Structure-activity relationships (SAR) of [D-Arg(2)]dermorphin(1-4) analogues, N(alpha)-amidino-Tyr-D-Arg-Phe-X.

Article abstract of DOI:10.1248/cpb.50.771

In investigating the development of compounds with potent analgesic effects after oral administration, 74 C-terminal analogues (N(alpha)-amidino-Tyr-D-Arg-Phe-X), based on the structure of N(alpha)-amidino-Tyr-D-Arg-Phe-Me beta Ala-OH (ADAMB), were synthesized. Their analgesic activity was evaluated using the mouse-tail pressure test after both subcutaneous and oral administration, and the structure-activity relationships (SAR) were examined in detail. The results clearly indicated that compounds containing beta-amino acid without a side chain at the X position are preferable for expression of potent analgesic activity, and that the free carboxyl group is superior in its analgesic activity to that of the esterified or amidated carboxy group at the C-terminal. In addition, N-methylation of the amide bond at the 4th position contributed to improved analgesic activity. These results indicated that the strong and long-lasting analgesic effect of ADAMB is expressed by the synergistic effects of N(alpha)-amidination, the N-methylation of the amide bond at the 4th position and the carbon chain length (beta-Ala) of the residue at the 4th position, and that this is the most suitable structure.

Full text of DOI:10.1248/cpb.50.771

June 2002
Chem. Pharm. Bull. 50(6) 771—780 (2002)
771
Structure–Activity Relationships (SAR) of [D-Arg2]Dermorphin(1—4)
Analogues, Na-Amidino-Tyr-D-Arg-Phe-X1)
Tadashi OGAWA,a Tetsuhisa MIYAMAE,a Toru OKAYAMA,a Masaki HAGIWARA,a Shinobu SAKURADA,b and
,a
*
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 January 7, 2002; accepted March 8, 2002
In investigating the development of compounds with potent analgesic effects after oral administration, 74 C-
terminal analogues (Na-amidino-Tyr-D-Arg-Phe-X), based on the structure of Na-amidino-Tyr-D-Arg-Phe-
MebAla-OH (ADAMB), were synthesized. Their analgesic activity was evaluated using the mouse-tail pressure
test after both subcutaneous and oral administration, and the structure–activity relationships (SAR) were exam-
ined in detail. The results clearly indicated that compounds containing b-amino acid without a side chain at the
X position are preferable for expression of potent analgesic activity, and that the free carboxyl group is superior
in its analgesic activity to that of the esterified or amidated carboxy group at the C-terminal. In addition, N-
methylation of the amide bond at the 4th position contributed to improved analgesic activity. These results indi-
cated that the strong and long-lasting analgesic effect of ADAMB is expressed by the synergistic effects of Na-
amidination, the N-methylation of the amide bond at the 4th position and the carbon chain length (b-Ala) of the
residue at the 4th position, and that this is the most suitable structure.
Key words D-Arg2-dermorphin analogue; opioid peptide; SAR; analgesic activity; oral administration; ADAMB
Dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2)2) iso-
tetrapeptide analogues. Antinociceptive activity of ADAMB
was assessed using the tail pressure test in mice after s.c. and
p.o. administration. ADAMB showed a strong oral antinoci-
ceptive activity, with an ED50 of 5.8 mg/kg versus 22.2 mg/kg
for morphine, as well as a 38-fold stronger activity after s.c.
administration. ADAMB also showed long-lasting antinoci-
ceptive activity, with 50% of the maximum effect persisting
in the tail-pressure test at 10 h after p.o. administration (10
mg/kg). In contrast, orally administered morphine (80 mg/
kg) showed a rapid decrease of activity in the same test and
its antinociceptive effect disappeared within 4 h.15)
lated from the skin of a South American frog (Phyllomedusa
sauvagei) exhibits a high affinity and selectivity with m-opi-
oid receptor leading to potent opioid activity. The minimum
active amino acid sequence of Dermorphin is derived from
the N-terminal tetrapeptide (H-Tyr-D-Ala-Phe-Gly-),3) and a
number of tetrapeptide analogues have been synthesized and
their SAR (structure activity relationship), including opioid
activity, receptor selectivity and affinity, were investigated.4,5)
Marked elevation of activity obtained by the replacement of
D-Ala with D-Arg at the 2nd position6) led to the synthesis of
a number of [D-Arg2]dermorphin derivatives, with their SAR
being studied in great detail.7—10) For example, H-Tyr-D-Arg-
Phe-Sar-OH (DAS-DER)11) investigated by Sasaki et al. is a
compound prepared by N-methylation of the 4th position of
H-Tyr-D-Arg-Phe-Gly-OH, the N-terminal tetrapeptide of [D-
Arg2]dermorphin, and is reported to have a higher affinity,
selectivity and analgesic activity than its parent compound.
H-Tyr-D-Arg-Phe-bAla-OH (TAPA)12,13) found by Chaki et
al. is prepared by replacement of the 4th Gly with b-Ala and
also exhibits a very strong antinociceptive action. In the
mouse-tail pressure test, in comparison to morphine, the
compound exhibited a comparative effect after oral (p.o.) ad-
ministration, 388-fold activity after intracerebroventricular
administration, 1440-fold activity after intrathecal adminis-
tration and 15-fold antinociceptive activities after subcuta-
neous (s.c.) administration, respectively, with these activity
being sustained over a long period of time. Furthermore,
Marastoni et al. investigated dermorphin derivatives in detail
and reported that those with an introduced amidino group at
the N-terminal show improved analgesic activity and reduced
receptor selectivity in comparison to N-terminal free deriva-
tives.14) Thus, the possibility of dermorphin derivatives as
analgesic agents was recognized, and we designed a novel
dermorphin tetrapeptide Na-amidino-Tyr-D-Arg-Phe-MebAla-
OH (ADAMB) based on the structures of several dermorphin
In the present study, 74 C-terminal analogues (Na-amidino-
Tyr-D-Arg-Phe-X), based on the structure of ADAMB, were
synthesized to further investigate the potent analgesic effects
after p.o. administration. Their analgesic activities were eval-
uated using the mouse-tail pressure test after both s.c. and
p.o. administration, and the SAR examined in detail.
Chemistry All analogues were synthesized by the solu-
tion method using 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (WSCI·HCl) with 1-hydroxy-ben-
zotriazole (HOBt) as a coupling reagent. As shown in Chart
1, the invariable tripeptide segment [Na-(N,NЈ-bis-Cbz)-
amidino-Tyr(Bn)-D-Arg(Cbz2)-Phe-OH (I)] was first pre-
pared by stepwise-elongation. This tripeptide (I) was subse-
quently condensed with the appropriate amino acid derivative
X(PG), where PG represents a protecting group if necessary.
Each of the resulting products was dissolved in acetic acid
and subjected to catalytic hydrogenation in the presence of
Pd-C. The catalyst was removed and the reaction mixture
was concentrated under reduced pressure. Final purification
was accomplished by a reverse phase flash column chro-
matography eluted with aqueous CH3CN containing 0.1 N–
acetic acid and subsequently lyophilized to give the desired
peptides in the form of powder (Ͼ95% purity by HPLC
analysis). The final structure of the product was confirmed by
1H-NMR and FAB mass spectra.
To whom correspondence should be addressed. e-mail: morikawa@daiichi-fcj.co.jp
© 2002 Pharmaceutical Society of Japan

772
Vol. 50, No. 6
Chart 1. Synthesis of C-Terminal Analogues of [D-Arg2]Dermorphin Tetrapeptide
Antinociceptive Assays The compounds synthesized analgesic activity compared. No significant analgesic activity
here were tested for their in vivo antinociceptive potency (after s.c. or p.o. administration) was found in the synthe-
using the tail pressure test in mice after s.c. and p.o. adminis- sized tripeptide analogues 1—16. However, the N-methylated
tration. The percent of maximum possible effect (% of MPE) compound 7 exhibited some improved analgesic activity in
value was measured at fixed doses, 1 mg/kg s.c. and 10 comparison to compound 6 without N-methylation, indicat-
mg/kg p.o., primarily to evaluate the activity of synthesized ing the positive effect of N-methylation at the 4th position.
compounds. Then, analogues with high potency were evalu-
(2) Substitution with Another Amino Acid SAR stud-
ated for their ED50 values by the method of Litchfield and ies of the N-terminal free tetrapeptide analogue, H-Tyr-D-
Wilcoxon35) to compare their antinociceptive activities. These Arg-Phe-X, have reported on the importance of a proper car-
values were calculated from the values obtained at the time bon chain length (C2—3) at position 4, existence of electron-
of peak effect after either peptide or morphine administra- withdrawing atoms (oxygen) which work as hydrogen bond-
tion.
ing acceptors, and an amide bond between Phe-X in biologi-
cal activity.20) From these observations, compounds with high
affinity, selectivity and analgesic activity such as TAPA were
Results and Discussion
A number of investigations of opioid peptides were carried found.12,13) These compounds exhibited weak analgesic activ-
out and several essential structural requirements for the im- ity after p.o. administration.12) Therefore, compounds with
provement of m-selectivity and analgesic activity summa- the amidino group, Na-amidino-Tyr-D-Arg-Phe-X derivatives,
rized.16,17) Among them, amidation of the C-terminal car- at the N-terminal amino group, were further substituted with
boxyl group, its reduction to alcohol residues and N-methyla- various substituents at the C-terminal amino acid residues,
tion of the 4th amide bond were found to be effective for 4th anticipating high analgesic activity. These analgesic activities
amino acid residue in the dermorphin tetrapeptide and struc- were evaluated after both s.c. and p.o. administration. Among
turally related peptides. Based on the strong antinociceptive the derivatives 17—35 at the X-position, marked analgesic
action observed in ADAMB after p.o. administration, C-ter- activity was observed in derivatives 21, 22 and 25 after s.c.
minal analogues (Na-amidino-Tyr-D-Arg-Phe-X) were syn- administration which were substituted with Sar-OH, Sar-
thesized to further investigate the SAR on the oral antinoci- OMe and Ala-OH, respectively, but with no marked activity
ception that would lead to the elucidation of the structural re- after p.o. administration. However, N-methylation at the 4th
quirements for oral activity as well as the development of position was effective to the analgesic activity increase in
more potent analogues.
comparison to compounds 17 and 21, compounds 18 and 22.
In the other substituted derivatives 36—50 with neutral
Table 1 enumerates the analgesic activity of dermorphin
derivatives 1—74 synthesized in the present study: Na- amino acids, compounds 43, 45, 47, 49 and 50 containing
amidino-Tyr-D-Arg-Phe-X, morphine, dermorphin and [D- cyclic or substituted b-amino acids exhibited superior anal-
Arg2]dermorphin(1—4)-NH2.
gesic activity to that of morphine after both s.c. and p.o. ad-
(1) Tripeptide Analogues As mentioned above, amida- ministration. A g-amino acid substituted compound 36 with
tion of the C-terminal carboxyl group or reduction to alcohol an elongated chain length showed a significant decline in ac-
moiety has been known to be effective in improving m-selec- tivity. The above results indicate that b-amino acid moiety at
tivity and analgesic activity among the structural require- the X-position in Na-amidino-Tyr-D-Arg-Phe-X is desirable
ments. Thus, tripeptide derivatives with various introduced for high analgesic activity.
amines and amino alcohols were synthesized based on the
Marked analgesic activity was observed in aminosulfonic
structure of ADAMB. Furthermore, SAR studies on [D- acid derivatives 55, 56 with no side chain among the ana-
Arg2]dermorphin with substituted D-Arg at the 2nd position logues having either basic amino acid (51, 52) or acidic
have shown that the N-terminal tripeptide (H-Tyr-D-Arg-Phe- amino acid (53—56).
