Structural studies of [2′,6′-dimethyl-L-tyrosine1]endomorphin-2 analogues: Enhanced activity and cis orientation of the Dmt-Pro amide bond

Article abstract of DOI:10.1016/S0968-0896(03)00068-3

Analogues of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH₂) (1) were designed to examine the importance of each residue on μ-opioid receptor interaction. Replacement of Tyr¹ by 2′,6′-dimethyl-L-tyrosine (Dmt) (9-12) exerted profound effects: [Dmt¹]EM-2 (9) elevated μ-opioid affinity 4.6-fold (K_iμ=0.15 nM) yet selectivity fell 330-fold as δ-affinity rose (K_iδ=28.2 nM). This simultaneous increased μ- and δ-receptor bioactivities resulted in dual agonism (IC₅₀=0.07 and 1.87 nM, respectively). While substitution of Phe⁴ by a phenethyl group (4) decreased μ affinity (K_iμ=13.3 nM), the same derivative containing Dmt (12) was comparable to EM-2 but also acquired weak δ antagonism (pA₂=7.05). ¹H NMR spectroscopy revealed a trans configuration (1:2 to 1:3, cis/trans) in the Tyr-Pro amide bond, but a cis configuration (5:3 to 13:7, cis/trans) with Dmt-Pro analogues.

Full text of DOI:10.1016/S0968-0896(03)00068-3

Bioorganic & Medicinal Chemistry 11 (2003) 1983–1994
Structural Studies of [20,60-Dimethyl-L-tyrosine1]endomorphin-2
Analogues: Enhanced Activity and cis Orientation of the
Dmt-Pro Amide Bond
Yoshio Okada,a,b,* Yoshio Fujita,a Takashi Motoyama,a Yuko Tsuda,a,b Toshio Yokoi,a,b
Tingyou Li,b Yusuke Sasaki,c Akihiro Ambo,c Yunden Jinsmaa,d Sharon D. Bryantd
and Lawrence H. Lazarusd
aFaculty of Pharmaceutical Sciences, Department of Medicinal Chemistry, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
bHigh Technology Research Center, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
cDepartment of Biochemistry, Tohoku Pharmaceutical University, 4-1, Komatsushima 4-chome, Aoba-ku, Sendai 981-8558, Japan
dPeptide Neurochemistry, LCBRA, National Institutes of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
Received 27 December 2002; accepted 27 January 2003
Abstract—Analogues of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH2) (1) were designed to examine the importance of each resi-
due on m-opioid receptor interaction. Replacement of Tyr1 by 20,60-dimethyl-l-tyrosine (Dmt) (9–12) exerted profound effects:
[Dmt1]EM-2 (9) elevated m-opioid affinity 4.6-fold (Kim=0.15 nM) yet selectivity fell 330-fold as d-affinity rose (Kid=28.2 nM). This
simultaneous increased m- and d-receptor bioactivities resulted in dual agonism (IC50=0.07 and 1.87 nM, respectively). While
substitution of Phe4 by a phenethyl group (4) decreased m affinity (Kim=13.3 nM), the same derivative containing Dmt (12) was
comparable to EM-2 but also acquired weak d antagonism (pA2=7.05). 1H NMR spectroscopy revealed a trans configuration (1:2
to 1:3, cis/trans) in the Tyr-Pro amide bond, but a cis configuration (5:3 to 13:7, cis/trans) with Dmt-Pro analogues.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
peptides differ structurally from previously known
endogenous opioid peptides, which contain the Tyr1-
Gly2 N-terminal sequence, by the presence of Pro2
comparable to the weakly active m-opioid casomor-
phin.9 Since Pro residues tend to form b bends in pro-
tein, this characteristic may contribute to the unusual
spectrum of activity we report with these endomorphin
derivatives.
The G-protein coupled d-, k- and m-opioid receptors are
located throughout the central nervous system and
peripheral tissues of various mammalian species, such as
in mouse vas deferens, guinea pig ileum, rabbit jejunum,
as well as in brain tissue of all vertebrates.1,2 These
receptors and their endogenous ligands, the enkepha-
lins,3 endorphins,4,5 dynorphins,6 and endomorphins7,8
appear to be involved in the modulation and perception
of pain. The endogenous neuropeptides, endomorphin-1
(EM-1: Tyr-Pro-Trp-Phe-NH2) and endomorphin-2
(EM-2: Tyr-Pro-Phe-Phe-NH2), were initially isolated
from bovine brain7 and later from human brain cortex.8
These endomorphins exhibited the highest affinity for
the m-opioid receptor and extraordinarily high selectiv-
ity relative to the d- and k-opioid receptor systems of all
known opioid substances.7 Furthermore, these tetra-
In order to develop novel analgesics mimicking the
endomorphins in lieu of morphine or other opioids, we
directed our attention to study the structure–activity
relationships of specific EM-2 analogues. One aspect
involved the influence of 20,60-dimethyl-l-tyrosine
(Dmt) in lieu of the natural N-terminal residue Tyr,
since it is known that Dmt markedly increases binding
affinity and bioactivity of numerous opioid peptide
agonists and antagonists.10ꢀ12 Therefore, we synthesized
a series of [Dmt1]EM-2 analogues and examined their
binding affinity, functional bioactivity, and solution
1
conformation by H NMR spectroscopy and circular
dichroism (CD).
*Corresponding author. Tel.: +81-78-974-1551; fax: +81-78-974-
5689; e-mail: okada@pharm.kobegakuin.ac.jp
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00068-3

1984
Y. Okada et al. / Bioorg. Med. Chem. 11 (2003) 1983–1994
Results and Discussion
synaptosomes (see Experimental) and summarized in
Table 2. In compounds 5–8, each constituent amino
acid was replaced with an Ala residue, which decreased
the binding affinity toward the m-opioid receptor. The
loss of Tyr in [Ala1]EM-2 (5) leads to a completely
inactive analogue substantiating the importance of Tyr1;
the OH function is considered an important component
in anchoring the ligand within opioid receptors
although it should be noted that an enkephalin ana-
logue lacking tyrosyl exhibits high m-receptor affinity.15
Furthermore, the m receptor prefers l-Tyr, since [d-
Tyr1]EM-2 had moderate m affinity (Ki=32.1 nM).16
Substitution of Pro2 by Ala (6) was equally deleterious;
however, d-Pro2 partially improved m affinity.16 In
addition, [d-Pro2]EM-2 was more potent than EM-2 in
increasing tail flick latencies with a longer duration of
action,17 apparently because it resisted enzymatic
degradation by dipeptidyl peptidase IV.18ꢀ22 In contrast
to EM-1, but similar to [d-Pro2]EM-2, [b-(R)-Pro2]EM-1
was 34-fold more potent than EM-1.23 In this case,
b-(R)-Pro might maintain the same spatial disposition
as natural Pro and offer greater stability as well.
Optically pure 20,60-dimethyl-l-tyrosine (Dmt) was pre-
pared as previously described.13 Optical purity (98%)
was ascertained both by HPLC using a chiral column
[CROWNPAC CR(+)] and by the reaction with d-
amino acid and l-amino acid oxidases, followed by
amino acid analysis.14 Boc-Dmt-OH was prepared in
the usual manner. Peptides, except for 1 and 2, were
synthesized by a solution method. All final products of
the peptide analogues were purified by semi-preparative
reverse-phase HPLC. Table 1 summarizes the analytical
data of compounds 3–12. Each compound exhibited a
single peak on analytical HPLC with a unique retention
time. Analysis by MALDI-TOF mass spectrometry
(MS) and by HPLC revealed that all compounds were
the desired product with greater than 98% purity.
Alanine scan
The binding affinity for each peptide analogue was
examined for d- and m-opioid receptors using rat brain
Table 1. Analytical data of synthetic peptides
Compd
TOF-MS
HPLC tR (min)
[a]2D0
Calcd
Found
H2O
ꢀ34.2ꢁ
ꢀ20.6ꢁ
ꢀ21.8ꢁ
ꢀ21.6ꢁ
ꢀ51.3ꢁ
ꢀ21.3ꢁ
ꢀ44.0ꢁ
ꢀ23.2ꢁ
+13.3ꢁ
+28.9ꢁ
+78.7ꢁ
ꢀ39.6ꢁ
c 1.0
16
1
2
3
4
5
6
7
8
YPFF-NH2
YPF-NH2
YP-NH2
16
c 0.67
c 3.22
c 1.0
c 1.0
c 1.0
c 1.0
c 1.0
c 0.87
c 0.93
c 0.38
c 1.0
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
278.3
529.5
479.6
546.6
497.6
497.6
600.7
453.5
306.4
556.9
278.1
529.1
480.7
546.9
497.0
498.1
600.5
453.1
306.5
556.9
7.7a
20.2b
19.2b
29.7c
22.5c
25.1b
19.1b
14.6b
8.4d
YPF-NH-C2H4-Ph
APFF-NH2
YAFF-NH2
YPAF-NH2
YPFA-NH2
Dmt-PFF-NH2
Dmt-PF-NH2
Dmt-Pro-NH2
Dmt-PF-NH-C2H4-Ph
9
10
11
12
22.5b
Y, tyrosine; P, proline; F, phenylalanine; A, alanine.
aHPLC A/B=90:10 to A/B=90:10 for 5 min, A/B=90:10 to A/B=50:50 for 20 min, A/B=50:50 to A/B=10:90 for 5 min.
bA/B=90:10 to A/B=40:60 for 25 min, A/B=40:60 to A/B=10:90 for 5 min.
cA/B=90:10 to A/B=50:50 for 40 min, A/B=50:50 to A/B=10:90 for 5 min.
dA/B=90:10 to A/B=90:10 for 5 min, A/B=90:10 to A/B=60:40 for 15 min, A/B=60:40 to A/B=10:90 for 5 min.
Table 2. Receptor binding data of endomorphin-2 analogues
Compd
[3H]DAMGO
Kim (nM)
[3H]DPDPE
Kid (nM)
Binding selectivity
R.P.
R.P.
Kid/Kim
7
C-term deletion analogues
1
2
3
4
YPFF-NH2
0.69ꢂ0.16
46.3ꢂ3.8
1
0.015
9230ꢂ200
15,900ꢂ2300
40,126ꢂ1115
4310ꢂ635
4722ꢂ650
3466ꢂ710
2658ꢂ738
3740ꢂ662
1
13,400
343
16
YPF-NH2
YP-NH2
YPF-NH-C2H4-Ph
0.58
0.23
2.1
26107ꢂ1339
(3)
(3)
2.6ꢃ10ꢀ6
(3)
(3)
1.5
324
13.3ꢂ2.6
0.052
Ala scanning mutants
5
6
7
8
APFF-NH2
YAFF-NH2
YPAF-NH2
YPFA-NH2
16750ꢂ6242
3272ꢂ283
257.4ꢂ9.7
65.7ꢂ23
(3)
(3)
(3)
(3)
4.1ꢃ10ꢀ6
2.1ꢃ10ꢀ5
0.0027
(4)
(4)
(4)
(4)
2.0
2.7
3.5
2.5
0.28
1.06
10
0.011
57
Dmt containing analogues
9
Dmt-PFF-NH2
Dmt-PF-NH2
Dmt-Pro-NH2
0.15ꢂ0.04
0.12ꢂ0.09
41.7ꢂ1.2
0.51ꢂ0.15
(3)
(3)
(3)
(3)
4.6
5.8
0.017
1.4
28.2ꢂ8.1
53.2ꢂ6.1
734.9ꢂ97
18.0ꢂ2.5
(3)
(3)
(4)
(3)
330
170
12.6
510
188
443
17.6
35
10
11
12
Dmt-PF-NH-C2H4-Ph
Y, tyrosine; P, proline; F, phenylalanine; A, alanine.
Displacement of [3H]DAMGO (m-selective) and [3H]DPDPE (d-selective) from rat brain membrane synaptosomes. Ki values are the meanꢂSE. The
potency is relative to that of EM-2.

