Design and synthesis of novel type somatostatin analogs with antiproliferative activities on A431 tumor cells

Article abstract of DOI:10.1016/j.tetlet.2004.06.105

It was reported that the somatostatin analog TT-232, d-Phe-c(Cys-Tyr-d-Trp- Lys-Cys)-Thr-NH₂, exhibited a highly potent antitumor activity in vitro and in vivo. Using pyrazinone analogs and aliphatic amino acids instead of the disulfide bond, we prepared novel type somatostatin analogs including the sequence essential for antitumor activities, Tyr-d-Trp-Lys. These analogs exhibited antiproliferative effect on A431 tumor cells.

Full text of DOI:10.1016/j.tetlet.2004.06.105

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 6323–6327
Design and synthesis of novel type somatostatin analogs with
antiproliferative activities on A431 tumor cells
Anna Miyazaki,a Toshio Yokoi,a,b Yoshifumi Tachibana,a Riyo Enomoto,a Eibai Lee,a,b
Gyongyi Bokonyi,c Gyorgy Keri,c Yuko Tsudaa,b and Yoshio Okadaa,b,
*
aFaculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
bHigh Technology Research Center, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
cPeptide Biochemistry Research Group of Hung. Acad. Sci., Department of Medical Chemistry, Semmelweis University,
Puskin Street 9, Budapest 1088, Hungary
Received 24 March 2004; revised 9 June 2004; accepted 18 June 2004
Abstract—It was reported that the somatostatin analog TT-232, D-Phe-c(Cys-Tyr-D-Trp-Lys-Cys)-Thr-NH2, exhibited a highly po-
tent antitumor activity in vitro and in vivo. Using pyrazinone analogs and aliphatic amino acids instead of the disulfide bond, we
prepared novel type somatostatin analogs including the sequence essential for antitumor activities, Tyr-D-Trp-Lys. These analogs
exhibited antiproliferative effect on A431 tumor cells.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Somatostatin is a cyclic tetradecapeptide, which was iso-
lated from bovine hypothalamus and known as a sup-
pressor of growth hormone (GH) secretion from the
anterior pituitary gland.1 Furthermore it also suppresses
the release of various hormones including glucagons,
insulin, gastrin, secretin, etc.2,3 Although many somato-
statin analogs have been prepared and studied in regard
to their biological activities, it is difficult to use them for
clinical purpose because of their enzymatic degradation
in vivo. Among them, however, sandostatin, D-Phe-
c(Cys-Phe-D-Trp-Lys-Thr-Cys)-Thr-ol, acts longer than
somatostatin in vivo and is widely used as the inhibitor
of hormone release and for the treatment for endocrine
tumors on the clinical level.4,5 On the other hand, TT-
232, D-Phe-c(Cys-Tyr-D-Trp-Lys-Cys)-Thr-NH2, has
no GH release inhibiting activity but has only highly
potent antitumor activity in vitro and in vivo, indicating
that the sequence essential for the antitumor activity
might be Tyr-D-Trp-Lys.6 For clinical purposes, the
enzymic stability and the passage through the physical
barriers including epithelial membrane and blood–brain
barrier of drugs are required. In this paper, we designed
and prepared peptidomimetic somatostatin analogs with
expectation of their high bioavailability in oral adminis-
tration.
We designed novel somatostatin analogs that include the
Tyr-D-Trp-Lys sequence and tested their antiprolifera-
tive activities on tumor cells. We prepared four somato-
statin analogs (I–IV) (Fig. 1). Two of them, I and II
have pyrazinone rings7 instead of disulfide bond. Previ-
ously we established a simple synthetic method for pyr-
azinone ring formation from dipeptidyl chloromethyl
ketone and the procedure has following advantages:
the selective introduction of amino and/or carboxyl
groups at position 3 and 6 on the pyrazinone ring.7 It
was expected that peptidomimetic containing pyrazi-
none ring would exhibit a longer period of activity than
somatostatin and its peptidic analogs owing to a stabil-
ity of the ring structures.7 The most attractive feature is
that peptidomimetics containing the pyrazinone rings
can pass the epithelial membrane of gastrointestinal
tracts and blood–brain barrier.8,9 These results provided
us with an idea to design effective drugs for oral admin-
istration. Thus we have prepared numerous pyrazinone
derivatives. On the other hand, III and IV have x-ali-
phatic amino acids with different chain lengths in order
to compare them with the pyrazinone ring-containing
compounds (I and II). Concerning the number of
atoms comprising the backbone in the analogs, the
(CH2)6 analog (IV) is almost same as that of TT-232.