NH2) is a minimally active structure18) and introduction of
(3) Modification of C-Terminal Carboxyl Group The
the methyl group to the C-terminal amide, including N- C-terminal substituted products of ADAMB, including C-ter-
methylamide or N,N-dimethylamide, provided a marked im- minal oxyesters 57—61 and amide derivatives 62—68, were
provement in activity.19) Further improvement of analgesic synthesized to explore better analgesic compounds after p.o.
activity was anticipated with the introduction of the amidino administration. The ester derivatives were anticipated to
group at the N-terminal.14,15) Thus, 16 tripeptide derivatives show improved intestinal absorption due to increased
with an introduced amidino group were synthesized and their lipophilicity and improved analgesic activity after p.o. ad-

June 2002
773
Table 1. Analgesic Activity of C-Terminal Analogues of Dermorphin Tetrapeptide: Na-Amidino-Tyr-D-Arg-Phe-X
MPE %a)
ED50 (mg/kg)a,b)
p.o.
No.
X
s.c.
p.o.
s.c.
1 mg/kg
10 mg/kg
Morphine
4.9
31.05
11.28
12.9
10.21
3.15
3.3
3.95
22.2
Dermorphin
(D-Arg2)-Dermorphin(1—4)-NH2
(1) Tripeptide analogues
1 Na-Amidino-Tyr-D-Arg-Phe-OH
0.34
6.36
4.57
3.67
0.00
13.30
46.50
14.74
7.58
12.48
7.73
0.52
5.96
1.71
6.61
0.00
8.93
12.68
8.35
6.35
5.84
4.93
10.80
3.62
2
3
-NHMe
-NHEt
4
5
6
-NH(n-Pr)
-NH(n-Hex)
-Gly-ol
7
-Sar-ol
8
9
-NH(CH2)2OMe
-Ala-ol
10
11
12
13
-D-Ala-ol
-NH(CH2)3OH
-NH(CH2)4OH
-NH(CH2)2C6H5
16.00
4.83
14
15
16
5.95
0.00
22.03
10.19
6.13
21.59
(2) Substitution by another amino acid
1) Gly substitution
17
18
19
20
21
22
23
24
-Gly-OH
-Gly-OMe
-Gly-NH2
-Gly-NHMe
-Sar-OH
-Sar-OMe
58.49
61.97
15.06
18.72
89.71
93.68
46.53
38.52
3.59
6.88
0.00
10.92
26.13
31.18
25.75
5.93
0.38
1.2
17.8
-(Et)Gly-OH
-(n-Pr)Gly-OH
Ͼ40.0
2) Ala substitution
25
26
27
28
29
30
31
32
33
34
35
-Ala-OH
-Ala-OMe
-Ala-NH2
100.00
82.36
39.91
8.03
4.54
30.44
8.14
10.08
9.40
22.72
43.06
0.00
5.27
5.28
9.51
2.20
7.49
3.08
5.86
1.18
6.39
3.55
0.18
0.40
Ͼ40.0
-Ala-NHMe
-MeAla-OH
-D-Ala-OH
-D-Ala-OMe
-D-Ala-NH2
-D-Ala-NHMe
-D-MeAla-OH
-D-MeAla-OMe
3) Substitution by neutral amino acid
36
-GABA-OH
28.43
11.45
11.91
7.14
37
38
39
40
41
42
-Leu-OH
-Nva-OH
-Phe-OH
-Pro-OH
-Pro-OMe
8.25
7.11
19.47
3.50
3.83
0.37
14.46
10.96
6.85
4.93
43
44
45
92.04
77.14
85.57
28.82
43.75
31.08
0.25
0.60
0.53
-(R,S)bPro-OH
40.0

774
Vol. 50, No. 6
Table 1. (Continued)
MPE %a)
ED50 (mg/kg)a,b)
s.c. p.o.
No.
X
s.c.
p.o.
1 mg/kg
10 mg/kg
46
47
48
49
50
-aAib-OH
19.14
83.14
10.32
73.25
77.95
8.17
11.86
0.11
12.53
34.98
-(R,S)bAib-OH
-(R)aAbu-OH
-(S)aAbu-OH
-(R,S)bAbu-OH
0.54
Ͼ40.0
0.46
0.69
Ͼ40.0
27.3
4) Substitution by basic amino acid
51
52
-Arg-NH2
-D-Arg-NH2
3.37
0.32
5.99
1.95
5) Substitution by acidic amino acid
53
54
55
56
-Asp-OH
-Asu-OH
-NH(CH2)2SO3H
-NHCH2SO3H
7.30
0.00
75.59
67.10
8.52
4.60
28.11
17.43
0.54
(3) Modification of C-terminal carboxyl group
ADAMB
57
58
59
60
61
62
63
64
65
66
67
-MebAla-OH
100.00
100.00
100.00
100.00
100.00
100.00
99.00
44.69
9.24
82.23
59.75
53.54
54.98
55.48
41.59
44.29
10.00
16.25
5.34
0.089
0.12
0.16
0.11
0.11
0.16
0.31
5.8
8.9
9.0
9.9
9.5
-MebAla-OMe
-MebAla-OEt
-MebAla-O(n-Pr)
-MebAla-O(n-Hex)
-MebAla-OPOM
-MebAla-NH2
14.2
24.6
-MebAla-NHMe
-MebAla-NHEt
-MebAla-N(Me)2
-MebAla-N(Et)2
-MebAla-NH(n-Hex)
-MebAla-NH(n-Pr)
17.54
5.62
2.74
13.98
0.00
5.27
68
8.99
(4) Substitution of N-methyl group
69
70
71
72
73
74
-bAla-OH
93.05
96.96
18.30
12.85
0.32
0.15
0.25
18.4
16.0
-bAla-OMe
-EtbAla-OH
-EtbAla-OMe
-(n-Pr)bAla-OH
-BnbAla-OH
59.72
100.00
8.23
6.86
16.29
0.00
a) Data are given as the mean value for groups of 10 mice. b) ED50 values were calculated from the values obtained at the time of peak effect after administration.
ministration. Methyl to n-hexyl esters of ADAMB were syn- zymic degradation, therefore, more potent and long-lasting
thesized, however, none of the anticipated superior analgesic analgesic activities can be expected. However, the amide de-
activity after p.o. administration compared to that of the free rivative 62 of ADAMB decreased analgesic activity. More-
carboxyl group, ADAMB, was found, although high activity over, the longer the alkyl chain length became and the greater
after s.c. administration was observed. The length of the the number of the N-substituents, the more analgesic activity
alkyl chain did not affected the activity. Derivative 61, having decreased. Amidation of the C-terminal carboxyl group
prodrug moiety of pivaloyloxymethyl ester (POM) also would therefore interrupt the peptide-receptor interaction.
showed no improved analgesic activity after p.o. administra-
(4) Substitution of N-Methyl Group The structural re-
tion. A slower onset of the antinociceptive activity would be quirement of N-methylation of the amide bond at the 4th po-
expected for these C-terminal ester derivatives due to a time sition for the improved m-selectivity and analgesic activity
lag in the metabolization to ADAMB. However, the times of mentioned above led to the introduction of various N-alkyl
peak effect after administration were almost the same as that groups based on the structure of ADAMB and the anticipated
of ADAMB (data not shown). Furthermore, extension of the improvement in analgesic activity. Thus, N-ethyl (71), which
analgesic effect expected of ester derivative was not ob- elongates the N-alkyl chain, and N-n-propyl (73) and N-ben-
served. It was thus thought that the C-terminal ester deriva- zyl (74) derivatives were synthesized. However, no improved
tive itself displayed analgesic activity, and that the ester moi- analgesic activity compared to the N-methylation was ob-
ety decreased analgesic activity compared with that of served, with activity particularly decreasing after p.o. admin-
ADAMB.
In amide derivatives 62—68 where improved analgesic ac-
istration.
Introduction of the N-alkyl group to the amide group at the
tivity and stability to enzymic degradation was anticipated, 4th position provided the highest activity in the methyl
potent analgesic activity was observed in the non-substituted group. The structural requirement for the improved analgesic
amide compound 62. However, overall, alkylamide deriva- activity of opioid peptides suggested improved resistance to
tives exhibited very slight activity. Unlike the ester deriva- enzymic degradation for N-methylation of the amide group at
tives, C-terminal amide derivatives could be resistant to en- the 4th position.21) However, [D-Arg2]dermorphin(1—4) de-

June 2002
775
the expression of potent and long-lasting analgesic activity
among the opioid peptides ever found. Future studies are
needed to evaluate the relationship between in vitro affinity
and selectivity to the opioid receptor and in vivo analgesic
activity, which can lead to elucidation of the mechanism of
analgesic action and the development of peptidergic anal-
gesics.
Experimental
Chemistry Commercial amino acid derivatives and 1-hydroxybenzotri-
azole (HOBt) were obtained from Kokusan Chemical Works, Ltd., Tokyo,
Japan. Other reagents and solvents were purchased from Aldrich Chemicals,
WI, U.S.A. Boc-D-Arg(Cbz2)-OH was prepared according to the procedure
reported by Jetten et al.33) Thin layer chromatography was performed on sil-
ica plates (0.25 mm; Merck, 60 F254) using the following solvent systems:
Rf1, n-butanol/acetic acid/water (4 : 1 : 5, v/v/v, supernatant); Rf2, n-butanol/
acetic acid/water/pyridine (15 : 3 : 10 : 12, v/v/v/v). Purification of the final
products was achieved by flash chromatography using a reverse phase silica
gel Chromatorex ODS DM1020T, Fuji Silysia Chemical Ltd., Aichi, Japan,
Fig. 1. Summary of Structural Requirements for Antinociceptive Activity
rivatives have already been reported to exhibit very high re- eluted by a stepwise gradient of acetonitrile (starting from 1—5% and step-
sistance to enzymic degradation,8,14,22) and TAPA with intro-
wise by 1—2%) in 0.1 M acetic acid. An analytical high-pressure liquid
chromatograph was on a Nucleosil 100 5C18 column (4.6ϫ150 mm) in an
Agilent 1100 HPLC system. The products were analyzed with a linear gradi-
ent of 1—70% acetonitrile in 0.1% aqueous trifluoroacetic acid (TFA) over
duced bAla to the 4th position was reported as being highly
stable.23) Thus, the N-methyl group in the 4th amide may
contribute not only to stability against enzymic degradation
but also to improved activity together with some other factors
such as interaction with a receptor. N-Methylamide binding
is more likely to take cis-configuration than that of conven-
tional amide bonds.24,25) The NMR spectra of some ana-
logues prepared in the present study, including N-methylated
Sar and MebAla at the 4th position, showed a splitting of
signals. This indicates two conformations of cis- and trans-
forms in the solution. The N-methyl proton signal of MebAla
of ADAMB split at an area ratio of 2 : 3. Results of NMR
studies suggested the presence of cis-isomers, indicating the
contribution of deviation to cis-amide bonds for elevated ac-
tivity,26) the active conformation of dermorphin type opioid
peptides binding to m-opioid receptor also remains unclear.
However, the primary structure of opioid receptors was re-
20 min at a flow rate of 1 ml/min. The chromatogram was recorded by UV
detection at 230 and 280 nm. H-NMR spectra were recorded with a JEOL
JMN-AL300 (300 MHz) spectrometer, using tetramethylsilane (TMS) as an
internal standard. Mass spectra (FAB-MS) were obtained with JEOL mass
spectrometer model JMS-700.
1
Method A (Coupling Procedure) To a solution of carboxy component
(1 mmol), the amino component (1.1 eq), HOBt (1.1 eq) in N,N-dimethylform-
amide (DMF) (ca. 20 ml), and triethylamine (TEA) (1 eq if the amino com-
ponent was in the protonated form) was added at 0 °C. Then, WSCI·HCl
(1.2 eq) was added to the solution at Ϫ10 °C. This mixture was stirred for
30 min at Ϫ10 °C and overnight at room temperature. The reaction mixture
was diluted with EtOAc. The solution or suspension 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 crystallized from appropri-
ate solvents or purified by column chromatography.
Method B (Hydrogenation) Hydrogenation was performed in AcOH
under H2 atomospheric pressure and at room temperature in the presence of
5% Pd-C (catalyst to peptide ratio, 1 : 2, w/w). The reaction mixture was fil-
cently elucidated27—32) and knowledge of its steric structure tered through a Celite bed and evaporated to dryness.