Y. Okada et al. / Bioorg. Med. Chem. 11 (2003) 1983–1994
1985
1
C-Terminal deletion analogues
1H NMR spectrometry
As shown in Figure 1, endomorphin-2 analogues were
designed and synthesized. The loss of Phe4 (2) and
Phe3–Phe4 (3) drastically reduced binding to m-opioid
receptors by orders of magnitude (Table 2). Replace-
ment of Phe4 by a phenethyl group (4) yielded a peptide
with about 5% of the m-receptor affinity of EM-2,
demonstrating that not only the second Phe residue is
critical, but also that the physical separation between
Phe3 and the third aromatic center appears important
for receptor interaction.
Table 4 summarizes the results of analyses of the H
NMR spectra of EM-2 analogues in DMSO-d6. The cis/
trans ratios, derived from a conformational equilibrium
around the Tyr-Pro amide bond and Dmt-Pro amide
bond, and calculated by integration values of 1H NMR,
clearly demonstrated differences between the Tyr- and
Dmt-containing analogues (Table 4). Published obser-
vations with EM-125 and EM-226 revealed that they
contain a trans configuration with cis/trans ratios of 1:3
and 1:2, respectively. The cis/trans ratio of Tyr-Pro-Phe-
NH2 (2) was also 1:2 (Table 4). On the other hand,
Dmt-containing peptides (9–12) existed predominantly
in the cis configuration, including the C-terminally
extended analogue (12) (Table 4). The cis/trans rotamers
around the Dmt-Pro amide bond were further identified
by NOESY observations. NOE cross peaks between
Dmt CaH and Pro CaH, and Dmt-dimethyl H and Pro
CaH, defining a cis conformation in the peptides are
summarized in Figure 2. The Pro CaH and Dmt CaH of
the cis isomer were observed at higher fields (3.6 and 2.9
ppm, respectively) than those of trans isomer (4.1 and
4.3 ppm, respectively). For the trans isomer, NOE cross
peaks between Dmt-dimethyl H and Pro CdH, and Dmt
CaH and Pro CdH were observed as shown in Figure 2.
Pro CdHA of the trans isomer was measured at a higher
field (2.3 ppm) than that of the cis isomer (3.2 ppm).
The CH3 groups of Dmt appear to sterically hinder the
adoption of the trans configuration at the Dmt-Pro
peptide bond, thereby pushing the conformational
equilibrium towards a cis configuration (Table 4). In
each cis and trans isomer, NOE cross peaks between the
N- and C-terminal residues were not observed.
Dmt-containing analogues
The exchange of Tyr1 in opioid peptides for Dmt resulted
in
a marked increase in receptor affinities and
bioactivity.10ꢀ12 Substitution of Dmt1 in EM-2 and in C-
terminal deletion analogues (9–12; see Fig. 1) profoundly
affected all the subsequently measured parameters
(Tables 2 and 3). In each case, Dmt1 enhanced the affi-
nities from about 5- to several hundred-fold relative to
their Tyr cognates (1–4) for both m and d receptors. The
functional bioactivity of [Dmt1]EM-2 (9) (Table 3)
revealed an increase in m- and d-bioactivities by 82- and
184-fold greater than EM-2, respectively. While Dmt-
Pro-Phe-NH2 (10) was about twice as active as EM-2 (1),
deletion of Phe3 (11) yielded a marked drop in both
bioactivities. In contrast, however, Dmt-Pro-Phe-NH-
phenethyl (12) had an agonist potency equivalent to EM-
2, but differs with the appearance of weak d antagonist
activity (pA2=7.05). In keeping with the low affinity of
Dmt-Pro-NH2 (11), about 2% relative to EM-2 (Table
2), it was essentially biologically inactive (Table 3). The
high affinity and pharmacological potency of Dmt in
most analogues (except 11) might be partly attributable
to resistance towards proteolysis24 at the N-terminus, or
to enhanced receptivity by either influencing the bioac-
tive or the molecular conformation of the peptide that
interacts with the m-receptor ligand-binding domain.
Based on NMR analyses, Podlogar et al.25 proposed an
extended conformation of trans EM-1 as the potential
bioactive form. However, a study of Tyr-Xaa[ꢀCH ,CH
3
3
Pro]EM-2 analogues by Keller et al.27 found pre-
dominately a cis conformation (>98%). Even though
both compounds were m agonists with high m affinities,
the data indicate that cis and trans conformations can
exist around the Tyr-Pro or Dmt-Pro peptide bond in
EM-2. Our results, showing a preference for cis con-
formation in the Dmt-Pro sites of analogues, represent
another variation to the data of Podlogar et al.25 Fur-
thermore, with the endomorphin-related morphiceptin
analogues, Yamasaki et al.28 demonstrated that [Val4]-
morphiceptin (Tyr-Pro-Phe-Val-NH2) and [d-Val4]mor-
phiceptin
containing
cis-2-aminocyclopentane
Figure 1. Structures of endomorphin 2 analogues.
Table 3. In vitro bioactivity data of [Dmt1]EM-2 and various analogues
Compd
GPI assaya
IC50 (nM)
MVD assayb
pA2 value
R.P.
IC50 (nM)
R.P.
versus Deltorphin-II
1
9
10
11
12
YPFF-NH2
5.79ꢂ0.4
0.07ꢂ0.02
2.33ꢂ0.49
>10,000
1
344ꢂ93
1.87ꢂ0.61
113ꢂ35
1
184
3.0
—
—
—
—
—
Dmt-PFF-NH2
Dmt-PF-NH2
Dmt-Pro-NH2
82.7
2.5
—
>10,000
>10,000
Dmt-PF-NH-C2H4-Ph
5.03ꢂ0.99
1.2
—
7.05
aValues are the mean of seven experiments ꢂSE.
bValues are the mean of six experimentsꢂSE. The potency is related to that of EM-2.

1986
Y. Okada et al. / Bioorg. Med. Chem. 11 (2003) 1983–1994
Table 4. The cis/trans ratios of [Dmt1]EM-2 and various analogues
in DMSO
Compd
cis/transa
EM-1
EM-2
YPF-NH2
1:325
1:226
1:2
1
2
9
[Dmt1]EM-2
Dmt-PF-NH2
Dmt-Pro-NH2
Dmt-PF-NH-C2H4-Ph
7:3
13:7
5:3
10
11
12
13:7
aDetermined by integration of 1H NMR data (see Experimental
methods).
carboxylic acid in place of Pro2 interacted only with
m-receptors, while the trans-2-aminocyclopentane carb-
oxylic acid substitution analogues were completely
inactive at both m- and d-receptor sites.28
Circular dichroism
The CD spectra of the Tyr analogues of EM-2 (1, 2, 4, 8)
and their Dmt-derivatives (9, 10, 12) are shown in Figure
3A and B, respectively. The EM-2 analogues exhibited
negative maxima (213.1–215.4 nm), while the Dmt-deri-
vatives had negative maxima near 203 nm, which were
stronger than those corresponding to the Tyr-containing
peptides. These results confirm that the Dmt-containing
analogues adopt a solution conformation different than
other EM-2 analogues,16 and further suggests that the
Dmt residue affects the solution conformation of the
peptide as suggested by the NOE data.
Figure 2. Characteristic NOE pairs of cis/trans configuration at the
Dmt1-Pro2 peptide bond.
indicate that Dmt as an N-terminal residue in opioid
peptides has the potential to readily contribute to the
development of novel types of bioactive opioidmimetics
for therapeutics and clinical applications.11,12
Experimental
General procedures
Dmt was prepared according to the method of Dygos et
al.,13 and its chirality was assessed by HPLC and amino
acid analysis. Peptides were synthesized using solution
methods. Peptides were coupled by a mixed anhydride
method using isobutyl chloroformate (IBCF), azide or
using PyBop. The Boc group was used for N-terminal
protection. Deprotection was performed using hydrogen
chloride in dioxane (HCl/dioxane) or TFA. Melting
points were determined on a Yanagimoto micro-melting
point apparatus and are uncorrected. Mass spectra were
measured with MALDI-TOF mass spectrometry
(KOMPACT MALDI IV mass spectra, Kratos Analy-
tical). On TLC (Kieselgel G Merck), R1f and Rf2 values
refer to the systems of CHCl3, MeOH and AcOH
(90:8:2) and CHCl3, MeOH and H2O (89:10:1), respec-
tively. These peptides exhibited greater than 98% purity
by analytical HPLC (Waters Model 600 E) using a
Cosmosil 5C18-AR column (4.6ꢃ250 mm), absorbance
monitored at 220 nm, and run in the following solvents:
A, 0.05% TFA in H2O; B, 0.05% TFA in MeCN.
Purified peptides were characterized by mass spectro-
metry. Abbreviations used in this report for amino
acids, peptides and their derivatives are those recom-
mended by the IUPAC-IUBCommission on Biochem-
ical Nomenclature: Biochemistry 1996, 5, 2485; 1966, 6,
362l; 1972, 11, 1726. The customary l-configuration for
amino acid residues is omitted. The following additional
abbreviations are used: AcOEt, ethyl acetate; AcOH,
acetic acid; Boc, tert-butyloxycarbonyl; CD, circular
Conclusions
The data clearly indicate that Dmt markedly enhanced
the biological properties of EM-2 analogues and as seen
in its effects on the Dmt-Tic pharmacophore family of d
antagonists.10ꢀ12 These changes in biological activity
may be mediated by difference in the configuration
about the Xaa-Pro amide bond, as both NMR and CD
data of Dmt-containing peptides show the preference
for cis conformation and different solution conforma-
tion than Tyr-containing peptides, respectively. Toge-
ther, the bioassays and structural data suggest that
conformational features of the peptide may be respon-
sible for different activities and selectivities in peptide-
receptor recognition. The methyl groups on the Tyr-
amine ring of Dmt undoubtedly play a dominant role in
the interaction within the opioid binding domain by
either direct interaction with hydrophobic side-chains of
receptor residues to align the critical OH group, or by
stabilization of a favored cis conformer in solution prior
to and during binding.11,12 Even though the m affinity of
[Dmt1]EM-2 rose substantially, the interaction with the
d receptor was elevated to an even greater degree (Table
2), producing peptides with unusual and high biological
effectiveness. These remarkable changes are comparable
to those observed in unrelated opioid peptides, such as
enkephalin24 and an analogue of deltorphin,29 dynor-
phin A (1-11),30 as well as the Dmt-Tic-R,31 and Dmt-
Tic-R-R0 family of peptides.32 Thus, these data clearly

