Synthesis and antinociceptive activity of orally active opioid peptides: Improvement of oral bioavailability by esterification

Article abstract of DOI:10.1248/cpb.51.759

To improve the oral bioavailability of a dermorphin tetrapeptide analog, N^α-1-iminoethyl-Tyr-D-MetO-Phe-MeβAla-OH (III), ¹) which has a potent analgesic activity after oral administration, various derivatives were synthesized to incr

Full text of DOI:10.1248/cpb.51.759

July 2003
Chem. Pharm. Bull. 51(7) 759—771 (2003)
759
Synthesis and Antinociceptive Activity of Orally Active Opioid Peptides:
Improvement of Oral Bioavailability by Esterification
Tadashi OGAWA,a Mamoru ARAKI,a Tetsuhisa MIYAMAE,a Toru OKAYAMA,a Masaki HAGIWARA,a
b
,a
*
Shinobu SAKURADA, and Tadanori MORIKAWA
a Research Institute, Daiichi Fine Chemical Co., Ltd.; 530 Chokeiji, Takaoka, Toyama 933–8511, Japan: and b Department
of Physiology and Anatomy, Tohoku Pharmaceutical University; 4–4–1 Komatsushima, Aoba-ku, Sendai 981–8558, Japan.
Received December 17, 2002; accepted April 24, 2003; published online May 6, 2003
To improve the oral bioavailability of a dermorphin tetrapeptide analog, Na-1-iminoethyl-Tyr-D-MetO-Phe-
MebAla-OH (III),1) which has a potent analgesic activity after oral administration, various derivatives were syn-
thesized to increase lipophilicity by esterification of the C-terminal carboxyl group and/or acylation of the phe-
nolic hydroxyl group on Tyr1. Antinociceptive activity was evaluated after subcutaneous or oral administration
using the mouse tail pressure test. As a result, increased antinociceptive activity after oral administration as well
as an improved ED50(p.o.)/ED50(s.c.) ratio, which is an indicator of oral bioavailability, were found for some com-
pounds. With regard to the improvement of bioavailability, derivatives with acylation of the phenolic hydroxyl
group on Tyr1 showed better results than derivatives with esterification of the C-terminal carboxyl group. In par-
ticular, an ED50(p.o.)/ED50(s.c.) ratio equivalent to that of morphine was found for an acetylated derivative, Na-1-
iminoethyl-Tyr(COMe)-D-MetO-Phe-MebAla-OH (7a), as well as for a methoxycarbonylated derivative, Na-1-
iminoethyl-Tyr(CO2Me)-D-MetO-Phe-MebAla-OH (7l).
Key words dermorphin tetrapeptide analog; oral bioavailability; lipophilicity; chemical modification
Dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2)2) was veloped, and some success has been achieved.20,21)
originally isolated from the skin of a South American frog
Dermorphin tetrapeptide analogues are known to be highly
(Phyllomedusa sauvagei). It shows a potent affinity and se- stable against enzymatic degradation.22—24) Considering the
lectivity towards opioid receptors exceeding that of mor- stability of the compound itself, the low oral bioavailability
phine, especially for m-opioid receptors, and possesses strong of compounds I—III was inferred to be due to low intestinal
opioid activity. The smallest dermorphin sequence that absorption. Compounds I—III have a very high water solu-
demonstrates activity is the N-terminal tetrapeptide (Tyr-D- bility and we assumed that this was related to low intestinal
Ala-Phe-Gly).3) A number of tetrapeptide analogs have al- absorption. This led to the assumption that intestinal absorp-
ready been synthesized and structure–activity relationship tion might be improved by modifying these peptides to in-
(SAR) studies have been performed on opioid activity, recep- crease lipophilicity. Here we report on improvement of the
tor affinity, and selectivity.4,5) Since replacement of D-Ala antinociceptive activity and oral bioavailability after s.c. and
with D-Arg at position 2 was found to lead to a dramatic p.o. administration in mice using various analogues that were
increase of activity,6) many [D-Arg2]-dermorphin derivatives synthesized to have increased lipophilicity by esterification
have been synthesized and intensive SAR studies have of the C-terminal carboxyl group or acylation of the phenolic
also been performed.7—10) Using the tail pressure test in hydroxyl group on Tyr1 of compound III (Fig. 1).
mice, we recently found that subcutaneous (s.c.) and oral
(p.o.) administration of Na-amidino-Tyr-D-Arg-Phe-MebAla- Chemistry
OH (I: ADAMB), Na-1-iminoethyl-Tyr-D-Arg-Phe-MebAla-
All analogues were synthesized via the solution method
OH (II) or Na-1-iminoethyl-Tyr-D-MetO-Phe-MebAla-OH using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy-
(III), achieved extremely strong and long-lasting antinoci- drochloride (EDC·HCl) with 1-hydroxybenzotriazole (HOBt)
ceptive activity after p.o. administration, which was superior as the coupling reagent.
to that of morphine.11,12) Although constipation is a common
Esterification of the C-Terminal Carboxyl Group C-
side effect of morphine, compound III shows a weaker cos- Terminal ester derivatives were prepared through 3ϩ1 con-
tive effect.12) However, these compounds with a strong an- densation by coupling the N-terminal tripeptide segment
tinociceptive activity show low oral bioavailability, which has
posed a problem.
Generally, peptide pharmaceuticals show very low intesti-
nal absorbability after p.o. administration. This is thought to
be due to the presence of many functional groups that form
hydrogen bonds, such as amide bonds. It may also be due to
the fact that membrane permeability is low for compounds
with a high molecular weight (ϾM.W. 500), or because pep-
tides are more susceptible to various hydrolytic enzymes in
the intestines.13—19) Accordingly, to improve the intestinal ab-
sorption of peptide pharmaceuticals, various absorption en-
hancers, peptidase inhibitors, and methods to alter molecular
structure of peptides by chemical modification have been de-
Fig. 1. Modification of the Chemical Structure of a [D-MetO2]dermorphin
Tetrapeptide Analogue (Compound III)
To whom correspondence should be addressed. e-mail: morikawa@daiichi-fcj.co.jp
© 2003 Pharmaceutical Society of Japan

760
Vol. 51, No. 7
Boc-Tyr-D-MetO-Phe-OH (1) with H-MebAla-OX (Chart 1).
A fully protected tetrapeptide (3a—u) was treated with 90%
trifluoroacetic acid (TFA) or 90% HCOOH to remove the
Boc group, and then the N-terminus was 1-iminoethylated by
treatment with ethyl acetimidate hydrochloride in the pres-
ence of triethylamine (TEA).
O-Acylation of the Hydroxy Group on Tyrosine De-
rivatives with O-acylation of Tyr1 were obtained via prepara-
tion of the tetrapeptide Boc-Tyr-D-MetO-Phe-MebAla-OBut
(5) and subsequent acylation of the phenol hydroxyl group
with acid chloride; Y-COCl or carboxylic acid; Y-COOH
(Chart 2). The fully protected tetrapeptide (6a—p) was
treated with 90% TFA to remove the Boc group, and then the
N-terminus was 1-iminoethylated.
Chart 1. Synthesis of C-Terminal Esterified [D-MetO2]dermorphin
Tetrapeptide Analogues
Dual-esterification of Tyr1 and the C-Terminus Ana-
logues having both O-acylation and C-terminal esterification
were prepared by condensation of the tripeptide Boc-Tyr-D-
MetO-Phe-OH (1) with H-MebAla-OX, followed by acyla-
tion with acid chloride; Y-COCl or carboxylic acid; Y-COOH
(Chart 3). The fully protected tetrapeptide (8a—g) was
treated with 90% TFA to remove the Boc group, and then the
N-terminus was 1-iminoethylated.
All crude products were purified by reversed-phase flush
column chromatography and eluted with aqueous CH3CN
containing 0.1% acetic acid to afford the purified products
(Ͼ95% purity by HPLC), which were subsequently
lyophilized to give the desired peptides in powder form. The
1
structure of each product was confirmed from the H-NMR
and FAB mass spectra.
Antinociceptive Assay The synthesized compounds
were tested for antinociceptive activity in vivo (Tables 1—3)
using the tail pressure test in mice after subcutaneous (s.c.)
or oral (p.o.) administration. The percentage of the maximum
possible effect (% MPE) value was measured at fixed doses
(1 mg/kg s.c. and 10 mg/kg p.o.) to initially evaluate the syn-
thesized compounds. Then, analogues with a high activity
underwent determination of the ED50 value by the method of
Litchfield and Wilcoxon25) to assess their antinociceptive ac-
tivity. All values were calculated from data obtained at the
time of peak effect after administration of either the peptides,
or morphine. Furthermore, the ED50 dose ratio (p.o./s.c.) was
calculated and compared with that of morphine or compound
III to assess oral bioavailability.
Chart 2. Synthesis of O-Acylated [D-MetO2]dermorphin Terapeptide Ana-
logues
Results and Discussion
Chart 3. Synthesis of Dual-esterified [D-MetO2]dermorphin Tetrapeptide
Analogues
Esterification of the C-Terminal Carboxyl Group The
carboxyl group located at the C-terminus of compound III is
easily ionized, leading to reduced lipophilicity of the com-
pound and substantially hindering gastrointestinal absorp-
Table 1 shows a comparison of the antinociceptive activity
tion. Therefore, it was expected that protection of this car- of compounds 4a—u with that of morphine. Relative oral
boxyl group by esterification would improve bioavailability bioavailability was also evaluated using the ED50 dose ratio
by preventing ionization and increasing lipophilicity. Since after s.c. and p.o. administration. From comparison of the
various esterases exist in the body, particularly in the in- ED50 values, the antinociceptive activity of compounds 4a—
testines, it was anticipated that the appropriately esterified u, synthesized by esterification of the C-terminal carboxyl
compound would be hydrolyzed and converted to the parent group of compound III, was lower than that of the parent
compound after intestinal absorption. Thus, esterification compound after both s.c. and p.o. administration. However,
was expected to increase gastrointestinal absorption while the ED50 values obtained after s.c. administration of hexade-
maintaining a strong antinociceptive activity like that of the cylated compound 4k and compound 4t (which has a 1Ј-
original compound. Accordingly, compounds 4a—u were ethoxycarbonyloxyethyl group26) and is a successful example
synthesized by esterification of the C-terminal carboxyl group of a prodrug developed from a b-lactam antibiotic) were
of compound III.
larger than that for compound III, although the values for

July 2003
761
Table 1. Analgesic Activity of Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-OX
MPE %
ED50 (mg/kg)a,b)
No.
X
s.c. 1 mg/kg
p.o. 10 mg/kg
s.c.
p.o.
p.o./s.c.
Morphine
4.9
100
12.9
96.2
3.3
0.08
22.2
3.1
7
39
III
-H
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
-Me
-Et
-Bu
-Pen
-Hex
-Hep
-Oct
-Non
-Dec
-Dde
-Hde
-CH2CF3
-Ph
96.9
100
100
100
100
98.8
100
100
100
98.7
78.1
93.4
100
100
100
96.9
91.0
100
100
97.4
99.7
81.0
93.6
58.4
66.3
79.8
62.5
80.8
21.7
56.1
54.4
95.8
75.3
75.1
63.2
37.6
38.4
44.5
12.9
88.5
94.4
79.2
0.12
0.27
0.23
0.17
0.22
0.13
0.24
0.22
0.25
0.26
0.38
0.15
0.23
Ϲ0.25
0.20
0.27
0.36
Ϲ0.5
0.28
0.21
0.32
5.2
5.6
43
21
6.0
25
3.6
4.1
10
27
-Bzl
-cHex
-CH2(cHex)
-But
-1-Ad
-POM
-CH(CH3)OCO2Et
-Phthalidyl
6.9
3.6
5.0
25
17
16
4t
4u
a) Data are given as the mean value for groups of 10 mice. b) ED50 values were calculated from data obtained at the time of peak effect after administration.
p.o. administration were about the same. The ED50(p.o.)/ED50 respectively, which were close to the value for morphine.
(s.c.) ratio was 10 and 17, respectively, showing improve- These compounds showed improved dose-activity ratios and
ment compared with the ratio of 39 for parent compound III. exhibited analgesic activity after oral administration that was
Therefore, it seems likely that esterification attenuated the equal to or greater than that of compound III. This was as-
antinociceptive activity, but oral antinociceptive activity was sumed to be due to the improvement of oral bioavailability.
maintained at a comparable level to that of compound III as On the other hand, activity was slightly decreased after s.c.
a consequence of improved oral bioavailability. As other ex- administration. This was assumed to be due to increased
amples of prodrugs, compound 4s with a pivaloyloxymethyl lipophilicity of the compound because of acylation, which
group27) and compound 4u with a phthalidyl group,28) also may have affected diffusion of the drug from the s.c. admin-
showed the same trend, although these compounds had a istration site, uptake into the blood, and binding with plasma
lower antinociceptive activity for after s.c. or p.o. administra- proteins.
tion than 4t.
Dualesterification at the Hydroxy Group of Tyrosine
O-Acylation of the Hydroxyl Group of Tyrosine It is and C-Terminal Carboxyl Group Esterification of the C-
well known that esterification of the phenolic hydroxyl group terminal carboxyl group or acylation of the Tyr1 phenolic hy-
in morphine-related compounds, such as heroin (synthesized droxyl group improved the oral bioavailability of some com-
by acetylation of the hydroxyl group of morphine), increases pounds. Combining the two substituents that improved oral
their lipophilicity and allows significantly improved perme- bioavailability was expected to have an augmented effect.
ation of the blood brain barrier (BBB), that leads to several- Therefore, to achieve further improvement, we synthesized
fold augmentation of analgesic activity.29,30) There also is a esterified and acylated compounds 9a—g.
phenolic hydroxyl group at Tyr1 of compound III. This hy-
Table 3 shows the antinociceptive activity and ED50(p.o.)/
drophilic group was thought to increase water solubility and ED50(s.c.) ratio of the esterified and acylated compounds
thus hinder gastrointestinal absorption. Therefore, as with es- 9a—g. Contrary to our expectations, comparison of the dose-
terification of the C-terminus, acylation of the hydroxyl activity ratios revealed that the antinociceptive activity of the
group of Tyr was expected to improve lipophilicity and gas- doubly modified compounds 9a—g was not increased. How-
trointestinal absorption. Accordingly, we synthesized com- ever, the ED50 value obtained after s.c. administration of
pounds with various acyl groups attached to the phenolic hy- compound 9c, with an acetylated phenolic hydroxyl at Tyr1
droxyl group of the Tyr1 residue.
and a 1Ј-ethoxycarbonyloxyethyl group at the C-terminal
Table 2 shows the antinociceptive activity and ED50(p.o.)/ carboxyl group, was larger than that of compound III, but
ED50(s.c.) ratio of O-acylated compounds 7a—p. From com- the ED50(p.o.)/ED50(s.c.) ratio was 11, showing an improve-
parison of the dose-activity ratios, it is apparent that the oral ment from that of parent compound III (which was 39).
bioavailability of these acylated compounds (7a—p) was
Comparison of the Duration of Antinociceptive Activ-
clearly higher than that of the parent compound, which had a ity Figure 2 shows the time course of antinociceptive activ-
value of 39. In particular, the acetylated compound 7a and ity after s.c. administration of morphine and compound III in
the methoxycarbonylated compound 7l had values of 9 and 5, the mouse tail pressure test. Compound III (Fig. 2B) showed

762
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Table 2. Analgesic Activity of Na-Iminoethyl-Tyr(COY)-D-MetO-Phe-MebAla-OH
MPE %
ED50 (mg/kg)a,b)
No.
Y
s.c. 1 mg/kg
p.o. 10 mg/kg
s.c.
p.o.
p.o./s.c.
Morphine
4.9
100
12.9
96.2
3.3
0.08
22.2
3.1
7
39
III
—
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
-Me
-Et
100
100
100
100
98.5
100
94.1
96.5
100
98.0
89.1
100
91.5
100
96.3
100
94.4
96.9
99.2
98.8
86.6
91.0
52.4
95.2
91.1
89.6
76.9
99.1
93.8
94.7
98.9
92.1
0.14
0.20
0.26
0.20
0.33
0.28
0.38
0.21
0.25
0.30
0.21
0.25
0.25
0.21
0.21
0.23
1.2
2.2
2.9
2.1
4.9
3.5
9
11
11
11
15
13
-Pr
-Pri
-Hep
-But
-1-Ad
-Ph
3.3
3.0
3.5
4.4
1.3
4.3
3.5
4.3
4.2
16
12
12
21
5
17
17
20
18
-(CH2)2Ph
-(CH2)3Ph
-cHex
-OMe
-OEt
-OPri
-OPh
-OBzl
a) Data are given as the mean value for groups of 10 mice. b) ED50 values were calculated from data obtained at the time of peak effect after administration.
Table 3. Analgesic Activity of Na-1-Iminoethyl-Tyr(COY)-D-MetO-Phe-MebAla-OX
MPE %
ED50 (mg/kg)a,b)
No.
Y
X
s.c. 1 mg/kg
p.o. 10 mg/kg
s.c.
p.o.
p.o./s.c.
Morphine
4.9
100
12.9
96.2
3.3
0.08
22.2
3.1
7
39
III
—
-H
9a
9b
9c
9d
9e
9f
-Me
-Me
-Me
-Me
-OEt
-OBui
-OBui
-Oct
-Hde
-CH(CH3)OCOOEt
-Phthalidyl
-Oct
90.2
88.5
93.8
99.7
83.7
76.2
63.2
94.9
79.2
38.1
60.4
30.4
0.34
6.0
18
0.25
0.32
0.50
0.22
2.7
5.0
11
16
-Bu
-Oct
100
44.0
9g
a) Data are given as the mean value for groups of 10 mice. b) ED50 values were calculated from data obtained at the time of peak effect after administration.
Fig. 2. Time Course of the Antinociceptive Effect of Subcutaneous Morphine (A) and Compound III (B) in the Mouse Tail Pressure Test
The doses used are shown in the figure. Data are given as the meanϮS.E.M. for groups of 10 mice.
a higher dose-dependent antinociceptive activity than mor- was even maintained at 10 h after administration of 5.0
phine (Fig. 2A). The duration of activity of compound III mg/kg, whereas the effect disappeared almost completely
was over 8 h after administration of 0.25 mg/kg, but the activ- within 6 h after 80 mg/kg of morphine (Fig. 3A).
ity of morphine ended within 4 h, even after administration
of 10 mg/kg.
Figure 4 shows the time course of antinociceptive activity
after s.c. administration of compound 4k (Fig. 4A) and com-
As can be seen in Fig. 3B, compound III showed a strong pound 7a (Fig. 4B), both of which showed improved ab-
dose-dependent antinociceptive effect after p.o. administra- sorbability. The time course of activity for compound 4k
tion. An antinociceptive effect equivalent to 30% of MPE (Fig. 4A) and compound 7a (Fig. 4B) resembled that of par-