Method C (Purification) The crude peptide was purified by ODS chro-
is progressing. In future, more detailed information is ex-
pected from the results of SAR studies at the receptor level.
In conclusion, an investigation into compounds exhibiting
potent analgesic activity after p.o. administration was carried
out and 74 C-terminal analogues were synthesized based on
the structure of ADAMB. Their analgesic activity was evalu-
ated using the mouse-tail pressure test (s.c. and p.o.) and
their SAR was investigated in detail. Although no analgesic
activity superior to ADAMB after p.o. administration was
observed, the results clearly demonstrated that participation
of a b-amino acid residue with no side chain at the X-posi-
tion, and a free carboxyl group at the C-terminal is more ad-
vantageous than that of ester or amide for improved analgesic
activity. Moreover, N-methylation of the amide bond at the
4th position provided not only stability with regards to en-
zymic degradation but also suggested improved analgesic ac-
tivity due to conformational factors. These results are sum-
marized in Fig. 1.
matography with a stepwise gradient of acetonitrile (starting from 1—5%
and stepwise by 1—2%) in 0.1 N acetic acid as the eluting solvent and then
lyophilized to give a white powder.
Preparation of Na-(N,N؅-bis-Cbz)amidino-Tyr(Bn)-D-Arg(Cbz2)-Phe-
OH (I) The starting material, Boc-Phe-OTce (237 g, 598 mmol), was
treated with 4 N-HCl/EtOAc (1000 ml) to remove the Boc group for 30 min
at room temperature. Ether was added to the solution and the precipitated
solid was separated by filtration and dried in a vacuum desiccator over solid
NaOH. This amine component was dissolved in DMF (1300 ml) on an ice
bath. To this solution were added Boc-D-Arg(Cbz2)-OH (295 g, 544 mmol)
and HOBt (81 g, 599 mmol), the mixture was then neutralized with TEA
(91 ml) followed by addition of WSCI·HCl (125 g, 652 mmol) at Ϫ10 °C.
The reaction mixture was stirred for 1 h at the same temperature, then
overnight at room temperature. The reaction mixture was diluted with
EtOAc. The solution 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 solidified from EtOAc and hexane to give Boc-D-Arg(Cbz2)-
Phe-OTce, 339 g (76%) as white amorphous solid.
Boc-D-Arg(Cbz2)-Phe-OTce (265 g, 323 mmol) was treated with 4 N-
HCl/EtOAc (500 ml) to remove the Boc group for 30 min at room tempera-
ture. Ether was added to the solution and the precipitated solid was separated
In a recent report of ours, very strong and long-lasting by filtration and dried in a vacuum desiccator over solid NaOH. This amine
component was dissolved in DMF (800 ml) on an ice bath. To the solution
were added Boc-Tyr(Bn)-OH (l09 g, 293 mmol) and HOBt (44 g, 326 mmol)
and the mixture was then neutralized with TEA (45 ml) and WSCI·HCl
(68 g, 352 mmol) was added at Ϫ10 °C. The reaction mixture was stirred for
analgesic activity of ADAMB after p.o. administration
showed expression of a synergistic effect of amidination at
N-terminal, N-methylation of the amide bond and homologa-
tion at the 4th position. The present results confirmed the
fact that ADAMB has one of the most suitable structures for
1 h at the same temperature, then overnight at room temperature. The mix-
ture was then diluted with EtOAc, and the solution was washed with 1 N-

776
Vol. 50, No. 6
HCl, sat. NaHCO3, and brine. The organic phase was dried over MgSO4, fil-
tered, and evaporated in vacuo. The residue was solidified from EtOAc and
hexane to give Boc-Tyr(Bn)-D-Arg(Cbz2)-Phe-OTce, 262 g (83%) as white
amorphous solid.
7.15 (5H, m), 6.96 (2H, d, Jϭ8.7 Hz), 6.62 (2H, d, Jϭ8.7 Hz), 4.50 (1H, dd,
Jϭ10.8, 4.5 Hz), 4.27 (1H, t, Jϭ7.5 Hz), 3.96 (1H, t, Jϭ7.2 Hz), 3.49 (2H,
m), 2.92—2.66 (6H, m), 1.82 (6H, s), 1.34 (2H, m), 1.07 (1H, br m), 0.90
(1H, br m). FAB-MS m/z: 570 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-Sar-ol·2AcOH (7) White powder (387
The Boc-Tyr(Bn)-D-Arg(Cbz2)-Phe-OTce (240 g, 223 mmol) was treated
with 4 N-HCl/EtOAc (500 ml) to remove the Boc group for 30 min at room
temperature. Ether was added to the solution and the precipitated solid was
separated by filtration and dried in a vacuum desiccator over solid NaOH.
This amine component was dissolved in DMF (800 ml) on an ice bath. The
solution was neutralized with TEA (34 ml), added with 1-(N,NЈ-bis(benzyl-
oxycarbonyl)amidino)pyrazole (93 g, 245 mmol) and stirred overnight at
room temperature. The reaction mixture was diluted with EtOAc. The solu-
tion 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
solidified from EtOAc and hexane to give Na-(N,NЈ-bis-Cbz)amidino-
Tyr(Bn)-D-Arg(Cbz2)-Phe-OTce, 215 g (75%) as white amorphous solid.
This fully-protected peptide (200 g, 156 mmol) was dissolved in 90%
AcOH (1000 ml). To this solution, zinc powder (50 g, 765 mmol) was added,
and vigorous stirring followed for 2 h at room temperature. After the zinc
powder was removed by filtration, the reaction mixture was concentrated
under reduced pressure. The residue was dissolved in EtOAc (1000 ml). The
solution 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 solidified from EtOAc and hexane to give Na-(N,NЈ-bis-Cbz)amidino-
Tyr(Bn)-D-Arg(Cbz2)-Phe-OH (I), 159 g (89%) as white amorphous solid.
1H-NMR (CDCl3) d: 11.6 (1H, br s), 9.26 (1H, br s), 8.96 (1H, d, Jϭ6.6 Hz),
7.50 (1H, br s), 7.37—7.05 (30H, m), 6.98 (2H, d, Jϭ8.4 Hz), 6.74 (2H, d,
Jϭ8.4 Hz), 5.19—4.96 (8H, m), 4.90 (2H, s), 4.69 (1H, dd, Jϭ12.8, 6.5 Hz),
4.52 (1H, t, Jϭ6.6 Hz), 3.90 (1H, br s), 3.02 (3H, m), 1.67 (1H, br m), 1.48
(2H, br m). FAB-MS m/z: 1154 (MϩH)ϩ.
1
mg, 55%): HPLC tR (min): 9.26. Rf1 0.42. Rf2 0.68. H-NMR (CD3OD) d:
7.14 (5H, m), 6.96 (2H, dd, Jϭ8.6, 2.0 Hz), 6.62 (2H, dd, Jϭ8.4, 1.5 Hz),
5.01 (1H, dt, Jϭ32, 7.2 Hz), 4.29—4.23 (1H, m), 4.18—4.12 (1H, m),
3.54—3.47 (2H, m), 3.32 (1H, t, Jϭ5.9 Hz), 3.01—2.76 (6H, m), 2.88
(1.5H, s), 2.82 (1.5H, s), 1.81 (6H,s), 1.48—1.06 (4H, br m). FAB-MS m/z:
584 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-NH(CH2)2OMe·2AcOH (8) White pow-
der (500 mg, 71%): HPLC tR (min): 9.68. Rf1 0.47. Rf2 0.70. 1H-NMR
(CD3OD) d: 7.15 (5H, m), 6.95 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
4.50 (1H, dd, Jϭ10.2, 4.8 Hz), 4.24 (1H, t, Jϭ7.5 Hz), 4.00 (1H, t,
Jϭ7.5 Hz), 3.31 (2H, m), 3.28—3.14 (2H, m), 2.97—2.66 (5H, m), 1.81
(6H, s), 1.35 (2H, m), 1.05 (1H, br m), 0.93 (1H, br m). FAB-MS m/z: 584
(MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-Ala-ol·2AcOH (9) White powder (549
1
mg, 78%): HPLC tR (min): 8.86. Rf1 050. Rf2 0.71. H-NMR (CD3OD) d:
7.13 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz), 4.50 (1H, dd,
Jϭ10.8, 4.5 Hz), 4.26 (1H, t, Jϭ7.5 Hz), 3.95 (1H, t, Jϭ7.2 Hz), 3.84 (1H,
dd, Jϭ12.3, 5.7 Hz), 3.40—3.16 (3H, m), 2.97—2.66 (5H, m), 1.81 (6H, s),
1.35 (2H, m), 1.08 (4H, d, Jϭ6.6 Hz), 0.88 (1H, br m). FAB-MS m/z: 584
(MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-D-Ala-ol·2AcOH (10) White powder
(507 mg, 72%): HPLC tR (min): 9.30. Rf1 0.50. Rf2 0.71. 1H-NMR (CD3OD)
d: 7.16 (5H, m), 6.96 (2H, d, Jϭ8.7 Hz), 6.62 (2H, d, Jϭ8.4 Hz), 4.47 (1H,
dd, Jϭ10.1, 5.3 Hz), 4.24 (1H, t, Jϭ7.1 Hz), 3.98 (1H, t, Jϭ7.2 Hz), 3.82
(1H, dd, Jϭ12.6, 6.0 Hz), 3.44—3.40 (2H, m), 3.20—3.13 (1H, m), 2.98—
2.68 (5H, m), 1.81 (6H, s), 1.37 (2H, br m), 1.08 (1H, br m), 0.94 (4H, d,
Jϭ6.9 Hz). FAB-MS m/z: 584 (MϩH)ϩ.
Preparation of Na-Amidino-Tyr-D-Arg-Phe-OH·2AcOH (1) Hydro-
genation of Na-(N,NЈ-bis-Cbz)amidino-Tyr(Bn)-D-Arg(Cbz2)-Phe-OH (I)
(1.15 g, 1.0 mmol) was carried out using method B, then purified using
method C. Compound 1 was obtained after lyophilization in the form of a
Na-Amidino-Tyr-D-Arg-Phe-NH(CH2)3OH·2AcOH (11) White pow-
der (500 mg, 71%): HPLC tR (min): 8.97. Rf1 0.49. Rf2 0.70. 1H-NMR
(CD3OD) d: 7.16 (5H, m), 6.97 (2H, d, Jϭ8.7 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
4.47 (1H, dd, Jϭ10.8, 4.8 Hz), 4.28 (1H, t, Jϭ7.2 Hz), 3.95 (1H, t, Jϭ7.5
Hz), 3.44 (2H, m), 2.97—2.65 (5H, m), 1.82 (6H, s), 1.60 (2H, m), 1.36
(2H, br m), 1.08 (1H, m), 0.92 (1H, m). FAB-MS m/z: 584 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-NH(CH2)4OH·2AcOH (12) White pow-
der (538 mg, 75%): HPLC tR (min): 10.84. Rf1 0.58. Rf2 0.74. 1H-NMR
(CD3OD) d: 7.16 (5H, m), 6.99 (2H, d, Jϭ8.4 Hz), 6.63 (2H, d, Jϭ8.4 Hz),
4.47 (1H, dd, Jϭ10.4, 4.8 Hz), 4.32 (1H, t, Jϭ7.8 Hz), 4.00 (1H, t,
Jϭ7.2 Hz), 3.95 (2H, t, Jϭ6.3 Hz), 3.17—3.09 (3H, m), 2.96—2.72 (5H,
m), 1.82 (6H, s), 1.46 (6H, m), 1.12 (1H, m), 0.99 (1H, m). FAB-MS m/z:
598 (MϩH)ϩ.
1
white powder (466 mg, 72%). HPLC tR (min): 9.35. Rf1 0.37. Rf2 0.62. H-
NMR (CD3OD) d: 7.12 (5H, m), 6.97 (2H, d, Jϭ8.6 Hz), 6.62 (2H, d,
Jϭ8.6 Hz), 4.54 (1H, dd, Jϭ9.4, 4.4 Hz), 4.22 (2H, q), 2.87 (5H, m), 1.81
(6H, s), 1.41 (1H, br m), 1.30 (1H, br m), 1.06 (2H, m). FAB-MS m/z: 527
(MϩH)ϩ.