Y. Okada et al. / Bioorg. Med. Chem. 11 (2003) 1983–1994
1987
Figure 3. (A) CD spectra of EM-2 and its analogues in neat TFE. 1.31 mM EM-2 (1) (ꢀ), 1.47 mM Tyr-Pro-Phe-NH2 (2) (*), 0.81 mM Tyr-Pro-
Phe-NH-Phenethyl (4) (+). and 1.24 mM Tyr-Pro-Phe-Ala-NH2 (8) (*). (B) 1.0 mM [Dmt1]EM-2 (9) (~), 1.45 mM Dmt-Pro-Phe-NH2 (10) (&)
and 0.79 mM Dmt-Pro-Phe-NH-Phenethyl (12) (^).
.
dichroism; DAMGO, Tyr-d-Ala-Gly-MePhe-Gly-ol;
DIPEA, N,N-diisopropylethylamine; DMF, N,N-di-
methylformamide; DMSO, dimethyl sulfoxide; Dmt,
20,60-dimethyl-l-tyrosine; DPDPE, Tyr-cyclo(d-Pen-
Gly-Phe-d-Pen); EDTA, ethylenediamine tetraacetic
acid; EM-2, endomorphin-2; Et3N, triethylamine; Et2O,
diethylether; GPI, guinea pig ileum; IBCF, isobutyl
chloroformate; MeOH, methanol; MeCN, acetonitrile;
MVD, mouse vas deference; NMM, N-methylmorpholine;
NMR, nuclear magnetic resonance; NOE, nuclear Over-
hauser effect; NOESY, nuclear Overhauser and exchange
spectroscopy; Ph, phenyl; PyBop, benzotriazol-1-yloxy-
tripyrrolidinophosphonium hexafluorophosphate; TFA,
trifluoroacetic acid; TFE, 2,2,2,-trifluoroethanol; THF,
tetrahydrofuran.
HCl H-Tyr-Pro-NH2 (3). Boc-Tyr-Pro-NH2 (185 mg,
0.49 mmol) was treated with 8.0 N HCl/dioxane (0.6
mL, 4.9 mmol) for 1 h at room temperature. Et2O was
added to the solution until the product precipitated. The
precipitate was collected by centrifugation, dried over
KOH pellets and purified by semipreparative reverse-
phase HPLC. The purified peptide was lyophilized from
1 N HCl to give amorphous powder: yield 120 mg
(78.3%), MH+ 278.1, [a]D20 ꢀ21.8 (c 3.2, H2O), Anal.
.
.
calcd for C14H19N3O3 HCl H2O: C, 50.7; H, 6.68; N,
12.2. Found: C, 50.5; H, 6.92; N, 12.1.
Boc-Phe-NH-Phenethyl. A mixed anhydride [prepared
from Boc-Phe-OH (3.0 g, 11.4 mmol), IBCF (1.66 mL,
12.8 mmol) and NMM (1.38 mL, 12.8 mmol) in the
usual way] in THF (30 mL) was added to a solution of
phenethylamine (1.61 mL, 12.8 mmol) in DMF (30 mL)
at ꢀ15 ꢁC. The reaction mixture was stirred at 0 ꢁC for
30 min and at 4 ꢁC overnight. After removal of the sol-
vent, the residue was extracted with AcOEt. The extract
was washed with 10% citric acid, 5% Na2CO3 and
water, dried over Na2SO4 and evaporated to dryness.
Petroleum ether was added to the residue to form crys-
tals, which were collected by filtration, yield 4.1 g
(97.7%), mp 133–135 ꢁC, Rf1=0.93, [a]2D0 ꢀ0.052 (c 1.0,
MeOH), Anal. calcd for C22H28N2O3: C, 71.2; H, 7.66;
.
Boc-Tyr-Pro-NH2. To a solution of H-Pro-NH2 HCl
[prepared from Boc-Pro-NH2 (300 mg, 1.4 mmol) and
6.9 N HCl/dioxane (2.0 mL, 1.4 mmol) in the usual way]
in DMF (30 mL) containing DIPEA (0.49 mL, 2.8
mmol), Boc-Tyr-OH (422 mg, 1.5 mmol) and PyBop
(780 mg, 1.5 mmol) were added. The reaction mixture
was stirred at 4 ꢁC overnight. After removal of the sol-
vent, the residue was extracted with AcOEt. The extract
was washed with 10% citric acid, 5% Na2CO3 and
water, dried over Na2SO4 and evaporated to dryness.
Petroleum ether was added to the residue to form crys-
tals, which were collected by filtration, yield 462 mg
(83.5%), mp 118–127ꢁC, Rf1=0.48, [a]2D0 ꢀ48.2 (c 1.0,
1
N, 7.60. Found: C, 71.6; H, 7.79; N, 7.66, H NMR
(CDCl3) d: 7.00–7.30 (10H, m, Ar–H), 5.91 (1H, br,
NHC2H4), 5.11 (1H, br, 1H, aNH), 4.26 (1H, br q,
J=7.0 Hz, aCH), 3.31–3.50 (2H, m, CH2CH2NH), 3.01
(2H, d, J=6.5 Hz, bCH2), 2.53–2.75 (2H, m,
CH2CH2NH), 1.38 (9H, s, But).
.
MeOH), Anal. calcd for C19H27N3O5 1/3H2O: C, 59.9;
1
H, 7.42; N, 10.1. Found: C, 59.5; H, 7.30; N, 10.0, H
NMR (CDCl3) d: 7.66 (1H, brs, NH), 6.89 (2H, d, Ar–
H), 6.53 (2H, d, Ar–H), 5.77–6.02 (2H, m, CONH2),
4.33–4.92 (2H, m, aCH of Tyr and Pro), 2.67–3.66 (4H,
m, bCH2 of Tyr, and dCH2 of Pro), 1.39–2.25 (13H, m,
But, gCH2 and bCH2 of Pro).
Boc-Pro-Phe-NH-Phenethyl. A mixed anhydride [pre-
pared from Boc-Pro-OH (1.3 g, 5.98 mmol), IBCF (0.78
mL, 5.98 mmol) and Et3N (0.65 mL, 5.98 mmol) in the

1988
Y. Okada et al. / Bioorg. Med. Chem. 11 (2003) 1983–1994
usual way] in THF (30 mL) was added to a solution of
.
H-Phe-NH-phenethyl HCl [prepared from Boc-Phe-
Pro d), 40.2 (s, NH–CH2–CH2–), 37.2 (s, Phe b), 35.5 (s,
Tyr b), 34.9 (s, NH–CH2–CH2–), 28.5 (s, Pro b), 28.1 (p,
But), 24.4 (s, Pro g).
NH-phenethyl (2.0 g, 5.43 mmol) and 6.9 N HCl/diox-
ane (7.3 mL, 54.3 mmol) in the usual way] in DMF (30
mL) containing NMM (0.65 mL, 5.98 mmol) at ꢀ15 ꢁC.
The reaction mixture was stirred at 0 ꢁC for 30 min and
at 4 ꢁC overnight. After removal of the solvent, the
residue was extracted with AcOEt. The extract was
washed with 10% citric acid, 5% Na2CO3 and water,
dried over Na2SO4 and evaporated to dryness. Petro-
leum ether was added to the residue to form crystals,
which were collected by filtration, yield 1.3 g (51.1%),
mp 157–160 ꢁC, Rf1=0.66, [a]2D0 ꢀ59.7 (c 1.0, MeOH),
Anal. calcd for C27H35N3O4: C, 69.7; H, 7.58; N, 9.03.
.
HCl H-Tyr-Pro-Phe-NH-Phenethyl (4). Boc-Tyr-Pro-
Phe-NH-phenethyl (242 mg, 0.38 mmol) was treated
with 8.0 N HCl/dioxane (0.48 mL, 3.8 mmol) for 1 h at
room temperature. Et2O was added to the solution until
the product precipitated. The precipitate was prepared
as described for the preparation of (3): yield 220 mg
(92%), MH+ 529.1, [a]D20 +43.0 (c 1.0, H2O), Anal.
.
.
calcd for C31H36N4O4 HCl 1/2H2O: C, 64.9; H, 6.67; N,
9.75. Found: C, 64.7; H, 6.72; N, 9.97.
1
Found: C, 69.7; H, 7.62; N, 9.10, H NMR (CDCl3) d:
Boc-Phe-Phe-NH2. A mixed anhydride [prepared from
Boc-Phe-OH (1.1 g, 4.17 mmol), IBCF (0.52 mL, 4.17
mmol) and Et3N (0.58 mL, 4.17 mmol) in the usual way]
in THF (30 mL) was added to a solution of H-Phe-
7.10–7.30 (10H, m, Ar–H), 6.71 (1H, br, NH–C2H4),
6.48 (1H, br, NH of Phe), 4.68 (1H, br, aCH of Phe),
3.05–3.51 (6H, m, dCH2 of Pro, and NH–C2H4), 4.14
(1H, br, aCH of Pro), 2.00–2.11 (2H, m, bCH2 of Pro),
1.63–1.89 (2H, m, gCH2 of Pro), 1.37 (9H, s, But). 13C
NMR (CDCl3) d: 171.6 (q), 170.4 (q), 155.7 (q), 139.0
(q), 136.6 (q), 129.3 (t, Ar), 128.7 (t, Ar), 128.6 (t, Ar),
128.5 (t, Ar), 127.1 (t, Ar), 126.4 (t, Ar), 80.8 (q, But),
60.9 (t, Pro a), 53.2 (t, Phe a), 47.2 (s, Pro d), 41.1 (s,
NH-CH2–CH2–), 35.6 (s, NH–CH2–CH2–), 37.3 (s, Phe
b), 29.3 (s, Pro b), 28.3 (p, But), 24.5 (s, Pro g).
.
NH2 HCl [prepared from Boc-Phe-NH2 (1.0 g, 3.79
mmol) and 7.2 N HCl/dioxane (5.1 mL, 37.89 mmol) in
the usual way] in DMF (30 mL) containing Et3N (0.58
mL, 4.17 mmol) at ꢀ15 ꢁC. The reaction mixture was
stirred at 0 ꢁC for 30 min and at 4 ꢁC overnight. After
removal of the solvent, the residue was extracted with
AcOEt. The extract was washed with 10% citric acid,
5% Na2CO3 and water, dried over Na2SO4 and evapo-
rated to dryness. Petroleum ether was added to the
residue to form crystals, which were collected by fil-
tration, yield 1.10 g (70.5%), mp 218–220 ꢁC, Rf1=0.59,
[a]2D0 ꢀ18.8 (c 1.0, MeOH), Anal. calcd for C23H29N3O4:
C, 67.1; H, 7.10; N, 10.2. Found: C, 67.3; H, 7.21; N,
Boc-Tyr-Pro-Phe-NH-Phenethyl. Boc-Tyr-NHNH2 (320
mg, 1.07 mmol) was dissolved in DMF (10 mL). Under
cooling with ice–NaCl, 6.9 N HCl/dioxane (0.29 mL,
2.14 mmol) and isoamylnitrite (0.16 mL, 1.18 mmol)
were added. Stirring was continued for 10 min, when
the hydrazine test became negative. The solution, after
neutralization with NMM (0.24 mL, 2.14 mmol), was
combinated with a solution of H-Pro-Phe-NH-phen-
1
10.5, H NMR (DMSO-d6) d: 8.01 (1H, d, J=8.3 Hz,
NH of Phe2), 7.44 (1H, brs, CONHB), 7.13–7.29 (10H,
m, Ar–H), 7.10 (1H, brs, CONHA), 6.93 (1H, d, J=8.5
Hz, NH of Phe1), 4.46 (1H, br, aCH of Phe2), 4.10 (1H,
br, aCH of Phe1), 3.03 (1H, dd, J=13.8, 5.0 Hz, bCHB
of Phe2), 2.83–2.80 (2H, m, bCHA of Phe,1 and bCHB of
Phe2), 2.68 (1H, dd, J=13.5, 10.2 Hz, bCHA of Phe1),
1.26 (9H, s, But). 13C NMR (DMSO-d6) d: 173.2 (q),
171.7 (q), 155.6 (q), 138.6 (q), 138.3 (q), 129.8 (t, Ar),
129.6 (t, Ar), 128.5 (t, Ar), 128.4 (t, Ar), 126.7 (t, Ar),
126.6 (t, Ar), 78.6 (q, But), 56.6 (t, Phe1 a), 54.1 (t, Phe2
a), 38.2 (s, Phe2 b), 37.9 (s, Phe1 b), 28.6 (p, But).
.
ethyl HCl [prepared from Boc-Pro-Phe-NH-phenethyl
(500 mg, 1.07 mmol) and 6.9 N HCl/dioxane (1.45 mL,
10.7 mmol) in the usual manner] and NMM (0.35 mL,
3.21 mmol) in DMF (20 mL). After the reaction mixture
was stirred at 4 ꢁC overnight, the solvent was evapo-
rated and the residue was extracted with AcOEt. The
extract was washed with 10% citric acid, 5% Na2CO3
and water, dried over Na2SO4 and evaporated to dry-
ness. Ether was added to the residue to form crystals,
which were collected by filtration to yield 381 mg
(56.0%), mp 203–207 ꢁC Rf1=0.39, [a]2D0 ꢀ28.6 (c 1.0,
Boc-Pro-Phe-Phe-NH2. A mixed anhydride [prepared
from Boc-Pro-OH (440 mg, 2.24 mmol), IBCF (0.27
mL, 2.24 mmol) and NMM (0.44 mL, 2.24 mmol) in the
usual way] in THF (30 mL) was added to a solution of
.
DMSO), Anal. calcd for C36H44N4O6 1/2H2O: C, 67.8;
1
H, 7.11; N, 8.78. Found: C, 67.7; H, 7.05; N, 9.30, H
.
NMR (DMSO-d6) d: 9.15 (1H, brs, Ar–OH), 7.85 (1H,
br, NHC2H4), 7.76 (1H, d, J=8.1 Hz, NH of Phe),
7.05–7.33 (12H, m, Ar–H), 6.98 (1H, d, J=8.3 Hz, NH
of Tyr), 6.65 (2H, d, J=8.2 Hz, Ar–H), 4.41 (1H, br,
aCH of Phe), 4.17 (2H, br, aCH of Tyr, and aCH of
Pro), 3.59 (1H, br, dCHB of Pro), 3.51 (1H, br, dCHA of
Pro), 3.08–3.41 (2H, m, NHCH2CH2), 3.01 (1H, dd,
J=13.7, 5.3 Hz, bCHB of Phe), 2.60–2.90 (5H, m, bCH2
of Tyr, bCHA of Phe, and NHCH2CH2), 1.65-1.98 (4H,
m, bCH2, and gCH2 of Pro), 1.30 (9H, s, But). 13C
NMR (DMSO-d6) d: 177.3 (q), 171.1 (q), 171.0 (q),
155.7 (q), 155.2 (q), 1139.2 (q), 137.8 (q), 130.1 (t, Ar),
129.0 (t, Ar), 128.5 (t, Ar), 128.2 (t, Ar), 127.9 (t, Ar),
126.1 (t, Ar), 126.0 (t, Ar), 114.8 (t, Ar), 77.9 (q, But),
59.8 (t, Pro a), 54.0 (t, Tyr a), 53.9 (t, Phe a), 46.7 (s,
H-Phe-Phe-NH2 HCl [prepared from Boc-Phe-Phe-NH2
(840 mg, 2.04 mmol) and 7.2 N HCl/dioxane (2.7 mL,
20.4 mmol) in the usual way] in DMF (30 mL) contain-
ing NMM (0.24 mL, 2.24 mmol) at ꢀ15 ꢁC. The reac-
tion mixture was stirred at 0 ꢁC for 30 min and at 4 ꢁC
overnight. After removal of the solvent, the residue was
extracted with AcOEt. The extract was washed with
10% citric acid, 5% Na2CO3 and water, dried over
Na2SO4 and evaporated to dryness. Petroleum ether
was added to the residue to form crystals, which were
collected by filtration, yield 810 mg (78.0%), mp 177–
183 ꢁC, Rf1=0.55, [a]2D0 ꢀ24.2 (c 1.0, DMSO), Anal.
.
calcd for C28H36N4O5 2H2O: C, 61.8; H, 7.40; N, 10.3.
1
Found: C, 61.4; H, 7.18; N, 10.8, H NMR (DMSO-d6)
d: 7.75–8.05 (2H, m, NH of Phe2,3), 7.06–7.29 (12H, m,

