46 (m, 3H), 5.22-5.25 (d, J
) 7.5, 1H), 6.97-6.99 (d, J ) 7.5, 2H), 7.10-7.29 (m, 7H), 7.40-
7.43 (d, J ) 7.5, 2H), 7.61-7.64 (d, J ) 7.5, 2H), 9.47 (s, 1H). 13C
NMR (CDCl3, 62.9 MHz): δ 25.4, 34.4, 35.8, 47.6, 61.6, 67.4, 120.5,
125.5, 127.5, 127.6, 128.2, 129.2, 129.8, 136.0, 141.8, 144.1, 156.3,
199.2. ES-MS: mass calcd for C24H22NO3 372.15 (MH+). Found
372.33.
(S)-N-r-(Fluorenylmethyloxycarbonyl)-leucinal (13b).57 This com-
pound was flash chromatographed on silica using petroleum ether/ethyl
acetate 4:1. Yield: 32%. Rf (petroleum ether/ethyl acetate 3:1) ) 0.37.
1H NMR (CDCl3, 250 MHz): δ 0.87 (d, J ) 4.5, 6H), 1.27-1.34 (m,
1H), 1.56-1.67 (m, 2H), 4.09-4.15 (t, J ) 6.67, 1H), 4.21-4.27 (br,
1H), 4.32-4.36 (d, J ) 6.74, 2H), 5.08-5.11 (br d, J ) 6.76, 1H),
7.15-7.33 (m, 4H), 7.48-7.51 (d, J ) 7.1, 2H), 7.64-7.67 (d, J )
7.41, 2H), 9.47 (s, 1H). 13C NMR (CDCl3, 62.9 MHz): δ 21.8, 23.0,
24.5, 38.0, 47.1, 58.7, 66.8, 119.9, 124.9, 127.0, 127.6, 141.2, 143.7,
156.1, 199.6. ES-MS: mass calcd for C21H24NO3 338.17 (MH+). Found
338.28.
1
white solid. Yield: 219 mg (49%). Rf ) 0.70. H NMR (CDCl3, 250
Solid-Phase Synthesis of Peptidomimetic Scaffold 1. Method A.
Fmoc-Rink-amide linker was coupled onto lyophilized PEGA800 resin
via TBTU-activation. The resin was washed with DMF (×6) and DCM
(×5), and then lyophilized overnight. The loading was measured to
0.27 mmol/g. The Fmoc group was removed, and the resin was washed
with DMF (×5). The first amino acid was coupled either as the Pfp
ester or via TBTU-activation, followed by washing with DMF (×5).
The Fmoc group was removed, and the resin was washed with DMF
(×5). NaBH3CN (6 equiv) and glacial acetic acid (18 equiv) were added
in DMF, followed by the Fmoc amino aldehyde (3 equiv). The reaction
was monitored, using a Kaiser test (reaction time usually 3-4 h). The
resin was then washed with 96% ethanol (×3), DCM (×3), and DMF
(×5). The Fmoc group was removed, and the resin was washed with
DMF (×5). Fmoc-Cys(SBut)-OH was then coupled via TBTU-
activation, followed by washing with DMF (×5) and DCM (×5).
Chloroacetic anhydride (25 equiv) and NEM (25 equiv) were added in
DCM. After 30 min, the resin was washed with DMF (×2), DCM (×5),
and THF (×2) and then treated with Bu3P (100 equiv) in THF:saturated
sodium acetate (aqueous) 19:1 for 1 h. The resin was washed with
96% ethanol (×2), DCM (×5), and DMF (×5), suspended in degassed,
dry DMF with NEM (5 equiv), and then heated to 55-60 °C for 7 h.
The resin was washed with DMF (×5). The Fmoc group was removed,
and the resin was washed with DMF (×5). NaBH3CN (6 equiv), glacial
acetic acid (18 equiv), and 1-naphthaldehyde (3 equiv) were added in
DMF. After 20 h, a Kaiser test was negative. The resin was washed
with DMF (×5) and DCM (×5), and the product was cleaved from
the resin by treatment with 95% TFA/TIPS 95:5 (a little DCM was
added to obtain a homogeneous solution) for 2 h. The resin was washed
five times with 95% TFA, three times with glacial acetic acid, and
finally twice with 96% ethanol. The combined washings were concen-
trated in vacuo and then lyophilized to give an oil. Diethyl ether was
added, and an off-white product was isolated. The crude products 1o
and 1p were purified on preparative HPLC.