July 2003
763
Fig. 3. Time Course of the Antinociceptive Effect of Oral Morphine (A) and Compound III (B) in the Mouse Tail Pressure Test
The doses used are shown in the figure. Data are given as the meanϮS.E.M. for groups of 10 mice.
Fig. 4. Time Course of the Antinociceptive Effect of Subcutaneous 4k (A)
and 7a (B) in the Mouse Tail Pressure Test
Fig. 5. Time Course of the Antinociceptive Effect of Oral 4k (A) and 7a
(B) in the Mouse Tail Pressure Test
The doses used are shown in the figure. Data are given as the meanϮS.E.M. for
groups of 10 mice.
The doses used are shown in the figure. Data are given as the meanϮS.E.M. for
groups of 10 mice.
ent compound III. Although a time lag before the onset of
antinociceptive activity and prolongation of its duration were
expected judging from the time necessary for metabolism of
ester and acyl derivatives involving the hydrolysis of ester
and acyl groups, no such effects were seen.
sary for metabolism of the compounds, such as hydrolysis of
the ester and acyl groups.
In conclusion, we synthesized various derivatives with im-
proved lipophilicity based on Na-1-iminoethyl-Tyr-D-MetO-
Phe-MebAla-OH (compound III), which has a strong an-
tinociceptive activity, by esterification of the C-terminal car-
boxyl group and acylation of the phenolic hydroxyl group on
Tyr1 in order to improve intestinal absorption. We then inves-
tigated the improvement of antinociceptive activity and as-
sessed the oral bioavailability of these compounds. As a re-
sult, an increase of antinociceptive activity after was seen
p.o. administration of some of the compounds. Acylation of
the phenolic hydroxyl group on Tyr1 improved absorption
more than esterification of the C-terminal carboxyl group. In
particular, short chain substituents such as acetyl groups
(compound 7a) and methoxycarbonyl groups (compound 7l)
achieved an oral bioavailability equivalent to that of mor-
phine. However, oral bioavailability was estimated using
ED50(p.o.)/ED50(s.c.) ratios in this study. In the future, there
Figure 5 shows the time course of antinociceptive activity
after p.o. administration of compound 4k (Fig. 5A) and com-
pound 7a (Fig. 5B). The peak antinociceptive effect of com-
pound III after a dose of 5.0 mg/kg was around 3 h, whereas
it was around 4 h for compound 4k (Fig. 5A). The maximum
%MPE for both compounds III and 4k was 65%. The maxi-
mum %MPE after administration of compound III at
2.5 mg/kg was around 40%, whereas it was about 90% for
compound 7a (Fig. 5B), and clearly showed an increase of
antinociceptive activity. Therefore, after p.o. administration,
compound 7a would show better absorption than compound
III. In addition, the peak antinociceptive effect of compound
4k and compound 7a was seen around 5 h after administra-
tion of 2.5 mg/kg. Thus, the peak was later than for com-
pound III and this was thought to be due to the time neces-

764
Vol. 51, No. 7
action mixture was stirred for 1 h at 0 °C and then stirred overnight at room
temperature. The reaction mixture was subsequently concentrated in vacuo,
and dissolved with EtOAc, after which the solution was washed with 1 N-
HCl, sat. NaHCO3, and brine. The organic phase was dried over MgSO4, fil-
tered, and evaporated in vacuo. The residue was purified by flush column
is the necessity of evaluating by the concentration measure-
ment in blood, the membrane permeability assay using Caco-
2 cell monolayer system, etc. On the other hand, when both
esterification and acylation were performed (anticipating a
synergistic effect), there was a significant decrease of an- chromatography.
Method D (Deprotection 1) The Boc-protected amino acid or peptide
tinociceptive activity after both s.c. and p.o. administration.
In general, passive diffusion across membranes, is highly de-
pendent on the molecular weight of a compound and if the
molecular weight is too high, membrane permeability will
decrease. Increasing the lipophilicity improves the BBB per-
meability and intestinal absorption of drugs. However, using
large substituents may increase the molecular weight and re-
duce the water solubility, resulting in poor intestinal absorp-
tion. Some of the ester and acyl groups investigated in this
study have a large enough ester moiety to markedly increase
the molecular weight, so our failure to obtain the anticipated
increase of absorption might be inevitable. Therefore, to
achieve higher oral bioavailability, it will be necessary to use
substituents with an appropriate balance between lipophili-
city and water solubility, along with concepts such as the
‘rule of 5’31) or ‘chemical delivery systems’.32) The knowl-
edge gained from this study about peptide chemical modifi-
cations and oral bioavailability will be useful for developing
future oral peptidergic analgesics.
was treated with 4 N-HCl/EtOAc (1—3% v/w) for 0.5—1 h at room tempera-
ture to remove the Boc group. Ether was added to the solution and the pre-
cipitated solid was separated by filtration and dried in a vacuum desiccator
over solid NaOH.
Method E (Deprotection 2) The Boc protected amino acid tert-butyl
ester or the N-Boc protected peptide with C-terminal tert-butyl ester was
treated with 90%-HCOOH (1—3% v/w) for 3—5 h at room temperature to
remove only the Boc group. The solvent was evaporated in vacuo at 20 °C.
The residue was dissolved in EtOAc, then washed with sat. NaHCO3, and
brine. The organic phase was dried over MgSO4, filtered, and evaporated in
vacuo. The residue was used for subsequent condensation without purifica-
tion.
Method F (O-Acylation 1) To a solution of the protected peptide with a
free hydroxy group of tyrosine (1.0 eq) in DMF (2—5% v/w), the acid chlo-
ride (1.1 eq) was added at 0 °C. Then TEA (1.2 eq) was added to the solution
at 0 °C. This mixture was stirred for 1 h at 0 °C and then stirred overnight at
room temperature. Then the reaction mixture was diluted with EtOAc, after
which the solution or suspension was washed with 1 N-HCl, sat. NaHCO3,
and brine. The organic phase was dried over MgSO4, filtered, and evaporated
in vacuo. The residue was crystallized from appropriate solvents or purified
by flush column chromatography.
Method G (O-Acylation 2) To a solution of the peptide with a free hy-
droxy group of tyrosine (1.0 eq) in DMF (2—5% v/w), Y-COOH (1.1 eq)
and DMAP (0.1 eq) were added at 0 °C. Then EDC·HCl (1.2 eq) was added
Experimental
Commercial amino acid derivatives, EDC·HCl and HOBt, were obtained to the solution at 0 °C, followed by stirring for 1 h at 0 °C and then stirred
from Kokusan Chemical Works, Ltd. (Tokyo, Japan) or Watanabe Chemical overnight at room temperature. Subsequently, the reaction mixture was di-
Industries, Ltd. (Hiroshima, Japan). Other reagents and solvents were pur- luted with EtOAc, after which the solution or suspension was washed with
chased from Aldrich Chemicals (WI, U.S.A.). Thin-layer chromatography 1 N-HCl, sat. NaHCO3, and brine. The organic phase was dried over MgSO4,
was performed on silica plates (0.25 mm; Merck, 60 F254) with the following filtered, and evaporated in vacuo. The residue was crystallized from appro-
solvent systems: Rf1 CHCl3/MeOH (10 : 1, v/v); Rf2 CHCl3/MeOH/acetic priate solvents or purified by flush column chromatography.
acid (95 : 5 : 3, v/v/v); Rf3 CHCl3/MeOH/acetic acid (80 : 10 : 5, v/v/v); Rf4 n-
Method H (1-Iminoethylation of the N-Terminus and Final Purifica-
butanol/acetic acid/water/pyridine (15 : 3 : 10 : 12, v/v/v/v). Flush chromatog- tion) The fully-protected peptide was treated with 90%–TFA (1—3% v/w)
raphy was performed on silica gel BW-300 from Fuji Silysia Chemical Ltd. or 90%–HCOOH (1—3% v/w, if it was tert-butyl ester) for 1—5 h at room
(Aichi, Japan). Purification of the final products was achieved by flush chro- temperature to remove the Boc group. The reaction mixture was concen-
matography using a reversed-phase silica gel Chromatorex ODS DM1020T trated in vacuo, and the residue was dissolved in DMF (2—5% v/w), after
from Fuji Silysia Chemical Ltd. (Aichi, Japan), which was eluted by a step- which ethyl acetimidate hydrochloride (2.0 eq) and TEA (3.0 eq) were
wise gradient of acetonitrile (starting from 1—5% and increasing stepwise added. This solution was stirred at room temperature for 2—4 h and then the
by 1—2%) in 0.1 M acetic acid. Analytical high-pressure liquid chromatogra- reaction mixture was concentrated in vacuo. The residue was purified by
phy was done on a Nucleosil 100 5C18 column (4.6ϫ150 mm) with an Agi-
lent 1100 HPLC system. The products were analyzed using a linear gradient
ODS chromatography with a stepwise gradient of acetonitrile (starting from
1—5% and increasing stepwise by 1—2%) in 0.1 N-acetic acid as the eluting
of 10—90% acetonitrile in 0.1% aqueous TFA over 20 min at a flow rate of solvent and then lyophilized to give a white powder.
1 ml/min and UV detection at 230 and 280 nm. 1H-NMR spectra were
Preparation of Boc-Tyr-D-MetO-Phe-OH (1) A mixture of Boc-D-
recorded with a JEOL JMN-AL300 (300 MHz) spectrometer, using tetra- MetO-OH (39.8 g, 150 mmol), HCl·H-Phe-OMe (32.4 g, 150 mmol) and
methylsilan (TMS) as the internal standard. Mass spectra (FAB-MS) were HOBt (22.3 g, 165 mmol) in DMF (400 ml) was stirred at 0 °C. TEA (15.1 g,
obtained with a JEOL mass spectrometer (model JMS-700).
150 mmol) was added, and then EDC·HCl (34.5 g, 180 mmol) was added to
Method A (Coupling Procedure 1) To a solution of a carboxy compo-
the solution at Ϫ10 °C. This mixture was stirred for 1 h at Ϫ10 °C and then
nent (1.0 eq), the amino component (1.1 eq), HOBt (1.1 eq) in DMF, and stirred overnight at room temperature. Subsequently, the reaction mixture
TEA (1 eq if the amino component was in the protonated form) were added was diluted with EtOAc, and the solution was washed with 1 N-HCl, sat.
at 0 °C. Then, EDC·HCl (1.2 eq) was added to the solution at Ϫ10 °C. This NaHCO3, and brine. The organic phase was dried over MgSO4, filtered, and
mixture was stirred for 30 min at Ϫ10 °C and then stirred overnight at room evaporated in vacuo. The residue was solidified from EtOAc and hexane to
temperature. Then the reaction mixture was diluted with EtOAc. The solu- give Boc-D-MetO-Phe-OMe (55.9 g, 87%) as a white amorphous solid.
tion or suspension was washed with 1 N-HCl, sat. NaHCO3, and brine. The
Boc-D-MetO-Phe-OMe (42.5 g, 100 mmol) was treated with 4 N-HCl/
organic phase was dried over MgSO4, filtered, and evaporated in vacuo. The EtOAc (500 ml) for 30 min at room temperature to remove the Boc group.
residue was crystallized from appropriate solvents or purified by flush col- Ether was added to the solution and the precipitated solid was separated by
umn chromatography.
filtration and dried in a vacuum desiccator over solid NaOH. This amine
Method B (Coupling Procedure 2) To a solution of a carboxy compo- component was dissolved in DMF (750 ml) on an ice bath. To this solution
nent (1.0 eq), HOBt (1.1 eq) in DMF (2—5% v/w), EDC·HCl (1.2 eq) was was added Boc-Tyr-OH (30.9 g, 110 mmol) and HOBt (27 g, 200 mmol).
added to the solution at Ϫ10 °C. This mixture was stirred for 30 min at Neutralization with TEA (12.1 g) was followed by addition of EDC·HCl
Ϫ10 °C. Then the amino component (1.1 eq) in DMF (2—5% v/w) was (23.0 g, 120 mmol) at Ϫ10 °C. The reaction mixture was stirred for 1 h at the
added at Ϫ10 °C, followed by stirring overnight at room temperature. The same temperature, and then stirred overnight at room temperature. Subse-
reaction mixture was diluted with EtOAc. The solution or suspension was quently, the reaction mixture was diluted with EtOAc, and the solution was
washed with 1 N-HCl, sat. NaHCO3, and brine. The organic phase was dried washed with 1 N-HCl, sat. NaHCO3, and brine. The organic phase was dried
over MgSO4, filtered, and evaporated in vacuo. The residue was crystallized over MgSO4, filtered, and evaporated in vacuo. The residue was solidified
from appropriate solvents or purified by flush column chromatography.
from EtOAc and hexane to give Boc-Tyr-D-MetO-Phe-OMe (51.4 g, 87%) as
Method C (Esterification) A mixture of the Boc-protected amino acid a white amorphous solid.
(1.0 eq), alcohol (1.1 eq), and DMAP (0.1 eq) in CH2Cl2 (2—5% v/w) was
stirred at 0 °C. Then, EDC·HCl (1.2 eq) was added to the solution. This re-
To a solution of Boc-Tyr-D-MetO-Phe-OMe (48.0 g, 81.4 mmol) in MeOH
(400 ml), 2 N-NaOH (62 ml) was added and the mixture was stirred at room