Preparation of Na-Amidino-Tyr-D-Arg-Phe-NHMe·2AcOH (2) Ac-
cording to method A, Na-(N,NЈ-bis-Cbz)amidino-Tyr(Bn)-D-Arg(Cbz2)-Phe-
OH (I) (1.15 g, 1.0 mmol) was coupled with HCl·NHMe (74 mg, 1.1 mmol),
yielding a fully-protected peptide, Na-(N,NЈ-bis-Cbz)amidino-Tyr(Bn)-D-
Arg(Cbz2)-Phe-NHMe, which was deprotected according to method B. The
crude compound was purified using method C and then lyophilized to give a
1
Na-Amidino-Tyr-
D-Arg-Phe-NH(CH2)2C6H5·2AcOH (13) White pow-
white powder (495 mg, 75%). HPLC tR (min): 9.03. Rf1 0.45. Rf2 0.67. H-
der (472 mg, 63%): HPLC tR (min): 12.77. Rf1 0.54. Rf2 0.76. 1H-NMR
(CD3OD) d: 7.10 (10H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
4.46 (1H, dd, Jϭ10.7, 5.1 Hz), 4.24 (1H, t, Jϭ7.8 Hz), 4.02 (1H, t,
Jϭ7.5 Hz), 3.36 (2H, t, Jϭ6.9 Hz), 3.15 (1H, m), 2.97—2.75 (4H, m),
2.69—2.60 (3H, m), 1.81 (6H, s), 1.33 (2H, m), 1.05 (1H, m), 0.90 (1H, m).
FAB-MS m/z: 630 (MϩH)ϩ.
NMR (CD3OD) d: 7.15 (5H, m), 6.95 (2H, d, Jϭ8.5 Hz), 6.62 (2H, d,
Jϭ8.5 Hz), 4.46 (1H, dd, Jϭ10.6, 4.6 Hz), 4.25 (1H, t, Jϭ7.7 Hz), 3.98 (1H,
t, Jϭ7.4 Hz), 2.97—2.56 (5H, m), 2.62 (3H, s), 1.81 (6H, s), 1.34 (2H,
br m), 1.06 (1H, br m), 0.92 (1H, br m). FAB-MS m/z: 540 (MϩH)ϩ.
Compounds 3—74 were prepared in the same manner from the corre-
sponding amino acid derivatives, respectively.
Na-Amidino-Tyr-D-Arg-Phe-NHEt·2AcOH (3) White powder (458
Na-Amidino-Tyr-
D-Arg-Phe
· 2AcOH (14) White powder (471
1
—N
mg, 68%): HPLC tR (min): 9.84. Rf1 0.49. Rf2 0.70. H-NMR (CD3OD) d:
7.15 (5H, m), 6.95 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.4 Hz), 4.46 (1H, dd,
Jϭ10.5, 4.8 Hz), 4.24 (1H, t, Jϭ8.0 Hz), 4.00 (1H, t, Jϭ7.2 Hz), 3.15 (2H,
dd, Jϭ16.2, 4.8 Hz), 3.03—2.65 (5H, m), 1.81 (6H, s), 1.35 (2H, br m),
1.10—0.80 (2H, br m), 0.98 (3H, t, Jϭ7.2 Hz). FAB-MS m/z: 554 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-NH(n-Pr)·2AcOH (4) White powder
(454 mg, 66%): HPLC tR (min): 10.50. Rf1 0.54. Rf2 0.70. 1H-NMR
(CD3OD) d: 7.14 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
4.47 (1H, dd, Jϭ10.5, 4.8 Hz), 4.26 (1H, t, Jϭ 7.1 Hz), 3.98 (1H, t,
Jϭ7.1 Hz), 3.03 (2H, t, Jϭ7.2 Hz), 2.95—2.65 (5H, m), 1.84 (6H, s), 1.44—
1.31 (4H, m), 1.06 (1H, br m), 0.95 (1H, br m), 0.76 (3H, t, Jϭ7.4 Hz). FAB-
MS m/z: 568 (MϩH)ϩ.
mg, 66%): HPLC tR (min): 11.79. Rf1 0.49. Rf2 0.76. 1H-NMR (CD3OD) d:
7.13 (5H, m), 6.97 (2H, d, Jϭ8.1 Hz), 6.62 (2H, d, Jϭ8.1 Hz), 4.98 (1H, t,
Jϭ7.8 Hz), 4.29 (1H, t, Jϭ7.5 Hz), 4.16 (1H, dd, Jϭ8.6, 5.1 Hz), 3.36 (3H,
m), 3.14 (1H, m), 2.96—2.89 (4H, m), 2.83—2.76 (2H, m), 1.82 (6H, s),
1.49—1.36 (7H, m), 1.17 (2H, m), 1.05 (1H, m). FAB-MS m/z: 594
(MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe
· 2AcOH (15) White powder
N
H
(524 mg, 72%): HPLC tR (min): 12.42. Rf1 0.54. Rf2 0.75. 1H-NMR
(CD3OD) d: 7.16 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.7 Hz),
4.45 (1H, dd, Jϭ10.2, 5.1 Hz), 4.24 (1H, t, Jϭ7.1 Hz), 3.98 (1H, t,
Jϭ7.1 Hz), 3.50 (1H, br m), 3.15 (1H, dd, Jϭ14.1, 5.1 Hz), 2.96—2.66 (5H,
m), 1.85 (6H, s), 1.72—1.49 (5H, br m), 1.37 (2H, br m), 1.23—0.92 (7H,
br m). FAB-MS m/z: 608 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-NH(n-Hex)·2AcOH (5) White powder
(453 mg, 62%): HPLC tR (min): 13.72. Rf1 0.60. Rf2 0.76. 1H-NMR
(CD3OD) d: 7.15 (5H, m), 6.96 (2H, d, Jϭ8.7 Hz), 6.61 (2H, d, Jϭ8.7 Hz),
4.47 (1H, dd, Jϭ10.4, 5.4 Hz), 4.25 (1H, t, Jϭ7.5 Hz), 4.01 (1H, t, Jϭ7.2
Hz), 3.15 (2H, dd, Jϭ15.4, 5.1 Hz), 3.05 (2H, t, Jϭ7.2 Hz), 2.98—2.65 (5H,
m), 1.81 (6H, s), 1.36 (2H, m), 1.17 (6H, m), 1.08 (1H, br m), 0.96 (1H,
br m), 0.80 (3H, t, Jϭ7.2 Hz). FAB-MS m/z: 610 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe —N
(411 mg, 53%): HPLC tR (min): 8.11. Rf1 0.28. Rf2 0.60. 1H-NMR (CD3OD) d:
NH · 3AcOH (16) White powder
Na-Amidino-Tyr-D-Arg-Phe-Gly-ol·2AcOH (6) White powder (504
1
mg, 73%): HPLC tR (min) 8.69. Rf1 0.45. Rf2 0.69. H-NMR (CD3OD) d: 7.13 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.4 Hz), 5.00 (1H, t,

June 2002
777
Jϭ8.2 Hz), 4.26 (1H, t, Jϭ7.2 Hz), 4.04 (1H, dd, Jϭ8.4, 5.4 Hz), 3.31 (3H,
3.88 (1H, t, Jϭ7.2 Hz), 3.27 (1H, dd, Jϭ14.0, 4.1 Hz), 2.97—2.78 (4H, m),
m), 3.14 (1H, m), 3.10—2.92 (4H, m), 2.88—2.65 (6H, m), 1.82 (9H, s), 2.70 (1H, dd, Jϭ14.0, 11.3 Hz), 1.82 (6H, s), 1.33 (5H, d, Jϭ7.2 Hz), 1.06
1.34 (2H, br m), 1.07 (1H, br m), 0.95 (1H, br m). FAB-MS m/z: 595
(1H, br m), 0.85 (1H, br m). FAB-MS m/z: 597(MϩH)ϩ.
(MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-Ala-NHMe·2AcOH (28) White powder
Na-Amidino-Tyr-D-Arg-Phe-Gly-OH·2AcOH (17) White powder (504 mg, 69%): HPLC tR (min): 9.26. Rf1 0.47. Rf2 0.71. 1H-NMR (CD3OD)
(535 mg, 76%): HPLC tR (min): 8.80. Rf1 0.35. Rf2 0.62. 1H-NMR (CD3OD) d: 7.16 (5H, m), 6.98 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz), 4.50 (1H,
d: 7.13 (5H, m), 6.96 (2H, d, Jϭ8.7 Hz), 6.61 (2H, d, Jϭ8.7 Hz), 4.58 (1H, dd, Jϭ11.3, 4.1 Hz), 4.35—4.25 (2H, m), 3.91 (1H, t, Jϭ7.1 Hz), 3.25 (1H,
dd, Jϭ11.0, 4.5 Hz), 4.23 (1H, t, Jϭ7.2 Hz), 4.01 (1H, t, Jϭ7.2 Hz), 3.67 dd, Jϭ14.9, 4.7 Hz), 2.92—2.83 (5H, m), 2.73 (1H, dd, Jϭ14.1, 11.4 Hz),
(2H, dd, Jϭ27.2, 17.1 Hz), 2.97—2.69 (5H, m), 1.81 (6H, s), 1.35 (2H, 2.58 (3H, s), 1.84 (6H, s), 1.36 (2H, m), 1.31 (3H, d, Jϭ7.2 Hz), 1.09 (1H,
br m), 1.05 (1H, br m), 0.91 (1H, br m). FAB-MS m/z: 584 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-Gly-OMe·2AcOH (18) White powder
br m), 0.89 (1H, br m). FAB-MS m/z: 611 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-MeAla-OH·2AcOH (29) White powder
(531 mg, 74%): HPLC tR (min): 9.66. Rf1 0.45. Rf2 0.74. 1H-NMR (CD3OD) (373 mg, 51%): HPLC tR (min): 10.44. Rf1 0.45. Rf2 0.70. 1H-NMR
d: 7.16 (5H, m), 6.95 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.4 Hz), 4.58 (1H, (CD3OD) d: 7.12 (5H, m), 6.99 (2H, d, Jϭ8.1 Hz), 6.64 (2H, d, Jϭ8.4 Hz),
dd, Jϭ10.8, 4.5 Hz), 4.25 (1H, t, Jϭ7.5 Hz), 4.01 (1H, t, Jϭ7.2 Hz), 3.85 5.00 (1H, m), 4.37 (1H, m), 4.19 (1H, m), 3.05—2.64 (9H, m), 1.83 (6H, s),
(2H, dd, Jϭ30.1, 17.4 Hz), 3.62 (3H, s), 2.98—2.68 (5H, m), 1.81 (6H, s),
1.33 (2H, br m), 1.05 (1H, br m), 0.90 (1H, br m). FAB-MS m/z: 598
(MϩH)ϩ.
