Identification of arginine analogues as antagonists and agonists for the melanocortin-4 receptor

Article abstract of DOI:10.1248/cpb.55.1232

In the present study, conducted to explore potent and small molecular melanocortin-4 (MC4) receptor ligands, we found that tripeptide 3a, containing a D-Phe-Arg-2-Nal (Nal; naphthylalanine) sequence, exhibited a moderate affinity for the MC4 receptor. Structural optimization led to the identification of a compound with a high affinity for the MC4 receptor, namely, tripeptide 3e, which showed a 70-fold higher affinity for the MC4 receptor than the lead compound 3a. Moreover, in an effort to further reduce the peptidic characters of tripeptide 3e, we found that dipeptide 3g exhibited a relatively high affinity for the MC4 receptor. Furthermore, in these analogues, the substituted position (1′ vs. 2′) of the naphthyl ring of Nal residue at position 7 was found to be important for the differentiation of agonist and antagonist activity. The synthesis and structure-activity relationships of the arginine analogues as MC4 receptor ligands were described in this paper.

Full text of DOI:10.1248/cpb.55.1232

1232
Chem. Pharm. Bull. 55(8) 1232—1239 (2007)
Vol. 55, No. 8
Identification of Arginine Analogues as Antagonists and Agonists for the
Melanocortin-4 Receptor
Dai NOZAWA,* Taketoshi OKUBO, Shigeyuki CHAKI, Shigeru OKUYAMA, and Atsuro NAKAZATO
Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd.; 1–403 Yoshino-cho, Kita-ku, Saitama, Saitama
331–9530, Japan. Received April 25, 2007; accepted May 10, 2007; published online May 23, 2007
In the present study, conducted to explore potent and small molecular melanocortin-4 (MC4) receptor lig-
ands, we found that tripeptide 3a, containing a D-Phe-Arg-2-Nal (Nal; naphthylalanine) sequence, exhibited a
moderate affinity for the MC4 receptor. Structural optimization led to the identification of a compound with a
high affinity for the MC4 receptor, namely, tripeptide 3e, which showed a 70-fold higher affinity for the MC4 re-
ceptor than the lead compound 3a. Moreover, in an effort to further reduce the peptidic characters of tripeptide
3e, we found that dipeptide 3g exhibited a relatively high affinity for the MC4 receptor. Furthermore, in these
analogues, the substituted position (1ꢀ vs. 2ꢀ) of the naphthyl ring of Nal residue at position 7 was found to be im-
portant for the differentiation of agonist and antagonist activity. The synthesis and structure–activity relation-
ships of the arginine analogues as MC4 receptor ligands were described in this paper.
Key words melanocortin-4 receptor; antagonist; agonist; depression; anxiety
Five subtypes of melanocortin receptors (MC1—MC5 re- His-Phe-Arg-Trp sequence is the minimal core sequence es-
ceptors) belonging to the superfamily of G-protein-coupled sential for the activation of the MCRs.1) The tetrapeptide His-
receptors (GPCRs) have been cloned and characterized. Phe-Arg-Trp sequence has thus been employed as a promis-