July 2003
765
temperature for 2 h. Then the solution was diluted with water, evaporated in g, 10.0 mmol) was reacted with 2,2,2-trifluoroethanol (1.10 g, 11.0 mmol) to
1
vacuo to remove the MeOH, and washed with Et2O. The aqueous layer was give 2l (2.78 g, 98 %). Rf1 0.80. H-NMR (CDCl3) d: 4.62 (2H, m), 3.50
acidified with 1 N-HCl and extracted with EtOAc. The extract was washed
(2H, m), 2.87 (3H, s), 2.52 (2H, t, Jϭ6.8 Hz), 1.42 (9H, s). FAB-MS m/z:
with brine, dried over MgSO4, filtered, and evaporated in vacuo. Finally, the 286 (MϩH)ϩ.
residue was solidified from EtOAc and hexane to give Boc-Tyr-D-MetO-Phe-
Boc-MebAla-OPh (2m) Using method C, Boc-MebAla-OH (1.22 g,
OH (1), 40.1 g (86%) as a white amorphous solid. Rf3 0.48. 1H-NMR 6.0 mmol) was reacted with phenol (678 mg, 7.2 mmol) to give 2m (1.68 g,
1
(CDCl3) d: 7.89—7.30 (1H, m), 7.30—7.10 (5H, m), 6.97 (2H, t, 100%). Rf1 0.80. H-NMR (CDCl3) d: 7.45—6.80 (5H, m), 3.63 (2H, br s),
Jϭ8.0 Hz), 6.72 (2H, t, Jϭ7.7 Hz), 4.82 (1H, m), 4.62 (1H, m), 4.23 (1H, 2.94 (3H, s), 2.51 (2H, t, Jϭ6.9 Hz), 1.48 (9H, s). FAB-MS m/z: 280 (Mϩ
m), 3.17 (1H, m), 2.87 (2H, d, Jϭ7.5 Hz), 2.72 (1H, m), 2.53 (2H, s), 2.50
(1H, s), 2.39 (2H, m), 2.10 (1H, br m), 1.60 (1H, br m), 1.39 (9H, s). FAB-
MS m/z: 576 (MϩH)ϩ.
Preparation of Boc-MebAla-OMe (2a) According to method C, Boc- (2.70 g, 92%). Rf1 0.81. H-NMR (CDCl3) d: 7.34 (5H, m), 5.10 (2H, m),
MebAla-OH (1.0 g, 4.9 mmol) was reacted with MeOH (172 mg, 5.4 mmol). 3.50 (2H, m), 2.87 (3H, s), 2.53 (2H, t, Jϭ6.9 Hz), 1.46 (9H, s). FAB-MS
The crude compound was purified by flush column chromatography eluted m/z: 294 (MϩH)ϩ.
H)ϩ.
Boc-MebAla-OBzl (2n) Using method C, Boc-MebAla-OH (2.03 g,
10.0 mmol) was reacted with benzyl alcohol (1.19 g, 11.0 mmol) to give 2n
1
with CH2Cl2/MeOH (50 : 1, v/v) to give 2a (958 mg, 90%) as an oil. Rf1
0.85. 1H-NMR (CDCl3) d: 3.66 (3H, s), 3.50 (2H, t, Jϭ6.8 Hz), 2.87 (3H, s),
2.54 (2H, t, Jϭ6.9 Hz), 1.46 (9H, s). FAB-MS m/z: 218 (MϩH)ϩ.
Boc-MebAla-OcHex (2o) Using method C, Boc-MebAla-OH (1.22 g,
6.0 mmol) was reacted with cyclohexanol (721 mg, 7.2 mmol) to give 2o
(1.70 g, 100%). Rf1 0.76. H-NMR (CDCl3) d: 4.76 (1H, m), 3.49 (2H, m),
1
Compounds 2b—r were prepared in the same manner (method C) from 2.87 (3H, s), 2.52 (2H, t, Jϭ6.8 Hz), 1.82 (2H, m), 1.72 (2H, m), 1.55—1.24
Boc-MebAla-OH and the corresponding alcohol (X-OH), respectively.
Boc-MebAla-OEt (2b) Using method C, Boc-MebAla-OH (1.22 g,
(6H, m), 1.46 (9H, s). FAB-MS m/z: 286 (MϩH)ϩ.
Boc-MebAla-OCH2cHex (2p) Using method C, Boc-MebAla-OH
6.0 mmol) was reacted with EtOH (304 mg, 6.60 mmol) to give 2b (1.33 g, (1.22 g, 6.0 mmol) was reacted with cyclohexylmethanol (822 mg, 7.2 mmol)
1
1
96%). Rf1 0.88. H-NMR (CDCl3) d: 4.14 (2H, t, Jϭ7.1 Hz), 3.50 (2H, t,
to give 2p (1.80 g, 100%). Rf1 0.79. H-NMR (CDCl3) d: 3.89 (2H, d, Jϭ
Jϭ6.8 Hz), 2.87 (3H, s), 2.54 (2H, t, Jϭ6.9 Hz), 1.46 (9H, s), 1.26 (3H, t, 6.3 Hz), 3.50 (2H, t, Jϭ6.9 Hz), 2.87 (3H, s), 2.54 (2H, t, Jϭ6.6 Hz), 1.71
Jϭ7.1 Hz). FAB-MS m/z: 232 (MϩH)ϩ.
(6H, m), 1.46 (9H, s), 1.20 (3H, m), 0.96 (2H, m). FAB-MS m/z: 300 (Mϩ
Boc-MebAla-O(Bu) (2c) Using method C, Boc-MebAla-OH (1.22 g, H)ϩ.
6.0 mmol) was reacted with 1-butanol (489 mg, 6.60 mmol) to give 2c
Boc-MebAla-OBut (2q) Using method C, Boc-MebAla-OH (2.03 g,
1
(1.50 g, 96%). Rf1 0.90. H-NMR (CDCl3) d; 4.08 (2H, t, Jϭ6.6 Hz), 3.50 10.0 mmol) was reacted with tert-butanol (815 mg, 11.0 mmol) to give 2q
1
(2H, t, Jϭ6.9 Hz), 2.87 (3H, s), 2.54 (2H, t, Jϭ7.1 Hz), 1.62 (2H, m), 1.46 (1.81 g, 70%). Rf1 0.84. H-NMR (CDCl3) d: 3.54 (2H, br s), 2.90 (3H, s),
(9H, s), 1.38 (2H, m), 0.94 (3H, t, Jϭ7.4 Hz). FAB-MS m/z: 260 (MϩH)ϩ.
2.51 (2H, t, Jϭ6.9 Hz), 1.49 (s, 9H), 1.42 (s, 9H). FAB-MS m/z: 260 (Mϩ
Boc-MebAla-O(Pen) (2d) Using method C, Boc-MebAla-OH (2.03 g, H)ϩ.
10.0 mmol) was reacted with 1-Pentanol (970 mg, 11.0 mmol) to give 2d
Boc-MebAla-O(1-Ad) (2r) Using method C, Boc-MebAla-OH (4.06 g,
1
(1.61 g, 98%). Rf1 0.90. H-NMR (CDCl3) d: 4.06 (2H, t, Jϭ6.8 Hz), 3.50 20.0 mmol) was reacted with 1-adamantanol (3.35 g, 22.0 mmol) to give 2r
1
(2H, t, Jϭ6.8 Hz), 2.85 (3H, s), 2.51 (2H, t, Jϭ6.8 Hz), 1.62 (2H, m), 1.46 (1.73 g, 26%). Rf1 0.90. H-NMR (CDCl3) d: 3.54 (2H, t, Jϭ6.9 Hz), 2.95
(9H, s), 1.27 (4H, m), 0.87 (3H, t, Jϭ6.8 Hz). FAB-MS m/z: 274 (MϩH)ϩ.
(3H, s), 2.45 (2H, t, Jϭ6.9 Hz), 2.16 (3H, br s), 2.10 (6H, br s), 1.66 (6H,
Boc-MebAla-O(Hex) (2e) Using method C, Boc-MebAla-OH (2.03 g, br s), 1.47 (9H, s). FAB-MS m/z: 338 (MϩH)ϩ.
10.0 mmol) was reacted with 1-hexanol (1.12 g, 11.0 mmol) to give 2e
Preparation of Boc-MebAla-OPOM (2s) To a solution of Boc-
1
(2.84 g, 99%). Rf1 0.90. H-NMR (CDCl3) d: 4.07 (2H, t, Jϭ6.8 Hz), 3.50 MebAla-OH (5.08 g, 25.0 mmol) in DMF (20 ml), chloromethyl pivalate
(2H, t, Jϭ7.1 Hz), 2.87 (3H, s), 2.54 (2H, t, Jϭ7.1 Hz), 1.62 (2H, m), 1.46 (POM-Cl, 7.53 g, 50.0 mmol), TEA (8.09 g, 80.0 mmol), and NaI (0.5 g)
(9H, s), 1.31 (6H, br s), 0.89 (3H, t, Jϭ6.8 Hz). FAB-MS m/z: 288 (MϩH)ϩ.
were added at 0 °C. This mixture was stirred for 1 h at 0 °C and then stirred
Boc-MebAla-O(Hep) (2f) Using method C, Boc-MebAla-OH (2.03 g, overnight at room temperature. After the reaction mixture was diluted with
10.0 mmol) was reacted with 1-heptanol (1.28 g, 11.0 mmol) to give 2f EtOAc, the solution was washed with 1 N-HCl, sat. NaHCO3, and brine. The
1
(2.65 g, 88%). Rf1 0.91. H-NMR (CDCl3) d: 4.07 (2H, t, Jϭ6.8 Hz), 3.51 organic phase was dried over MgSO4, filtered, and evaporated in vacuo. The
(2H, t, Jϭ7.1 Hz), 2.86 (3H, s), 2.52 (2H, t, Jϭ7.2 Hz), 1.61 (2H, m), 1.46 residue was purified by flush column chromatography eluted with hexane/
(9H, s), 1.31 (8H, br s), 0.87 (3H, t, Jϭ6.9 Hz). FAB-MS m/z: 302 (MϩH)ϩ.
EtOAc (2 : 1, v/v) to give 2s (8.17 g, 100%). Rf1 0.80. H-NMR (CDCl3) d:
1
Boc-MebAla-O(Oct) (2g) Using method C, Boc-MebAla-OH (2.03 g, 5.76 (2H, s), 3.52 (2H, t, Jϭ6.9 Hz), 2.86 (3H, s), 2.58 (2H, t, Jϭ6.9 Hz),
10.0 mmol) was reacted with octyl alcohol (1.43 g, 11.0 mmol) to give 2g 1.45 (9H, s), 1.20 (9H, s). FAB-MS m/z: 318 (MϩH)ϩ.
1
(2.90 g, 92%). Rf1 0.92. H-NMR (CDCl3) d: 4.08 (2H, t, Jϭ6.8 Hz), 3.50
Preparation of Boc-MebAla-OCH(Me)OCOOEt (2t) To a solution of
(2H, t, Jϭ6.8 Hz), 2.85 (3H, s), 2.51 (2H, t, Jϭ7.1 Hz), 1.62 (2H, m), 1.42 Boc-MebAla-OH (2.0 g, 9.8 mmol) in DMF (30 ml), 1-bromoethyl ethyl car-
(9H, s), 1.26 (10H, br s), 0.88 (3H, t, Jϭ6.8 Hz). FAB-MS m/z: 316 (Mϩ bonate33) (2.12 g, 10.8 mmol) and KHCO3 (1.2 g, 12.0 mmol) were added at
H)ϩ.
0 °C. This mixture was stirred overnight at 50 °C. Then the reaction mixture
was diluted with EtOAc, and the solution was washed with 1 N-HCl, sat.
Boc-MebAla-O(Non) (2h) Using method C, Boc-MebAla-OH (2.03 g,
10.0 mmol) was reacted with nonyl alcohol (1.59 g, 11.0 mmol) to give 2h NaHCO3, and brine. The organic phase was dried over MgSO4, filtered, and
1
(2,90 g, 88%). Rf1 0.91. H-NMR (CDCl3) d: 4.06 (2H, t, Jϭ6.9 Hz), 3.49 evaporated in vacuo. The residue was purified by flush column chromatogra-
(2H, t, Jϭ6.9 Hz), 2.85 (3H, s), 2.51 (2H, t, Jϭ6.8 Hz), 1.61 (2H, t,
phy eluted with CH2Cl2/MeOH (90 : 1, v/v) to give 2t (3.0 g, 95%). Rf1 0.77.
Jϭ6.6 Hz), 1.39 (9H, s), 1.26 (12H, br s), 0.88 (3H, t, Jϭ6.8 Hz). FAB-MS 1H-NMR (CDCl3) d: 6.77 (1H, q, Jϭ5.4 Hz), 4.22 (2H, q, Jϭ7.1 Hz), 3.50
m/z: 330 (MϩH)ϩ.
(2H, br s), 2.87 (3H, s), 2.58 (2H, t, Jϭ6.9 Hz), 1.52 (3H, d, Jϭ5.7 Hz), 1.46
Boc-MebAla-O(Dec) (2i) Using method C, Boc-MebAla-OH (2.03 g, (9H, s), 1.32 (3H, t, Jϭ7.2 Hz). FAB-MS m/z: 320 (MϩH)ϩ.
10.0 mmol) was reacted with decyl alcohol (1.74 g, 11.0 mmol) to give 2i
Preparation of Boc-MebAla-O(Phthalidyl) (2u) To a solution of Boc-
1
(3.12 g, 99%). Rf1 0.92. H-NMR (CDCl3) d: 4.07 (2H, t, Jϭ6.9 Hz), 3.50 MebAla-OH (1.0 g, 4.93 mmol) in DMF (20 ml), bromophthalide34) (2.3 g,
(2H, t, Jϭ6.9 Hz), 2.87 (3H, s), 2.54 (2H, t, Jϭ6.9 Hz), 1.62 (2H, m), 1.46 10.8 mmol) and KHCO3 (1.18 g, 1.18 mmol) were added at 0 °C. The reac-
(9H, s), 1.27 (14H, br s), 0.88 (3H, t, Jϭ6.8 Hz). FAB-MS m/z: 344
(MϩH)ϩ.
tion mixture was stirred for 1 h at 0 °C and then stirred overnight at room
temperature. Subsequently, the reaction mixture was diluted with EtOAc,
and the solution was washed with 1 N-HCl, sat. NaHCO3, and brine. The or-
Boc-MebAla-O(Dde) (2j) Using method C, Boc-MebAla-OH (2.03 g,
10.0 mmol) was reacted with dodecyl alcohol (2.05 g, 11.0 mmol) to give 2j ganic phase was dried over MgSO4, filtered, and evaporated in vacuo. The
1
(3.51 g, 100%). Rf1 0.93. H-NMR (CDCl3) d: 4.07 (2H, t, Jϭ6.8 Hz), 3.50 residue was purified by flush column chromatography eluted with CH2Cl2/
1
(2H, t, Jϭ6.8 Hz), 2.87 (3H, s), 2.54 (2H, t, Jϭ6.8 Hz), 1.62 (2H, m), 1.46 MeOH (90 : 1, v/v) to give 2u (2.18 g, 66%). Rf1 0.74. H-NMR (CDCl3) d:
(9H, s), 1.26 (18H, br s), 0.88 (3H, t, Jϭ6.6 Hz). FAB-MS m/z: 372
8.00—7.30 (5H, m), 3.56 (2H, t, Jϭ6.9 Hz), 2.88 (3H, s), 2.68 (2H, t,
(MϩH)ϩ.
Jϭ7.1 Hz), 1.44 (9H, s). FAB-MS m/z: 336 (MϩH)ϩ.
Boc-MebAla-O(Hde) (2k) Using method C, Boc-MebAla-OH (2.03 g,
Preparation of Boc-Tyr-D-MetO-Phe-MebAla-OMe (3a) Compound
10.0 mmol), was reacted with 1-hexadecanol (2.67 g, 11.0 mmol) to give 2k 1 (2.88 g, 5.0 mmol) and HCl·H-MebAla-OMe, obtained from 2a by depro-
1
(3.04 g, 71%). Rf1 0.92. H-NMR (CDCl3) d: 4.06 (2H, t, Jϭ6.8 Hz), 3.50 tection using method D, were coupled (method A) to yield 3a (2.76 g, 82%)
1
(2H, t, Jϭ6.8 Hz), 2.85 (3H, s), 2.51 (2H, t, Jϭ6.8 Hz), 1.24 (26H, m), 1.42
as a white powder. Rf3 0.50. H-NMR (CDCl3) d: 7.30—7.10 (5H, m), 7.04
(2H, d, Jϭ7.6 Hz), 6.80 (2H, m), 5.95—5.65 (1H, m), 5.04 (1H, m), 4.64
(9H, s), 0.88 (3H, t, Jϭ6.8 Hz). FAB-MS m/z: 428 (MϩH)ϩ.
Boc-MebAla-OCH2CF3 (2l) Using method C, Boc-MebAla-OH (2.03 (1H, br s), 4.22 (1H, br s), 3.65 (4H, m), 3.35 (1H, br s), 2.96 (4H, m), 2.86