MHz): δ 1.42 (br s, 18H), 1.58 (br s, 2H), 1.96 (br s, 2H), 3.32-3.40
(m, 2H), 4.11-4.17 (t, J ) 6.7, 1H), 4.24-4.26 (br s, 1H), 4.31-4.38
(d, J ) 6.8, 2H), 5.91-5.94 (d, J ) 7.1, 1H), 7.18-7.35 (m, 4H),
7.51-7.53 (m, 2H), 7.67-7.70 (d, J ) 7.3, 2H), 8.31 (br s, 1H), 9.52
(s, 1H), 11.40 (s, 1H). 13C NMR (CDCl3, 62.9 MHz): δ 24.5, 27.1,
27.2, 38.9, 46.2, 58.9, 65.9, 78.4, 82.3, 119.0, 124.1, 126.1, 126.7, 140.3,
142.7, 152.3, 155.4, 162.3, 198.4. ES-MS: mass calcd for C31H41N4O7
581.31 (MH+). Found 581.51, 599.38 (MH+ + H2O).
General Procedure for Preparation of Fmoc Amino Esters (10
mmol Scale). The Fmoc amino acid was refluxed in absolute ethanol
(50 mL) with concentrated H2SO4 (0.5 mL) for 4 h. Water (200 mL)
was added, and the mixture was extracted three times with ethyl acetate.
The combined organic phases were washed with saturated NaHCO3
(aqueous) and brine and dried over MgSO4. Evaporation left a white
solid which was dried in vacuo. This material was used directly.
Ethyl (S)-N-r-(Fluorenylmethyloxycarbonyl)-phenylalaninate (12a).
White solid. Yield: 86%. Rf (silica, petroleum ether/ethyl acetate 3:1)
1
) 0.58. H NMR (CDCl3, 250 MHz): δ 1.12-1.19 (t, J ) 7.1, 3H),
3.01-3.05 (m, 2H), 4.02-4.39 (m, 5H), 4.53-4.61 (q, J ) 6, 1H),
5.20-5.23 (d, J ) 8, 1H), 7.01-7.03 (d, J ) 5.9, 2H), 7.14-7.34 (m,
7H), 7.45-7.50 (m, 2H), 7.65-7.69 (d, J ) 7.4, 2H). 13C NMR (CDCl3,
62.9 MHz): δ 15.4, 39.6, 48.5, 56.1, 62.8, 68.2, 121.3, 126.4, 128.4,
128.5, 128.9, 129.0, 129.5, 137.1, 142.6, 145.1, 156.8, 172.8. ES-MS:
mass calcd for C26H26NO4 416.18 (MH+). Found 416.35. HPLC purity
> 95%, Rt (B) ) 18.96.
Ethyl (S)-N-r-(Fluorenylmethyloxycarbonyl)-leucinate (12b). White
1
solid. Yield: 83%. Rf (petroleum ether/ethyl acetate 3:1) ) 0.51. H
NMR (CDCl3, 250 MHz): δ 0.84-0.86 (m, 6H), 1.14-1.19 (t, J )
7.12, 3H), 1.33-1.69 (m, 3H), 4.05-4.14 (m, 3H), 4.28-4.35 (m, 3H),
5.21-5.24 (d, J ) 8.7, 1H), 7.16-7.31 (m, 4H), 7.47-7.51 (m, 2H),
7.62-7.65 (d, J ) 7.5, 2H). 13C NMR (CDCl3, 62.9 MHz): δ 14.6,
22.3, 23.3, 25.2, 42.2, 47.6, 53.0, 61.7, 67.4, 119.9, 120.4, 121.5, 125.5,
127.5, 128.1, 141.7, 144.2, 156.4, 173.6. ES-MS: mass calcd for C23H28-
NO4 382.19 (MH+). Found 382.42. HPLC purity > 95%, Rt (B) )
19.13.
(59) Rich, D. H.; Sun, E. T.; Boparai, A. S. J. Org. Chem. 1978, 43, 3624-
3626.
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J. AM. CHEM. SOC. VOL. 124, NO. 37, 2002 11053