*
Corresponding author. Tel.: +81-78-974-1551; fax: +81-78-974-
5689; e-mail: okada@pharm.kobegakuin.ac.jp
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.06.105

6324
A. Miyazaki et al. / Tetrahedron Letters 45 (2004) 6323–6327
N
( )4
O
*
I
*
*
*
R
( )2
O
N
H
NH
O
HN
HC
H
N
O
( )4
*
.
CH (CH2)4 NH2 HCl
II
HO
C
H2
R
*
( )2
*
NH
N
O
HN
CH
*
*
O
III
(CH2)4
CH2
*
IV
(CH2)6
HN
Figure 1. Somatostatin analogs. R: I, a pyrazinone ring prepared from Boc-Lys(Z)-Glu(OBzl)-CH2Cl; II, a pyrazinone ring prepared from Boc-
Glu(OBzl)-Lys(Z)-CH2Cl; III, an aliphatic amino acid (n=4); IV, an aliphatic amino acid (n=6).
The antiproliferative activities of these compounds were
examined on tumor cells by methylene blue test.10 Fur-
thermore to clarify their structure–activity relationships,
CD and NMR spectroscopy measurements were per-
formed.
9-fluorenylmethanol, WSCIÆHCl and 4-dimethylamino-
pyridine to give Boc-Lys(Z)-OFm. After removal of
Boc group by HCl/dioxane, the resulting HCl salt was
coupled with Boc-D-Trp-OH by a mixed anhydride
method to give Boc-D-Trp-Lys(Z)-OFm. After removal
of Boc group from the dipeptide by HCl/dioxane, the
resulting amine was coupled with Boc-Tyr-OH to give
crude tripeptide. The crude crystal was purified by silica
gel column chromatography (3% methanol/chloroform)
to give Boc-Tyr-D-Trp-Lys(Z)-OFm (Scheme 1, A). The
Boc group was removed from the tripeptide by HCl/
The compounds (I–IV) were synthesized by solution-
phase method using fragment condensation (Scheme
1). The Boc–pyrazinones were prepared by a method de-
scribed previously.7 The tripeptide was prepared starting
from Boc-Lys(Z)-OH. Boc-Lys(Z)-OH was reacted with
(ii)
(i)
(ii), (iii)
(iv)
A. Boc-Lys(Z)-OH
Boc-Lys(Z)-OFm
Boc-D-Trp-Lys(Z)-OFm
Boc-Tyr-D-Trp-Lys(Z)-OFm
(v)
Boc-D-Trp-OH
O
C
O
(ii)
(iii), (iv)
B. Boc
N
H
*
Boc
N
H
*
*
OH
*
C
Tyr-D-Trp-Lys(Z)-OFm
R
R
(i)
Boc-Tyr-D-Trp-Lys(Z)-OFm
*
*
R
O
NH
O
HN
HC
(v)
(vi), (vii)
.
CH (CH2)4 NH2 HCl
HO
C
H2
NH
O
HN
CH
O
CH2
HN
I - IV
Scheme 1. Reagents and conditions. A: (i) 9-fluorenylmethanol, WSCIÆHCl, 4-dimethylaminopyridine, DMF; (ii) HCl/dioxane; (iii) triethylamine,
DMF; (iv) triethylamine, isobutyl chloroformate, THF, ꢀ15ꢁC; (v) Boc-Tyr-OH, PyBop, N,N-diisopropylethylamine, in DMF. B: (i) HCl/dioxane;
(ii) PyBop, N,N-diisopropylethylamine, in DMF; (iii) 20% piperidine/DMF; (iv) trifluoroacetic acid, 1,2-ethanedithiol; (v) triethylamine, DPPA, in
0.5mM DMF; (vi) 25% HBr/acetic acid, thioanisole, 1,2-ethanedithiol; (vii) 1M HCl. (R: see Fig. 1).