Y. Okada et al. / Bioorg. Med. Chem. 11 (2003) 1983–1994
1989
Ar–H and CONH2), 4.39–4.55 (2H, m, aCH of Phe2,3),
4.03 (1H, br, aCH of Pro), 3.19–3.32 (2H, m, dCH2 of
Pro), 2.75–3.05 (4H, m, bCH2 of Phe2,3), 1.98 (1H, br,
bCHB of Pro), 1.58–1.73 (3H, m, gCH2 and bCHA of
Pro), 1.38 (3H, s, trans But), 1.13 (6H, s, cis But). 13C
NMR (DMSO-d6) d: 172.5 (q), 172.1 (q), 170.8 (q),
170.5 (q), 153.9 (q), 153.2 (q), 137.6 (q), 129.0 (t, Ar),
128.0 (t, Ar), 127.9 (t, Ar), 126.1 (t, Ar), 78.7 (q, trans
But), 78.3 (q, cis But), 59.4 (t, Pro a), 53.8 (t, cis-Phe a),
53.5 (t, trans-Phe a), 46.5 (s, trans-Pro d), 46.3 (s, cis-
Pro d), 37.5 (s, cis-Phe b), 37.1 (s, trans-Phe b), 30.6 (s,
cis-Pro b), 29.3 (s, trans-Pro b), 28.1 (p, trans But), 27.7
(p, cis But), 23.7 (s, trans-Pro g), 22.8 (s, cis-Pro g).
IBCF (0.55 mL, 4.2 mmol) and NMM (0.46 mL, 4.2
mmol) in the usual way] in THF (30 mL) was added to a
.
solution of H-Ala-Phe-Phe-NH2 TFA [prepared from
Boc-Ala-Phe-Phe-NH2 (2.0 g, 4.2 mmol) and TFA (4.62
mL, 62 mmol) in the usual way] in DMF (30 mL) con-
taining NMM (0.46 mL, 4.2 mmol) at ꢀ15 ꢁC. The
reaction mixture was stirred at 0 ꢁC for 30 min and at
4 ꢁC overnight. After removal of the solvent, the residue
was extracted with AcOEt. The extract was washed with
10% citric acid, 5% Na2CO3 and water, dried over
Na2SO4 and evaporated to dryness. Petroleum ether
was added to the residue to form crystals, which were
collected by filtration, yield 2.4 g (81.1%), mp 251–
253 ꢁC Rf1=0.44, [a]2D0 ꢀ14.3 (c 1.0, DMF), Anal. calcd
for C39H51N5O7: C, 66.4; H, 7.34; N, 9.92. Found: C,
66.2; H, 7.31; N, 9.84.
Boc-Ala-Pro-Phe-Phe-NH2. A mixed anhydride [pre-
pared from Boc-Ala-OH (760 mg, 4.0 mmol), IBCF (0.52
mL, 4.0 mmol) and NMM (0.44 mL, 4.0 mmol) in the
usual way] in THF (30 mL) was added to a solution of H-
HCl H-Tyr-Ala-Phe-Phe-NH2 (6). Boc-Tyr(But)-Ala-
.
.
Pro-Phe-Phe-NH2 TFA [prepared from Boc-Pro-Phe-
Phe-Phe-NH2 (1.0 g, 1.4 mmol) was treated with TFA
(2.66 mL, 36 mmol) and anisole (0.27 mL, 2.5 mmol) for
1 h at room temperature. Et2O was added to the solution
until the product precipitated. The precipitate was pre-
pared as described for the preparation of (3): yield 800
mg (98.3%), MH+ 546.9, [a]D20 ꢀ21.3 (c 1.0, H2O), Anal.
Phe-NH2 (2.4 g, 4.0 mmol) and TFA (4.47 mL, 60 mmol)
in the usual way] in DMF (30 mL) containing NMM
(0.44 mL, 4.0 mmol) at ꢀ15 ꢁC. The reaction mixture was
stirred at 0 ꢁC for 30 min and at 4 ꢁC overnight. After
removal of the solvent, the residue was extracted with
AcOEt. The extract was washed with 10% citric acid, 5%
Na2CO3 and water, dried over Na2SO4 and evaporated
to dryness. Petroleum ether was added to the residue to
form crystals, which were collected by filtration, yield 1.3
g (56.5%), mp 180–184ꢁC Rf2=0.12, [a]2D0 ꢀ54.2 (c 1.0,
.
.
calcd for C30H35N5O5 HCl 5/2H2O: C, 57.5; H, 6.59; N,
11.2. Found: C, 57.7; H, 6.21; N, 11.1, amino acid anal.
Tyr/Ala/Phe=0.94:0.90:2.15 (average recovery 80.0%).
Boc-Ala-Phe-NH2. A mixed anhydride [prepared from
Boc-Ala-OH (2.84 g, 15 mmol), IBCF (2.0 mL, 15
mmol) and NMM (1.65 mL, 15 mmol) in the usual way]
in THF (30 mL) was added to a solution of H-Phe-
.
MeOH), Anal. calcd for C31H41N5O6 1/4H2O: C, 63.7;
H, 7.16; N, 12.0. Found: C, 63.7; H, 7.00; N, 11.8.
.
HCl H-Ala-Pro-Phe-Phe-NH2 (5). Boc-Ala-Pro-Phe-
.
NH2 TFA [prepared from Boc-Phe-NH2 (4.0 g, 15
Phe-NH2 (1.0 g, 1.5 mmol) was treated with TFA (2.24
mL, 30 mmol) and anisole (0.22 g, 2 mmol) for 1 h at
room temperature. Et2O was added to the solution until
the product precipitated. The precipitate was prepared
as described for the preparation of (3): yield 500 mg
(83.5%), MH+ 480.9, [a]D20 ꢀ51.3 (c 1.0, H2O), Anal.
mmol) and TFA (17.1 mL, 0.23 mol) in the usual way]
in DMF (30 mL) containing NMM (1.65 mL, 15 mmol)
at ꢀ15 ꢁC. The reaction mixture was stirred at 0 ꢁC for
30 min and at 4 ꢁC overnight. After removal of the sol-
vent, the residue was extracted with AcOEt. The extract
was washed with 10% citric acid, 5% Na2CO3 and
water, dried over Na2SO4 and evaporated to dryness.
Petroleum ether was added to the residue to form crys-
tals, which were collected by filtration, yield 4.0 g
(77.3%), mp 154–155 ꢁC Rf2=0.14, [a]2D0 ꢀ34.7 (c 1.0,
MeOH), Anal. calcd for C17H25N3O4: C, 59.3; H, 7.60;
N, 12.2. Found: C, 58.8; H, 7.26; N, 12.2.
.
.
calcd for C26H33N5O4 HCl 3/2H2O: C, 57.1; H, 6.87; N,
12.8. Found: C, 57.4; H, 6.44; N, 12.8, amino acid anal.
Ala/Pro/Phe=0.80:1.15:1.96 (average recovery 83.3%).
Boc-Ala-Phe-Phe-NH2. A mixed anhydride [prepared
from Boc-Ala-OH (1.9 g, 10 mmol), IBCF (1.31 mL, 10
mmol) and NMM (1.1 mL, 10 mmol) in the usual way]
in THF (30 mL) was added to a solution of H-Phe-Phe-
Boc-Pro-Ala-Phe-NH2. A mixed anhydride [prepared
from Boc-Pro-OH (2.1 g, 9.0 mmol), IBCF (1.2 mL, 9.0
mmol) and NMM (1.0 mL, 9.0 mmol) in the usual way]
in THF (30 mL) was added to a solution of H-Ala-Phe-
.
NH2 TFA [prepared from Boc-Phe-Phe-NH2 (4.1 g, 10
mmol) and TFA (11.2 mL, 150 mmol) in the usual way]
in DMF (30 mL) containing NMM (1.1 mL, 10 mmol)
at ꢀ15 ꢁC. The reaction mixture was stirred at 0 ꢁC for
30 min and at 4 ꢁC overnight. After removal of the sol-
vent, the residue was extracted with AcOEt. The extract
was washed with 10% citric acid, 5% Na2CO3 and
water, dried over Na2SO4 and evaporated to dryness.
Petroleum ether was added to the residue to form crys-
tals, which were collected by filtration, yield 2.5 g
(51.3%), mp 204–205 ꢁC Rf2=0.20, [a]2D0 ꢀ37.3 (c 1.0,
MeOH), Anal. calcd for C26H34N4O5: C, 64.1; H, 7.13;
N, 11.50. Found: C, 64.0; H, 7.07; N, 11.4.
.
NH2 TFA [prepared from Boc-Ala-Phe-NH2 (3.0 g, 9.0
mmol) and TFA (10.1 mL, 0.14 mol) in usual way] in
DMF (30 mL) containing NMM (1.0 mL, 9.0 mmol) at
ꢀ15 ꢁC. The reaction mixture was stirred at 0 ꢁC for 30
min and at 4 ꢁC overnight. After removal of the solvent,
the residue was extracted with AcOEt. The extract was
washed with 10% citric acid, 5% Na2CO3 and water,
dried over Na2SO4 and evaporated to dryness. Petro-
leum ether was added to the residue to form crystals,
which were collected by filtration, yield 2.0 g (52.5.%),
mp 199–200 ꢁC Rf2=0.11, [a]2D0 ꢀ73.7 (c 1.0, MeOH),
Anal. calcd for C22H32N4O5: C, 60.9; H, 7.51; N, 12.5.
Found: C, 60.9; H, 7.46; N, 12.5.
Boc-Tyr(But)-Ala-Phe-Phe-NH2. A mixed anhydride
[prepared from Boc-Tyr(But)-OH (1.42 g, 4.2 mmol),