1.52 (2H, br m), 1.33 (1H, m), 1.24 (2H, d, Jϭ7.2 Hz), 1.07 (3H, d,
Jϭ7.2 Hz). FAB-MS m/z: 612 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-D-Ala-OH·2AcOH (30) White powder
Na-Amidino-Tyr-D-Arg-Phe-Gly-NH2·2AcOH (19) White powder (495 mg, 69%): HPLC tR (min): 9.54. Rf1 0.40. Rf2 0.68. 1H-NMR (CD3OD)
(499 mg, 71%): HPLC tR (min): 8.40. Rf1 0.38. Rf2 0.68. 1H-NMR (CD3OD) d: 7.15 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.7 Hz), 4.58 (1H,
d: 7.16 (5H, m), 6.95 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.4 Hz), 4.50 (1H, dd, Jϭ9.9, 5.1 Hz), 4.28 (1H, t, Jϭ7.1 Hz), 4.08 (2H, m), 3.14 (1H, dd,
dd, Jϭ10.7, 4.5 Hz), 4.26 (1H, t, Jϭ7.2 Hz), 3.98 (1H, t, Jϭ7.2 Hz), 3.75 Jϭ13.8, 5.1 Hz), 3.89 (3H, m), 2.82—2.72 (2H, m), 1.83 (6H, s), 1.40 (2H,
(2H, dd, Jϭ20.3, 16.8 Hz), 2.94—2.71 (5H, m), 1.82 (6H, s), 1.36 (2H, br m), 1.17 (3H, d, Jϭ6.9 Hz), 1.13 (1H, br m), 1.02 (1H, br m). FAB-MS
br m), 1.09 (1H, br m), 0.96 (1H, br m). FAB-MS m/z: 583 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-Gly-NHMe·2AcOH (20) White powder
m/z: 598 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-D-Ala-OMe·2AcOH (31) White powder
(437 mg, 61%): HPLC tR (min): 8.86. Rf1 0.41. Rf2 0.71. 1H-NMR (CD3OD) (476 mg, 65%): HPLC tR (min): 10.56. Rf1 0.51. Rf2 0.77. 1H-NMR
d: 7.16 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz), 4.48 (1H, (CD3OD) d: 7.15 (5H, m), 6.95 (2H, d, Jϭ8.1 Hz), 6.61 (2H, d, Jϭ6.9 Hz),
dd, Jϭ10.5, 4.8 Hz), 4.32 (1H, t, Jϭ7.2 Hz), 3.99 (1H, t, Jϭ7.5 Hz), 3.73 4.55 (1H, dd, Jϭ10.7, 5.0 Hz), 4.28 (2H, m), 3.98 (1H, t, Jϭ7.2 Hz), 3.60
(2H, dd, Jϭ30.5, 16.8 Hz), 2.96—2.75 (5H, m), 2.62 (3H, s), 1.82 (6H, s),
1.38 (2H, br m), 1.12 (1H, br m), 1.01 (1H, br m). FAB-MS m/z: 597
(MϩH)ϩ.
(3H, s), 3.17 (1H, m), 2.97—2.68 (5H, m), 1.81 (6H, s), 1.36 (2H, br m),
1.36 (2H, br m), 1.23 (3H, d, Jϭ6.9 Hz), 1.08 (1H, br m), 0.92 (1H, br m).
FAB-MS m/z: 612 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-Sar-OH·2AcOH (21) White powder
Na-Amidino-Tyr-D-Arg-Phe-D-Ala-NH2·2AcOH (32) White powder
(467 mg, 65%): HPLC tR (min): 8.49. Rf1 0.34. Rf2 0.63. 1H-NMR (CD3OD) (437 mg, 61%): HPLC tR (min): 9.04. Rf1 0.44. Rf2 0.72. 1H-NMR (CD3OD)
d: 7.11 (5H, m), 6.97 (2H, d, Jϭ8.7 Hz), 6.62 (2H, dd, Jϭ8.6, 2.1 Hz), 5.03 d: 7.13 (5H, m), 6.96 (2H, d, Jϭ8.1 Hz), 6.62 (2H, d, Jϭ8.4 Hz), 4.42 (1H,
(0.5H, dd, Jϭ8.1, 6.0 Hz), 4.92 (0.5H, dd, Jϭ8.9, 5.2 Hz), 4.27 (1H, dd, dd, Jϭ9.6, 6.0 Hz), 4.28 (1H, t, Jϭ7.1 Hz), 4.17 (1H, dd, Jϭ14.4, 7.2 Hz),
Jϭ15.3, 8.1 Hz), 4.18 (0.5H, dd, Jϭ8.1, 5.4 Hz), 4.13 (0.5H, dd, Jϭ7.2, 4.00 (1H, t, Jϭ7.1 Hz), 3.11 (1H, dd, Jϭ13.7, 6.2 Hz), 2.89 (3H, br m), 2.80
6.6 Hz), 4.02 (0.5H, d, Jϭ16.5 Hz), 3.81 (1H, d, Jϭ2.4 Hz), 3.49 (0.5H, d, (2H, m), 1.82 (6H, s), 1.41 (2H, br m), 1.15 (4H, d, Jϭ7.2 Hz), 1.04 (1H,
Jϭ16.5 Hz), 3.03—2.74 (6H, m), 2.89 (1.5H, s), 2.83 (1.5H, s), 1.82 (6H, s),
br m). FAB-MS m/z: 597 (MϩH)ϩ.
1.49—1.06 (4H, br m). FAB-MS m/z: 598 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-D-Ala-NHMe·2AcOH (33) White pow-
Na-Amidino-Tyr-
D-Arg-Phe-Sar-OMe·2AcOH (22) White powder (490
der (417 mg, 57%): HPLC tR (min): 9.40. Rf1 0.48. Rf2 0.73. 1H-NMR
1
mg, 67%): HPLC tR (min): 10.48. Rf1 0.41. Rf2 0.70. H-NMR (CD3OD) d: (CD3OD) d: 7.14 (5H, m), 6.95 (2H, d, Jϭ9.0 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
7.15 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz), 5.03 (0.5H, 4.41 (1H, dd, Jϭ9.6, 6.0 Hz), 4.29 (1H, t, Jϭ6.9 Hz), 4.15 (1H, dd, Jϭ14.3,
dd, Jϭ8.4, 6.3 Hz), 4.27 (1H, t, Jϭ7.8 Hz), 4.17—4.10 (1H, m), 3.99 (2H,
7.1 Hz), 3.99 (1H, t, Jϭ7.1 Hz), 3.11 (1H, dd, Jϭ13.5, 6.0 Hz), 2.90 (3H,
dd, Jϭ42.4, 17.1 Hz), 3.63 (1H s), 3.60 (2H, s), 3.04—2.73 (6H, m), 2.97 br m), 2.79 (2H, m), 2.60 (3H, s), 1.82 (6H, s), 1.42 (2H, br m), 1.12 (4H, d,
(2H, s), 2.83 (1H, s), 1.82 (6H, s), 1.47 (1H, br m), 1.33 (1H, br m), 1.12 Jϭ7.2 Hz), 1.04 (1H, br m). FAB-MS m/z: 611 (MϩH)ϩ.
(2H, br m). FAB-MS m/z: 612 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-D-MeAla-OH·2AcOH (34) White pow-
Na-Amidino-Tyr-D-Arg-Phe-EtGly-OH·2AcOH (23) White powder der (388 mg, 53%): HPLC tR (min): 10.28. Rf1 0.45. Rf2 0.66. 1H-NMR
(424 mg, 58%): HPLC tR (min): 9.99. Rf1 0.36. Rf2 0.65. 1H-NMR (CD3OD) (CD3OD) d: 7.13 (5H, m), 6.95 (2H, m), 6.64 (2H, m), 5.01 (1H, t,
d: 7.12 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.62 (2H, dd, Jϭ8.7, 2.1 Hz), Jϭ7.4 Hz), 4.26 (2H, m), 3.11—2.82 (6H, m), 2.77 (1H, s), 2.67 (2H, s),
5.01—4.85 (1H, m), 4.24 (1H, dd, Jϭ15.6, 7.8 Hz), 4.14 (1H, t, Jϭ6.6 Hz), 1.86 (6H, s), 1.40 (2H, br m), 1.33—1.18 (3H, m), 1.09 (2H, d, Jϭ7.2 Hz),
3.73 (1H, dd, Jϭ25.7, 18.0 Hz), 3.53—3.37 (2H, m), 3.12—2.72 (7H, m),
1.81 (6H, s), 1.32 (2H, br m), 1.15 (1H, br m), 1.07 (1H, br m), 0.96—0.88
(1H, br m). FAB-MS m/z: 612 (MϩH)ϩ.
0.98 (1H, d, Jϭ7.2 Hz). FAB-MS m/z: 612 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-D-MeAla-OMe·2AcOH (35) White pow-
der (418 mg, 56%): HPLC tR (min): 11.26. Rf1 0.54. Rf2 0.75. 1H-NMR
Na-Amidino-Tyr-D-Arg-Phe-(n-Pr)Gly-OH·2AcOH (24) White pow- (CD3OD) d: 7.15 (5H, m), 6.94 (2H, m), 6.63 (2H, m), 4.98 (1H, m), 4.70
der (403 mg, 54%): HPLC tR (min): 10.94. Rf1 0.42. Rf2 0.68. 1H-NMR (1H, dd, Jϭ14.7, 7.2 Hz), 4.28 (1H, t, Jϭ7.4 Hz), 4.18 (1H, dd, Jϭ8.5,
(CD3OD) d: 7.12 (5H, m), 6.97 (2H, d, Jϭ8.1 Hz), 6.62 (2H, dd, Jϭ8.4, 5.4 Hz), 3.60 (0.5H, s), 3.56 (2.5H, s), 2.97—2.90 (4H, m), 2.84—2.79 (2H,
2.7 Hz), 5.00—4.87 (1H, m), 4.27—3.96 (2H, m), 3.74 (1H, s), 3.49—3.25 m), 2.77 (2.5H, s), 2.69 (0.5H, s), 1.83 (6H, s), 1.52 (1H, br m), 1.36 (1H,
(1H, m), 3.06—2.75 (7H, m), 1.82 (6H, s), 1.70—1.00 (6H, m), 0.71 (3H, br m), 1.32 (1H, d, Jϭ6.6 Hz), 1.16 (4H, d, Jϭ7.2 Hz). FAB-MS m/z: 626
dd, Jϭ13.4, 7.2 Hz). FAB-MS m/z: 626 (MϩH)ϩ.
Na-Amidino-Tyr-
(MϩH)ϩ.
D
-Arg-Phe-Ala-OH·2AcOH (25) White powder (481
Na-Amidino-Tyr-D-Arg-Phe-GABA-OH·2AcOH (36) White powder
mg, 67%): HPLC tR (min): 9.19. Rf1 0.40. Rf2 0.68. H-NMR (CD3OD) d: (498 mg, 68%): HPLC tR (min): 9.26. Rf1 0.50. Rf2 0.70. 1H-NMR (CD3OD)
1
7.13 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.60 (2H, d, Jϭ8.4 Hz), 4.53 (1H, dd, d: 7.12 (5H, m), 6.98 (2H, d, Jϭ8.7 Hz), 6.62 (2H, d, Jϭ8.4 Hz), 4.49 (1H,
Jϭ11.1, 3.9 Hz), 4.26—4.15 (2H, m), 3.98 (1H, t, Jϭ7.0 Hz), 2.87—2.67
dd, Jϭ10.5, 4.2 Hz), 3.95 (1H, t, Jϭ7.1 Hz), 3.26 (1H, m), 3.08 (1H, m),
(5H, m), 1.84 (6H, s), 1.34 (2H, br m), 1.30 (3H, d, Jϭ7.2 Hz), 1.05 (1H, m), 2.97—2.65 (5H, m), 2.16 (1H, t, Jϭ6.6 Hz), 1.83 (6H, s), 1.72 (2H, m), 1.39
0.88 (1H, br m). FAB-MS m/z: 598 (MϩH)ϩ.
(2H, br m), 1.08 (1H, br m), 0.85 (1H, br m). FAB-MS m/z: 612 (MϩH)ϩ.
Na-Amidino-Tyr-
D
-Arg-Phe-Ala-OMe·2AcOH (26) White powder (505
a
–N
N -Amidino-Tyr-D-Arg-Phe
· 2AcOH (37) White powder
1
mg, 69%): HPLC tR (min): 10.22. Rf1 0.51. Rf2 0.75. H-NMR (CD3OD) d:
7.15 (5H, m), 6.96 (2H, d, Jϭ8.1 Hz), 6.61 (2H, d, Jϭ8.1 Hz), 4.57 (1H, dd,
Jϭ10.7, 4.2 Hz), 4.35—4.24 (2H, m), 4.01 (1H, t, Jϭ7.5 Hz), 3.59 (3H, s),
2.96—2.67 (5H, m), 1.82 (6H, s), 1.37 (2H, br m), 1.32 (3H, d, Jϭ7.2 Hz),
1.06 (1H, br m), 0.97 (1H, br m). FAB-MS m/z: 612 (MϩH)ϩ.