A. Miyazaki et al. / Tetrahedron Letters 45 (2004) 6323–6327
6325
20
15
10
5
40
30
20
10
0
0
-5
-10
-20
-30
-10
-15
185
210
235
260
185
210
235
260
Wavelength [nm]
Wavelength [nm]
(A)
(B)
Figure 2. The CD spectra pattern of I–IV in A (trifluoroethanol) and B (methanol). I: j, II: r, III: N, IV: ꢁ.
dioxane and the resulting amine was coupled with Boc–
pyrazinones or Boc–aliphatic amino acids by PyBop
reagent as described above. The Fm and Boc groups
were removed using 20% piperidine/DMF and trifluoro-
acetic acid containing 1,2-ethanedithiol, respectively.
The compounds purified by reversed-phase HPLC, were
cyclized by diphenyl phosphoryl azide (DPPA) and tri-
ethylamine in 0.5mM DMF solution at room tempe-
rature, monitoring the reaction by reversed-phase
HPLC. The crude crystal was recrystallized. Finally,
the Z group was removed by HBr/acetic acid containing
thioanisole and 1,2-ethanedithiol. The crude compounds
were purified by reversed-phase HPLC [column: COS-
MOSIL C18 (4.6·250nm); solvents: from 0.05% tri-
fluoroacetic acid in water (90), 0.05% trifluoroacetic
acid in CH3CN (10) to 0.05% trifluoroacetic acid in
water (50), 0.05% trifluoroacetic acid in CH3CN (50)
for 40min at a flow rate of 1mL/min with detection at
220nm], (Scheme 1, B) and identified by NMR, MALDI
TOF-MS, and elemental analysis.11–14
chemical shifts. The side chains of amino acids were flex-
ible, such that the interactions between them were not
observed. CD spectra were recorded in methanol and
trifluoroethanol (Fig. 2). In the both cases, all analogs
showed random structures and the spectra patterns of
analogs containing pyrazinone rings were different from
those including aliphatic amino acids. As shown in Fig-
ure 2, insertion of this constraint extremely changed the
backbone conformation of the cyclic peptide.
Finally, the antiproliferative effects of analogs I–IV on
A431 cells (human epithelial tumor cells) in which the
somatostatin receptor is expressed were measured by
methylene blue test (Fig. 3).10 The concentrations of
the analogs were 0.08, 0.4, 2.0, 10, and 50lmol/L. The
cycloheximide inducing apoptosis by inhibiting protein
synthesis was used as positive control. After incubation
of A431 cells with I–IV and cycloheximide for 6 or 48h,
the cells dyed by methylene blue were counted. On the
120
100
80
60
40
20
0
Generally, somatostatin, sandostatin, and many other
somatostatin analogs form b-turn structures. For exam-
ple, sandostatin forms an antiparallel b-pleated sheet
with a type b-turn spanning residues D-Trp4 and Lys5,
and is stabilized by the hydrogen bond between Thr6
(NaH) and Phe3 (C@O). This is supported by the strong
NOE between CaH of D-Trp4 and NaH of Lys5, the
average NOE between NaH of Lys5, and NaH of Thr6
and the low temperature coefficient measured for the
Thr6NaH resonance.15,16 In the above case, the side
chain of D-Trp4 and Lys5 were in juxtaposition, which
was supported by the upfield chemical shift (<0.4ppm)
0.08
0.4
2
10
50
Concentration (µmol/L)
5
16,17
of c-methylene protons of Lys .