1990
Y. Okada et al. / Bioorg. Med. Chem. 11 (2003) 1983–1994
Boc-Tyr(But)-Pro-Ala-Phe-NH2. A mixed anhydride
[prepared from Boc-Tyr(But)-OH (1.0 g, 2.9 mmol),
IBCF (0.38 mL, 2.9 mmol) and NMM (0.32 mL, 2.9
mmol) in the usual way] in THF (30 mL) was added to a
(93.3%), mp 215–217 ꢁC Rf2=0.06, [a]2D0 ꢀ59.0 (c 1.0,
MeOH), Anal. calcd for C22H32N4O5: C, 61.1; H, 7.41;
N, 13.0. Found: C, 60.8; H, 7.38; N, 13.0.
Boc-Tyr(But)-Pro-Phe-Ala-NH2. A mixed anhydride
[prepared from Boc-Tyr(But)-OH (648 mg, 1.92 mmol),
IBCF (0.25 mL, 1.92 mmol) and NMM (0.20 mL, 1.92
mmol) in the usual way] in THF (30 mL) was added to a
.
solution of H-Pro-Ala-Phe-NH2 TFA [prepared from
Boc-Pro-Ala-Phe-NH2 (1.30 g, 2.9 mmol) and TFA (3.2
mL, 0.04 mol) in the usual way] in DMF (30 mL) con-
taining NMM (0.32 mL, 2.9 mmol) at ꢀ15 ꢁC. The reac-
tion mixture was stirred at 0 ꢁC for 30 min and at 4 ꢁC
overnight. After removal of the solvent, the residue was
extracted with AcOEt. The extract was washed with 10%
citric acid, 5% Na2CO3 and water, dried over Na2SO4
and evaporated to dryness. Petroleum ether was added to
the residue to form crystals, which were collected by fil-
tration, yield 1.8 g (93.8%), mp 143–145 ꢁC Rf1=0.44,
[a]2D0 ꢀ34.2 (c 1.0, DMF), Anal. calcd for C35H49N5O7:
C, 63.6; H, 7.62; N, 10.6. Found: C, 63.3; H, 7.48; N,
10.8.
.
solution of H-Pro-Phe-Ala-NH2 TFA [prepare from
Boc-Pro-Phe-Ala-NH2 (830 mg, 1.92 mmol) and TFA
(2.85 mL, 38.4 mmol) in the usual way] in DMF (30
mL) containing NMM (0.20 mL, 1.92 mmol) at ꢀ15 ꢁC.
The reaction mixture was stirred at 0 ꢁC for 30 min and
at 4 ꢁC overnight. After removal of the solvent, the
residue was extracted with AcOEt. The extract was
washed with 10% citric acid, 5% Na2CO3 and water,
dried over Na2SO4 and evaporated to dryness. Petro-
leum ether was added to the residue to form crystals,
which were collected by filtration, yield 0.7 g (56.0%),
mp 120–124 ꢁC Rf2=0.44, [a]2D0 ꢀ39.0 (c 1.0, MeOH),
Anal. calcd for C35H49N5O7: C, 64.5; H, 7.58; N, 10.8.
Found: C, 64.5; H, 7.52; N, 11.0.
HCl H-Tyr-Pro-Ala-Phe-NH2 (7). Boc-Tyr(But)-Pro-
.
Ala-Phe-NH2 (650 mg, 1.0 mmol) was treated with TFA
(1.86 mL, 25 mmol) and anisole (0.19 mL, 1.7 mmol) for
1 h at room temperature. Et2O was added to the solu-
tion until the product precipitated. The precipitate was
prepared as described for the preparation of (3): yield
410 mg (83.1%), MH+ 479.0, [a]D20 ꢀ44.0 (c 1.0, H2O),
HCl H-Tyr-Pro-Phe-Ala-NH2 (8). Boc-Tyr(But)-Pro-
.
Phe-Ala-NH2 (400 mg, 0.6 mmol) was treated with TFA
(0.89 mL, 12 mmol) and anisole (0.09 g, 0.8 mmol) for 1
h at room temperature. Et2O was added to the solution
until the product precipitated. The precipitate was pre-
pared as described for the preparation of (3): yield 300
mg (58.0%), MH+ 496.5, [a]D20 ꢀ23.2 (c 1.0, H2O),
.
.
Anal. calcd for C26H33N5O5 HCl 2H2O: C, 55.0; H,
6.74; N, 12.3; Found: C, 55.0; H, 6.32; N, 12.5, amino
acid anal. Tyr/Ala/Pro/Phe=0.90:1.09:0.92:1.02 (aver-
age recovery 77.1%).
.
.
Anal. calcd for C26H33N5O5 HCl 3/2H2O: C, 55.9; H,
6.67; N, 12.5. Found: C, 55.7; H, 6.32; N, 12.4, amino
acid anal. Tyr/Pro/Phe/Ala=0.96:1.05:1.01:0.97 (aver-
age recovery 87.5%).
Boc-Phe-Ala-NH2. A mixed anhydride [prepared from
Boc-Phe-OH (5.3 g, 20 mmol), IBCF (2.62 mL, 20
mmol) and NMM (2.2 mL, 20 mmol) in the usual way]
in THF (30 mL) was added to a solution of H-Ala-
.
NH2 TFA [prepared from Boc-Ala-NH2 (3.76 g, 20
Boc-Dmt-OH. HCl H-Dmt-OH (4.7 g, 17.8 mmol) was
dissolved in H2O/dioxane and Et3N (4.9 mL, 35.6
mmol) and (Boc)2O (4.27 g, 19.6 mmol) were added.
The mixture was stirred at room temperature over
night. After removal of the solvent, the residue was
extracted with AcOEt. The extract was washed with
10% citric acid, and water, dried over Na2SO4 and
evaporated to dryness. Petroleum ester was added to
the residue to form crystals, which were collected by
filtration: 5.4 g (97%) yield; mp 178–181 ꢁC, Rf2=0.33,
[a]2D0 ꢀ11.7 (c 1.0, MeOH), Anal. calcd for
mmol) and TFA (22.4 mL, 0.3 mol) in the usual way] in
DMF (30 mL) containing NMM (2.2 mL, 20 mmol) at
ꢀ15 ꢁC. The reaction mixture was stirred at 0 ꢁC for 30
min and at 4 ꢁC overnight. After removal of the solvent,
the residue was extracted with AcOEt. The extract was
washed with 10% citric acid, 5% Na2CO3 and water,
dried over Na2SO4 and evaporated to dryness. Petro-
leum ether was added to the residue to form crystals,
which were collected by filtration, yield 3.9 g (58.0%),
mp 173–177 ꢁC Rf2=0.13, [a]2D0 ꢀ68.8 (c 1.0, MeOH),
Anal. calcd for C17H25N3O4: C, 60.9; H, 7.51; N, 12.5.
Found: C, 60.9; H, 7.46; N, 12.5.
.
C38H42N4O8 1/10H2O: C, 61.8; H, 7.53; N, 4.50.
1
Found: C, 61.8; H, 7.40; N, 4.43, H NMR (DMSO-d6)
d: 8.95 (1H, brs, Ar–OH), 7.09 (1H, d, J=8.5 Hz,
NH), 6.38 (1H, s, Ar–H), 3.98 (1H, brq, J=8.0 Hz,
aCH), 2.95 (1H, dd, J=14.3, 6.6 Hz, bCHB), 2.78 (1H,
dd, J=14.1, 8.3 Hz, bCHA), 2.18 (6H, s, Me), 1.32
(9H, s, But).
Boc-Pro-Phe-Ala-NH2. A mixed anhydride [prepared
from Boc-Pro-OH (1.5 g, 7.0 mmol), IBCF (0.92 mL,
7.0 mmol) and NMM (0.77 mL, 7.0 mmol) in the usual
way] in THF (30 mL) was added to a solution of H-Phe-
.
Ala-NH2 TFA [prepared from Boc-Phe-Ala-NH2 (2.3 g,
7.0 mmol) and TFA (7.8 mL, 105 mmol) in the usual
way] in DMF (30 mL) containing NMM (0.77 mL, 7.0
mmol) at ꢀ15 ꢁC. The reaction mixture was stirred at
0 ꢁC for 30 min and at 4 ꢁC overnight. After removal of
the solvent, the residue was extracted with AcOEt. The
extract was washed with 10% citric acid, 5% Na2CO3
and water, dried over Na2SO4 and evaporated to dry-
ness. Petroleum ether was added to the residue to form
crystals, which were collected by filtration, yield 2.8 g
Boc-Dmt-Pro-Phe-Phe-NH2. To a solution of H-Pro-
.
Phe-Phe-NH2 HCl [prepared from Boc-Pro-Phe-Phe-
NH2 (329 mg, 0.65 mmol) and 6.9 N HCl/dioxane (0.9
mL, 6.47 mmol) in the usual way] in DMF (30 mL)
containing DIPEA (0.25 mL, 1.30 mmol), Boc-Dmt-OH
(200 mg, 0.65 mmol) and PyBop (370 mg, 0.65 mmol)
were added. The reaction mixture was stirred at 4 ꢁC
overnight. After removal of the solvent, the residue was
extracted with AcOEt. The extract was washed with