766
Vol. 51, No. 7
s), 1.26 (26H, br s), 0.88 (3H, t, Jϭ6.9 Hz). FAB-MS m/z: 886 (MϩH)ϩ.
(1H, s), 2.76 (2H, s), 2.50 (5H, m), 2.22 (2H, br s), 1.94 (1H, br s), 1.80 (1H,
br s), 1.42 (9H, s). FAB-MS m/z: 675 (MϩH)ϩ.
Compounds 3b—p, 3s—u were prepared in the same manner from com-
pound 1 and the corresponding Boc-MebAla-OX (2b—p, 2s—u), respec-
tively.
Boc-Tyr- -MetO-Phe-MebAla-OCH2CF3 (3l) A white powder (3.08 g,
D
83%). Rf3 0.63. 1H-NMR (CDCl3) d: 7.35—7.10 (5H, m), 7.03 (2H, d,
Jϭ7.8 Hz), 6.79 (2H, m), 5.90—5.64 (1H, m), 5.02 (1H, m), 4.67 (1H, m),
4.62 (2H, m), 4.23 (1H, m), 3.65 (1H, m), 3.36 (1H, m), 2.99 (4H, m), 2.85
(1H, s), 2.78 (2H, s), 2.46 (5H, m), 2.21 (2H, m), 1.93 (1H, br s), 1.78 (1H,
m), 1.41 (9H, s), 1.23 (3H, t, Jϭ7.2 Hz). FAB-MS m/z: 743 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-OPh (3m) A white powder (2.54 g,
Boc-Tyr-D-MetO-Phe-MebAla-OEt (3b)
A white powder (2.65 g,
77%). Rf3 0.52. 1H-NMR (CDCl3) d: 7.35—7.10 (5H, m), 7.03 (2H, d,
Jϭ7.8 Hz), 6.79 (2H, m), 5.90—5.64 (1H, m), 5.02 (1H, m), 4.67 (1H, br s),
4.23 (1H, br s), 4.10 (2H, q, Jϭ7.1 Hz), 3.67 (1H, m), 3.38 (1H, br s), 2.98
(4H, m), 2.86 (1H, s), 2.77 (2H, s), 2.48 (5H, m), 2.20 (2H, br s), 1.92 (1H,
br s), 1.78 (1H, br s), 1.41 (9H, s), 1.24 (3H, t, Jϭ7.1 Hz). FAB-MS m/z: 689
(MϩH)ϩ.
1
69%). Rf3 0.46. H-NMR (CDCl3) d: 7.31—6.94 (12H, m), 6.64 (2H, m),
5.72 (1H, br m), 5.10 (2H, m), 4.87 (1H, m), 4.52 (1H, m), 4.15 (1H, m),
3.63 (1H, m), 3.35 (1H, m), 3.05—2.74 (6H, m), 2.66 (2H, m), 2.45 (3H,
m), 2.31—2.15 (2H, m), 1.89 (1H, m), 1.71 (1H, m), 1.41 (9H, s). FAB-MS
m/z: 737 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-O(Bu) (3c) A white powder (2.69 g,
75%). Rf3 0.58. 1H-NMR (CDCl3) d: 7.35—7.15 (5H, m), 7.03 (2H, d,
Jϭ8.1 Hz), 6.80 (2H, m), 5.90—5.60 (1H, m), 5.00 (1H, m), 4.68 (1H, m),
4.24 (1H, m), 4.05 (2H, t, Jϭ6.8 Hz), 3.70 (1H, m), 3.38 (1H, m), 3.01 (3H,
m), 2.87 (1H, s), 2.78 (2H, s), 2.49 (6H, m), 2.27 (2H, br s), 1.91 (1H, br s),
1.78 (1H, br s), 1.59 (2H, m), 1.42 (9H, s), 0.93 (3H, t, Jϭ7.2 Hz). FAB-MS
m/z: 717 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-OBzl (3n) A white powder (2.36 g,
63%). Rf3 0.46. 1H-NMR (CDCl3) d: 7.97—7.82 (2H, m), 7.34 (5H, m),
7.24 (3H, m), 7.16 (2H, m), 7.04 (2H, d, Jϭ8.1 Hz), 6.77 (2H, m), 5.73 (1H,
br m), 5.10 (2H, m), 4.87 (1H, m), 4.50 (1H, m), 4.14 (1H, m), 3.65 (1H, m),
3.51 (2H, s), 3.38 (2H, m), 3.05—2.65 (6H, m), 2.49 (5H, m), 1.96 (1H, m),
1.73 (1H, m), 1.40 (9H, s). FAB-MS m/z: 751 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-OcHex (3o) A white powder (2.63 g,
Boc-Tyr-D-MetO-Phe-MebAla-O(Pen) (3d) A white powder (2.92 g,
80%). Rf3 0.55. 1H-NMR (CDCl3) d: 7.35—7.15 (5H, m), 7.03 (2H, d,
Jϭ8.1 Hz), 6.79 (2H, m), 5.90—5.60 (1H, m), 5.02 (1H, m), 4.66 (1H, m),
4.28 (1H, br s), 4.04 (2H, m), 3.70 (1H, m), 3.38 (2H, br s), 2.88 (4H, m),
2.88 (1H, s), 2.79 (2H, s), 2.65—2.38 (5H, m), 2.29 (2H, m), 2.06 (2H, m),
1.61 (2H, t, Jϭ7.1 Hz), 1.39 (9H, s), 1.41 (4H, br s), 0.90 (3H, m). FAB-MS
m/z: 731 (MϩH)ϩ.
1
71%). Rf3 0.54. H-NMR (CDCl3) d: 8.61 (1H, br m), 8.18—7.94 (1H, m),
7.43—7.33 (1H, m), 7.29—7.16 (5H, m), 7.04 (2H, d, Jϭ8.1 Hz), 6.79 (2H,
dd, Jϭ8.4, 4.8 Hz), 6.08—5.98 (1H, m), 4.71 (2H, m), 4.26 (1H, br s), 3.72
(0.66H, m), 3.36 (1.33H, m), 2.98 (8H, m), 2.86 (1H, s), 2.77 (2H, s), 2.48
(3H, m), 2.42 (2H, m), 2.16 (2H, m), 1.90 (1H, m), 1.80 (3H, m), 1.70 (2H,
m), 1.54 (1H, m), 1.41 (9H, s), 1.37 (5H, m). FAB-MS m/z: 743 (MϩH)ϩ.
Boc-Tyr- -MetO-Phe-MebAla-OCH2cHex (3p) A white powder (2.65
D
Boc-Tyr-D-MetO-Phe-MebAla-O(Hex) (3e) A white powder (3.09 g,
83%). Rf3 0.54. 1H-NMR (CDCl3) d: 7.35—7.10 (5H, m), 7.03 (2H, d,
Jϭ8.1 Hz), 6.78 (2H, dd, Jϭ8.4, 4.8 Hz), 6.10—5.66 (1H, m), 5.02 (1H, m),
4.73 (1H, br s), 4.25 (1H, br s), 4.03 (2H, m), 3.74 (1H, m), 3.36 (1H, m),
3.01 (4H, m), 2.88 (1.5H, s), 2.79 (1.5H, s), 2.50 (5H, m), 2.20 (2H, br s),
1.90 (1H, br s), 1.60 (1H, t, Jϭ7.2 Hz), 1.42 (9H, s), 1.30 (6H, br s), 0.88
(3H, m). FAB-MS m/z: 745 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-O(Hep) (3f) A white powder (2.62 g,
69%). Rf3 0.57. 1H-NMR (CDCl3) d: 7.45—7.15 (5H, m), 7.02 (2H, d,
Jϭ8.4 Hz), 6.81 (2H, m), 5.90—5.60 (1H, m), 5.02 (1H, m), 4.65 (1H, m),
4.28 (1H, br s), 4.04 (2H, t, Jϭ6.8 Hz), 3.70 (1H, m), 3.40 (2H, br s), 2.98
(4H, m), 2.87 (1H, s), 2.78 (2H, s), 2.65—2.43 (5H, m), 2.29 (2H, m), 2.05
(2H, m), 1.61 (2H, t, Jϭ6.8 Hz), 1.39 (9H, s), 1.31 (8H, br s), 0.88 (3H, t,
Jϭ6.8 Hz). FAB-MS m/z: 759 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-O(Oct) (3g) A white powder (2.78 g,
72%). Rf3 0.60. 1H-NMR (CDCl3) d: 7.40—7.15 (5H, m), 7.02 (2H, d,
Jϭ8.4 Hz), 6.81 (2H, m), 5.88—5.60 (1H, m), 5.00 (1H, m), 4.64 (1H, m),
4.27 (1H, br s), 4.04 (2H, t, Jϭ6.8 Hz), 3.70 (1H, m), 3.39 (2H, br s), 2.99
(4H, m), 2.86 (1H, s), 2.79 (2H, s), 2.65—2.40 (5H, m), 2.29 (2H, m), 2.07
(2H, m), 1.61 (2H, t, Jϭ6.8 Hz), 1.39 (9H, s), 1.29 (10 br s), 0.88 (3H, t,
Jϭ6.8 Hz). FAB-MS m/z: 773 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-O(Non) (3h) A white powder (2.94 g,
75%). Rf3 0.62. 1H-NMR (CDCl3) d: 7.40—7.15 (5H, m), 7.03 (2H, d,
Jϭ8.4 Hz), 6.80 (2H, m), 5.88—5.58 (1H, m), 5.01 (1H, m), 4.65 (1H, m),
4.27 (1H, br s), 4.04 (2H, t, Jϭ6.8 Hz), 3.70 (1H, m), 3.41 (2H, br s), 3.00
(4H, m), 2.87 (1H, s), 2.79 (2H, s), 2.65—2.44 (5H, m), 2.30 (2H, m), 2.08
(2H, m), 1.61 (2H, t, Jϭ6.6 Hz), 1.39 (9H, s), 1.26 (12H, br s), 0.88 (3H, t,
Jϭ6.8 Hz). FAB-MS m/z: 787 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-O(Dec) (3i) A white powder (2.64 g,
66%). Rf3 0.67. 1H-NMR (CDCl3) d: 7.30—7.10 (5H, m), 7.03 (2H, d,
Jϭ7.5 Hz), 6.79 (2H, dd, Jϭ8.3, 4.5 Hz), 6.10—5.73 (1H, m), 5.04 (1H, m),
4.72 (1H, br s), 4.24 (1H, br s), 4.03 (2H, t, Jϭ6.6 Hz), 3.77 (1H, m), 3.36
(1H, m), 3.10 (2H, m), 2.88 (1.5H, s), 2.80 (1.5H, s), 2.50 (5H, m), 2.20
(2H, br s), 1.92 (1H, br s), 1.81 (1H, br s), 1.61 (2H, t, Jϭ6.8 Hz), 1.42 (9H,
s), 1.26 (14H, br s), 0.88 (3H, t, Jϭ6.5 Hz). FAB-MS m/z: 802 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-O(Dde) (3j) A white powder (2.57 g,
62%). Rf3 0.69. 1H-NMR (CDCl3) d: 7.35—7.15 (5H, m), 7.11 (2H, d,
Jϭ8.4 Hz), 6.91 (2H, m), 6.10—5.73 (1H, m), 5.03 (1H, m), 4.67 (1H, m),
4.29 (1H, br s), 4.04 (2H, m), 3.75 (1H, m), 3.39 (1H, m), 3.00 (4H, m), 2.88
(1H, s), 2.80 (2H, m), 2.60—2.35 (6H, m), 2.26 (1H, m), 2.06 (2H, m), 2.20
(2H, br s), 1.60 (2H, m), 1.81 (1H, br s), 1.61 (2H, t, Jϭ6.8 Hz), 1.39 (9H,
s), 1.26 (18H, br s), 0.89 (3H, t, Jϭ6.8 Hz). FAB-MS m/z: 830 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-O(Hde) (3k) A white powder (2.79 g,
63%). Rf3 0.70. 1H-NMR (CDCl3) d: 7.35—7.15 (5H, m), 7.04 (2H, d,
Jϭ8.4 Hz), 6.85 (2H, m), 6.10—5.73 (1H, m), 5.01 (1H, m), 4.65 (1H, m),
4.28 (1H, br s), 4.05 (2H, m), 3.73 (1H, m), 3.40 (1H, m), 3.02 (4H, m), 2.89
(1H, s), 2.79 (2H, m), 2.60—2.35 (6H, m), 2.24 (1H, m), 2.05 (2H, m), 2.22
(2H, br s), 1.62 (2H, m), 1.80 (1H, br s), 1.61 (2H, t, Jϭ6.8 Hz), 1.38 (9H,
1
g, 70%). Rf3 0.61. H-NMR (CDCl3) d: 8.50 (1H, br m), 8.14—7.90 (1H,
m), 7.38 (1H, m), 7.29—7.16 (5H, m), 7.04 (2H, d, Jϭ8.1 Hz), 6.79 (2H, dd,
Jϭ8.3, 5.0 Hz), 5.97—5.69 (1H, m), 5.00 (1H, m), 4.68 (1H, m), 4.26 (1H,
m), 3.85 (2H, d, Jϭ6.0 Hz), 3.71 (0.66H, m), 3.36 (1.33H, m), 3.12—2.70
(11H, m), 2.48—2.40 (5H, m), 2.19 (2H, m), 1.90 (1H, m), 1.70 (7H, m),
1.41 (9H, s), 1.20 (3H, m), 0.95 (2H, m). FAB-MS m/z: 757 (MCϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-OBut (3q) Compound 1 (2.88 g, 5.0
mmol) and H-MebAla-OBut, obtained from 2q by deprotection using
method E, were coupled (method A) to yield 3q (2.79 g, 78%) as a white
powder. Rf3 0.57. 1H-NMR (CDCl3) d: 7.35—7.10 (5H, m), 7.03 (2H, d,
Jϭ8.1 Hz), 6.97 (2H, dd, Jϭ8.4, 4.8 Hz), 6.12—5.76 (1H, m), 5.03 (1H, m),
4.73 (1H, m), 4.25 (1H, br s), 3.70 (1H, m), 3.36 (1H, m), 2.96 (4H, m), 2.88
(1.5H, s), 2.79 (1.5H, s), 2.48 (3H, m), 2.36 (2H, m), 2.17 (2H, m), 1.92
(1H, br s), 1.76 (1H, br s), 1.42 (18H, s). FAB-MS m/z: 717 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-O(1-Ad) (3r) Compound 1 (2.88 g, 5.0
mmol) and H-MebAla-O(1-Ad), obtained from 2r by deprotection using
method E, were coupled (method A) to yield 3r (2.50 g, 63%) as a white
powder. Rf3 0.62. 1H-NMR (CDCl3) d: 7.30—7.10 (5H, m), 7.04 (2H, d,
Jϭ7.6 Hz), 6.80 (2H, m), 5.95—5.65 (1H, m), 5.04 (1H, m), 4.64 (1H, br s),
4.22 (1H, br s), 3.63 (1H, m), 3.33 (1H, m), 2.95 (4H, m), 2.87 (1H, s), 2.76
(2H, s), 2.50 (5H, m), 2.22 (2H, br s), 2.16 (3H, br s), 2.10 (6H, br s), 1.94
(1H, br s), 1.80 (1H, br s), 1.66 (6H, br s), 1.42 (9H, s). FAB-MS m/z: 795
(MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-O(POM) (3s) A white powder (2.75 g,
71%). Rf3 0.64. 1H-NMR (CDCl3) d: 7.30—7.10 (5H, m), 7.04 (2H, d,
Jϭ7.6 Hz), 6.80 (2H, m), 5.95—5.65 (3H, m), 5.04 (1H, m), 4.64 (1H, m),
4.22 (1H, m), 3.65 (1H, m), 3.35 (1H, br s), 2.96 (4H, m), 2.87 (1H, s), 2.76
(2H, s), 2.53 (5H, m), 2.25 (2H, m), 1.96 (1H, m), 1.80 (1H, br s), 1.42 (9H,
s), 1.20 (9H, s). FAB-MS m/z: 775 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-OCH(Me)OCOOEt (3t) A white pow-
der (2.52 g, 65%). Rf3 0.62. 1H-NMR (CDCl3) d: 8.60—8.47 (1H, m),
8.24—8.03 (2H, m), 7.28—7.17 (6H, m), 7.03 (2H, d, Jϭ8.1 Hz), 6.80—
6.70 (3H, m), 6.17—5.87 (1H, m), 5.04 (1H, m), 4.78 (1H, m), 4.27 (1H,
br m), 4.25—4.08 (2H, m), 3.75 (1H, m), 3.36 (1H, m), 3.27 (1H, br m),
3.01 (2H, m), 2.94 (2H, m), 2.87 (1H, s), 2.82 (2H, s), 2.48 (5H, m), 2.24—
2.08 (2H, m), 1.90 (1H, br m), 1.79 (1H, br m), 1.50 (3H, d, Jϭ5.4 Hz), 1.41
(9H, s), 1.34—1.20 (3H, m). FAB-MS m/z: 777 (MϩH)ϩ.
Boc-Tyr-D-MetO-Phe-MebAla-O(Phthalidyl) (3u) A white powder
(2.46 g, 62%). Rf3 0.61. 1H-NMR (CDCl3) d: 7.80—7.55 (4H, m), 7.35 (2H,
d, Jϭ5.4 Hz), 7.12 (5H, m), 6.97 (2H, m), 6.60 (2H, m), 4.50 (1H, m), 4.32
(2H, m), 3.60 (1H, m), 3.50 (1H, m), 3.20—2.65 (7H, m), 2.55 (2H, m),
2.45 (3H, m), 2.27 (2H, m), 1.83 (1H, m), 1.70 (1H, m), 1.42 (9H, s). FAB-
MS m/z: 793 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-OMe (4a) As described
in method H, compound 3a (2.02 g, 3.0 mmol) was treated with 90%–TFA
(30 ml) for 1 h at room temperature to remove the Boc group. The reaction
mixture was concentrated in vacuo, and the residue was dissolved in DMF