H
CO2H
(414 mg, 54%): HPLC tR (min): 12.45. Rf10.58. Rf2 0.80.1H-NMR (CD3OD)
d: 8.48 (1H, t, Jϭ9.2 Hz), 7.93 (1H, m), 7.30 (1H, m), 7.15 (6H, m), 6.98
(2H, t, Jϭ8.6 Hz), 6.61 (2H, t, Jϭ7.8 Hz), 4.66 (1H, m), 4.37 (1H, m), 4.23
(1H, dt, Jϭ24.0, 6.6 Hz), 3.30 (1H, m), 3.05 (1H, m), 2.92 (3H, m), 2.84—
2.72 (1H, m), 1.87 (1H, br m), 1.80 (6H, s), 1.59 (2H, br m), 1.15 (2H, br m).
FAB-MS m/z: 646 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-Ala-NH2·2AcOH (27) White powder
(459 mg, 64%): HPLC tR (min): 8.83. Rf1 0.43. Rf2 0.72. 1H-NMR (CD3OD)
d: 7.16 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.4 Hz), 4.50 (1H,
dd, Jϭ11.0, 4.1 Hz), 4.34 (1H, dd, Jϭ14.4, 7.2 Hz), 4.25 (1H, t, Jϭ7.1 Hz),
Na-Amidino-Tyr-
D-Arg-Phe-Leu-OH·2AcOH (38) White powder (509

778
Vol. 50, No. 6
1
mg, 67%): HPLC tR (min): 11.41. Rf1 0.50. Rf2 0.77. H-NMR (CD3OD) d:
7.15 (5H, m), 6.98 (2H, d, Jϭ8.1 Hz), 6.61 (2H, d, Jϭ8.4 Hz), 4.48 (1H, dd,
Jϭ11.4, 3.6 Hz), 4.28 (1H, t, Jϭ6.9 Hz), 4.13 (1H, t, Jϭ7.2 Hz), 3.89 (1H, t,
Jϭ7.4 Hz), 3.31 (1H, dd, Jϭ14.3, 3.5 Hz), 2.79 (4H, m), 2.64 (1H, dd,
Jϭ13.8, 11.7 Hz), 1.81 (6H, s), 1.58 (3H, m), 1.26 (2H, br m), 0.97 (1H,
br m), 0.85 (6H, dd, Jϭ6.0, 2.1 Hz), 0.75 (1H, br m). FAB-MS m/z: 640
(MϩH)ϩ.
(6H, s), 1.32 (2H, br m), 1.02 (4H, dd, Jϭ7.1, 4.4 Hz), 0.80 (1H, br m), 0.71
(1H, br m). FAB-MS m/z: 612 (MϩH)ϩ.
a
N -amidino-Tyr-D-Arg-Phe-(R) Abu-OH·2AcOH (48) White pow-
der (505 mg, 69%): HPLC tR (min): 10.24. Rf1 0.42. Rf2 0.70. 1H-NMR
(CD3OD) d: 7.13 (5H, m), 6.97 (2H, d, Jϭ8.7 Hz), 6.61 (2H, d, Jϭ8.4 Hz),
4.65 (1H, dd, Jϭ9.6, 5.4 Hz), 4.29 (1H, t, Jϭ7.2 Hz), 4.09 (2H, dd, Jϭ14.9,
7.4 Hz), 3.13 (1H, dd, Jϭ14.0, 5.6 Hz), 2.90 (3H, m), 2.83—2.73 (2H, m),
1.83 (6H, s), 1.70 (6H, m), 1.57 (1H, m), 1.41 (2H, br m), 1.11 (1H, br m),
a
Na-Amidino-Tyr-D-Arg-Phe-Nva-OH·2AcOH (39) White powder
(530 mg, 71%): HPLC tR (min): 10.66. Rf1 0.49. Rf2 0.77. 1H-NMR
(CD3OD) d: 7.13 (5H, m), 6.96 (2H, d, Jϭ8.1 Hz), 6.60 (2H, d, Jϭ8.1 Hz),
4.52 (1H, dd, Jϭ11.1, 3.6 Hz), 4.21 (2H, t, Jϭ6.3 Hz), 3.96 (1H, t, Jϭ7.2
Hz), 3.27 (1H, dd, Jϭ14.3, 3.8 Hz), 2.83 (4H, t, Jϭ6.8 Hz), 2.71 (1H, dd,
Jϭ14.0, 11.3 Hz), 1.84 (6H, s), 1.78—1.55 (2H, m), 1.34—1.27 (4H, m),
1.03 (1H, br m), 0.85 (3H, t, Jϭ7.2 Hz), 0.80 (1H, br m). FAB-MS m/z: 626
(MϩH)ϩ.
1.02 (1H, br m), 0.67 (3H, t, Jϭ7.2 Hz). FAB-MS m/z: 612 (MϩH)ϩ.
a
N -Amidino-Tyr-D-Arg-Phe-(S) Abu-OH·2AcOH (49) White pow-
der (476 mg, 65%): HPLC tR (min): 9.65. Rf1 0.44. Rf2 0.79. 1H-NMR
(CD3OD) d: 7.14 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.60 (2H, d, Jϭ8.4 Hz),
4.51 (1H, dd, Jϭ11.3, 3.8 Hz), 4.18 (2H, m), 3.99 (1H, t, Jϭ7.2 Hz), 3.28
(1H, dd, Jϭ14.1, 3.9 Hz), 2.83 (4H, m), 2.71 (1H, dd, Jϭ14.1, 11.4 Hz),
1.81 (6H, s), 1.35 (2H, m), 1.78—1.63 (2H, m), 1.33 (2H, br m), 1.02 (1H,
br m), 0.84 (1H, t, Jϭ7.4 Hz). FAB-MS m/z: 612 (MϩH)ϩ.
a
Na-Amidino-Tyr-
D-Arg-Phe-Phe-OH·2AcOH (40) White powder (508
1
Na-Amidino-Tyr-
D
-Arg-Phe-(R,S) Abu-OH·2AcOH (50) White pow-
b
mg, 64%): HPLC tR (min): 11.91. Rf1 0.47. Rf2 0.76. H-NMR (CD3OD) d:
7.14 (10H, m), 6.98 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.4 Hz), 4.42 (2H,
m), 4.16 (1H, t, Jϭ7.5 Hz), 4.00 (1H, t, Jϭ7.1 Hz), 3.15 (2H, m), 3.00—
2.71 (5H, m), 2.54 (1H, t, Jϭ12.9 Hz), 1.81 (6H, s), 1.35 (1H, br m), 1.24
(1H, br m), 0.99 (1H, br m), 0.80 (1H, br m). FAB-MS m/z: 674 (MϩH)ϩ.
1
der (520 mg, 71%): HPLC tR (min): 9.29, 9.60. Rf1 0.44. Rf2 0.74. H-NMR
(CD3OD) d: 7.13 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.61 (2H, dd, Jϭ8.4,
2.4 Hz), 4.45 (1H, m), 4.27 (1H, br m), 4.12 (1H, br m), 3.99 (1H, br m),
3.20 (1H, br m), 2.96—2.67 (5H, br m), 2.28 (2H, m), 1.84 (6H, s), 1.36
(2H, br m), 1.13 (2H, d, Jϭ6.6 Hz), 1.07 (1H, br m), 1.00 (1H, d, Jϭ6.6 Hz),
0.86 (1H, br m). FAB-MS m/z: 612 (MϩH)ϩ.
Na-Amidino-Tyr-
D-Arg-Phe-Pro-OH·2AcOH (41) White powder (424
1
mg, 57%): HPLC tR (min): 9.94. Rf1 0.40. Rf2 0.68. H-NMR (CD3OD) d:
7.12 (5H, m), 6.98 (2H, t, Jϭ7.7 Hz), 6.62 (2H, dd, Jϭ8.7, 6.6 Hz), 4.70
(1H, dd, Jϭ8.3, 6.3 Hz), 4.29 (1H, dd, Jϭ8.7, 6.6 Hz), 4.22 (1H, t,
Jϭ6.6 Hz), 4.13 (1H, br m), 3.65 (1H, br m), 3.45 (1H, br m), 2.98—2.71
(5H, m), 2.06—1.89 (2H, br m), 1.85 (6H, s), 1.67—1.45 (3H, br m), 1.23
(1H, br m), 1.11 (1H, br m). FAB-MS m/z: 624 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-Arg-NH2·3AcOH (51) White powder
(543 mg, 63%): HPLC tR (min): 8.21. Rf1 0.25. Rf2 0.70. 1H-NMR (CD3OD)
d: 7.16 (5H, m), 6.96 (2H, d, Jϭ8.7 Hz), 6.61 (2H, d, Jϭ8.7 Hz), 4.50 (1H,
dd, Jϭ11.3, 4.4 Hz), 4.33 (1H, dd, Jϭ9.2, 5.0 Hz), 4.25 (1H, t, Jϭ7.1 Hz),
3.88 (1H, t, Jϭ7.4 Hz), 3.26 (1H, m), 3.12 (2H, t, Jϭ7.4 Hz), 2.85 (4H, t,
Jϭ7.1 Hz), 2.75 (1H, dd, Jϭ14.1, 11.1 Hz), 1.81 (6H, s), 1.72 (1H, br m),
1.58 (1H, br m), 1.34 (1H, br m), 1.07 (1H, br m), 0.88 (1H, br m). FAB-MS
m/z: 682 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-Pro-OMe·2AcOH (42) White powder
(440 mg, 58%): HPLC tR (min): 11.22. Rf1 0.49. Rf2 0.78. 1H-NMR
(CD3OD) d: 7.17 (5H, m), 6.97 (2H, d, Jϭ8.0 Hz), 6.63 (2H, t, Jϭ6.8 Hz),
4.30 (2H, m), 4.12 (1H, dd, Jϭ8.4, 5.4 Hz), 3.63 (1H, m), 3.61 (3H, s), 3.36
(1H, br m), 3.06—2.73 (6H, m), 2.11 (1H, br m), 1.82 (6H, s), 1.60 (1H,
br m), 1.42 (1H, br m), 1.33 (1H, br m), 1.12 (1H, br m). FAB-MS m/z: 638
(MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-D-Arg-NH2·3AcOH (52) White powder
(526 mg, 61%): HPLC tR (min): 8.24. Rf1 0.22. Rf2 0.67. 1H-NMR (CD3OD)
d: 7.15 (5H, m), 6.96 (2H, d, Jϭ8.7 Hz), 6.62 (2H, d, Jϭ8.4 Hz), 4.44 (1H,
dd, Jϭ8.9, 7.1 Hz), 4.29 (1H, t, Jϭ6.8 Hz), 4.16 (1H, dd, Jϭ9.6, 4.5 Hz),
4.05 (1H, t, Jϭ7.1 Hz), 3.08 (1H, m), 2.99—2.79 (7H, m), 1.81 (9H, s), 1.74
(1H, br m), 1.44 (3H, br m), 1.22 (1H, br m), 1.06 (1H, br m). FAB-MS m/z:
682 (MϩH)ϩ.
Na-Amidino-Tyr-
D-Arg-Phe
· 2AcOH (43) White pow-
—N
CO2H
Na-Amidino-Tyr-D-Arg-Phe-Asp-OH·2AcOH (53) White powder
(480 mg, 63%): HPLC tR (min): 8.65. Rf1 0.31. Rf2 0.57. 1H-NMR (CD3OD)
d: 7.13 (5H, m), 6.98 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.4 Hz), 4.56 (1H,
dd, Jϭ11.0, 4.1 Hz), 4.46 (1H, t, Jϭ5.7 Hz), 4.24 (1H, t, Jϭ7.4 Hz), 4.00
(1H, t, Jϭ6.9 Hz), 2.97—2.76 (4H, m), 2.70 (3H, br m), 1.83 (6H, s), 1.38
(2H, br m), 1.07 (1H, br m), 0.88 (1H, br m). FAB-MS m/z: 642 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-Asu-OH·2AcOH (54) White powder
(556 mg, 68%): HPLC tR (min): 10.26. Rf1 0.49. Rf2 0.72. 1H-NMR
(CD3OD) d: 7.12 (5H, m), 6.91 (2H, d, Jϭ10.2 Hz), 6.60 (2H, d, Jϭ8.7 Hz),
4.47 (1H, dd, Jϭ11.1, 3.9 Hz), 4.20 (1H, t, Jϭ7.4 Hz), 4.14 (1H, dd, Jϭ7.1,
4.7 Hz), 3.98 (1H, t, Jϭ7.2 Hz), 3.21 (1H, dd, Jϭ11.1, 4.2 Hz), 2.84—2.64
(6H, m), 2.11 (2H, t, Jϭ7.2 Hz), 1.82 (6H, s), 1.70 (2H, br m), 1.47 (2H,
br m), 1.36 (2H, br m), 1.24 (4H, br m), 1.04 (1H, br m), 0.92 (1H, br m).
