6650 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21
Moore et al.
(4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-
NH2 (2) was synthesized by manual SPPS, on Rink amide
resin (substitution level 0.54 mmol/g), 0.2 mmol scale, to yield
228 mg of crude peptide. A portion of the product was purified
using 25% B at λ ) 280 nm to yield 16.9 mg of pure pep-
tide. Analytical conditions were the following: gradient 10-
90% B, tR ) 14.33 min and isocratic 26% B, tR ) 11.13 min.
MALDI-FTMS (m/z): [MH]+ calcd for C50H68N12O10S2 expected
1061.4695, found 1061.4733.
(4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-
NH2 (3) was synthesized by manual SPPS on Rink amide
MBHA resin (0.54 mmol/g), 0.37 g, 0.2 mmol scale, as noted
above. The crude yield was 143.8 mg. A portion of the peptide
(40 mg) was purified with 25% B isocratic at λ ) 280 nm to
yield 13.3 mg of pure peptide. Analytical conditions were the
following: gradient 10-90% B over 30 min, tR ) 17.12 min
and isocratic 26% B, tR ) 8.72 min. MALDI-FTMS (m/z):
[MH]+ calcd for C50H66N12O11S2 expected 1075.4488, found
1075.4448.
(4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Thr-
NH2 (4) was synthesized as noted above, on 0.1 mmol scale,
and 115 mg of crude was obtained. Purification of a portion of
material was carried out using isocratic conditions of 23% B
at λ ) 220 nm, and an amount of 5 mg of pure product was
obtained. Analytical conditions were the following: gradient
10-90% B over 30 min, tR ) 13.58 min and isocratic 20% B,
tR ) 11.59 min. MALDI-FTMS (m/z): [MH]+ calcd for
C50H68N12O10S2 expected 1061.4695, found 1061.4650.
(4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Asp-
NH2 (5) was synthesized manually, as noted above, on a Rink
amide MBHA resin (0.54 mmol/g), 0.19 g, 0.1 mmol scale, and
88.9 mg was obtained. A portion of the peptide was purified
using isocratic conditions (20% B) at λ ) 220 nm, and 6.16 mg
of pure peptide was obtained. Analytical HPLC conditions were
the following: gradient 10-90% B over 30 min, tR ) 13.59 min,
and isocratic 20%, tR ) 11.26 min. MALDI-FTMS (m/z): [MH]+
calcd for C50H66N12O11S2 expected 1075.4488, found 1075.4492.
(4-Amino)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-
Thr-NH2 (6) was synthesized by manual SPPS, 0.2 mmol
scale, 0.37 g of resin. Fmoc-(3-I)-Tyr-OH (0.26 g, 0.5 mmol,
2.5 equiv), PyBOP (0.26 g, 0.5 mmol, 2.5 equiv), HOBt (0.07
g, 0.5 mmol, 2.5 equiv), and DIEA (0.17 mL, 1.0 mmol, 5 equiv)
were mixed in DMF (20 mL) and added over the deprotected
peptide-resin. The resulting mixture was opalescent because
the amino acid was not completely soluble. Despite this
problem, the coupling of this building block was successful.
The resin was moved from the reaction vessel into a scintil-
lation vial, and coupling was carried out overnight. The resin
was removed from the scintillation vial, washed in a Buchner
funnel several times with DMF and DCM, and then trans-
ferred back to the reaction vessel for subsequent couplings.
The Kaiser test was used to assess the completion of the
reaction, and the resin was washed again with DMF (4 × 20
mL), DCM (2 × 20 mL), and DMF (4 × 20 mL). The building
of the peptide chain was resumed as usual. The last amino
acid added was Boc-(4-Fmoc-amino)-D-Phe-OH (0.25 g, 0.5
mmol, 2.5 equiv) preactivated in the presence of PyBOP (0.42
g, 0.5 mmol, 2.5 equiv), HOBt (0.12 g, 0.5 mmol, 2.5 equiv),
and DIEA (0.21 mL, 0.5 mmol, 5 equiv), in 20 mL of DMF.
This residue was coupled for 18 h. For cyclization, only 5 equiv
of iodine (0.25 g, 1.0 mmol) was used, for 3 h, to prevent
iodination of the aromatic residues. The peptide-resin was
then extensively washed as described in General Notes and
dried for 2 h in a desiccator under high vacuum to eliminate
the smallest trace of iodine. The side chain Fmoc protecting
group was removed using 20% piperidine in DMF, for 1 h. The
peptide-resin was washed thoroughly and dried in a desic-
cator overnight in preparation for final deprotection/cleavage
from the resin. Final treatment with TFA/H2O/anisole (9.5/
2.5/2.5 mL) and usual workup resulted in 237.7 mg of crude
peptide. A portion of 23 mg of the crude product was purified
using isocratic conditions, 25% B, λ ) 210 and 220 nm, to yield
8.1 mg of pure product. Analytical conditions were the follow-
ing: gradient 10-90% B, 30 min, tR ) 18.04 min, isocratic 26%
B, tR ) 16.45 min. MALDI-FTMS (m/z): [MH]+ calcd for
C50H67IN12O10S2 expected 1187.3662, found 1187.378; [M +
Na]+ expected 1209.3481, found 1209.3533.