With I–IV, we ex-
Figure 3. The result of antiproliferative activity of I–IV on A431
tumor cells. The dotted line and solid line show the case of incubation
for 6 and 48h, respectively. The d, s, m, n, and j show I–IV, and
cycloheximide, respectively.
pected similar results; however, they failed to exhibit the
above NOE. The aromatic side chain orientation of D-
Trp4 was not determined due to the overlap of the

6326
A. Miyazaki et al. / Tetrahedron Letters 45 (2004) 6323–6327
1.0, H2O); Rf (n-butanol/acetic acid/pyridine/H2O=
4:1:1:2)=0.50; 1H NMR (500MHz, DMSO-d6) d: 10.85
(1H, s, 1-NH of indole), 8.45 (1H, d, J=7.8Hz, a-NH of
D-Trp), 8.19 (1H, d, J=8.3Hz, a-NH of Lys), 7.88 (2H, s,
e-NH2 of Lys), 7.78 (1H, d, J=8.5Hz, a-NH of Tyr), 7.69
(1H, d, J=7.8Hz, 4-CH of indole), 7.65 (1H, t, J=5.0Hz,
3-CH2CH2CH2CH2NH of pyrazinone), 7.31 (1H, d,
J=8.0Hz, 7-CH of indole), 7.18 (1H, d, J=1.8Hz, 2-CH
of indole), 7.04 (1H, t, J=7.3Hz, 6-CH of indole), 6.98
(1H, t, J=7.3Hz, 5-CH of indole), 6.78 (2H, d, J=8.3Hz,
2, 6-OH of Tyr), 6.51 (2H, d, J=8.3Hz, 3, 5-CH of Tyr),
4.60–4.52 (2H, m, a-CH of D-Trp and a-CH of Tyr), 4.13
(1H, td, J=8.5, 5.7Hz, a-CH of Lys), 3.18 (1H, m,
3-CH2CH2CH2CH2NH of pyrazinone), 3.01 (1H, dd,
J=14.5, 5.8Hz, b-CH2 of Tyr), 2.89–2.72 (4H, m,
3-CH2CH2CH2CH2NH of pyrazinone, 6-CH2CH2CO of
pyrazinone and b-CH2 of Tyr), 2.66 (2H, m, e-CH2 of
Lys), 2.56–2.17 (6H, m, 3-CH2CH2CH2CH2NH of pyraz-
inone, 6-CH2CH2CO of pyrazinone and b-CH2 of D-Trp),
2.14 (3H, s, 5-CH3 of pyrazinone), 1.60–1.23 (8H, m,
3-CH2CH2CH2CH2NH of pyrazinone, b-CH2 and d-CH2
of Lys),1.05 (2H, m, c-CH2 of Lys); MS (M+H)+ Calcd
713.8. Found. 713.8. Anal. Calcd for C38H49ClN8O6
(+4.5H2O): C, 54.9; H, 7.04; N, 13.5. Found: C, 54.7; H,
6.95; N, 13.7.
incubation for 48h, their activities increase depending
on concentration and all analogs (I–IV) have the highest
efficient at 50lmol/L (90%, 90%, 30%, and 50% inhibi-
tion, respectively). The analogs containing pyrazinone
ring (I and II) showed highly potent activities at
50lmol/L although the activity was weaker than that
of cycloheximide at low concentration. Interestingly I
and II were more potent than analogs containing ali-
phatic amino acid (III and IV). The differences of activ-
ity between I and II were not observed, on the other
hand, the (CH2)6 analog (IV), the number of atoms
comprising the backbone was same as that of TT-232,
was slightly stronger than the (CH2)4 analog (III).
In conclusion, we designed and prepared novel type so-
matostatin analogs I–IV. As shown in Figure 3, these
analogs exhibited high antiproliferative effects on A431
cells, although their conformations determined by
NMR and CD were different from those of typical so-
matostatin analogs. Compounds I and II had high de-
gree of antiproliferative activity at 50lmol/L and we
can expect the more effective uptake by oral administra-
tion owing to the stability and physicochemical features
in vivo8,9 as well as their potent antiproliferative activ-
ity.