July 2003
767
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-O(Dec) (4i) Compound 3i
(2.40 g, 3.0 mmol) was treated according to method H, yielding 4i (334 mg,
15%) as a white powder. HPLC tR (min): 16.54. Rf4 0.71. 1H-NMR
(CD3OD) d: 7.27—7.19 (5H, m), 7.07 (2H, d, Jϭ8.4 Hz), 6.72 (2H, d, Jϭ
8.4 Hz), 5.12 (0.5H, m), 5.04 (0.5H, m), 4.40 (2H, m), 4.06 (2H, t,
Jϭ6.8 Hz), 3.64 (1H, m), 3.46 (1H, m), 3.15—2.99 (3H, m), 2.97—2.81
(4H, m), 2.56 (3H, m), 2.48 (1H, m), 2.43—2.25 (3H, m), 2.18 (3H, m),
1.92 (1H, m), 1.77 (1H, m), 1.60 (2H, m), 1.29 (14H, m), 0.89 (3H, t,
Jϭ6.8 Hz). FAB-MS m/z: 742 (MϩH)ϩ.
(60 ml), and then ehtyl acetimidate hydrochloride (741 mg, 6.0 mmol) and
TEA (1.25 ml, 9.0 mmol) were added. This solution was stirred at room tem-
perature for 2 h. The crude peptide was purified by ODS chromatography
with a stepwise gradient of acetonitrile (starting from 1% and increasing
stepwise by 2%) in 0.1 N acetic acid as the eluting solvent, and then was
lyophilized to give 4a (867 mg, 47%) as a white amorphous powder. HPLC
1
tR (min): 8.39. Rf4 0.66. H-NMR (CD3OD) d: 7.21—7.10 (5H, m), 6.99
(2H, d, Jϭ8.4 Hz), 6.64 (2H, d, Jϭ8.4 Hz), 5.05 (0.5H, m), 4.93 (0.5H, m),
4.29 (2H, m), 3.56 (3H, s), 3.52 (1H, m), 3.40 (1H, m), 3.07—2.91 (3H, m),
2.87—2.93 (m, 2H), 2.87—2.76 (4H, m), 2.47 (3H, m), 2.40 (1H, m), 2.31
(1H, m), 2.20 (1H, m), 2.09 (3H, d, Jϭ2.4 Hz), 1.87 (1H, m), 1.71 (1H, m).
FAB-MS m/z: 616 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-O(Dde) (4j) Compound
3j (2.49 g, 3.0 mmol) was treated according to method H, yielding 4j
(809 mg, 35%) as a white powder. HPLC tR (min): 18.56. Rf4 0.70. 1H-NMR
(CD3OD) d: 7.21—7.10 (5H, m), 6.99 (2H, d, Jϭ7.5 Hz), 6.63 (2H, d,
Jϭ8.4 Hz), 5.02 (0.5H, m), 4.93 (0.5H, m), 4.32 (2H, m), 3.96 (2H, t,
Jϭ6.8 Hz), 3.57 (1H, m), 3.36 (1H, m), 3.06—2.90 (3H, m), 2.88—2.76
(4H, m), 2.47 (3H, m), 2.39 (1H, m), 2.34—2.16 (3H, m), 2.09 (3H, m),
1.86 (1H, m), 1.70 (1H, m), 1.53 (2H, m), 1.19 (18H, m), 0.80 (3H, t,
Jϭ6.8 Hz). FAB-MS m/z: 770 (MϩH)ϩ.
Compounds 4b—p, 4s—u were prepared in the same manner.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-OEt (4b) Compound 3b
(2.07 g, 3.0 mmol) was treated according to method H, yielding 4b (661 mg,
35%) as a white powder. HPLC tR (min): 9.92. Rf4 0.66. 1H-NMR (CD3OD)
d: 7.20—7.10 (5H, m), 7.00 (2H, d, Jϭ7.8 Hz), 6.74 (2H, m), 5.02 (0.5H,
m), 4.94 (0.5H, m), 4.32 (2H, m), 4.08 (2H, q, Jϭ7.2 Hz), 3.60 (1H, m),
3.38 (1H, m), 2.98 (4H, m), 2.86 (1H, s), 2.77 (2H, s), 2.48 (3H, m), 2.27
(1H, m), 2.19 (1H, m), 2.10 (3H, d, Jϭ2.4 Hz), 1.90 (1H, m), 1.74 (1H, m),
1.24 (3H, t, Jϭ7.2 Hz). FAB-MS m/z: 630 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-O(Hde) (4k) Compound
3k (2.66 g, 3.0 mmol) was treated according to method H, yielding 4k
(818 mg, 33%) as a white powder. HPLC tR (min): Ͼ20.0. Rf4 0.73. 1H-
NMR (CD3OD) d: 7.28—7.19 (5H, m), 7.08 (2H, m), 6.73 (2H, m), 5.11
(0.5H, m), 5.03 (0.5H, m), 4.38 (2H, m), 4.06 (2H, m), 3.66 (1H, m), 3.46
(1H, m), 3.15—3.00 (3H, m), 2.97—2.77 (4H, m), 2.63 (1H, s), 2.56 (2H,
s), 2.50—2.20 (4H, m), 2.19 (3H, m), 1.94 (1H, m), 1.81 (1H, m), 1.62 (1H,
m), 1.28 (26H, m), 0.89 (3H, t, Jϭ6.8 Hz). FAB-MS m/z: 827 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-O(Bu) (4c) Compound 3c
(2.15 g, 3.0mmol) was treated according to method H, yielding 4c (809
mg, 41%) as a white powder. HPLC tR (min): 11.01. Rf4 0.68. 1H-NMR
(CD3OD) d: 7.21—7.09 (5H, m), 6.99 (2H, d, Jϭ7.8 Hz), 6.63 (2H, d, Jϭ
7.8 Hz), 5.01 (0.5H, m), 4.93 (0.5H, m), 4.36 (1H, m), 4.29 (1H, m), 3.97
(2H, t, Jϭ7.2 Hz), 3.53 (1H, m), 3.37 (1H, m), 3.05—2.90 (3H, m), 2.87—
2.75 (4H, m), 2.47 (3H, m), 2.40 (2H, m), 2.29 (1H, m), 2.21 (1H, m), 2.09
(3H, d, Jϭ2.4 Hz), 1.85 (1H, m), 1.71 (1H, m), 1.51 (2H, m), 1.29 (2H, m),
0.84 (3H, t, Jϭ7.4 Hz). FAB-MS m/z: 658 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-
-MetO-Phe-MebAla-OCH2CF3 (4l) Compound
D
3l (2.23 g, 3.0 mmol) was treated according to method H, yielding 4l (617
mg, 30%) as a white powder. HPLC tR (min): 10.68. Rf4 0.71. 1H-NMR
(CD3OD) d: 7.25—7.10 (5H, m), 6.99 (2H, m), 6.64 (2H, m), 5.13 (0.5H,
m), 4.93 (0.5H, m), 4.30 (2H, m), 3.57 (1H, m), 3.33 (1H, m), 3.05—2.89
(3H, m), 2.87—2.74 (4H, m), 2.47 (3H, m), 2.29 (3H, m), 2.09 (3H, s), 1.87
(1H, m), 1.60 (1H, m). FAB-MS m/z: 686 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-O(Pen) (4d) Compound
3d (2.19 g, 3.0 mmol) was treated according to method H, yielding 4d
(705 mg, 35%) as a white powder. HPLC tR (min): 11.99. Rf4 0.69. 1H-NMR
(CD3OD) d: 7.22—7.11 (5H, m), 6.99 (2H, d, Jϭ8.4 Hz), 6.63 (2H, d,
Jϭ8.7 Hz), 5.01 (0.5H, m), 4.90 (0.5H, m), 4.31 (2H, m), 3.96 (2H, t,
Jϭ6.6 Hz), 3.54 (1H, m), 3.37 (2H, m), 3.05—2.90 (3H, m), 2.87—2.76
(4H, m), 2.47 (3H, m), 2.41 (1H, m), 2.32—2.18 (3H, m), 2.09 (3H, m),
1.85 (1H, m), 1.68 (1H, m), 1.53 (2H, m), 1.23 (4H, m), 0.82 (3H, t,
Jϭ6.5 Hz). FAB-MS m/z: 672 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-OPh (4m) Compound 3m
(2.21 g, 3.0 mmol) was treated according to method H, yielding 4m (691
mg, 34%) as a white powder. HPLC tR (min): 10.83. Rf4 0.66. 1H-NMR
(CD3OD) d: 7.31—6.94 (12H, m), 6.64 (2H, m), 5.08 (0.5H, m), 4.97 (0.5H,
m), 4.32 (2H, m), 3.63 (1H, m), 3.55 (1H, m), 3.05—2.94 (3H, m), 2.93—
2.74 (4H, m), 2.66 (2H, m), 2.45 (3H, m), 2.31—2.15 (2H, m), 2.06 (3H,
m), 1.84 (1H, m), 1.71 (1H, m). FAB-MS m/z: 678 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-O(Hex) (4e) Compound
3e (2.23 g, 3.0 mmol) was treated according to method H, yielding 4e
(473 mg, 42%) as a white powder. HPLC tR (min): 12.95. Rf4 0.71. 1H-NMR
(CD3OD) d: 7.21—7.08 (5H, m), 6.98 (2H, d, Jϭ8.4 Hz), 6.63 (2H, d,
Jϭ8.4 Hz), 5.02 (0.5H, m), 4.93 (0.5H, m), 4.28 (2H, m), 3.97 (2H, t,
Jϭ6.6 Hz), 3.54 (1H, m), 3.37 (2H, m), 3.06—2.90 (3H, m), 2.88—2.72
(4H, m), 2.47 (3H, m), 2.40 (1H, m), 2.32—2.18 (3H, m), 2.09 (3H, m),
1.83 (1H, m), 1.70 (1H, m), 1.53 (2H, m), 1.23 (6H, m), 0.81 (3H, t,
Jϭ6.8 Hz). FAB-MS m/z: 686 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-OBzl (4n) Compound 3n
(2.25 g, 3.0 mmol) was treated according to method H, yielding 4n (789 mg,
38%) as a white powder. HPLC tR (min): 11.32. Rf4 0.68. 1H-NMR
(CD3OD) d: 7.28—7.22 (5H, m), 7.17—7.07 (5H, m), 6.98 (2H, m), 6.64
(2H, m), 5.01 (2H, s), 4.91 (1H, m), 4.49 (1H, m), 4.28 (1H, m), 3.54 (1H,
m), 3.40 (1H, m), 3.05—2.86 (3H), 2.84—2.70 (4H, m), 2.51—2.40 (4H,
m), 2.32—2.13 (2H, m), 2.06 (3H, m), 1.86 (1H, m), 1.69 (1H, m). FAB-MS
m/z: 692 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-OcHex (4o) Compound
3o (2.23 g, 3.0 mmol) was treated according to method H, yielding 4o (451
mg, 22%) as a white powder. HPLC tR (min): 11.86. Rf4 0.70. 1H-NMR
(CD3OD) d: 7.21—7.11 (5H, m), 6.99 (2H, d, Jϭ7.8 Hz), 6.63 (2H, m,
Jϭ8.1 Hz), 5.06 (0.5H, m), 4.93 (0.5H, m), 4.63 (1H, m), 4.29 (2H, m), 3.54
(1H, m), 3.38 (1H, m), 3.06—2.90 (3H, m), 2.87—2.76 (4H, m), 2.47 (3H,
m), 2.38 (1H, m), 2.31—2.19 (3H, m), 2.09 (3H, m), 1.86 (1H, m), 1.73
(2H, m), 1.63 (2H, m), 1.45 (1H, m), 1.39—1.23 (6H, m). FAB-MS m/z: 684
(MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-O(Hep) (4f) Compound
3f (2.28 g, 3.0 mmol) was treated according to method H, yielding 4f
(777 mg, 37%) as a white powder. HPLC tR (min): 13.81. Rf4 0.71. 1H-NMR
(CD3OD) d: 7.21—7.08 (5H, m), 6.99 (2H, d, Jϭ8.4 Hz), 6.63 (2H, d,
Jϭ8.4 Hz), 5.01 (0.5H, m), 4.93 (0.5H, m), 4.38—4.26 (2H, m), 3.96 (2H, t,
Jϭ6.8 Hz), 3.54 (1H, m), 3.35 (2H, m), 3.05—2.90 (3H, m), 2.88—2.73
(4H, m), 2.47 (3H, m), 2.39 (1H, m), 2.32—2.16 (3H, m), 2.09 (3H, m),
1.84 (1H, m), 1.70 (1H, m), 1.52 (2H, m), 1.22 (8H, m), 0.81 (3H, t,
Jϭ6.6 Hz). FAB-MS m/z: 700 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-OCH2cHex (4p) Com-
pound 3p (2.27 g, 3.0 mmol) was treated according to method H, yielding 4p
(691 mg, 33%) as a white powder. HPLC tR (min): 12.87. Rf4 0.70. 1H-NMR
(CD3OD) d: 7.22—7.09 (5H, m), 6.98 (2H, d, Jϭ8.4 Hz), 6.63 (2H, m, Jϭ
8.4 Hz), 5.03 (0.5H, m), 4.93 (0.5H, m), 4.29 (2H, m), 3.78 (2H, d,
Jϭ6.3 Hz), 3.54 (1H, m), 3.36 (1H, m), 3.06—2.90 (3H, m), 2.87—2.72
(4H, m), 2.48 (3H, m), 2.39 (1H, m), 2.31—2.17 (3H, m), 2.09 (3H, m),
1.86 (1H, m), 1.63 (6H, m), 1.15 (3H, m), 0.91 (2H, m). FAB-MS m/z: 698
(MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-O(Oct) (4g) Compound
3g (2.32 g, 3.0 mmol) was treated according to method H, yielding 4g
(750 mg, 35%) as a white powder. HPLC tR (min): 14.67. Rf4 0.70. 1H-NMR
(CD3OD) d: 7.20—7.09 (5H, m), 7.00 (2H, d, Jϭ8.1 Hz), 6.63 (2H, d,
Jϭ8.1 Hz), 5.04 (0.5H, m), 4.93 (0.5H, m), 4.31 (2H, m), 3.96 (2H, t,
Jϭ6.8 Hz), 3.56 (1H, m), 3.36 (2H, m), 3.10—2.75 (7H, m), 2.48 (3H, s),
2.35—2.15 (4H, m), 2.09 (3H, s), 1.86 (1H, m), 1.71 (1H, m), 1.52 (2H, m),
1.21 (10H, m), 0.80 (3H, t, Jϭ6.6 Hz). FAB-MS m/z: 714 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-O(Non) (4h) Compound
3h (2.36 g, 3.0 mmol) was treated according to method H, yielding 4h
(699 mg, 32%) as a white powder. HPLC tR (min): 15.52. Rf4 0.71. 1H-NMR
(CD3OD) d: 7.21—7.12 (5H, m), 7.00 (2H, d, Jϭ7.5 Hz), 6.63 (2H, d,
Jϭ8.4 Hz), 5.03 (0.5H, m), 4.94 (0.5H, m), 4.30 (2H, m), 3.96 (2H, t,
Jϭ6.8 Hz), 3.55 (1H, m), 3.35 (1H, m), 3.07—2.90 (3H, m), 2.87—2.76
(4H, m), 2.47 (3H, m), 2.40 (1H, m), 2.35—2.17 (3H, m), 2.09 (3H, m),
1.86 (1H, m), 1.71 (1H, m), 1.52 (2H, m), 1.20 (12H, m), 0.80 (3H, t,
Jϭ6.8 Hz). FAB-MS m/z: 728 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-OBut (4q) Compound 3q
(2.15 g, 3.0 mmol) was treated according to method H, yielding 4q (592
mg, 30%) as a white powder. HPLC tR (min): 10.76. Rf4 0.69. 1H-NMR
(CD3OD) d: 7.28—7.17 (5H, m), 7.08 (2H, d, Jϭ7.2 Hz), 6.72 (2H, d,
Jϭ8.4 Hz), 5.13 (0.5H, m), 5.03 (0.5H, m), 4.40 (2H, m), 3.59 (1H, m), 3.42
(1H, m), 3.15—3.00 (3H, m), 2.95—2.81 (4H, m), 2.56 (4H, m), 2.40 (2H,
m), 2.32 (1H, m), 2.18 (3H, d, Jϭ2.4 Hz), 1.96 (1H, m), 1.80 (1H, m), 1.42
(9H, s). FAB-MS m/z: 658 (MϩH)ϩ.