Y. Okada et al. / Bioorg. Med. Chem. 11 (2003) 1983–1994
1991
10% citric acid, 5% Na2CO3 and water, dried over
Na2SO4 and evaporated to dryness. Petroleum ether
was added to the residue to form crystals, which were
collected by filtration, yield 276 mg (61.0%), mp 235–
237 ꢁC, Rf1=0.40, [a]D20 ꢀ38.5 (c 1.0, CHCl3), Anal.
calcd for C39H49N5O7: C, 66.9; H, 7.06; N, 10.0, Found:
(1.8H, s, Me of trans Dmt), 2.04 (4.2H, s, Me of cis
Dmt), 1.81 (0.3H, br, bCHB of trans Pro), 1.50–1.65
(1.6H, m, bCHB of cis Pro, gCH2 and bCHA of trans
Pro), 1.42 (0.7H, br, gCHB of cis Pro), 1.21 (0.7H, br,
bCHA of cis Pro), 1.08 (0.7H, br, gCHA of cis Pro). 13C
NMR (DMSO-d6) d: 173.62 (q), 173.06 (q), 171.34 (q),
170.88 (q), 170.54 (q), 168.32 (q), 167.93 (q), 156.50 (q),
156.24 (q), 139.02 (q), 138.73 (q), 138.39 (q), 138.22 (q),
138.07 (q), 129.83 (t, Ar), 129.70 (t, Ar), 129.63 (t, Ar),
128.58 (t, Ar), 128.52 (t, Ar), 128.51(t, Ar), 128.43 (t,
Ar), 126.80 (t, Ar), 126.71 (t, Ar), 126.67 (t, Ar), 121.96
(q), 121.55 (q), 115.62 (t, Ar), 115.56 (t, Ar), 60.28 (t,
trans-Pro a), 59.60 (t, cis-Pro a), 54.68 (t, cis-Phe a),
54.52 (t, trans-Phe a), 54.20 (t, cis-Phe a and trans-Phe
a), 50.38 (t, trans-Dmt a), 50.30 (t, cis-Dmt a), 47.22 (s,
cis-Pro d), 46.82 (s, trans-Pro d), 38.20 (s, trans-Phe b),
38.01 (s, cis-Phe b), 37.91 (s, trans-Phe b), 36.74 (s, cis-
Phe b), 31.63 (s, cis-Pro b), 31.22 (s, cis-Pro b), 30.80 (s,
trans-Dmt b), 29.40 (s, trans-Pro b), 24.67 (s, trans-Pro
g), 21.76 (s, cis-Pro g), 20.61 (p, trans-Dmt Me), 19.83
(p, cis-Dmt Me).
1
C, 66.7; H, 7.13; N, 9.84, H NMR (DMSO-d6) d: 9.05
(0.4H, s, cis CONHB), 8.86 (0.4H, s, cis CONHA), 8.25
(0.6H, d, J=8.0 Hz, trans NH), 7.89 (0.4H, d, 0.4H,
J=8.4 Hz, cis NH), 7.84 (0.4H, d, J=7.6 Hz, cis NH),
7.75 (0.6H, d, J=8.2 Hz, trans NH), 7.01–7.30 (11.8H,
m, trans CONH2, trans NH, and Ar–H of Phe3,4), 6.70
(0.4H, d, J=8.3 Hz, cis NH), 6.38 (1.2H, s, Ar–H of
trans Dmt), 6.36 (0.8H, s, Ar–H of cis Dmt), 4.25–4.48
(3H, m, aCH of Phe3,4, aCH of cis Dmt, and aCH of
trans Pro), 4.12 (0.6H, br, aCH of trans Dmt), 3.47
(0.4H, br, dCHB of cis Pro), 2.65–3.18 (8.0H, m, aCH
and dCHA of cis Pro, bCH2 of Phe3,4, dCH2 of trans
Pro, and bCH2 of Dmt), 2.15 (2.4H, s, Me of cis Dmt),
2.08 (3.6H, s, Me of trans Dmt), 1.62–1.93 (2.8H, m,
gCH2 and bCH2 of trans Pro, and bCHB of cis Pro),
1.35–1.44 (5.8H, m, trans But, and gCHB of cis Pro),
1.25 (3.6H, s, cis But), 0.97 (0.4H, br, bCHA of cis Pro),
0.77 (0.4H, br, gCHA of cis Pro). 13C NMR (DMSO-d6)
d: 173.0 (q), 172.8 (q), 171.9 (q), 171.8 (q), 171.6 (q),
171.2 (q), 170.9 (q), 170.7 (q), 170.6 (q), 156.1 (q), 156.0
(q), 155.5 (q), 155.2 (q), 138.4 (q), 138.3 (q), 138.2 (q),
138.1 (q), 138.0 (q), 129.7 (t, Ar), 129.6 (t, Ar), 129.5 (t,
Ar), 129.3 (t, Ar), 128.5 (t, Ar), 126.8 (t, Ar), 126.7 (t,
Ar), 124.9 (q), 123.6 (q), 115.4 (t, Ar), 115.2 (t, Ar), 79.1
(q, cis But), 78.6 (q, trans But), 60.3 (t, trans-Pro a), 59.7
(t, cis-Pro a), 55.3 (t, trans-Phe a), 54.7 (t, cis-Phe a),
54.3 (t, trans-Phe a), 54.1 (t, cis-Phe a), 52.1 (t, trans-
Dmt a), 51.9 (t, cis-Dmt a), 46.5 (s, trans-Pro d), 46.3 (s,
cis-Pro d), 38.5 (s, cis-Phe b), 37.9 (s, trans-Phe b), 37.4
(s, trans-Phe b), 36.7 (s, cis-Phe b), 31.8 (s, trans-Dmt b),
31.5 (s, cis-Dmt b), 30.6 (s, cis-Pro b), 29.2 (s, trans-Pro
b), 28.8 (p, trans But), 228.6 (p, cis But), 4.8 (s, trans-Pro
g), 21.6 (s, cis-Pro g), 20.7 (p, cis-Dmt Me), 20.0 (p,
trans-Dmt Me).
Boc-Pro-Phe-NH2. A mixed anhydride [prepared from
Boc-Pro-OH (1.68 g, 7.58 mmol), IBCF (0.99 mL, 7.58
mmol) and NMM (1.63 mL, 15.16 mmol) in the usual
way] in THF (30 mL) was added to a solution of H-Phe-
.
NH2 HCl [prepared from Boc-Phe-NH2 (2.0 g, 7.58
mmol) and 7.2 N HCl/dioxane (10.0 mL, 75.8 mmol) in
the usual way] in DMF (30 mL) containing NMM (0.90
mL, 8.34 mmol) at ꢀ15 ꢁC. The reaction mixture was
stirred at 0 ꢁC for 30 min and at 4 ꢁC overnight. After
removal of the solvent, the residue was extracted with
AcOEt. The extract was washed with 10% citric acid,
5% Na2CO3 and water, dried over Na2SO4 and evapo-
rated to dryness. Petroleum ether was added to the
residue to form crystals, which were collected by fil-
tration, yield 2.29 g (90.5%), mp 71–76 ꢁC, Rf1=0.47,
[a]2D0 ꢀ98.5 (c 1.0, DMSO), Anal. calcd for C19H27N3O4:
C, 62.8; H, 7.34; N, 11.5. Found: C, 63.1; H, 7.53; N,
1
11.6, H NMR (CDCl3) d: 7.15–7.33 (5H, m, Ar–H),
6.71 (1H, br, CONHB), 6.39 (1H, br, CONHA), 5.44
(1H, br, NH of Phe), 4.74 (1H, br, aCH of Phe), 4.18
(1H, dd, J=8.6, 3.8 Hz, aCH of Pro), 3.21 (3H, m,
bCHB of Phe, and dCH2 of Pro), 3.09 (1H, br, bCHA of
Phe), 1.93–2.14 (2H, m, bCH2 of Pro), 1.82 (1H, br,
gCHB of Pro), 1.68 (1H, br, gCHA of Pro), 1.35 (9H, s,
But). 13C NMR (CDCl3) d: 173.4 (q), 172.2 (q), 155.8
(q), 136.5 (q), 129.3 (t, Ar), 128.8 (t, Ar), 127.2 (t, Ar),
81.0 (q, But), 52.7 (t, Pro a), 47.2 (t, Phe a), 47.2 (s, Pro
d), 36.9 (s, Phe b), 29.4 (s, Pro g), 28.3 (p, But), 24.5 (s,
Pro b).
1
.
HCl [Dmt ]EM-2 (9). Boc-Dmt-Pro-Phe-Phe-NH2 (200
mg, 0.29 mmol) was treated with 8.0 N HCl/dioxane
(0.38 mL, 3.0 mmol) for 1 h at room temperature. Et2O
was added to the solution until the product precipitated.
The precipitate was prepared as described for the pre-
paration of (3): yield 159 mg (89.1%), MH+ 600.5, [a]D20
ꢀ5.60
(c
0.67,
H2O),
Anal.
calcd
for
.
.
C34H41N5O6 HCl 3/2H2O: C, 60.1; H, 6.67; N, 10.3.
1
Found: C, 60.3; H, 6.33; N, 10.3. H NMR (DMSO-d6)
d: 9.27 (0.7H, brs, cis Ar–OH), 9.13 (0.3H, s, trans Ar–
OH), 8.55 (2.1H, br, NH+3 of cis Dmt), 8.29–8.36 (1.6H,
m, NH of cis Phe4, and NH3+ of trans Dmt), 8.15 (0.7H,
d, J=8.4 Hz, NH of cis Phe3), 8.00 (0.6H, d, J=7.7 Hz,
NH of trans Phe3,4), 7.60 (0.7H, brs, cis CONHB), 7.10–
7.32 (11H, m, Ar–H of Phe3,4, cis CONHA, and trans
CONHB), 7.08 (0.3H, brs, trans CONHA), 6.43 (2H, s,
Ar–H of Dmt), 4.32–4.52 (2.3H, m, aCH of trans Pro,
and aCH of Phe3,4), 4.10 (0.3H, br, aCH of trans Dmt),
3.57 (0.7H, br, aCH of cis Dmt), 3.27–3.40 (1.0H, m,
dCHB of Pro), 3.17 (0.7H, br, dCHA of cis Pro), 2.80–
3.07 (6.7H, m, aCH of cis Pro, bCH2 of Dmt, and bCH2
of Phe3,4), 2.31 (0.3H, br, dCHA of trans Pro), 2.16
Boc-Dmt-Pro-Phe-NH2. To a solution of H-Pro-Phe-
.
NH2 HCl [prepared from Boc-Pro-Phe-NH2 (300 mg,
0.90 mol) and 6.9 N HCl/dioxane (1.3 mL, 8.99 mmol)
in the usual way] in DMF (30 mL) containing DIPEA
(0.33 mL, 1.89 mmol), Boc-Dmt-OH (277 mg, 0.90
mmol) and PyBop (467 mg, 0.90 mmol) were added.
The reaction mixture was stirred at 4 ꢁC overnight.
After removal of the solvent, the residue was extracted
with AcOEt. The extract was washed with 10% citric
acid, 5% Na2CO3 and water, dried over Na2SO4 and
evaporated to dryness. Petroleum ether was added to