12. c[(Glu-Lys)-Tyr-D-Trp-Lys]ÆHCl (II): (Glu-Lys) shows 6-
(4-aminobutyl)-3-(2-carboxyethyl)-5-methyl-2(1H)-pyrazi-
25
none. Yield 7.00mg (14.4%): amorphous; ½aꢂD þ 32:1 (c
1.0,
H2O);
Rf
(n-butanol/acetic
acid/pyridine/
H2O=4:1:1:2)=0.55; 1H NMR (500MHz, DMSO-d6) d:
10.85 (1H, s, 1-NH of indole), 9.05 (1H, s, 4-OH of Tyr),
8.42 (1H, d, J=8.3Hz, a-NH of Lys), 8.19 (1H, d,
J=8.7Hz, a-NH of D-Trp), 7.96 (2H, s, e-NH2 of Lys),
7.81 (1H, d, J=6.4Hz, 6-CH2CH2CH2CH2NH of pyraz-
inone), 7.73 (1H, d, J=7.8Hz, 4-CH of indole), 7.50 (1H,
d, J=8.8Hz, a-NH of Tyr), 7.30 (1H, d, J=8.0Hz, 7-CH
of indole), 7.18 (1H, d, J=1.8Hz, 2-CH of indole), 7.05
(1H, t, J=7.4Hz, 6-CH of indole), 6.98 (1H, t, J=7.4Hz,
5-CH of indole), 6.70 (2H, d-like, J=8.3Hz, 2, 6-OH of
Tyr), 6.47 (2H, d-like, J=8.3Hz, 3, 5-CH of Tyr), 4.72
(1H, td, J=8.9, 5.3Hz, a-CH of Tyr), 4.59 (1H, td, J=8.6,
4.8Hz, a-CH of D-Trp), 4.15 (1H, td, J=8.3, 5.9Hz, a-CH
of Lys), 3.57 (1H, m, 6-CH2CH2CH2CH2NH of pyrazi-
none), 2.97(1H, dd, J=14.3, 5.1Hz, b-CH2 of D-Trp),
2.91–2.87 (1H, m, 3-CH2CH2CO of pyrazinone), 2.84 (1H,
dd, J=14.3, 9.6Hz, b-CH2 of D-Trp), 2.69 (2H, br, e-CH2
of Lys), 2.63–2.50 (3H, m, 3-CH2CH2CO of pyrazinone
and 6-CH2CH2CH2CH2NH of pyrazinone), 2.39 (1H,
dd, J=13.5, 4.5Hz, b-CH2 of Tyr), 2.31 (1H, dd, J=
13.5, 8.5Hz, b-CH2 of Tyr), 2.14 (1H, m, 6-
CH2CH2CH2CH2NH of pyrazinone), 2.04 (1H, d,
J=14.4Hz, 3-CH2CH2CO of pyrazinone), 1.90 (3H, s,
5-CH3 of pyrazinone), 1.55–1.30 (8H, m, 6-
CH2CH2CH2CH2NH of pyrazinone, b-CH2 and d-CH2
of Lys), 1.22–1.07 (2H, m, c-CH2 of Lys); MS (M+H)+
Calcd 713.8. Found. 713.2. Anal. Calcd for C38H49ClN8O6
(+3H2O): C, 56.8; H, 6.90; N, 14.0. Found: C, 56.7; H,
6.88; N, 14.2.
Acknowledgement
The authors thank to Dr. Lawrence H. Lazarus of Na-
tional Institute of Environmental Health Sciences, USA
for his helpful advice to prepare this manuscript.
References and notes
1. Brazeau, P.; Vale, W.; Burgus, R.; Ling, N.; Bucher, M.;
River, J.; Guillemin, R. Science 1973, 179, 77–99.
2. Reichlin, D. N. Engl. J. Med. 1983, 309, 1495–1501.
3. Moreau, S. C.; Murphy, W. A.; Coy, D. H. Drug Develop.
Res. 1991, 22, 79–93.
4. Bauer, W.; Briner, U.; Doepfner, W.; Haller, R.; Hugue-
nin, R.; Marbach, P.; Petcher, T. J. Life Sci. 1982, 31,
1133–1140.