FAB-MS m/z: 698 (MϩH)ϩ.
der (477 mg, 63%): HPLC tR (min): 10.39. Rf1 0.48. Rf2 0.73. 1H-NMR
(CD3OD) d: 7.12 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.61 (2H, dd, Jϭ8.0, 3.5
Hz), 4.51 (1H, br m), 4.26 (1H, br m), 4.10 (1H, m), 3.73 (1H, br m), 3.53
(1H, br m), 2.94 (7H, br m), 2.49 (1H, br m), 2.04 (2H, br m), 1.81 (6H, s),
1.53—1.14 (7H, br m). FAB-MS m/z: 638 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-(R,S)bPro-OH·2AcOH (44) White pow-
der (491 mg, 66%): HPLC tR (min): 9.84. Rf1 0.43. Rf2 0.70. 1H-NMR
(CD3OD) d: 7.13 (5H, m), 6.97 (2H, d, Jϭ9.0 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
4.70 (1H, m), 4.24 (1H, m), 4.13 (1H, m), 3.72 (1H, m), 3.56 (2H, m),
3.44—3.25 (2H, m), 2.92 (4H, br m), 2.81 (3H, br m), 2.68 (1H, br m),
2.13—1.93 (3H, m), 1.86 (6H, s), 1.44 (2H, br m), 1.13 (2H, br m). FAB-MS
m/z: 624 (MϩH)ϩ.
a
—N
N -Amidino-Tyr-D-Arg-Phe
· 2AcOH (45) White powder
Na-Amidino-Tyr-
D-Arg-Phe-Taurine·2AcOH (55) White powder (415
1
mg, 55%): HPLC tR (min): 8.27. Rf1 0.39. Rf2 0.69. H-NMR (CD3OD) d:
7.11 (5H, m), 6.96 (2H, d, Jϭ8.1 Hz), 6.60 (2H, d, Jϭ8.4 Hz), 4.45 (1H, dd,
Jϭ10.8, 4.2 Hz), 4.29 (1H, t, Jϭ6.9 Hz), 3.93 (1H, t, Jϭ6.8 Hz), 3.49 (2H, t,
Jϭ6.5 Hz), 2.86 (6H, br m), 2.70 (1H, m), 1.81 (6H, s), 1.32 (2H, br m), 1.03
(1H, br m), 0.90 (1H, br m). FAB-MS m/z: 634 (MϩH)ϩ.
CO2H
(523 mg, 69%): HPLC tR (min): 10.12. Rf1 0.46. Rf2 0.70. 1H-NMR
(CD3OD) d: 7.12 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.4 Hz),
4.50 (1H, dd, Jϭ10.8, 4.5 Hz), 4.27 (1H, m), 4.10 (1H, m), 3.36 (1H, t, Jϭ
5.9 Hz), 3.40—3.16 (3H, m), 2.95 (1H, d, Jϭ15.1 Hz), 2.33 (1H, dd,
Jϭ15.1, 8.4 Hz), 2.05—1.65 (4H, m), 1.81 (6H, s), 1.35 (2H, m), 1.08 (1H,
br m), 0.88 (1H, br m). FAB-MS m/z: 638 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-NHCH2SO3H·2AcOH (56) White pow-
der (429 mg, 58%): HPLC tR (min): 8.01. Rf1 0.35. Rf2 0.64. 1H-NMR
(CD3OD) d: 7.13 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
4.55 (3H, m), 4.26 (1H, t, Jϭ7.5 Hz), 2.86 (6H, br m), 2.73 (1H, m), 1.82
(6H, s), 1.35 (2H, br m), 1.00 (1H, br m), 0.92 (1H, br m). FAB-MS m/z: 620
(MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-aAib-OH·2AcOH (46) White powder
(527 mg, 72%): HPLC tR (min): 9.97. Rf1 0.45. Rf2 0.73. 1H-NMR (CD3OD)
d: 7.13 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.7 Hz), 4.42 (1H,
dd, Jϭ10.2, 4.5 Hz), 4.24 (1H, dd, Jϭ8.1, 6.3 Hz), 4.24 (1H, t, Jϭ7.1 Hz),
3.15 (1H, dd, Jϭ14.1, 4.5 Hz), 2.97—2.87 (3H, m), 2.80—2.69 (2H, m),
1.81 (6H, s), 1.49 (2H, br m), 1.39 (6H, s), 1.13 (1H, br m), 1.05 (1H, br m).
FAB-MS m/z: 612 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-MebAla-OMe·2AcOH (57) White pow-
der (530 mg, 71%): HPLC tR (min): 10.89. Rf1 0.48. Rf2 0.77. 1H-NMR
(CD3OD) d: 7.13 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
4.95 (1H, dt, Jϭ27.9, 7.4 Hz), 4.50 (1H, br s), 4.25 (1H, t, Jϭ7.1 Hz), 4.17
(1H, dd, Jϭ8.1, 6.0 Hz), 3.55 (3H, s), 3.50—3.33 (2H, m), 2.92 (4H, m),
2.82 (3H, m), 2.75 (2H, m), 2.38 (2H, t, Jϭ7.2 Hz), 1.81 (6H, s), 1.46 (1H,
br m), 1.37 (1H, br m), 1.16 (2H, br m). FAB-MS m/z: 626 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-MebAla-OEt·2AcOH (58) White pow-
der (555 mg, 73%): HPLC tR (min): 11.77. Rf1 0.51. Rf2 0.77. 1H-NMR
(CD3OD) d: 7.13 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
Na-Amidino-Tyr-D-Arg-Phe-(R,S)bAib-OH·2AcOH (47) White pow-
der (512 mg, 70%): HPLC tR (min): 9.50. Rf1 0.47. Rf2 0.75. 1H-NMR
(CD3OD) d: 7.14 (5H, m), 6.98 (2H, dd, Jϭ8.7, 2.7 Hz), 6.61 (2H, dd,
Jϭ8.4, 1.5 Hz), 4.52 (2H, br m), 4.48 (1H, t, Jϭ4.1 Hz), 4.21 (1H, m), 3.93
(1H, dt, Jϭ38.5, 7.4 Hz), 3.32 (1H, dd, Jϭ13.8, 3.9 Hz), 3.10 (1H, m), 2.98
(1H, m), 2.82 (3H, m), 2.67 (1H, dd, Jϭ24.9, 12.9 Hz), 2.38 (1H, br m), 1.81

June 2002
779
4.95 (1H, dt, Jϭ27.3, 7.5 Hz), 4.53 (1H, br s), 4.26 (1H, t, Jϭ7.2 Hz), 4.17 3.00 (2H, t, Jϭ7.2 Hz), 2.94 (4H, m), 2.80 (4H, m), 2.76 (1H, s), 2.25 (2H,
(1H, dd, Jϭ8.3, 5.3 Hz), 4.01 (2H, dd, Jϭ14.3, 7.1 Hz), 3.55—3.00 (3H, m),
2.91 (4H, m), 2.82 (3H, m), 2.75 (2H, m), 2.37 (2H, t, Jϭ7.4 Hz), 1.81 (6H,
s), 1.47 (1H, br m), 1.34 (1H, br m), 1.17 (2H, br m), 1.14 (3H, dt, Jϭ2.4,
7.2 Hz). FAB-MS m/z: 640 (MϩH)ϩ.
m), 1.82 (6H, s), 1.46—1.34 (4H, m), 1.17 (2H, m), 0.80 (3H, dt, Jϭ3.0,
7.4 Hz). FAB-MS m/z: 653 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-bAla-OH·2AcOH (69) White powder
(517 mg, 72%): HPLC tR (min): 9.05. Rf1 0.43. Rf2 0.66. 1H-NMR (CD3OD)
d: 7.13 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.7 Hz), 4.49 (1H,
Na-Amidino-Tyr-
-Arg-Phe-MebAla-O(n-Pr)·2AcOH (59) White pow-
D
der (542 mg, 70%): HPLC tR (min): 13.45. Rf1 0.54. Rf2 0.79. 1H-NMR dd, Jϭ11.1, 4.2 Hz), 4.23 (2H, t Jϭ7.2 Hz), 3.89 (1H, t, Jϭ7.2 Hz), 3.38
(CD3OD) d: 7.15 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz), (2H, m), 3.28 (2H, dd, Jϭ14.1, 4.2 Hz), 2.94 (1H, m), 2.84 (3H, m), 2.67
4.94 (1H, dt, Jϭ31.3, 7.5 Hz), 4.31 (1H, m), 4.13 (1H, m), 3.47 (1H, m), (1H, dd, Jϭ14.0, 11.3 Hz), 2.28 (2H, t, Jϭ6.2 Hz), 1.82 (6H, s), 1.33 (2H,
3.38 (1H, m), 3.00 (2H, t, Jϭ7.2 Hz), 2.91 (4H, m), 2.82 (3H, m), 2.76 (2H,
m), 2.25 (2H, m), 1.82 (6H, s), 1.41 (4H, m), 1.17 (2H, m), 0.80 (3H, dt,
Jϭ3.0, 7.0 Hz). FAB-MS m/z: 654 (MϩH)ϩ.
br m), 1.06 (1H, br m), 0.79 (1H, br m). FAB-MS m/z: 598 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-bAla-OMe·2AcOH (70) White powder
(534 mg, 73%): HPLC tR (min): 10.05. Rf1 0.50. Rf2 0.75. 1H-NMR
Na-Amidino-Tyr-D-Arg-Phe-MebAla-O(n-Hex)·2AcOH (60) White (CD3OD) d: 7.14 (5H, m), 6.95 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
1
powder (563 mg, 69%): HPLC tR (min): 15.74. Rf1 0.58. Rf2 0.80. H-NMR 4.46 (1H, dd, Jϭ10.5, 4.8 Hz), 4.26 (2H, t, Jϭ7.1 Hz), 3.97 (1H, t,
(CD3OD) d: 7.13 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.7 Hz), Jϭ7.2 Hz), 3.54 (3H, s), 3.34 (2H, dt, Jϭ2.4, 6.9 Hz), 3.17 (1H, m), 2.95—
4.94 (1H, dt, Jϭ27.6, 7.5 Hz), 4.28 (1H, t, Jϭ7.2 Hz), 4.15 (1H, dd, Jϭ8.4,
2.76 (4H, m), 2.67 (1H, dd, Jϭ14.0, 11.0 Hz), 2.42 (2H, t, Jϭ6.9 Hz), 1.81
5.1 Hz), 3.96 (2H, t, Jϭ6.6 Hz), 3.53 (1H, m), 3.32 (1H, m), 2.91 (4H, m), (6H, s), 1.36 (2H, br m), 1.08 (1H, br m), 0.90 (1H, br m). FAB-MS m/z: 612
2.82 (3H, m), 2.75 (2H, m), 2.37 (2H, t, Jϭ7.2 Hz), 1.82 (6H, s), 1.52 (3H, (MϩH)ϩ.
br m), 1.37 (1H, br m), 1.23 (8H, m), 0.81 (3H, t, Jϭ6.8 Hz). FAB-MS m/z :
696 (MϩH)ϩ.