768
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-O(1-Ad) (4r) Compound
3r (2.38 g, 3.0 mmol) was treated according to method H, yielding 4r
(508 mg, 23%) as a white powder. HPLC tR (min): 12.45. Rf4 0.69. 1H-NMR
(CD3OD) d: 7.30—7.15 (5H, m), 7.04 (2H, d, Jϭ7.6 Hz), 6.80 (2H, m), 5.12
(0.5H, m), 5.01 (0.5H, m), 4.42 (2H, m), 3.61 (1H, m), 3.39 (1H, m), 3.15—
3.00 (3H, m), 2.95—2.81 (4H, m), 2.57 (4H, m), 2.38 (2H, m), 2.32 (1H,
m), 2.19 (3H, d, Jϭ2.4 Hz), 2.16 (3H, m), 2.10 (6H, m), 1.96 (1H, m), 1.80
(1H, m), 1.66 (6H, m). FAB-MS m/z: 736 (MϩH)ϩ.
Vol. 51, No. 7
Boc-Tyr(CO2Me)-D-MetO-Phe-MebAla-OBut (6l) Using method F,
compound 5 (2.15 g, 3.00 mmol) was reacted with methyl chloroformate
(311 mg, 3.3 mmol) to give 6l (2.12 g, 91%) as a white powder. Rf1 0.59. 1H-
NMR (CDCl3) d: 7.42 (2H, m), 7.27—7.17 (7H, m), 7.09 (2H, m), 5.32—
5.20 (1H, m), 5.01 (1H, m), 4.54 (1H, m), 4.30 (1H, m), 3.87 (3H, s), 3.62
(0.67 H, m), 3.41 (1.33H, m), 3.08 (1H, m), 3.00 (3H, m), 2.85 (1H, s), 2.79
(2H, s), 2.58 (2H, m), 2.54 (2H, s), 2.46 (1H, s), 2.40 (2H, m), 2.03 (2H, m),
1.43 (9H, s), 1.40 (9H, s). FAB-MS m/z: 775 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-OPOM (4s) Compound
3s (2.32 g, 3.0 mmol) was treated according to method H, yielding 4s
(537 mg, 25%) as a white powder. HPLC tR (min): 11.44. Rf4 0.67. 1H-NMR
(CD3OD) d: 7.21—7.03 (7H, m), 6.98 (2H, m), 6.63 (2H, d, Jϭ8.1 Hz), 5.63
(2H, m), 5.04—4.91 (1H, m), 4.28 (2H, m), 3.53 (1H, m), 3.40 (1H, m),
3.07—2.72 (7H, m), 2.48 (5H, m), 2.31—2.15 (2H, m), 2.10 (3H, d,
Jϭ2.4 Hz), 1.85 (1H , m), 1.71 (1H, m). FAB-MS m/z: 716 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-OCH(Me)OCOOEt (4t)
Compound 3t (2.33 g, 3.0 mmol) was treated according to method H, yield-
ing 4t (431 mg, 20%) as a white powder. HPLC tR (min): 10.74. Rf4 0.67.
1H-NMR (CD3OD) d:7.16 (5H, m), 6.98 (2H, d, Jϭ8.4 z), 6.65 (3H, m),
5.04 (0.5H, m), 4.95 (0.5H, m), 4.51 (1H, m), 4.28 (2H, m), 4.06 (2H, m),
3.55 (1H,m), 3.41 (1H, m), 3.10—2.70 (7H, m), 2.46 (5H, m), 2.25 (2H, m),
2.09 (3H, s), 1.85 (2H, m), 1.69 (1H, m), 1.38 (3H, d, Jϭ5.7 Hz), 1.18 (3H,
m). FAB-MS m/z: 718 (MϩH)ϩ.
Boc-Tyr(CO2Et)-D-MetO-Phe-MebAla-OBut (6m) Using method F,
compound 5 (2.15 g, 3.00 mmol) was reacted with ethyl chloroformate (358
1
mg, 3.9 mmol) to give 6m (2.12 g, 90%) as a white powder. Rf1 0.57. H-
NMR (CDCl3) d: 7.84—7.66 (1H, m), 7.35—7.17 (8H, m), 7.09 (2H, m),
5.72—5.43 (1H, m), 5.02 (1H, m), 4.66 (1H, m), 4.33 (1H, m), 4.29 (2H, q,
Jϭ7.1 Hz), 3.66 (0.66H, m), 3.38 (1.33H, m), 3.16—2.90 (4H, m), 2.87 (1H,
s), 2.64—2.51 (2H, m), 2.45 (3H, m), 2.40—2.32 (5H, m), 2.20—1.92 (2H,
m), 1.43 (9H, m), 1.40 (9H, s), 1.38 (3H, t, Jϭ6.9 Hz). FAB-MS m/z: 789
(MϩH)ϩ.
Boc-Tyr(CO2Bui)-D-MetO-Phe-MebAla-OBut (6n) Using method F,
compound 5 (1.79 g, 2.50 mmol) was reacted with isobutyl chloroformate
(376 mg, 2.75 mmol) to give 6n (1.85 g, 91%) as a white powder. Rf1 0.58.
1H-NMR (CDCl3) d: 7.93—7.74 (1H, m), 7.35—7.18 (8H, m), 7.09 (2H,
m), 5.85—5.52 (1H, m), 5.13—5.01 (1H, m), 4.69 (1H, m), 4.34 (1H, m),
4.01 (2H, d, Jϭ6.9 Hz), 3.70 (0.66H, m), 3.45 (0.33H, m), 3.37 (1H, m),
3.16—3.02 (2H, m), 2.96 (2H, m), 2.87 (1H, s), 2.80 (2H, s), 2.64—2.51
(2H, m), 2.45 (3H, m), 2.40—2.32 (3H, m), 2.20—1.92 (3H, m), 1.43 (9H,
m), 1.40 (9H, s), 1.00 (6H, d, Jϭ6.9 Hz). FAB-MS m/z: 817 (MϩH)ϩ.
Boc-Tyr(CO2Ph)-D-MetO-Phe-MebAla-OBut (6o) Using method F,
compound 5 (2.15 g, 3.00 mmol) was reacted with phenyl chloroformate
(516 mg, 3.3 mmol) to give 6o (2.34 g, 93%) as a white powder. Rf1 0.60. 1H-
NMR (CDCl3) d: 7.96—7.73 (1H, m), 7.41 (2H, m), 7.30—7.15 (12H, m),
5.88—5.56 (1H, m), 5.13—5.00 (1H, m), 4.69 (1H, m), 4.35 (1H, m), 3.69
(0.66H, m), 3.47 (0.33H, m), 3.37 (1H, m), 3.10 (2H, m), 2.99 (2H, m), 2.87
(1H, s), 2.80 (2H, s), 2.57—2.32 (8H, m), 2.26—1.93 (3H, m), 1.43 (9H,
m), 1.40 (9H, s). FAB-MS m/z: 837 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr-D-MetO-Phe-MebAla-O(Phthalidyl) (4u) Com-
pound 3u (2.38 g, 3.0 mmol) was treated according to method H, yielding 4u
(594 mg, 27%) as a white powder. HPLC tR (min): 10.15. Rf4 0.66. 1H-NMR
(CD3OD) d: 7.81—7.55 (4H, m), 7.33 (2H, d, Jϭ5.4 Hz), 7.14 (5H, m), 6.97
(2H, mb, 6.62 (2H, m), 4.95 (1H, m), 4.30 (2H, m), 3.61 (1H, m), 3.48 (1H,
m), 3.10—2.70 (7H, m), 2.54 (2H, m), 2.46 (3H, m), 2.25 (2H, m), 2.09
(3H, m), 1.85 (1H, m), 1.71 (1H, m). FAB-MS m/z: 734 (MϩH)ϩ.
Preparation of Boc-Tyr-D-MetO-Phe-MedAla-OBut (5) Compound 1
(17.3 g, 30.0 mmol) and H-MebAla-OBut, obtained from 2q by deprotection
using method E, were coupled (method A) to yield 5 (17.8 g, 83 %) as a
1
white powder. Rf1 0.46. H-NMR (CDCl3) d: 8.45—7.90 (2H, m), 7.35—
Boc-Tyr(CO2Bzl)-D-MetO-Phe-MebAla-OBut (6p) Using method F,
compound 5 (2.15 g, 3.00 mmol) was reacted with benzyl chloroformate
(563 mg, 3.3 mmol) to give 6p (2.20 g, 86%) as a white powder. Rf1 0.58.
1H-NMR (CDCl3) d: 7.88—7.53 (1H, m), 7.45—7.34 (5H, m), 7.30—7.17
(7H, m), 7.10 (2H, dd, Jϭ8.7, 2.4 Hz), 5.75—5.47 (1H, m), 5.24 (2H, s),
5.02 (1H, m), 4.64 (1H, m), 4.32 (1H, m), 3.67 (0.66H, m), 3.38 (1.33H, m),
3.08 (2H, m), 2.99 (2H, m), 2.86 (1H, s), 2.79 (2H, s), 2.58—2.42 (7H, m),
2.37 (2H, m), 2.19—1.94 (3H, m), 1.43 (9H, m), 1.39 (9H, s). FAB-MS m/z:
851 (MϩH)ϩ.
7.15 (5H m), 7.03 (2H, d, Jϭ8.1 Hz), 6.79 (2H, dd, Jϭ8.4, 4.8 Hz), 6.11—
5.76 (1H, m), 5.03 (1H, m), 4.73 (1H, s), 4.25 (1H, m), 3.70 (1H, m), 3.36
(1H, m), 3.10—2.90 (4H, m), 2.88 (1.5H, s), 2.79 (1.5H, s), 2.48 (3H, m),
2.36 (2H, m), 2.16 (2H, br m), 1.92 (1H, br m), 1.76 (1H, br m), 1.42 (18H,
s). FAB-MS m/z: 717 (MϩH)ϩ.
Boc-Tyr(COMe)-D-MetO-Phe-MebAla-OBut (6a) As described in
method F, compound 5 (6.0 g, 8.37 mmol) was reacted with acetyl chloride
(723 mg, 9.21 mmol) to yield 6a (6.0 g, 95%) as a white powder. Rf2 0.45.
1H-NMR (CDCl3) d: 7.89 (1H, m), 7.68 (1H, m), 7.27—7.21 (7H, m),
7.01—6.97 (2H, m), 5.48 (1H, br s), 5.02 (1H, br s), 4.64 (1H, m), 4.38 (1H,
br s), 3.07—2.96 (4H, m), 2.83—2.76 (3H, m), 2.53 (3H, s), 2.49 (3H, s),
2.26 (3H, s), 2.11—2.05 (2H, s), 1.43 (9H, s), 1.39 (9H, s). FAB-MS m/z:
759 (MϩH)ϩ.
Boc-Tyr(COEt)-D-MetO-Phe-MebAla-OBut (6b) Using method G,
compound 5 (3.58 g, 5.0 mmol) was reacted with propionic acid (407 mg,
1
5.5 mmol) to give 6b (3.63 g, 94%) as a white powder. Rf1 0.51. H-NMR
(CDCl3) d: 7.50 (1H, m), 7.24 (7H, m), 7.00 (2H, dd, Jϭ8.6, 2.0 Hz), 5.30—
5.24 (1H, m), 4.99 (1H, m), 4.57 (1H, m), 4.28 (1H, m), 3.63 (0.5H, m),
3.37 (1.5H, m), 3.08 (1H, m), 2.99 (3H, m), 2.86 (1.5H, s), 2.77 (1.5H, s),
2.60—2.53 (6H, m), 2.46—2.44 (1H, m), 2.37 (2H, m), 2.25—2.01 (2H, m),
1.92 (2H, br s), 1.43 (9H, s), 1.41 (9H, s), 1.24 (3H, t, Jϭ7.5 Hz). FAB-MS
m/z: 773 (MϩH)ϩ.
Compounds 6c, d, h, l—p were prepared in the same manner from com-
pound 5 and the corresponding YCO-Cl, respectively.
Boc-Tyr(COPrn)-D-MetO-Phe-MebAla-OBut (6c) Using method F,
compound 5 (2.50 g, 3.49 mmol) was reacted with n-butyryl chloride (409
1
mg, 3.84 mmol) to give 6c (1.38 g, 50%) as a white powder. Rf3 0.68. H-
NMR (CDCl3) d: 7.75—7.49 (2H, m), 7.26 (7H, m), 7.00 (2H, dd, Jϭ8.4,
2.1 Hz), 5.51—5.35 (1H, m), 5.01 (1H, m), 4.63 (1H, m), 4.30 (1H, m), 3.65
(0.5H, m), 3.39 (1.5H, m), 3.06 (1H, m), 2.99 (3H, m), 2.87 (1H, s), 2.78
(2H, s), 2.65—2.45 (8H, m), 2.36 (2H, m), 2.09 (4H, m), 1.76 (3H, m), 1.43
(9H, s), 1.40 (9H, s), 1.03 (3H, t, Jϭ7.4 Hz). FAB-MS m/z: 787 (MϩH)ϩ.
Boc-Tyr(COPri)-D-MetO-Phe-MebAla-OBut (6d) Using method F,
compound 5 (2.50 g, 3.49 mmol) was reacted with isobutyryl chloride (409
Compounds 6e—g, i—k were prepared in the same manner from com-
pound 5 and the corresponding YCOOH, respectively.
Boc-Tyr(COHep)-D-MetO-Phe-MebAla-OBut (6e) Using method G,
compound 5 (4.00 g, 5.58 mmol) was reacted with octanoic acid (885 mg,
1
6.14 mmol) to give 6e (4.25 g, 90%) as a white powder. Rf2 0.54. H-NMR
(CDCl3) d: 7.52 (1H, m), 7.25 (7H, m), 7.02 (2H, m), 5.25—5.21 (1H, m),
4.96 (1H, m), 4.46 (1H, m), 4.21 (0.5H, m), 3.85—3.65 (1H, m), 3.25 (1H,
m), 3.04 (2H, m), 2.93—2.89 (4H, m), 2.56—2.53 (5H, m), 2.37 (4H, m),
2.20 (3H, s), 2.10—2.80 (2H, m), 1.72 (2H, m), 1.43 (9H, s), 1.33 (8H, m),
0.92 (3H, t, Jϭ6.8 Hz). FAB-MS m/z: 843 (MϩH)ϩ.
1
mg, 3.84 mmol) to give 6d (1.79 g, 65%) as a white powder. Rf2 0.39. H-
NMR (CDCl3) d: 7.78—7.48 (1H, m), 7.26 (7H, m), 7.00 (2H, dd, Jϭ8.4,
2.4 Hz), 5.60—5.42 (1H, m), 5.02 (1H, m), 4.64 (1H, m), 4.31 (1H, br s),
3.67 (1H, m), 3.38 (1H, m), 3.06 (1H, m), 2.99 (3H, m), 2.87 (1H, s), 2.82—
2.73 (3H, m), 2.59—2.44 (3H, m), 2.36 (2H, m), 2.27 (2H, m), 2.20—1.90
(2H, m), 1.43 (9H, s), 1.40 (9H, s), 1.29 (3H, t, Jϭ6.9 Hz). FAB-MS m/z:
787 (MϩH)ϩ.
Boc-Tyr(COBut)-D-MetO-Phe-MebAla-OBut (6f) Using method G,
compound 5 (2.15 g, 3.00 mmol) was reacted with pivalic acid (337 mg,
1
3.3 mmol) to give 6f (1.22 g, 51%) as a white powder. Rf1 0.52. H-NMR
(CDCl3) d: 7.75—7.45 (1H, m), 7.31—7.18 (7H, m), 6.98 (2H, m), 5.55—
5.32 (1H, m), 5.01 (1H, m), 4.59 (1H, m), 4.29 (1H, m), 3.63 (0.5H, m),
3.37 (1.5H, m), 3.08 (1H, m), 2.99 (3H, m), 2.87 (1.5H, s), 2.79 (1.5H, s),
2.61—2.51 (4H, m), 2.46—2.44 (1H, m), 2.37 (2H, m), 2.25—2.01 (2H, m),
1.92 (2H, br s), 1.43 (9H, s), 1.41 (9H, s), 1.33 (9H, s). FAB-MS m/z: 801
(MϩH)ϩ.
Boc-Tyr(COPh)-D-MetO-Phe-MebAla-OBut (6h) Using method F,
compound 5 (2.15 g, 3.00 mmol) was reacted with benzoyl chloride (464 mg,
1
3.3 mmol) to give 6h (2.28 g, 93%) as a white powder. Rf1 0.58. H-NMR
(CDCl3) d: 8.16 (2H, d, Jϭ7.5 Hz), 7.62 (1H, t, Jϭ7.4 Hz), 7.49 (3H, m),
7.30—7.10 (8H, m), 5.32—5.22 (1H, m), 5.01 (1H, m), 4.56 (1H, m), 4.32
(1H, m), 3.64 (0.67H, m), 3.39 (1.33H, m), 3.11 (1H, m), 2.99 (3H, m), 2.85
(1H, s), 2.78 (2H, s), 2.59 (2H, m), 2.55 (2H, s), 2.47 (1H, m), 2.38 (2H, m),
2.14 (2H, m), 1.43 (9H, s), 1.42 (9H, s). FAB-MS m/z: 821 (MϩH)ϩ.
Boc-Tyr(CO(1-Ad))-D-MetO-Phe-MebAla-OBut (6g) Using method
G, compound 5 (2.15 g, 3.0 mmol) was reacted with 1-adamantane car-
boxylic acid (594 mg, 3.30 mmol) to give 6g (1.64 g, 62%) as a white pow-