1992
Y. Okada et al. / Bioorg. Med. Chem. 11 (2003) 1983–1994
the residue to form crystals, which were collected by fil-
tration, yield 356 mg (71.7%), mp 211–213 ꢁC,
R1f =0.42, [a]D20 ꢀ27.3 (c 1.0, CHCl3), Anal. calcd for
C30H40N4O6: C, 65.2; H, 7.30; N, 10.1. Found: C, 64.9;
(0.35H, br, bCHB of trans Dmt), 3.08 (0.65H, dd,
J=13.9, 4.3 Hz, bCHB of cis Phe), 3.02 (1.3H, d, J=4.7
Hz, bCH2 of cis Dmt), 2.99–3.01 (1.7H, m, bCHA of
trans Dmt, bCHA of cis Phe, and bCH2 of trans Phe),
2.87 (0.65H, dd, J=8.0, 2.7 Hz, aCH of cis Pro), 2.30–
2.39 (0.35H, m, dCHA of trans Pro), 2.18 (2.1H, s, Me
of trans Dmt), 2.07 (3.9H, s, Me of cis Dmt), 1.81–1.90
(0.35H, m, bCHB of trans Pro), 1.40–1.68 (2.35H, m,
gCHB and bCHB of cis Pro, gCH2 and bCHA of trans
Pro), 1.13–1.31 (1.3H, m, gCHA and bCHA of cis Pro).
13C NMR (DMSO-d6) d: 173.24 (q), 172.52 (q), 170.26
(q), 170.24 (q), 1167.67 (q), 167.49 (q), 155.95 (q),
155.68 (q), 138.45 (q), 138.13 (q), 138.09 (q), 137.79 (q),
129.14 (t, Ar), 128.85 (t, Ar), 128.10 (t, Ar), 127.95 (t,
Ar), 1126.19 (t, Ar), 126.09 (t, Ar), 121.41 (q), 121.00
(q), 115.02 (t, Ar), 115.00 (t, Ar), 59.92 (t, trans-Pro a),
59.27 (t, cis-Pro a), 54.22 (t, cis-Phe a), 53.83 (t, trans-
Phe a), 49.81 (t, trans-Dmt a), 49.72 (t, cis-Dmt a),
46.70 (s, cis-Pro d), 46.29 (s, trans-Pro d), 37.39 (s, trans-
Phe b), 36.47 (s, cis-Phe b), 30.94 (s, cis-Pro b), 30.52 (s,
cis-Pro b), 30.03 (s, trans-Dmt b), 28.82 (s, trans-Pro b),
24.15 (s, trans-Pro g), 21.41 (s, cis-Pro g), 20.09 (p,
trans-Dmt Me), 19.32 (p, cis-Dmt Me).
1
H, 7.20; N, 9.95, H NMR (CDCl3) d: 7.50 (0.6H, d,
J=7.5 Hz, NH of cis Phe), 7.08–7.37 (5H, m, Ar–H of
Phe), 6.80 (0.6H, br s, cis CONHB), 6.62 (0.4H, brs,
trans CONHB), 6.59 (0.8H, s, Ar–H of trans Dmt), 6.52
(1.2H, s, Ar–H of cis Dmt), 6.13 (0.4H, d, J=8.0 Hz,
NH of trans Phe), 5.94 (0.4H, brs, trans CONHA), 5.61
(0.6H, brs, cis CONHA), 5.51 (0.6H, d, J=6.7 Hz, NH
of cis Dmt), 5.25 (0.4H, d, J=8.9 Hz, NH of trans
Dmt), 4.66–4.74 (0.8H, m, aCH of trans Phe, and aCH
of trans Dmt), 4.40–4.50 (1H, m, aCH of cis Phe, and
aCH of trans Pro), 4.15 (0.6H, br, aCH of cis Dmt),
3.60 (0.4H, m, dCHB of trans Pro), 3.30 (0.4H, dd,
J=14.0, 4.3 Hz, bCHB of cis Phe), 2.93–3.25 (4.8H, m,
bCH2 of cis Dmt, dCHA of trans Pro, dCH2 of cis Pro,
bCH2 of trans Phe, aCH of cis Pro, and bCHA of cis
Phe), 2.81–2.89 (1H, m, bCHA of cis Dmt, and bCHB of
trans Dmt), 2.76 (0.4H, dd, J=13.9, 7.0 Hz, bCHA of
trans Dmt), 2.30 (2.4H, s, Me of trans Dmt), 2.19 (3.6H,
s, Me of cis Dmt), 1.63–1.94 (1.8H, m, bCHB of cis Pro,
and bCH2 and gCHB trans Pro), 1.47 (5.4H, s, cis But),
1.22–1.41 (4.6H, m, gCHA of trans Pro, gCHB of cis
Pro, and trans But), 1.11–1.18 (0.6H, m, bCHA of cis
Pro), 0.70 (0.6H, br, gCHA of cis Pro). 13C NMR
(CDCl3) d: 174.0 (q), 173.6 (q), 173.1 (q), 172.2 (q),
171.6 (q), 171.5 (q), 156.5 (q), 155.4 (q), 155.1 (q), 155.0
(q), 138.7 (q), 138.1 (q), 137.2 (q), 136.9 (q), 129.0 (t,
Ar), 128.9 (t, Ar), 128.8 (t, Ar), 128.6 (t, Ar), 127.0 (t,
Ar), 126.9 (t, Ar), 124.1 (q), 123.4 (q), 115.7 (t, Ar),
115.6 (t, Ar), 81.0 (q, cis But), 80.1 (q, trans But), 61.0 (t,
trans-Pro a), 60.1 (t, cis-Pro a), 56.0 (t, cis-Phe a), 53.8
(t, trans-Phe a), 52.2 (t, cis-Dmt a), 51.3 (t, trans-Dmt
a), 47.5 (s, trans-Pro d), 47.0 (s, cis-Pro d), 36.9 (s, cis-
Phe b), 36.3 (s, trans-Phe b), 32.3 (s, trans-Dmt b), 31.9
(s, cis-Dmt b), 31.0 (s, cis-Pro b), 28.9 (s, trans-Pro b),
28.4 (p, cis But), 28.4 (p, trans But), 24.3 (s, trans-Pro g),
21.4 (s, cis-Pro g), 20.5(p, trans-Dmt Me), 19.9 (p, cis-
Dmt Me ).
.
Boc-Dmt-Pro-NH2. To a solution of H-Pro-NH2 HCl
[prepared from Boc-Pro-NH2 (139 mg, 0.65 mmol) and
6.9 N HCl/dioxane (0.9 mL, 6.47 mmol) in the usual
way] in DMF (30 mL) containing DIPEA (0.25 mL,
1.30 mmol), Boc-Dmt-OH (200 mg, 0.65 mmol) and
PyBop (370 mg, 0.65 mmol) were added. The reaction
mixture was stirred at 4 ꢁC overnight. After removal of
the solvent, the residue was extracted with AcOEt. The
extract was washed with 10% citric acid, 5% Na2CO3
and water, dried over Na2SO4 and evaporated to dry-
ness. Petroleum ether was added to the residue to form
crystals, which were collected by filtration, yield 307 mg
(74.2%), mp 129–132 ꢁC, Rf1=0.42, [a]2D0 ꢀ9.82 (c 1.0,
CHCl3), Anal. calcd for C21H31N3O5: C, 62.2; H, 7.71;
1
N, 10.3. Found: C, 62.4; H, 7.89; N, 10.50, H NMR
(CDCl3) d: 7.38 (1H, brs, NH), 6.53 (2H, s, Ar–H),
5.51–5.86 (2H, m, CONH2), 4.46–4.79 (2H, m, aCH of
Dmt and Pro), 2.89–3.70 (4H, m, bCH2 of Dmt, and
dCH2 of Pro), 2.32 (6H, s, Me), 1.62–2.20 (4H, m, gCH2
and bCH2 of Pro), 1.42 (9H, s, But).
.
HCl H-Dmt-Pro-Phe-NH2 (10). Boc-Dmt-Pro-Phe-NH2
(563 mg, 1.07 mmol) was treated with 8.0 N HCl/diox-
ane (1.25 mL, 10 mmol) for 1 h at room temperature.
Et2O was added to the solution until the product pre-
cipitated. The precipitate was prepared as described for
the preparation of (3): yield 356 mg (71.7%), MH+
453.1, [a]2D0 ꢀ13.3 (c 0.87, H2O), Anal. calcd for
.
HCl H-Dmt-Pro-NH2 (11). Boc-Dmt-Pro-NH2 (142 mg,
0.35 mmol) was treated with 8.0 N HCl/dioxane (0.44
mL, 3.5 mmol) for 1 h at room temperature. Et2O was
added to the solution until the product precipitated. The
precipitate was prepared as described for the prepara-
tion of (3): yield 75 mg (63.2%), MH+ 306.5, [a]D20
.
.
C25H32N4O4 HCl H2O: C, 59.2; H, 6.96; N, 11.1.
1
Found: C, 59.6; H, 6.82; N, 10.7. H NMR (DMSO-d6)
d: 9.30 (0.65H, br, Ar–OH of cis Dmt), 9.17 (0.35H, br,
Ar–OH of trans Dmt), 8.55 (1.95H, br, NH+3 of cis
Dmt), 8.42 (1.05H, br, NH+3 of trans Dmt), 7.98 (0.65H,
d, J=8.7 Hz, NH of cis Phe), 7.93 (0.35H, d, J=8.3 Hz,
NH of trans Phe), 7.77 (0.65H, brs, cis CONHB), 7.37
(0.35H, brs, trans CONHB), 7.15–7.30 (5.65H, m, Ar–
H, and cis CONHA), 7.04 (0.35H, brs, trans CONHA),
6.44 (2H, s, Ar–H of Dmt), 4.39–4.47 (1H, m, aCH of
Phe), 4.36 (0.35H, dd, J=8.2, 3.6 Hz, aCH of trans
Pro), 4.11 (0.35H, br, aCH of trans Dmt), 3.60 (0.65H,
br, aCH of cis Dmt), 3.32-3.47 (1H, m, dCHB of Pro),
3.15 (0.65H, dt, J=11.7, 8.0 Hz, dCHA of cis Pro), 3.10
+78.7
(c
0.38,
H2O),
Anal.
calcd
for
.
C16H23N3O3 HCl 1/2H2O: C, 54.8; H, 7.18; N, 12.0.
.
1
Found: C, 55.1; H, 7.32; N, 12.1. H NMR (DMSO-d6)
d: 9.25 (0.63H, s, cis CONHB), 9.13 (0.37H, s, trans
CONHB), 8.34 (3H, br, NH+3 of Dmt), 7.38 (0.63H, s,
cis CONHA) 7.25 (0.37H, s, trans CONHA), 6.45
(1.26H, s, Ar–H of cis Dmt), 6.40 (0.74H, s, Ar–H of
trans Dmt), 4.23 (0.37H, dd, J=8.3, 4.3 Hz, aCH of
trans Pro), 4.16 (0.63H, dd, J=9.7, 6.0 Hz, aCH of cis
Pro), 2.88–3.63 (5H, m, aCH of Dmt, bCH2 of Dmt,
dCHB of trans Pro, and dCH2 of cis Pro), 2.41 (0.37H,
m, dCHA of trans Pro), 2.19 (2.22H, s, Me of trans