Na-Amidino-Tyr-D-Arg-Phe-EtbAla-OH·2AcOH (71) White powder
(462 mg, 62%): HPLC tR (min): 10.84. Rf1 0.44. Rf2 0.70. 1H-NMR
(CD3OD) d: 7.14 (5H, m), 6.95 (2H, d, Jϭ8.7 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
Na-Amidino-Tyr-
-Arg-Phe-MebAla-OPOM·2AcOH (61) White pow-
D
der (524 mg, 62%): HPLC tR (min): 14.95. Rf1 0.57. Rf2 0.63. 1H-NMR 4.96 (1H, m), 4.30 (1H, t, Jϭ6.6 Hz), 4.23 (1H, dd, Jϭ8.6, 4.9 Hz), 3.66—
(CD3OD) d: 7.13 (5H, m), 6.97 (2H, dd, Jϭ8.4, 4.5 Hz), 6.62 (2H, dd, 3.37 (2H, m), 3.05 (4H, m), 2.95—2.87 (2H, m), 2.61—2.30 (2H, m), 1.82
Jϭ8.7, 1.8 Hz), 5.75 (2H, s), 5.00 (1H, dt, Jϭ61.3, 7.2 Hz), 4.26 (1H, dt, (6H, s), 1.57 (1H, br m), 1.48 (1H, br m), 1.25 (1H, br m), 1.05 (3H, m).
Jϭ24.9, 7.2 Hz), 4.17 (1H, dd, Jϭ13.2, 6.9 Hz), 3.75 (1H, m), 3.57 (1H, m),
FAB-MS m/z: 626 (MϩH)ϩ.
2.94 (4H, m), 2.80 (3H, m), 2.73 (2H, m), 2.24 (2H, m), 1.83 (6H, s), 1.50
Na-Amidino-Tyr-D-Arg-Phe-EtbAla-OMe·2AcOH (72) White pow-
(1H, br m), 1.40 (1H, br m), 1.23 (9H, s), 1.11 (2H, br m). FAB-MS m/z: 726 der (494 mg, 65%): HPLC tR (min): 11.65. Rf1 0.49. Rf2 0.72. 1H-NMR
(MϩH)ϩ.
(CD3OD) d: 7.15 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
Na-Amidino-Tyr-D-Arg-Phe-MebAla-NH2·2AcOH (62) White pow- 4.96 (1H, m), 4.35 (1H, t, Jϭ6.6 Hz), 4.26 (1H, dd, Jϭ8.4, 5.2 Hz), 3.65
der (541 mg, 74%): HPLC tR (min): 9.10. Rf1 0.30. Rf2 0.64. 1H-NMR (3H, s), 3.62—3.40 (2H, m), 3.03 (4H, m), 2.93—2.85 (2H, m), 2.57—2.39
(CD3OD) d: 7.14 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.61 (2H, d, Jϭ8.4 Hz), (2H, m), 1.81 (6H, s), 1.55 (1H, br m), 1.47 (1H, br m), 1.25 (1H, br m), 1.04
4.90 (1H, dt, Jϭ33.1, 7.2 Hz), 4.50 (1H, br s), 4.19 (1H, m), 4.05 (1H, m),
3.46—3.25 (2H, m), 2.92 (4H, m), 2.81 (3H, m), 2.20 (2H, m), 1.81 (6H, s),
1.44 (1H, br m), 1.35 (1H, br m), 1.03 (2H, br m). FAB-MS m/z: 611
(MϩH)ϩ.
(3H, m). FAB-MS m/z: 640 (MϩH)ϩ.
Na-Amidino-Tyr-
D
-Arg-Phe-(n-Pr)bAla-OH·2AcOH (73) White pow-
der (479 mg, 63%): HPLC tR (min): 11.18. Rf1 0.51. Rf2 0.74. 1H-NMR
(CD3OD) d: 7.14 (5H, m), 6.97 (2H, dd, Jϭ8.4, 3.6 Hz), 6.62 (2H, d,
Jϭ7.8 Hz), 4.96 (1H, dt, Jϭ60.7, 7.5 Hz), 4.27 (1H, dt, Jϭ21.3, 7.4 Hz),
Na-Amidino-Tyr-
-Arg-Phe-MebAla-NHMe·2AcOH (63) White pow-
D
der (544 mg, 73%): HPLC tR (min): 9.49. Rf1 0.32. Rf2 0.67. 1H-NMR 4.16 (1H, br s), 3.48 (1H, m), 3.32 (2H, m), 2.93 (4H, m), 2.87—2.72 (3H,
(CD3OD) d: 7.16 (5H, m), 6.96 (2H, d, Jϭ8.1 Hz), 6.62 (2H, d, Jϭ8.4 Hz), m), 2.28 (2H, m), 1.83 (6H, s), 1.38 (3H, br m), 1.15 (3H, br m), 0.70 (3H,
4.94 (1H, dt, Jϭ31.6, 7.4 Hz), 4.52 (1H, br s), 4.27 (1H, m), 4.15 (1H, m),
dt, Jϭ9.9, 7.4 Hz). FAB-MS m/z: 640 (MϩH)ϩ.
3.52—3.32 (2H, m), 2.92 (4H, m), 2.80 (3H, m), 2.59 (3H, s), 2.24 (2H, m),
Na-Amidino-Tyr-D-Arg-Phe-(Bn)bAla-OH·2AcOH (74) White pow-
1.82 (6H, s), 1.46 (1H, br m), 1.37 (1H, br m), 1.16 (2H, br m). FAB-MS der (460 mg, 57%): HPLC tR (min): 12.42. Rf1 0.53. Rf2 0.75. 1H-NMR
m/z: 625 (MϩH)ϩ.
Na-Amidino-Tyr-
(CD3OD) d: 7.14 (7H, m), 6.96 (5H, m), 6.63 (2H, d, Jϭ8.1 Hz), 5.02 (1H,
dt, Jϭ93.8, 7.2 Hz), 4.52 (1H, br s), 4.43—4.30 (2H, m), 4.21 (2H, m), 3.67
D
-Arg-Phe-MebAla-NHEt·2AcOH (64) White pow-
der (539 mg, 71%): HPLC tR (min): 10.00. Rf1 0.41. Rf2 0.70. 1H-NMR (1H, dm, Jϭ110 Hz), 3.04—2.89 (3H, m), 2.84—2.78 (2H, m), 2.60 (1H,
(CD3OD) d: 7.16 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz), dm, Jϭ96.2 Hz), 2.29 (1H, m), 1.82 (6H, s), 1.47 (1H, br m), 1.35 (1H,
4.94 (1H, dt, Jϭ31.0, 7.5 Hz), 4.53 (1H, br s), 4.28 (1H, m), 4.14 (1H, m),
3.07 (2H, dd, Jϭ14.6, 7.4 Hz), 2.92 (4H, m), 2.82 (3H, m), 2.76 (2H, m),
br m), 1.12 (2H, br m). FAB-MS m/z: 688 (MϩH)ϩ.
Antinociceptive Assays Male mice of ddY strain weighing 10—32 g
2.24 (2H, m), 1.81 (6H, s), 1.46 (1H, br m), 1.37 (1H, br m), 1.16 (2H, br m), were used in the experiment. They were purchased from Japan SLC Inc.
1.00 (3H, dt, Jϭ2.7, 7.2 Hz). FAB-MS m/z: 639 (MϩH)ϩ.
Na-Amidino-Tyr-
(Shizuoka, Japan) and housed in cages of 5—6 animals matched for weight
and placed in a colony room. Animals were given standard food (MM-3,
D
-Arg-Phe-MebAla-N(Me)2·2AcOH (65) White pow-
der (501 mg, 66%): HPLC tR (min): 10.20. Rf1 0.33. Rf2 0.67. 1H-NMR Japan SLC Inc.) and tap water ad libitum in an air-conditioned room at
(CD3OD) d: 7.14 (5H, m), 6.96 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz), 23Ϯ2 °C and 55Ϯ20% relative humidity with a standard 12-h light–dark
4.92 (1H, m), 4.52 (1H, br s), 4.25 (1H, t, Jϭ7.2 Hz), 4.17 (1H, dd, Jϭ8.0,
5.3 Hz), 3.50 (1H, m), 3.33 (1H, m), 2.95 (3H, m), 2.92 (3H, s), 2.86 (2H, s),
cycle (lights on 6 : 00—18 : 00). Antinociception was examined by the tail
pressure test according to the reported method by Sakurada et al.34) with a
2.83 (2H, s), 2.80 (3H, s), 2.76 (2H, s), 2.40 (1H, m), 1.81 (6H, s), 1.47 (1H, slight modification. Thus, mechanical pressure was applied to the base of the
br m), 1.34 (1H, br m), 1.17 (1H, br m). FAB-MS m/z: 639 (MϩH)ϩ.
Na-Amidino-Tyr-
tail at a rate of 32 g/s using an automated tail-pressure unit (Ugo Basile,
Italy). Biting or struggling behavior in mice was used as an indication of re-
D
-Arg-Phe-MebAla-N(Et)2·2AcOH (66) White pow-
der (488 mg, 62%): HPLC tR (min): 11.58. Rf1 0.43. Rf2 0.72. 1H-NMR sponse threshold and only mice responding behaviorally to a tail-pressure of
(CD3OD) d: 7.14 (5H, m), 6.96 (2H, d, Jϭ8.7 Hz), 6.63 (2H, d, Jϭ8.7 Hz), 100 to 300 g were selected for this experiment. The trials were terminated at
4.93 (1H, m), 4.51 (1H, br s), 4.26 (1H, t, Jϭ7.2 Hz), 4.17 (1H, dd, Jϭ8.3, the level of 500 g to prevent tail tissue damage. The meanϮS.E.M. of the
5.3 Hz), 3.52 (1H, m), 3.26 (1H, m), 2.94 (4H, m), 2.83 (4H, s), 2.76 (1H, s),
pressure level was plotted. To obtain the response curve, the dose was plot-
2.38 (2H, m), 1.81 (6H, s), 1.47 (1H, br m), 1.37 (1H, br m), 1.17 (2H, br m), ted against percentage of maximum possible effect (%MPE) as follows:
1.04 (6H, dt, Jϭ24.6, 7.1 Hz), . FAB-MS m/z: 667 (MϩH)ϩ.
Na-Amidino-Tyr-
-Arg-Phe-MebAla-NH(n-Hex)·2AcOH (67) White
powder (546 mg, 67%): HPLC tR (min): 13.83. Rf1 0.55. Rf2 0.73. H-NMR tration (g). The peptides administered were dissolved in saline solution
(CD3OD) d: 7.16 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz), (Fuso Chemical Industries, Osaka, Japan). Saline solution was used as the
4.95 (1H, dt, Jϭ33.4, 7.4 Hz), 4.29 (1H, m), 4.14 (1H, m), 3.52 (1H, m), control. ED50 values were obtained by the method of Litchfield and
%MPEϭ(P2ϪP1/500ϪP1)ϫ100, where P1 is the response pressure before
drug administration (g) and P2 is the response pressure after drug adminis-
D
1
3.35 (1H, m), 3.04 (2H, t, Jϭ6.5 Hz), 2.92 (4H, m), 2.81 (3H, m), 2.76 (2H,
Wilcoxon35) to compare their antinociceptive activities. These values were
m), 2.24 (2H, m), 1.82 (6H, s), 1.37 (4H, m), 1.21 (8H, br s), 0.80 (3H, t, calculated from the values obtained at the time of peak effect after either
Jϭ6.2 Hz). FAB-MS m/z: 695 (MϩH)ϩ.
peptide or morphine administration.
Na-Amidino-Tyr-D-Arg-Phe-MebAla-NH(n-Pr)·2AcOH (68) White
1
powder (541 mg, 70%): HPLC tR (min): 10.80. Rf1 0.47. Rf2 0.70. H-NMR References and Notes
(CD3OD) d: 7.15 (5H, m), 6.97 (2H, d, Jϭ8.4 Hz), 6.62 (2H, d, Jϭ8.4 Hz),
4.94 (1H, dt, Jϭ31.3, 7.5 Hz), 4.31 (1H, m), 4.13 (1H, m), 3.38 (1H, m),
1) Symbols and abbreviations are in accordance with recommendations
of IUPAC-IUB Joint Commission on Biochemical Nomenclature:

780
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