When stimulated by agonist ligands, the melanocortin recep- ing template for structure–activity relationship (SAR) studies
tors (MCRs) activate the cyclic adenosine monophosphate of MCR ligands.6—9) Moreover, cyclic peptide analogues
(cAMP) signal transduction pathway. The naturally occurring with reduced conformational freedom were designed to in-
agonists of MCRs are melanocyte-stimulating hormones (a- crease the activity and in vivo stability of the peptide ligands.
MSH; Fig. 1, b-MSH and g-MSH) and adrenocorticotropic Thus, both MT-II and SHU-9119 (Fig. 1), which were de-
hormone (ACTH), which are derived from proopiome- signed based on the cyclization of the tetrapeptide His-D-
lanocortin (POMC) by enzymatic processing. In addition, Phe-Arg-Trp sequence, were discovered as cyclic peptides
two endogenous antagonistic peptides, agouti and agouti-re- with potent activities on the MCRs.10,11)
lated protein (AGRP), have also been identified. The MCRs
Among the MCRs, numerous studies have suggested that
mediate a variety of physiological responses that include skin the MC4 receptor is involved in the regulation of feeding
pigmentation (MC1 receptor), steroidogenesis (MC2 recep- behavior and energy homeostasis.12—18) It has also been re-
tor), feeding behavior (MC3 and MC4 receptors), and ex- portedly involved in the regulation of sexual functions19,20)
ocrine gland secretion (MC5 receptor). Consequently, the and protection against tumor-induced decreases in body
melanocortin system has become an attractive therapeutic weight.21,22) In addition, the MC4 receptor has been a focus
target for drug development.1—4)
of interest for its possible relationship to stress and the regu-
Over the last 30 years, significant progress has been made lation of emotional behavior in relation to conditions such as
in the design of peptidic ligands as potential therapeutic depression and anxiety.23—29) These findings till date indicate
agents for the treatment of melanocortin-mediated diseases. that the MC4 receptor could be a promising target for the de-
Among the first significant research findings on a-MSH was velopment of drugs for the above-mentioned conditions, and
the discovery of [Nle4, D-Phe7]a-MSH (NDP-MSH; Fig. 1).5) numerous ligands of the MC4 receptor have been re-
Inverting Phe at position 7 of a-MSH to the D-isomer, in ad- ported.30—56)
dition to replacing Met at position 4 with Nle, resulted in the
Stress is considered to play a pivotal role in both anxiety
formation of the analogue, NDP-MSH, that exhibited an en- and depression. Indeed, dysfunction of the hypothalamic-pi-
hanced potency for the MCRs and enzymatic stability.5) Fur- tuitary-adrenal (HPA) axis, which is caused by continuous
ther studies on melanocortins indicate that the tetrapeptide exposure to stress, has been reported in patients with major
Fig. 1. Endogenous and Synthetic Peptidyl Ligands of the MC4 Receptor
∗ To whom correspondence should be addressed. e-mail: dai.nozawa@po.rd.taisho.co.jp
© 2007 Pharmaceutical Society of Japan