July 2003
769
1
Na-1-Iminoethyl-Tyr(COBut)-D-MetO-Phe-MebAla-OH (7f) Com-
pound 6f (1.10 g, 1.42 mmol) was treated according to method H, yielding 7f
(330 mg, 34%) as a white powder. HPLC tR (min): 13.77. Rf4 0.65. 1H-NMR
(CD3OD) d: 7.25 (7H, m), 7.04 (2H, d, Jϭ7.2 Hz), 5.25 (0.5 H, m), 5.02
(0.5H, m), 4.56 (0.5H, m), 4.45 (1H, m), 4.40 (0.5H, m), 3.70 (1H, m), 3.42
(1H, m), 3.25 (1H, m), 2.94 (1H, m), 2.88 (2H, m), 2.54 (3H, m), 2.39
(4h, m), 2.19 (3H, s), 2.10—1.80 (2H, m), 1.41 (9H, s). FAB-MS m/z: 686
(MϩH)ϩ.
der. Rf1 0.43. H-NMR (CDCl3) d: 7.48 (1H, m), 7.33—7.15 (7H, m), 6.99
(2H, m), 5.28—5.20 (1H, m), 4.96 (1H, m), 4.53 (1H, m), 4.27 (1H, m),
3.60 (0.5H, m), 3.34 (1.5H, m), 3.05 (1H, m), 2.97 (3H, m), 2.87 (1.5H, s),
2.77 (1.5H, s), 2.60—2.51 (4H, m), 2.46—2.44 (1H, m), 2.35 (2H, m),
2.28—2.03 (5H,m), 1.96 (10H, m), 1.87—1.76 (2H, m), 1.71 (8H, m), 1.42
(9H, s), 1.40 (9H, s). FAB-MS m/z: 879 (MϩH)ϩ.
Boc-Tyr(COCH2CH2Ph)-D-MetO-Phe-MebAla-OBut (6i) Using
method G, compound 5 (2.00 g, 2.79 mmol) was reacted with hydrocinnamic
acid (461 mg, 3.07 mmol) to give 6i (2.24 g, 95%) as a white powder. Rf3
Na-1-Iminoethyl-Tyr(CO(1-Ad))-
-MetO-Phe-MebAla-OH (7g) Com-
D
1
0.76. H-NMR (CDCl3) d: 7.73—7.50 (2H, m), 7.34—7.17 (12H, m), 6.93
pound 6g (1.60 g, 1.82 mmol) was treated according to method H, yielding
1
(2H, dd, Jϭ8.6, 2.9 Hz), 5.57—5.37 (1H, m), 5.01 (1H, m), 4.62 (1H, m),
4.29 (1H, br s), 3.67 (1H, m), 3.36 (2H, m), 3.09—2.96 (6H, m), 2.88—2.83
(3H, m), 2.78 (2H, s), 2.58—2.45 (3H, m), 2.42—2.31 (4H, m), 2.17 (3H,
s), 2.11—2.02 (2H, m), 1.43 (9H, s), 1.40 (9H, s), FAB-MS m/z: 849
(MϩH)ϩ.
7g (487 mg, 35%) as a white powder. HPLC tR (min): 14.31. Rf4 0.63. H-
NMR (CD3OD) d: 7.24—7.08 (7H, m), 6.90 (2H, m), 5.13 (0.5H, m), 4.93
(0.5H, m), 4.44 (0.5H, m), 4.35 (1H, m), 4.30 (0.5H, m), 3.60 (1H, m), 3.32
(1H, m), 3.13 (1H, m), 2.95 (2H, m), 2.86—2.76 (4H, m), 2.46 (1.5H, s),
2.43 (1.5H, s), 2.36—2.17 (4H, m), 2.10 (3H, m), 1.95 (8H, br s), 1.87—
1.76 (2H, m), 1.71 (8H, m). FAB-MS m/z: 764 (MϩH)ϩ.
Boc-Tyr(COCH2CH2CH2Ph)-D-MetO-Phe-MebAla-OBut (6j) Using
method G, compound 5 (3.00 g, 4.18 mmol) was reacted with 4-phenylbu-
tyric acid (756 mg, 4.60 mmol) to give 6j (3.51 g, 97%) as a white powder.
Na-1-Iminoethyl-Tyr(COPh)-D-MetO-Phe-MebAla-OH (7h) Com-
pound 6h (2.00 g, 2.44 mmol) was treated according to method H, yielding
1
1
Rf3 0.82. H-NMR (CDCl3, TMS) d:7.62—6.69 (17H, m), 6.45—5.36 (1H,
7h (1.01 g, 59%) as a white powder. HPLC tR (min): 12.11. Rf4 0.60. H-
m), 5.01 (1H, m), 4.63 (1H, m), 4.29 (1H, m), 3.40—3.34 (4H, m), 3.10—
2.94 (4H, m), 2.86—2.70 (3H, m), 2.59—2.40 (9H, m), 2.28—2.01 (6H, m),
1.43 (9H, s), 1.40 (9H, s). FAB-MS m/z: 863 (MϩH)ϩ.
NMR (CD3OD) d: 8.05 (2H, m), 7.59 (1H, t, Jϭ7.4 Hz), 7.45 (2H, t, Jϭ
7.5 Hz), 7.28 (2H, m), 7.16 (7H, m), 5.14 (0.5H, t, Jϭ7.3 Hz), 4.92 (0.5H,
m), 4.53 (0.5H, m), 4.45 (1H, m), 4.37 (1H, m), 3.57 (1H, m), 3.34 (1H, m),
3.19 (1H, m), 2.95 (2H, m), 2.84—2.75 (4H, ms), 2.47 (2H, s), 2.44 (1H, s),
2.30 (4H, m), 2.11 (3H, m), 2.00—1.60 (2H, m). FAB-MS m/z: 706
(MϩH)ϩ.
Boc-Tyr(COcHex)-D-MetO-Phe-MebAla-OBut (6k) Using method G,
compound 5 (2.00 g, 2.79 mmol) was reacted with cyclohexanecarboxylic
acid (393 mg, 3.07 mmol) to give 6k (2.18 g, 94%) as a white powder. Rf3
1
Na-1-Iminoethyl-Tyr(COCH2CH2Ph)-D-MetO-Phe-MebAla-OH (7i)
Compound 6i (2.00 g, 2.36 mmol) was treated according to method H, yield-
ing 7i (778 mg, 45%) as a white powder. HPLC tR (min): 12.67. Rf4 0.59. 1H-
NMR (CD3OD) d: 7.28 (12H, m), 6.95 (2H, d, Jϭ8.4 Hz), 5.23 (0.5H, t,
Jϭ6.4 Hz), 5.02 (0.5H, m), 4.53 (0.5H, m), 4.46 (1H, m), 4.39 (0.5H, m),
3.71 (1H, m), 3.41 (1H, m), 3.23 (1H, m), 3.07—3.00 (4H, m), 2.96—2.84
(6H, m), 2.60—2.25 (7H, m), 1H), 2.18 (3H, s), 2.10—1.60 (2H, m). FAB-
MS m/z: 734 (MϩH)ϩ.
0.86. H-NMR (CDCl3) d: 7.76—7.40 (2H, m), 7.31—7.17 (7H, m), 6.98
(2H, dd, Jϭ8.6, 2.3 Hz), 5.51—5.34 (1H, m), 5.02 (1H, m), 4.64 (1H, m),
4.30 (1H, br s), 3.65 (1H, m), 3.38 (1H, m), 3.06 (1H, m), 2.99 (3H, m), 2.86
(1H, s), 2.78 (2H, s), 2.60—2.25 (5H, m), 2.25—1.90 (3H, m), 1.79 (2H,
m), 1.75—1.45 (3H, m), 1.43 (9H, s), 1.41 (9H, s), 1.38—1.22 (3H, m).
FAB-MS m/z: 827 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr(COMe)-D-MetO-Phe-MebAla-OH (7a) Com-
pound 6a (6.00 g, 7.91 mmol) was treated according to method H, yielding
7a (2.14 g, 42%) as a white powder. HPLC tR (min): 8.50. Rf4 0.62. 1H-NMR
(CD3OD) d: 7.16 (7H, m), 6.96 (2H, m), 5.12 (0.5H, t, Jϭ7.3 Hz), 4.93
(0.5H, t, Jϭ7.3 Hz), 4.50—4.30 (2H, m), 3.51 (1H, m), 3.42 (1H, m), 3.15
(1H, m), 2.93 (2H, m), 2.85—2.78 (4H, m), 2.46 (1.5H, s), 2.44 (1.5H, s),
2.32 (2H, m), 2.16 (3H, s), 2.09 (3H, s), 2.00—1.55 (2H, m). FAB-MS m/z:
644 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr(COCH2CH2CH2Ph)-D-MetO-Phe-MebAla-OH
(7j) Compound 6j (3.00 g, 3.48 mmol) was treated according to method H,
yielding 7j (177 mg, 6.8%) as a white powder. HPLC tR (min): 13.91. Rf4
1
0.65. H-NMR (CD3OD) d: 7.26 (12H, m), 7.03 (2H, d, Jϭ7.5 Hz), 5.24
(0.5H, t, Jϭ6.4 Hz), 5.01 (0.5H, m), 4.56 (0.5H, m), 4.48 (1H, m), 4.38
(0.5H, m), 3.73 (1H, m), 3.35 (1H, m), 3.23 (1H, m), 3.04 (2H, m), 2.96—
2.88 (4H, m), 2.72 (2H, t, Jϭ7.5 Hz), 2.40 (4H, m), 2.19 (3H, s), 2.02 (2H,
m), 1.91—1.60 (2H, m). FAB-MS m/z: 748 (MϩH)ϩ.
Compounds 7b—o, 7s—u were prepared in the same manner.
Na-1-Iminoethyl-Tyr(COEt)-D-MetO-Phe-MebAla-OH (7b) Com-
pound 6b (3.00 g, 3.88 mmol) was treated according to method H, yielding
Na-1-Iminoethyl-Tyr(COcHex)-D-MetO-Phe-MebAla-OH (7k) Com-
pound 6k (2.00 g, 2.42 mmol) was treated according to method H, yielding
1
7b (561 mg, 22%) as a white powder. HPLC tR (min): 9.37. Rf4 0.64. H-
1
NMR (CD3OD) d: 7.19 (7H, m), 6.95 (2H, d, Jϭ6.9 Hz), 5.15 (0.5H, t,
Jϭ7.3 Hz), 4.92 (0.5H, t, Jϭ7.3 Hz), 4.49 (0.5H, m), 4.38 (1H, m), 4.31
(0.5H, m), 3.65 (1H, m), 3.26 (1H, m), 3.15 (1H, m), 2.95 (2H, m), 2.86—
2.74 (4H, m), 2.53—2.43 (5H, m), 2.37—2.24 (3H, m), 2.09 (3H, s), 1.93
(1H, m), 1.77 (1H, m), 1.62 (1H, m), 1.11 (3H, t, Jϭ7.5 Hz). FAB-MS m/z:
658 (MϩH)ϩ.
7k (430 mg, 25%) as a white powder. HPLC tR (min): 14.38. Rf4 0.65. H-
NMR (CD3OD) d: 7.25 (7H, m), 7.02 (2H, d, Jϭ7.8 Hz), 5.24 (0.5H, m),
5.01 (0.5H, m), 4.57 (0.5H, m), 4.47 (1H, m), 4.38 (0.5H, m), 3.73 (1H, m),
3.36 (1H, m), 3.24 (1H, m), 3.04 (2H, m), 2.92—2.89 (4H, m), 2.62—2.52
(5H, m), 2.37 (4H, m), 2.19 (3H, s), 2.10—1.20 (13H, m). FAB-MS m/z:
712 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr(COPr)-D-MetO-Phe-MebAla-OH (7c) Com-
pound 6c (1.20 g, 1.52 mmol) was treated according to method H, yielding
Na-1-Iminoethyl-Tyr(CO2CH3)-D-MetO(RS)-Phe-MebAla-OH (7l)
Compound 6l (2.00 g, 2.58 mmol) was treated according to method H, yield-
ing 7l (800 mg, 47%) as a white powder. HPLC tR (min): 8.67. Rf4 0.60. 1H-
NMR (CD3OD) d: 7.30—7.10 (7H, m), 7.04 (2H, dd, Jϭ8.6, 1.7 Hz), 5.15
(0.5H, t, Jϭ6.4 Hz), 4.93 (0.5H, m), 4.46 (0.5H, m), 4.38 (1H, m), 4.31
(0.5H, m), 3.76 (3H, s), 3.63 (1H, m), 3.30 (1H, m), 3.20 (1H, m), 2.93 (2H,
m), 2.84—2.79 (4H, m), 2.46 (1.5H, s), 2.79 (1.5H, s), 2.28 (4H, m), 2.09
(3H, s), 2.00—1.55 (2H, m). FAB-MS m/z: 660 (MϩH)ϩ.
1
7c (440 mg, 43%) as a white powder. HPLC tR (min): 10.72. Rf4 0.67. H-
NMR (CD3OD) d: 7.25 (7H, m), 7.04 (2H, d, Jϭ7.2 Hz), 5.25 (0.5H, m),
5.02 (0.5H, m), 4.56 (0.5H, m), 4.45 (1H, m), 4.40 (0.5H, m), 3.70 (1H, m),
3.42 (1H, m), 3.25 (1H, m), 3.04 (2H, m), 2.94 (1H, m), 2.88 (2H, m), 2.54
(3H, m), 2.39 (4h, m), 2.19 (3H, s), 2.10—1.80 (2H, m), 1.74 (2H, m), 1.03
(3H, t, Jϭ7.5 Hz, 2H). FAB-MS m/z: 672 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr(COPri)-D-MetO-Phe-MebAla-OH (7d) Com-
pound 6d (1.60 g, 2.03 mmol) was treated according to method H, yielding
Na-1-Iminoethyl-Tyr(CO2Et)-D-MetO-Phe-MebAla-OH (7m) Com-
pound 6m (2.00 g, 2.54 mmol) was treated according to method H, yielding
1
1
7d (464 mg, 34%) as a white powder. HPLC tR (min): 11.03. Rf4 0.63. H-
7m (700 mg, 41%) as a white powder. HPLC tR (min): 9.46. Rf4 0.63. H-
NMR (CD3OD) d: 7.25—7.12 (7H, m), 6.94 (2H, d, Jϭ7.0 Hz), 5.25 (0.5H,
t, Jϭ6.4 Hz), 5.04 (0.5H, t, Jϭ6.4 Hz), 4.57 (0.5H, t, Jϭ8.4 Hz), 4.51—4.40
(1.5H, m), 3.74 (1H, m), 3.47—3.22 (2H, m), 3.06 (2H, m), 2.95 (1H, s),
2.93 (1H, s), 2.90 (3H, s), 2.87—2.77 (3H, m), 2.57 (2H, s), 2.54 (2H, s),
2.44—2.33 (4H, m), 2.20 (3H, s), 2.06—1.70 (3H, m), 1.30 (6H, d,
Jϭ7.0 Hz). FAB-MS m/z: 672 (MϩH)ϩ.
NMR (CD3OD) d: 7.25—7.02 (9H, m), 7.04 (2H, dd, Jϭ8.6, 1.7 Hz), 5.13
(0.5H, m), 4.92 (0.5H, m), 4.45—4.28 (1.5H, m), 4.17 (2H, q, Jϭ7.2 Hz),
3.91 (0.5H, m), 3.54 (1H, m), 3.36 (1H, m), 3.14 (1H, m), 3.20 (1H, m),
2.97—2.89 (3H, m), 2.86—2.74 (4H, m), 2.56—2.39 (4H, m), 2.36—2.17
(4H, m), 2.09 (3H, s), 2.00—1.55 (2H, m), 1.24 (3H, t, Jϭ7.2 Hz). FAB-MS
m/z: 674 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr(COHep)-D-MetO-Phe-MebAla-OH (7e) Com-
pound 6e (1.70 g, 2.02 mmol) was treated according to method H, yielding
Na-1-Iminoethyl-Tyr(CO2Bui)-D-MetO-Phe-MebAla-OH (7n) Com-
pound 6n (1.80 g, 2.20 mmol) was treated according to method H, yielding
1
1
7e (82 mg, 5.6%) as a white powder. HPLC tR (min): 13.94. Rf4 0.65. H-
7n (588 mg, 38%) as a white powder. HPLC tR (min): 10.12. Rf4 0.66. H-
NMR (CD3OD) d: 7.26 (7H, m), 7.04 (2H, m), 5.25 (0.5H, t, Jϭ6.4 Hz),
5.01 (0.5H, m), 4.56 (0.5H, m), 4.46 (1H, m), 4.41 (0.5H, m), 3.85—3.65
(1H, m), 3.25 (1H, m), 3.04 (2H, m), 2.93—2.89 (4H, m), 2.56—2.53 (5H,
m), 2.37 (4H, m), 2.20 (3H, s), 2.10—2.80 (2H, m), 1.72 (2H, m), 1.33 (8H,
m), 0.92 (3H, t, Jϭ6.8 Hz). FAB-MS m/z: 728 (MϩH)ϩ.
NMR (CD3OD) d: 7.16 (7H, m), 6.94 (2H, d, Jϭ7.0 Hz), 5.14 (0.5H, m),
4.92 (0.5H, m), 4.46 (0.5H, m), 4.38 (1H, m), 4.32 (0.5H, m), 3.63 (1H, m),
3.30 (1H, m), 3.15 (1H, m), 2.95 (2H, m), 2.95—2.65 (5H, m), 2.46 (1.5H,
s), 2.43 (1.5H, s), 2.28 (4H, m), 2.09 (3H, s), 1.87 (1H, m), 1.74 (2H, m),
1.60 (1H, m), 1.19 (6H, d, Jϭ6.9 Hz). FAB-MS m/z: 702 (MϩH)ϩ.