5. Kaupmann, K.; Bruns, C.; Raulf, F.; Weber, H. P.;
Mattes, H.; Lubbert, H. EMBO J. 1995, 14, 727–735.
6. Keri, G.; Mezo, I.; Vadasz, Z.; Horvath, A.; Idei, M.;
Vantus, T.; Balogh, A.; Bokonyui, G.; Bajor, T.; Teplan,
I.; Tamas, J.; Mak, M.; Horvath, J.; Csuka, O. Pept. Res.
1993, 6, 281–288.
7. Okada, Y.; Fukumizu, A.; Takahashi, M.; Yamazaki, J.;
Yokoi, T.; Tsuda, Y.; Bryant, D. S.; Lazarus, H. L.
Tetrahedron 1999, 55, 14391–14406.
8. Jinsmaa, Y.; Okada, Y.; Tsuda, Y.; Shiotani, K.; Sasaki,
Y.; Ambo, A.; Bryant, D. S.; Lazarus, H. L. J. Pharmacol.
Exp. Ther. 2004, 309, 432–438.
9. Jinsmaa, Y.; Miyazaki, A.; Fujita, Y.; Fujisawa, Y.;
Shiotani, K.; Li, T.; Tsuda, Y.; Yokoi, T.; Ambo, A.;
Sasaki, Y.; Bryant, D. S.; Lazarus, H. L.; Okada, Y.
J. Med. Chem. 2004, 47, 2599–2610.
10. Oliver, M. H.; Harrison, N. K.; Bishop, J. E.; Cole, P. J.;
Laurent, G. J. J. Cell Sci. 1989, 92, 513–518.
11. c[(Lys-Glu)-Tyr-D-Trp-Lys]ÆHCl (I): (Lys-Glu) shows 3-
(4-aminobutyl)-6-(2-carboxyethyl)-5-methyl-2(1H)-pyrazin-
13. c[(CH2)4-Tyr-D-Trp-Lys]ÆHCl (III): Yield 14.7mg (49.2%):
25
amorphous; ½aꢂD ꢀ 22:3 (c 1.0, H2O); Rf (n-butanol/acetic
acid/pyridine/H2O=4:1:1:2)=0.60; 1H NMR (500MHz,
DMSO-d6) d: 10.93 (1H, s, 1-NH of indole), 9.22 (1H, s, 4-
OH of Tyr), 8.66 (1H, d, J=6.9Hz, a-NH of D-Trp), 8.32
(1H, d, J=8.1Hz, a-NH of Lys), 8.00 (1H, d, J=8.7Hz, a-
NH of Tyr), 7.51 (1H, d, J=7.8Hz, 4-CH of indole), 7.34
(1H, d, J=8.0Hz, 7-CH of indole), 7.20 (1H, t, J=5.5Hz,
NH of Val), 7.17 (1H, d, J=1.3Hz, 2-CH of indole), 7.06
(1H, t, J=7.5Hz, 6-CH of indole), 7.01 (2H, d, J=8.4Hz,
2, 6-OH of Tyr), 6.98 (1H, t, J=7.4Hz, 5-CH of indole),
25
one. Yield 75.5mg (34.0%): amorphous; ½aꢂD þ 75:4 (c

A. Miyazaki et al. / Tetrahedron Letters 45 (2004) 6323–6327
6327
6.65 (2H, d, J=8.4Hz, 3, 5-CH of Tyr), 4.53 (1H, td,
J=9.1, 5.3Hz, a-CH of Tyr), 4.40 (1H, q-like, J=7.0Hz,
a-CH of D-Trp), 3.90 (1H, m, a-CH of Lys), 3.11 (1H, dd,
J=14.3, 8.7Hz, b-CH2 of D-Trp), 3.04 (1H, dd, J=13.0,
6.5Hz, d-CH2 of Val), 2.97 (1H, dd, J=13.0, 5.8Hz, d-
CH2 of Val), 2.84 (1H, dd, J=14.3, 6.5Hz, b-CH2 of D-
Trp), 2.74 (1H, dd, J=13.8, 5.2Hz, b-CH2 of Tyr), 2.64
(1H, dd, J=13.8, 9.0Hz, b-CH2 of Tyr), 2.63–2.59 (1H, m,
e-CH2 of Lys), 2.18 (1H, m, a-CH2 of Val), 1.84 (1H, dt,
J=12.7, 4.6Hz, a-CH2 of Val), 1.67–1.52 (2H, m, b-CH2
of Lys and c-CH2 of Val), 1.48–1.30 (5H, m, b-CH2, d-
CH2 of Lys and b-CH2 of Val), 1.28–1.18 (1H, m, c-CH2
of Val), 0.92 (2H, q, J=7.6Hz, c-CH2 of Lys); MS
(M+H)+ Calcd 577.7. Found. 577.8. Anal. Calcd for
C31H41ClN6O5 (+3H2O): C, 60.7; H, 6.74; N, 13.7. Found:
C, 61.0; H, 6.75; N, 14.0.