Y. Okada et al. / Bioorg. Med. Chem. 11 (2003) 1983–1994
1993
Dmt), 2.13 (3.78H, s, Me of cis Dmt), 1.36–1.90 (2H, m,
bCH2 and gCH2 of Pro).
NMR (DMSO-d6) d: 8.39 (1.95H, br, NH+3 of cis Dmt),
8.22–8.31 (1.7H, m, cis NH–C2H4, and NH+3 of trans
Dmt), 7.97–8.04 (1.35H, m, NH of Phe, and trans NH–
C2H4), 7.15–7.33 (10H, m, Ar–H), 6.43 (2H, s, Ar–H),
4.40–4.49 (1H, m, aCH of Phe), 4.37 (0.35H, dd, J=8.6,
4.6 Hz, aCH of trans Pro), 4.17 (0.35H, br, aCH of
trans Dmt), 3.56 (0.65H, br, aCH of cis Dmt), 3.12–3.48
(3.65H, m, dCHB of trans Pro, NH–CH2–CH2–, and
dCH2 of cis Pro), 2.81–3.00 (4.65H, m, aCH of cis Pro,
bCH2 of Phe, and bCH2 of Dmt), 2.71 (1.3H, t, J=7.2
Hz, cis-CH2–CH2–Ph), 2.64 (0.7H, t, J=7.2 Hz, trans-
CH2–CH2–Ph), 2.30–2.37 (0.35H, m, dCHA of trans-
Pro), 2.18 (2.1H, s, Me of trans Dmt), 2.06 (3.9H, s, Me
of cis Dmt), 1.80–1.90 (0.35H, m, bCHB of trans Pro),
1.28–1.67 (3.65H, m, gCH2 and bCH2 of cis Pro, gCH2
and bCHA of trans Pro). 13C NMR (DMSO-d6) d: 171.1
(q), 170.4 (q), 170.3 (q), 170.2 (q), 167.6 (q), 167.4 (q),
158.2 (q), 157.8 (q), 155.9 (q), 155.7 (q), 139.3 (q), 139.2
(q), 138.5 (q), 138.2 (q), 137.7 (q), 137.6 (q), 129.1 (t,
Ar), 128.9 (t, Ar), 128.8 (t, Ar), 128.7 (t, Ar), 128.6 (t,
Ar), 128.5 (t, Ar), 128.2 (t, Ar), 128.0 (t, Ar), 126.3 (t,
Ar), 126.1 (t, Ar), 126.0 (t, Ar), 125.9 (t, Ar), 121.4 (q),
120.1 (q), 118.4 (q), 115.4 (q), 115.0 (t, Ar), 59.8 (t,
trans-Pro a), 59.2 (t, cis-Pro a), 54.0 (t, Phe a), 49.9 (t,
trans-Dmt a), 49.8 (t, cis-Dmt a), 46,9 (s, cis-Pro d), 46.2
(s, trans-Pro d), 40.3 (s, cis-NH–CH2–CH2–), 40.0 (s,
trans-NH–CH2–CH2–), 37.8 (s, trans-Phe b), 36.9 (s, cis-
Phe b), 34.9 (s, trans–CH2–CH2–Ph), 34.8 (s, cis–CH2–
CH2–Ph), 31.1 (s, cis-Pro b), 30.6 (s, cis-Dmt b), 30.1 (s,
trans-Dmt b), 28.9 (s, trans-Pro b), 24.2 (s, cis-Pro g),
21.6 (s, trans-Pro g), 20.0 (p, trans-Dmt Me), 19.2 (p,
cis-Dmt Me).
Boc-Dmt-Pro-Phe-NH-Phenethyl. To a solution of H-
.
Pro-Phe-NH-Phenethyl HCl [prepared from Boc-Pro-
Phe-NH-phenethyl (500 mg, 1.1 mmol) and 6.9 N HCl/
dioxane (1.6 mL, 11.1 mmol) in the usual way] in DMF
(30 mL) containing DIPEA (0.57 mL, 3.3 mmol), Boc-
Dmt-OH (340 mg, 1.1 mmol) and PyBop (570 mg, 1.1
mmol) were added. The reaction mixture was stirred at
4 ꢁC overnight. After removal of the solvent, the residue
was extracted with AcOEt. The extract was washed with
10% citric acid, 5% Na2CO3 and water, dried over
Na2SO4 and evaporated to dryness. Petroleum ether
was added to the residue to form crystals, which were
collected by filtration, yield 430 mg (60.0%), mp 178–
180 ꢁC R1f =0.29, [a]2D0 ꢀ39.3 (c 1.0, MeOH), Anal. calcd
for C38H48N4O6: C, 69.5; H, 7.37; N, 8.53. Found: C,
1
69.6; H, 7.25; N, 8.51, H NMR (DMSO-d6) d: 9.05
(0.55H, s, Ar–OH of trans Dmt), 8.86 (0.45H, s, Ar–OH
of cis Dmt), 8.15 (0.55H, d, J=8.3 Hz, NH of trans
Phe), 7.84 (1H, br, ꢀNH–C2H4), 7.77 (0.45H, d, J=7.9
Hz, NH of cis Phe), 7.12–7.35 (10H, m, Ar–H), 7.07
(0.55H, d, J=5.5 Hz, NH of trans Dmt), 6.74 (0.45H, d,
J=7.7 Hz, NH of cis Dmt), 6.40 (1.1H, s, Ar–H), 6.37
(0.9H, s, Ar–H), 4.12–4.48 (2,45H, m, aCH of Dmt,
aCH of Phe, and aCH of trans Pro), 3.48 (0.45H, br,
dCHB of cis Pro), 3.25–3.33 (1H, m, –NH–CHB–CH2–),
2.97–3.25 (4H, m, bCHB of Phe, –NH–CHA–CH2–,
aCH and dCHA of cis Pro, and dCH2 of trans Pro),
2.63–2.95 (5H, m, bCH2 of Dmt, bCHA of Phe, and
–NH–CH2–CH2–Ph), 2.18 (2.7H, s, Me of cis Dmt),
2.11 (3.3H, s, Me of trans Dmt), 1.62–1.95 (2.65H, m,
bCH2 and gCH2 of trans Pro, and bCHB of cis Pro),
1.35–1.45 (5.4H, m, trans But, and gCHB of cis Pro),
1.26 (4.05H, s, cis But), 1.03 (0.45H, br, bCHA of cis
Pro), 0.84 (0.45H, br, gCHA of cis Pro). 13C NMR
(DMSO-d6) d: 171.10 (q), 170.9 (q), 170.8 (q), 170.4 (q),
170.2 (q), 170.1 (q), 155.5 (q), 155.0 (q), 154.1 (q), 139.2
(q), 138.1 (q), 138.0 (q), 137.9 (q), 129.0 (t, Ar), 128.8 (t,
Ar), 128.4 (t, Ar), 128.4 (t, Ar), 128.3 (t, Ar), 128.2 (t,
Ar), 127.9 (t, Ar), 126.2 (t, Ar), 126.1 (t, Ar), 126.0 (t,
Ar), 125.9 (t, Ar), 124.3 (q), 123.0 (q), 114.8 (t, Ar),
114.6 (t, Ar), 78.5 (q, But), 77.9 (q, But), 59.9 (t, trans-
Pro a), 59.2 (t, cis-Pro a), 54.4 (t, trans-Phe a), 53.9 (t,
cis-Dmt a), 51.4 (t, trans-Dmt a), 46.6 (s, cis-Pro d), 46.2
(s, trans-Pro d), 40.2 (s, –NH–CH2–CH2–), 37.0 (s,
trans-Phe b), 36.6 (s, cis-Phe b), 35.0 (s, trans-CH2–
CH2–Ph), 34.9 (s, cis–CH2–CH2–Ph), 31.4 (s, trans-Dmt
b), 30.8 (s, cis-Dmt b), 30.1 (s, cis-Pro b), 28.6 (s, trans-
Pro b), 28.2 (p, trans But), 27.9 (p, cis But), 24.3 (s,
trans-Pro g), 21.0 (s, cis-Pro g), 20.1 (p, cis-Dmt Me),
19.4 (p, trans-Dmt Me).
Nuclear magnetic resonance spectroscopy
1H (400 or 500 MHz) and 13C (100 or 125 MHz) NMR
spectra were recorded on a Bruker DPX-400 or ARX-
500 spectrometers, respectively. Chemical shift values
expressed as ppm downfield from tetramethylsilane, used
as an internal standard (d value). The J values are given
in Hz. The 13C signals were assigned with the aid of dis-
tortionless enhancement by polarization transfer (DEPT)
and two-dimensional experiments, and multiplicities are
indicated by p (primary), s (secondary), t (tertiary) or q
(quaternary). Final compounds (30 mg) were dissolved in
1.0 mL DMSO-d6 (99.9% isotopic purity).
Circular dichroism spectroscopy
CD spectra were recorded at room temperature using a
JASCO J-725 spectrapolarimeter in 0.02 cm cylindrical
cell. Two scans were collected for each sample over a
wavelength range of 185–260 nm. The peptide solutions
were prepared in neat TFE (2,2,2-trifuluoroethanol)
with peptide concentration ranging from 0.79 to 1.45
mM. Band intensities are expressed as molar ellipti-
cities, [y]mꢃ103 (deg cm2/dmol).
.
HCl H-Dmt-Pro-Phe-NH-Phenethyl (12). Boc-Dmt-
Pro-Phe-NH-phenethyl (297 mg, 0.47 mmol) was trea-
ted with 8.0 N HCl/dioxane (0.59 mL, 4.7 mmol) for 1 h
at room temperature. Et2O was added to the solution
until the product precipitated. The precipitate was pre-
pared as described for the preparation of (3): yield 240
mg (90.2%), MH+ 556.9, [a]D20 +13.3 (c 1.0, H2O),
Radioligand binding
Synaptosomal brain membrane P2 preparations from
Sprague–Dawley rats were prepared and used as the
source for m- and d-receptors after the removal of
.
.
Anal. calcd for C33H40N4O4 HCl 1/2H2O: C, 65.8; H,
6.98; N, 9.30. Found: C, 65.7; H, 6.91; N, 9.47. 1H

1994
Y. Okada et al. / Bioorg. Med. Chem. 11 (2003) 1983–1994
endogenous opioid.33,34 The competitive displacement
assay used 5.57 nM [3H]-DPDPE (NEN-DuPont) and
3.5 nM [3H]DAMGO (Amersham) for d- and m-sites,
respectively as published.33,34 Affinity constants (Ki)
were determined as given earlier.34
10. Salvadori, S.; Attila, M.; Balboni, G.; Bianchi, C.;
Bryant, S. D.; Crescenzi, O.; Guessiini, R.; Picone, D.;
Tancredi, T.; Temussi, P. A.; Lazarus, L. H. Mol. Med.
1995, 1, 678.
11. Bryant, S. D.; Salvadori, S.; Cooper, P. S.; Lazarus, L. H.
Trends. Pharmcol. Sci. 1998, 19, 42.
12. Lazarus, L. H.; Bryant, S. D.; Cooper, P. S.; Guerrini, R.;
Balboni, G.; Salvadori, S. Drug Discov. Today 1998, 3, 284.
13. Dygos, J. H.; Yonan, E. E.; Scaros, M. G.; Goodmonson,
O. J.; Getman, D. P.; Periana, R. A.; Beck, G. R. A. Synthesis
1992, 8, 741.
14. Ishii, S.; Witkop, B.; Gramicidin, A. I. J. Am. Chem. Soc.
1963, 85, 1832.
15. Mosberg, H. I.; Ho, J. C.; Sobczyk-Kojiro, K. Bioorg.
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16. Okada, Y.; Fukumizu, A.; Takahashi, M.; Shimizu, Y.;
Tsuda, Y.; Bryant, S. D.; Lazarus, L. H. Biochem. Biophys.
Res. Commun. 2000, 7, 276.
17. Shane, R.; Wilk, S.; Bodnar, R. J. Brain Res. 1999, 815,
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18. Kato, T.; Nagatsu, T.; Kimura, T.; Sakakibara, S.
Experientia 1978, 15, 319.
19. Mentlein, R. Regul. Pept. 1999, 85, 9.
20. Sugimoto-Watanabe, A.; Kubota, K.; Fujibayashi, K.;
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In vitro bioactivity assay
For the GPI assay, the myenteric plexus-longitudinal
muscle was obtained from male Hartley strain guinea-
pig (250–300 g) ileum as described by Rang,35 and the
tissue was mounted in a 10-mL bath that contained
aerated (95% O2, 5% CO2) Krebs–Henseleit solution at
35 ꢁC. The tissue was stimulated transmurally between
the platinum wire electrodes using pulses of 0.5 ms
duration with a frequency of 0.1 Hz at the supramax-
imal voltage. Longitudinal contractions were recorded
via an isometric transducer. For the MVD assay, the vas
deferens of male ddY strain mouse (25–35 g) were pre-
pared, as described by Hughes et al.36 A pair of vasa was
mounted in a 10-mL bath that contained aerated (95%
CO2, 5% O2), modified Mg2+-free Krebs solution con-
.
taining ascorbic acid (0.1 mM) and EDTA 4Na (0.027
mM) at 35 ꢁC. The tissue was stimulated transmurally
with trains of rectilinear pulses of 1 ms. Stimulation
trains were given at intervals of 20 s and consisted of
seven stimuli of 1 ms duration with intervals of 10 ms.
21. Ronai, A. Z.; Timar, J.; Mako, E.; Erdo, F.; Gyarmati, Z.;
Toth, G.; Orosz, G.; Furst, S.; Szekely, J. I. Life Sci. 1999, 64,
145.
22. Peter, A.; Toth, G.; Tomboly, C.; Laus, G.; Tourwe, D. J.
Chromat. 1999, 846, 39.
23. Cardillo, G.; Gentilucci, L.; Melchiorre, P.; Spampinato,
S. Bioorg. Med. Chem. Lett. 2000, 10, 2755.
24. Sasaki, Y.; Sato, T.; Ambo, A.; Ouchi, H.; Yamamoto, Y.
Chem. Pharm. Bull. 1999, 47, 1506.
25. Podlogar, B. L.; Paterline, M. G.; Ferguson, D. M.; Leo,
G. C.; Demeter, D. A.; Brown, F. K.; Reitz, A. B. FEBS Lett.
1998, 439, 13.
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gauchi, M.; Sugiura, M.; Ishida, T. J. Pept. Res. 2001, 58, 399.
27. Keller, M.; Boissard, C.; Patiny, L.; Chung, N. N.;
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In both assays, three to four washings were done with
intervals of 15 min between each dose. Dose–response
curves were constructed and IC50 values (concentration
causing a 50% reduction of the electrically induced
twitches) were calculated graphically. For antagonism
experiments, the test sample was added to the bath 15
min prior to addition of deltorphin II as agonist. The
concentration–response curves of the agonist (deltor-
phin II) were performed in the absence and in the pres-
ence of increasing concentrations of the sample, and the
pA2 values were calculated according to the method of
Arunlakshana and Schild.37
28. Yamazaki, T.; Probstl, A.; Schiller, P. W.; Goodman, M.
Int. J. Pept. Res. 1991, 37, 164.
29. Guerrini, R.; Capasso, A.; Sorrentino, L.; Anacardio, R.;
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30. Guerrini, R.; Capasso, A.; Marastoni, A.; Bryant, S. D.;
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References and Notes
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6. Goldstein, A. G.; Fischil, W.; Lowney, L. I.; Hunkapilier,
M.; Hood, L. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 7219.
7. Zadina, J. E.; Hackler, L.; Ge, L. J.; Kastin, A. J. Nature
1997, 386, 499.
8. Hackler, L.; Zadina, J. E.; Ge, L. J.; Kastin, A. J. Peptides
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9. Henschen, A.; Lottspeich, F.; Brantl, V.; Teschemacher, H.
Hoppe-Seyler’s Z. Physiol. Chem. 1979, 360, 1217.
37. Arunlakshana, O.; Schild, H. O. Br. J. Pharmacol. 1959,
14, 48.