(4-Amino-3-iodo)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-
Thr-NH2 (7) was built as noted above, on 0.2 mmol scale, but
only an amount of 90 mg of peptide-resin was used to couple
the last amino acid. The coupling mixture contained Boc-(4-
amino-3-iodo)-D-Phe-OH (0.044 g, 0.11 mmol, 1.5 equiv), DIC
(0.017 mL, 0.11 mmol, 1.5 equiv), and HOBt (0.017 g, 0.11
mmol, 1.5 equiv) in 10 mL of DMF, and the reaction was
allowed to proceed for 10 h. Cyclization was carried out with
iodine (0.16 g, 2.0 mmol, 10 equiv) for 3 h, and the peptide-
resin was carefully washed and dried. The final deprotection/
cleavage procedure was accomplished in 1 h, and after
lyophilization, 30.6 mg of crude peptide resulted. Purification
was accomplished with 24% B in isocratic mode. The yield was
9.9 mg (13% based on theoretical loading level of the resin).
Analytical HPLC conditions were as follows: gradient 10-
90% B, λ ) 220 nm, tR ) 18.9 min, isocratic 25% B, tR ) 9.32
min. MALDI-FTMS (m/z): [MH]+ calcd for C50H67IN12O10S2
expected 1187.3662, found 1187.3689; [M + Na]+ expected
1209.3481, found 1209.3470.
(4-Amino-3-iodo)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-
Asp-NH2 (8) was synthesized as above. A portion of 90 mg of
peptide-resin was used for coupling of the final building block,
which resulted in 19.4 mg of crude peptide. The peptide was
purified with 24% B at λ ) 220 nm. The yield was 4.1 mg (7%
overall). Analytical HPLC conditions were as follows: gradient
10-90% B, 30 min, tR ) 18.74 min and isocratic 22% B, tR
)
10.65 min. MALDI-FTMS (m/z): [MH]+ calcd for C50H65-
IN12O11S2 expected 1201.3454, found 1201.3542; [M + Na]+
expected 1223.3274, found 1223.3296.
(4-Amino-3-iodo)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-
Val-Cys]-Thr-NH2 (9) was synthesized as above, using 90 mg
of peptide-resin. The couplings of the unusual building blocks
were carried out as described above. The crude yield was 41
mg. Purification was carried out with 25-50% B over 30 min
at λ ) 220 nm. The yield was 4.1 mg (7.4% overall). Analytical
conditions were as follows: gradient 10-90% B, 30 min, tR
)
20.4 min and isocratic 25% B, tR ) 11.17 min. MALDI-FTMS
(m/z): [MH]+ calcd for C50H66I2N12O10S2 expected 1313.2628,
found 1313.2644; [M + Na]+ expected 1335.2448, found
1335.2471.
(4-Amino-3-iodo)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-
Val-Cys]-Asp-NH2 (10) was synthesized by manual SPPS as
described above, and an amount of 38.7 mg of crude was
obtained. Purification conditions were as follows: 25-40% B,
30 min, λ ) 220 nm. The yield was 5 mg (7.8% overall).
Analytical conditions were as follows: gradient 10-90% B over
40 min, tR ) 17.62 min, isocratic 25% B, tR ) 10.41 min.
MALDI-FTMS (m/z): [MH]+ calcd for C50H64I2N12O11S2 ex-
pected 1327.2421, found 1327.2423; [M + Na]+ expected
1349.224, found 1349.2274.
Biological Testing. The biological assays were carried out
in the laboratories of Professor Steven W. J. Lamberts at
University Hospital, Rotterdam, The Netherlands, by Joost
van der Hoek, Peter M. van Koetsveld, and Leo J. Hofland.
The binding assays were executed on membranes from CC531
cells (hsst2) and CHO-K1 cells (hsst1, hsst3, and hsst5),
transfected with individual human somatostatin receptor
subtypes. The functional assays (inhibition of GH and PRL
secretion) were carried out with dispersed rat anterior pitu-
itary cells. The results are presented in the text above, and
the experimental details are found below.
Animals. Female Wistar rats (Harlan, The Netherlands),
weighing 180-200 g, were kept in an artificially illuminated
room (08.30 to 20.30 h) with food and water ad libitum. The
animals were killed between 09.00 and 10.00 h by decapitation.
The pituitary glands were removed within 5 min after killing,
the neurointermediate lobe was discarded, and the anterior
lobes were collected in calcium and magnesium Hank’s bal-
anced salt solution (HBSS) supplemented with 1% human
serum albumin (HSA), penicillin (100 U/mL), streptomycin