CH of indole), 7.20 (1H, d, J=1.1Hz, 2-CH of indole),
7.06 (1H, t, J=7.5Hz, 6-CH of indole), 6.99 (1H, t,
J=7.5Hz, 5-CH of indole), 6.97 (2H, d, J=8.3Hz, 2, 6-
CH of Tyr), 6.61 (2H, J=8.3Hz, 3, 5-OH of Tyr), 4.54
(1H, td, J=9.0, 4.9Hz, a-CH of Tyr), 4.28 (1H, q,
J=7.1Hz, a-CH of D-Trp), 3.91 (1H, td, J=10.2, 3.9Hz,
a-CH of Lys), 3.14–3.09 (1H, m, x-CH2 of hep), 3.07 (1H,
dd, J=14.1, 7.8Hz, b-CH2 of D-Trp), 2.91 (1H, dd,
J=14.1, 6.8Hz, b-CH2 of D-Trp), 2.90–2.87 (1H, m, x-
CH2 of hep), 2.75–2.70 (1H, m, a-CH2 of hep), 2.64–
2.59(1H, m, a-CH2 of hep), 1.70–1.62 (1H, m, b-CH2 of
Lys), 1.58–1.34 (5H, m, b-CH2, e-CH2 of hep and d-CH2
of Lys), 1.31–1.05 (6H, m, c-CH2, d-CH2, e-CH2 of hep
and b-CH2 of Lys), 0.85 (2H, q, J=7.2Hz, c-CH2 of Lys);
MS (M+H)+ Calcd 605.7. Found. 605.9. Anal. Calcd for
C33H45ClN6O5 (+1.4H20): C, 59.5; H, 7.23; N, 12.6.
Found: C, 59.2; H, 7.34; N, 12.8.
14. c[(CH2)6-Tyr-D-Trp-Lys]ÆHCl (IV): Yield 7.10mg (55.5%):
25
amorphous; ½aꢂD ꢀ 167 (c 1.0, H2O); Rf (n-butanol/acetic
15. Wynants, C.; Van, B. G.; Loosli, H. R. Int. J. Pept.
Protein Res. 1985, 25, 622–627.
16. Melacini, G.; Zhu, Q.; Goodman, M. Biochemistry 1997,
36, 1233–1241.
17. Freidinger, M. R.; Perlow, S. D.; Randall, C. W.;
Saperstein, R.; Arison, H. B.; Veber, F. D. Int. J. Pept.
Protein Res. 1984, 23, 142–150.
acid/pyridine/H2O=4:1:1:2)=0.37; 1H NMR (500MHz,
DMSO-d6) d: 10.94 (1H, s, 1-NH of indole), 9.20 (1H, s, 4-
OH of Tyr), 8.46 (1H, d, J=5.7Hz, a-NH of D-Trp), 8.32
(1H, d, J=8.3Hz, a-NH of Lys), 7.93 (1H, d, J=8.2Hz, a-
NH of Tyr), 7.54 (1H, d, J=7.8Hz, 4-CH of indole), 7.45
(1H, t, J=5.7Hz, NH of hep), 7.35 (1H, d, J=8.0Hz, 7-