770
Vol. 51, No. 7
Na-1-Iminoethyl-Tyr(CO2Ph)-D-MetO-Phe-MebAla-OH (7o) Com-
m), 4.30 (1H, m), 4.02 (4H, m), 3.71 (0.67H, m), 3.49 (0.33H, m), 3.40 (1H,
m), 3.09 (2H, m), 2.99 (2H, m), 2.87 (1H, s), 2.79 (2H, s), 2.60—2.21 (8H,
m), 2.04 (4H, m), 1.61 (2H, t, Jϭ5.4 Hz), 1.39 (9H, s), 1.28 (10H, m), 0.88
(3H, m). FAB-MS m/z: 873 (MϩH)ϩ.
pound 6o (2.00 g, 2.39 mmol) was treated according to method H, yielding
1
7o (483 mg, 28%) as a white powder. HPLC tR (min): 11.08. Rf4 0.63. H-
NMR (CD3OD) d: 7.30—7.04 (9H, m), 6.80—6.70 (5H, m), 5.19 (0.5H, m),
5.04 (0.5H, m), 4.53—4.27 (1.5H, m), 3.98 (0.5H, m), 3.63—3.39 (2H, m),
3.15—2.94 (3H, m), 2.92—2.82 (4H, m), 2.63—2.50 (4H, m), 2.38 (3H, m),
2.17 (3H, m), 1.90 (1H, m), 1.75 (1H, m). FAB-MS m/z: 722 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr(CO2Bzl)-D-MetO-Phe-MebAla-OH (7p) Com-
pound 6p (2.00 g, 2.35 mmol) was treated according to method H, yielding
Na-1-Iminoethyl-Tyr(COMe)-
D
-MetO-Phe-Me Ala-O(Oct) (9a) Com-
b
pound 8a (2.00 g, 2.45 mmol) was treated according to method H, yielding
1
9a (464 mg, 25%) as a white powder. HPLC tR (min): 15.89. Rf4 0.70. H-
NMR (CD3OD) d: 7.35—7.20 (7H, m), 7.04 (2H, d, Jϭ8.4 Hz), 5.15 (0.5H,
m), 5.03 (0.5H, m), 4.53 (1H, m), 4.41 (1H, m), 4.03 (2H, t, Jϭ6.8 Hz), 3.65
(1H, m), 3.42 (1H, m), 3.22 (1H, m), 3.10—2.97 (3H, m), 2.96—2.83 (4H,
m), 2.57 (3H, m), 2.53—2.35 (4H, m), 2.25 (3H, s), 2.19 (3H, m), 1.95 (1H,
m), 1.81 (1H, m), 1.62 (2H, m), 1.29 (10H, m), 0.89 (3H, t, Jϭ6.8 Hz).
1
7p (415 mg, 24%) as a white powder.HPLC tR (min): 11.59. Rf4 0.64. H-
NMR (CD3OD) d: 7.32—7.02 (14H, m), 6.94 (2H, d, Jϭ7.0 Hz), 5.15 (2H,
s), 5.06 (0.5H, m), 4.93 (0.5H, m), 4.38—4.25 (2H, m), 3.55 (1H, m), 3.36
(1H, m), 3.15 (1H, m), 2.99—2.91 (3H, m), 2.85—2.73 (4H, m), 2.54—2.36
(4H, m), 2.29 (3H, m), 2.20 (1H, m), 2.09 (3H, s), 1.81 (1H, m), 1.66 (1H,
m). FAB-MS m/z: 736 (MϩH)ϩ.
Boc-Tyr(COMe)-D-MetO-Phe-MebAla-O(Oct) (8a) As described in
method F, compound 3g (2.32 g, 3.0 mmol) was reacted with acetyl chloride
(260 mg, 3.30 mmol) to yield 8a (2.20 g, 90%) as a white powder. Rf3 0.67.
1H-NMR (CDCl3) d: 7.74—7.52 (1H, m), 7.22 (7H, m), 7.01 (2H, dd,
Jϭ8.4, 1.8 Hz), 5.58—5.31 (1H, m), 4.99 (1H, m), 4.58 (1H, m), 4.29 (1H,
m), 4.04 (2H, t, Jϭ6.8 Hz), 3.68 (1H, m), 3.40 (1H, m), 3.08 (1H, m), 2.99
(3H, m), 2.87 (1H, s), 2.78 (2H, s), 2.65—2.30 (7H, m), 2.27 (3H, s), 2.05
(2H, m), 1.61 (2H, t, Jϭ7.2 Hz), 1.40 (9H, s), 1.29 (10H, m), 0.88 (3H, m).
FAB-MS m/z: 815 (MϩH)ϩ.
Boc-Tyr(COMe)-D-MetO-Phe-MebAla-OHde (8b) As described in
method F, compound 3k (2.66 g, 3.0 mmol) was reacted with acetyl chloride
(260 mg, 3.3 mmol) to yield 8b (2.31 g, 83%) as a white powder. Rf3 0.69.
1H-NMR (CDCl3) d: 7.72—7.60 (1H, m), 7.22 (7H, m,), 7.00 (2H, dd,
Jϭ8.4, 1.8 Hz), 5.55—5.30 (1H, m), 4.97 (1H, m), 4.80 (1H, m), 4.30 (1H,
m), 4.03 (2H, t, Jϭ6.8 Hz), 3.66 (1H, m), 3.42 (1H, m), 3.06 (1H, m), 2.98
(3H, m), 2.88 (1H, s), 2.78 (2H, s), 2.65—2.30 (7H, m), 2.27 (3H, s), 2.05
(2H, m), 1.61 (2H, t, Jϭ7.2 Hz), 1.40 (9H, s), 1.29 (26H, m), 0.88 (3H, m).
FAB-MS m/z: 928 (MϩH)ϩ.
FAB-MS m/z: 756 (MϩH)ϩ.
a
N -1-Iminoethyl-Tyr(COMe)-D-MetO-Phe-Me Ala-O(Hde) (9b)
Compound 8b (2.10 g, 2.26 mmol) was treated according to method H,
yielding 9b (433 mg, 22%) as a white powder. HPLC tR (min): Ͼ20.0. Rf4
b
1
0.71. H-NMR (CD3OD) d: 7.32—7.20 (7H, m), 7.06 (2H, d, Jϭ8.4 Hz),
5.14 (0.5H, m), 5.05 (0.5H, m), 4.53 (1H, m), 4.41 (1H, m), 4.05 (2H, t, Jϭ
6.8 Hz), 3.66 (1H, m), 3.44 (1H, m), 3.23 (1H, m), 3.10—2.99 (3H, m),
2.96—2.83 (4H, m), 2.57 (3H, m), 2.53—2.35 (4H, m), 2.25 (3H, s), 2.19
(3H, m), 1.96 (1H, m), 1.81 (1H, m), 1.61 (2H, m), 1.31 (26H, m), 0.89 (3H,
t, Jϭ6.8 Hz). FAB-MS m/z: 869 (MϩH)ϩ.
a
N -1-Iminoethyl-Tyr(COMe)-D-MetO-Phe-Me Ala-OCH(Me)-
OCOOEt (9c) Compound 8c (2.00 g, 2.44 mmol) was treated according to
method H, yielding 9c (538 mg, 29%) as a white powder. HPLC tR (min):
11.69. Rf4 0.69. 1H-NMR (CD3OD) d: 7.27 (7H, m), 7.06 (2H, d, Jϭ8.4 Hz),
6.71 (1H, m), 5.05 (1H, m), 4.43 (1H, m), 4.16 (2H, m), 3.66 (1H, m), 3.48
(1H, m), 3.23 (1H, m), 3.10—2.80 (7H, m), 2.70—2.15 (4H, m), 2.55 (3H,
s), 2.26 (3H, s), 2.19 (3H, s), 1.96 (1H, m), 1.81 (1H, m), 1.47 (3H, d,
b
Jϭ5.4 Hz), 1.24 (3H, m). FAB-MS m/z: 760 (MϩH)ϩ.
a
N -1-Iminoethyl-Tyr(COMe)-D-MetO-Phe-Me Ala-O(Phthalidyl)
(9d) Compound 8d (2.00 g, 2.40 mmol) was treated according to method
H, yielding 9d (390 mg, 21%) as a white powder. HPLC tR (min): 11.13. Rf4
b
1
Boc-Tyr(COMe)-D-MetO-Phe-MebAla-OCH(Me)OCOOEt (8c) As
described in method F, compound 3t (2.33 g, 3.0 mmol) was reacted with
acetyl chloride (260 mg, 3.3 mmol) to yield 8c (2.09 g, 85%) as a white pow-
0.67. H-NMR (CD3OD) d: 7.95—7.65 (4H, m), 7.43 (2H, d, Jϭ6.6 Hz),
7.27 (7H, m), 7.05 (2H, m), 5.23 (0.5H, t, Jϭ6.4 Hz), 5.06 (0.5H, t,
Jϭ6.4 Hz), 4.45 (2H, m), 3.80—3.40 (2H, m), 3.23 (1H, m), 3.10—2.80
(6H, m), 2.64 (2H, m), 2.54 (3H, m), 2.41 (2H, m), 2.25 (3H, s), 2.18 (3H,
s), 1.97 (1H, m), 1.85 (1H, m). FAB-MS m/z: 776 (MϩH)ϩ.
b
1
der. Rf3 0.66. H-NMR (CDCl3) d: 7.74—7.52 (1H, m), 7.22 (7H, m), 7.01
(2H, dd, Jϭ8.4, 1.8 Hz), 6.77 (1H, q, Jϭ5.4 Hz), 5.60—5.35 (1H, m), 5.00
(1H, m), 4.55 (1H, m), 4.27 (1H, m), 4.22 (2H, q, Jϭ7.1 Hz), 3.68 (1H, m),
3.40 (1H, m), 3.08 (1H, m), 2.99 (3H, m), 2.87 (1H, s), 2.78 (2H, s), 2.65—
2.30 (7H, m), 2.27 (3H, s), 2.05 (2H, m), 1.52 (3H, d, Jϭ5.7 Hz), 1.40 (9H,
s), 1.32 (3H, t, Jϭ7.2 Hz). FAB-MS m/z: 819 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr(CO2Et)-
D-MetO-Phe-Me Ala-O(Oct) (9e) Com-
pound 8e (1.90 g, 2.25 mmol) was treated according to method H, yielding
1
9e (424 g, 24%) as a white powder. HPLC tR (min): 16.18. Rf4 0.72. H-
NMR (CD3OD) d: 7.23—7.10 (7H, m), 7.04 (2H, d, Jϭ8.4 Hz), 5.03 (0.5H,
m), 4.95 (0.5H, m), 4.39 (1H, m), 4.32 (1H, m), 4.18 (2H, q, Jϭ7.2 Hz),
3.96 (2H, t, Jϭ6.6 Hz), 3.55 (1H, m), 3.35 (1H, m), 3.15 (1H, m), 3.00—
2.91 (3H, m), 2.88—2.72 (4H, m), 2.54—2.45 (3H, m), 2.39 (2H, m), 2.32
(2H, m), 2.09 (3H, m), 1.88 (1H, m), 1.72 (1H, m), 1.52 (2H, m), 1.24 (3H,
t, Jϭ7.2 Hz), 1.20 (10H, m), 0.80 (3H, t, Jϭ6.9 Hz). FAB-MS m/z: 786
(MϩH)ϩ.
Boc-Tyr(COMe)-D-MetO-Phe-MebAla-O(Phthalidyl) (8d) As de-
scribed in method F, compound 3u (2.38 g, 3.0 mmol) was reacted with
acetyl chloride (260 mg, 3.3 mmol) to yield 8d (2.08 g, 83%) as a white
powder. Rf3 0.67. 1H-NMR (CDCl3) d: 7.95—7.65 (4H, m), 7.43 (2H, d,
Jϭ6.6 Hz), 7.27 (7H, m), 7.05 (2H, m), 5.58—5.30 (1H, m), 4.99 (1H, m),
4.58 (1H, m), 4.29 (1H, m), 3.68 (1H, m), 3.40 (1H, m), 3.08 (1H, m), 2.99
(3H, m), 2.87 (1H, s), 2.78 (2H, s), 2.65—2.30 (7H, m), 2.27 (3H, s), 2.05
(2H, m), 1.85 (1H, m), 1.40 (9H, s). FAB-MS m/z: 835 (MϩH)ϩ.
Na-1-Iminoethyl-Tyr(CO2Bui)-
D
-MetO-Phe-Me Ala-O(Bu) (9f) Com-
b
pound 8f (1.80 g, 2.20 mmol) was treated according to method H, yielding 9f
(317 mg, 19%) as a white powder. HPLC tR (min): 14.26. Rf4 0.71. 1H-NMR
(CD3OD) d: 7.24—7.12 (7H, m), 7.05 (2H, d, Jϭ8.4 Hz), 5.03 (0.5H, m),
4.95 (0.5H, m), 4.38 (1H, m), 4.32 (1H, m), 3.97 (2H, t, Jϭ6.6 Hz), 3.91
(2H, d, Jϭ6.6 Hz), 3.54 (1H, m), 3.37 (1H, m), 3.15 (1H, m), 3.00—2.91
(3H, m), 2.88—2.72 (4H, m), 2.54—2.45 (3H, m), 2.39 (2H, m), 2.32 (2H,
m), 2.09 (3H, s), 1.89 (3H, m), 1.72 (1H, m), 1.51 (2H, m), 1.29 (2H, m),
0.89 (6H, d, Jϭ6.6 Hz), 0.85 (3H, t, Jϭ7.5 Hz). FAB-MS m/z: 758 (MϩH)ϩ.
Boc-Tyr(CO2Et)-D-MetO-Phe-MebAla-O(Oct) (8e) As described in
method F, compound 3g (2.32 g, 3.0 mmol) was reacted with Ethyl chloro-
formate (357 mg, 3.3 mmol) to give 8e (2.00 g, 79%) as a white powder. Rf3
1
0.67. H-NMR (CDCl3) d: 7.92—7.70 (1H, m), 7.30—7.18 (7H, m), 7.09
(2H, dd, Jϭ8.4, 2.4 Hz), 5.78—5.45 (1H, m), 5.05 (1H, m), 4.66 (1H, m),
4.33 (1H, m), 4.29 (2H, q, Jϭ7.2 Hz), 4.00 (2H, m), 3.72 (0.67H, m), 3.52
(0.33H, m), 3.38 (1H, m), 3.10 (2H, m), 2.97 (2H, m), 2.88 (1H, s), 2.80
(2H, s), 2.60—2.21 (8H, m), 2.01 (2H, m), 1.61 (2H, t, Jϭ5.4 Hz), 1.40 (9H,
s), 1.38 (3H, t, Jϭ7.2 Hz), 1.28 (10H, m), 0.88 (3H, m). FAB-MS m/z: 845
(MϩH)ϩ.
a
i
N -1-Iminoethyl-Tyr(CO2Bu )-D-MetO-Phe-Me Ala-O(Oct) (9g)
Compound 8g (1.80 g, 2.06 mmol) was treated according to method H,
yielding 9g (436 mg, 26%) as a white powder. HPLC tR (min): 17.12. Rf4
b
Boc-Tyr(CO2Bui)-D-MetO-Phe-MebAla-O(Bu) (8f) As described in
method F, compound 3c (2.15 g, 3.0 mmol) was reacted with Isobutyl chlo-
roformate (451 mg, 3.30 mmol) to give 8f (1.84 g, 75%) as a white powder.
Rf3 0.74. 1H-NMR (CDCl3) d: 7.83—7.63 (1H, m), 7.30—7.17 (7H, m),
7.09 (2H, dd, Jϭ8.4, 2.4 Hz), 5.69—5.38 (1H, m), 5.03 (1H, m), 4.66 (1H,
m), 4.30 (1H, m), 4.08 (2H, t, Jϭ6.6 Hz), 3.71 (0.67H, m), 3.49 (0.33H, m),
3.40 (1H, m), 3.09 (2H, m), 2.99 (2H, m), 2.87 (1H, s), 2.79 (2H, s), 2.60—
2.21 (8H, m), 2.04 (4H, m), 1.62 (2H, m), 1.40 (9H, s), 1.35 (2H, m), 0.95
(3H, t, Jϭ7.4 Hz). FAB-MS m/z: 817 (MϩH)ϩ.
1
0.73. H-NMR (CD3OD) d: 7.33—7.19 (7H, m), 7.12 (2H, d, Jϭ8.4 Hz),
5.13 (0.5H, m), 5.04 (0.5H, m), 4.53 (1H, m), 4.43 (1H, m), 4.05 (2H, t,
Jϭ6.6 Hz), 4.00 (2H, d, Jϭ6.6 Hz), 3.64 (1H, m), 3.44 (1H, m), 3.24 (1H,
m), 3.08—3.00 (3H, m), 2.96—2.82 (4H, m), 2.64—2.54 (3H, m), 2.48 (2H,
m), 2.43 (2H, m), 2.18 (3H, m), 2.00 (3H, m), 1.81 (1H, m), 1.61 (2H, m),
1.30 (10H, m), 0.98 (6H, d, Jϭ6.6 Hz), 0.89 (3H, t, Jϭ6.0 Hz). FAB-MS
m/z: 814 (MϩH)ϩ.
Antinociceptive Assay Male ddY mice weighing 10—32 g were used.
The animals were purchased from Japan SLC Inc. (Shizuoka, Japan) and
were housed in cages (5—6 matched for weight) in a colony room. Animals
were given standard food (MM-3, Japan SLC Inc.) and tap water ad libitum.
The air-conditioned room was maintained at 23Ϯ2 °C and 55Ϯ20% relative
humidity, with a standard 12-h light-dark cycle (lights on from 6:00 to
18:00). Antinociceptive activity was examined by the tail pressure test ac-
Boc-Tyr(CO2Bui)-D-MetO-Phe-MebAla-O(Oct) (8g) As described in
method F, compound 3g (2.32 g, 3.0 mmol) was reacted with Isobutyl chlo-
roformate (450 mg, 3.3 mmol) to give 8g (1.89 g, 72%) as a white powder.
Rf3 0.76. 1H-NMR (CDCl3) d: 7.83—7.63 (1H, m), 7.30—7.17 (7H, m),
7.09 (2H, dd, Jϭ8.4, 2.4 Hz), 5.69—5.38 (1H, m), 5.03 (1H, m), 4.66 (1H,

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771
cording to the method of Sakurada et al.35) with slight modification. In brief,
mechanical pressure was applied to the base of the tail at 32 g/s using an au-
tomated tail-pressure unit (Ugo Basile, Italy). Biting or struggling behavior
of the mice was used as an indication of the response threshold and only
mice responding to a tail pressure of 100 to 300 g were selected for this ex-
periment. The trial was terminated at a level of 500 g to prevent tissue dam-
age to the tail. The meanϮS.E.M. of the pressure was plotted. To obtain the
dose–response curve, the dose was plotted against the percentage of the
maximum possible effect (%MPE), which was calculated as follows:
%MPEϭ(P2ϪP1/500ϪP1)ϫ100, where P1 is the response pressure before
drug administration (g) and P2 is the response pressure after drug adminis-
tration (g). The peptides were dissolved in saline (Fuso Chemical Industries,
Osaka, Japan) for administration and saline alone was used as the control.
ED50 values were obtained by the method of Litchfield and Wilcoxon25) to
compare the antinociceptive activity of the compounds. The values were cal-
culated from data that were obtained at the time of peak effect after either
peptide or morphine administration.
11) Ogawa T., Miyamae T., Okayama T., Hagiwara M., Sakurada S.,
Morikawa T., Chem. Pharm. Bull., 50, 771—780 (2002).
12) Ogawa T., Miyamae T., Murayama K., Okuyama K., Okayama T.,
Hagiwara M., Sakurada S., Morikawa T., J. Med. Chem., 45, 5081—
5089 (2002).
13) Humphrey M. J., Ringrose P. S., Drug Metab. Rev., 17, 283—310
(1986).
14) Lee V. H. L., Yamamoto A., Adv. Drug Deliv. Rev., 4, 171—207
(1990).
15) Bocci V., Adv. Drug Deliv. Rev., 4, 149—169 (1990).
16) Modi N. B., J. Control. Release, 29, 269—281 (1994).
17) Zhou X. H., J. Control. Release, 29, 239—252 (1994).
18) Amidon G. L., Lee H. J., Ann. Rev. Pharmacol. Toxicol., 34, 321—341
(1994).
19) Pauletti G. M., Gangwar S., Knipp G. T., Nerurkar M. M., Okumu F.
W., Tamura K., Siahaan T. J., Borchardt R. T., J. Control. Release, 41,
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References and Notes
1) Symbols and abbreviations are in accordance with recommendations
of IUPAC-IUB Joint Commission on Biochemical Nomenclature:
Nomenclature and Symbolism for Amino Acids and Peptides.
Biochem. J., 219, 345—373 (1984). The other abbreviations are as fol-
lows: AcOH, acetic acid; Ad, adamantyl; Bu, n-butyl; Bui, isobutyl;
But, tert-butyl; Bzl, benzyl; Boc, tert-butyloxycarbonyl; cHex, cyclo-
hexyl; Dec, decyl; Dde, dodecyl; DMF, N,N-dimethylformamide;
EtOAc, ethyl acetate; Hde, hexadecyl; Hep, heptyl; Hex, hexyl; HOBt,
1-hydroxybenzotriazole; MebAla, N-methyl-b-alanine; MetO, methio-
nine (R,S)-sulfoxide; Non, nonyl; Oct, octyl; Pen, pentyl; Ph, phenyl;
Pr, n-propyl; Pri, isopropyl; TEA, triethylamine; EDC·HCl, 1-ethyl-3-
(3-dimethylaminopropyl)-carbodiimide hydrochloride.
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(1949).
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C. H., Sjoberg B., Antimicrob. Agents Chemother., 8, 518—525
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8) Sato T., Sakurada S., Sakurada T., Furuta S., Chaki K., Kisara K.,
Sasaki Y., Suzuki K., J. Pharmacol. Exp. Ther., 242, 654—659 (1987).
9) Kisara K., Sakurada S., Sakurada T., Sasaki Y., Sato T., Suzuki K.,
Watanabe H., Br. J. Pharmacol., 87, 183—189 (1986).
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M., Chem. Pharm. Bull., 38, 2197—2200 (1990).
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K., J. Pharm. Pharmacol., 34, 750—751 (1982).