August 2007
1233
depressive disorders. Stress responses are initiated by the re- synthetic method similar to Method A. The removal of the
lease of corticotropin-releasing factor (CRF). In addition of Boc group of 5a under acidic conditions, followed by con-
CRF system activation, several lines of evidence suggest that densation with 2-naphthyl-(CH2)2-CO2H 15 in the presence
melanocortins mediate important behavioral and biochemical of HOBt and EDC, yielded 2-naphthyl-(CH2)2-Arg(Z)2-2-
responses to stress and, consequently, stress-induced disor- Nal-NH2 8-1. Compound 9 was obtained by the removal of
ders.
the Z groups of 8-1 using hydrogenolysis. Compound 10 was
We previously reported that a tripeptide MC4 receptor an- also obtained from 5a by the same synthetic method as that
tagonist 3e (MCL0020) exhibited antidepressant, anxiolytic, used for 9. Furthermore, compound 20 was prepared from
and anti-stress effects in a variety of animal models (Table Boc-Arg(Z)2-NH-(CH2)2-2-naphthyl 5c using a synthetic
1).26) These findings suggest that blockade of the MC4 recep- method similar to that used for 9 (Method B, Chart 2).
tor may be a useful approach for treating subjects with de-
pressive and anxiety disorders. This hypothesis is supported Results and Discussion
by the demonstration in various rodent modes of depression
and anxiety that other MC4 receptor antagonists also exert were evaluated based on their binding affinity to the mem-
antidepressant and anxiolytic effects.27,28)
branes of COS-1 cells expressing the human MC4 receptor;
In the present study, to identify small molecular MC4 re- the affinities were calculated from the inhibition curve of
ceptor ligands, we focused our attention on a tripeptide se-
125I]NDP-a-MSH binding,27) and the IC50 values are shown
quence, D-Phe-Arg-Trp, and modified both the D-Phe and Trp in Table 1.
The affinities of all the compounds for the MC4 receptor
[
residues to increase the affinity for the MC4 receptor. More-
All endogenous agonists for the MCRs contain a tetrapep-
over, to reduce the peptidic character and molecular weight, tide sequence, His-Phe-Arg-Trp, which has been identified as
the effects of the removing the N- or C-terminus of the the minimal peptide sequence crucial for MCRs activation.
tripeptides on the binding affinity for the receptor were ex- Further studies have shown that the inversion of the chirality
plored. The synthesis, SARs and biological evaluation of the of Phe to D-Phe results in an increase in the binding affinity
arginine analogues as MC4 receptor ligands are described in for the MC4 receptor, as described above. Moreover, the His
this study.
residue in tetrapeptide 1 (Fig. 1), which showed a moderate
Chemistry All the compounds were prepared from Boc- affinity (IC50ꢀ1153 nM) for the MC4 receptor, can be deleted
Arg(Z)2-OH 4 according to the general synthetic methods without much loss in binding affinity (tripeptide 2;
(Methods A and B) described as Charts 1 and 2. The reaction IC50ꢀ2081 nM, Fig. 1).8) Therefore, in the present study, to
of 4 with H-2-Nal-NH2·HCl 11a in the presence of HOBt explore potent and small molecular MC4 receptor ligands,
and EDC yielded Boc-Arg(Z)2-2-Nal-NH2 5a. After the re- we focused our attention on mimicking the tripeptide, D-Phe-
moval of the Boc group of 5a by TFA, condensation with Arg-Trp sequence.
Boc-D-2-Nal-OH 12 in the presence of HOBt and EDC
It has been suggested that the chemically reactive indol in
yielded Boc-D-2-Nal-Arg(Z)2-2-Nal-NH2 6a-1. The removal the Trp residue could be replaced with a non-reactive naph-
of the Boc group of 6a-1 by TFA, followed by the treatment thyl moiety in the design of peptide melanocortin receptor
with Ac2O and pyridine, yielded Ac-D-2-Nal-Arg(Z)2-2-Nal- ligands.6) In our attempt to identify further potent tripeptide
NH2 7a-1. Compound 3e was obtained by the removal of the analogues, we initially anticipated that 2-Nal could be used
Z groups of 7a-1 using hydrogenolysis with Pd(OH)2 as a as an alternative to the Trp in the tripeptide, Ac-D-Phe-Arg-
catalyst. Compounds 3a—d and 3f—h were also obtained Trp-NH2 2. We prepared the tripeptides 3a (containing 2-
from 4 by using similar synthetic methods to those of 3e Nal) and 3b (containing D-2-Nal), as shown in Chart 1, and
(Method A, Chart 1).
evaluated their affinity for the MC4 receptor. The resultant
Compounds 9 and 10 were synthesized from 5a using a 3a and 3b were found to exhibit a similar affinity (IC50ꢀ
Reagents and conditions: (a) N-methyl morpholine, EDC-HCl, HOBt-H2O, DMF, rt (81%); (b) 5a-1, TFA, CH2Cl2, rt; then 12, EDC-HCl , HOBt-H2O, DMF, rt (38%); (c) 6a-1,
TFA, CH2Cl2, rt; then Ac2O, pyridine, CHCl3, rt (74%); (d) H2, Pd(OH)2/C, MeOH, rt (95%).
Chart 1. Synthesis of Tripeptide 3e (General Method of Synthesis for 3a—h, Method A)

1234
Vol. 55, No. 8
Table 1. Binding Affinity for the MC4 Receptor and cAMP Stimulation
cAMP
Compound R1
R2
IC50 (nM)
1070
formationa)
3a
3b
2170
Reagents and conditions: (e) 5a, TFA, CH2Cl2, rt; then 15, EDC-HCl, HOBt-H2O,
DMF, rt (58%); (f) H2, Pd(OH)2/C, MeOH, rt (96%).
3c
3d
3e
327
690
15.4
2.8
1.9
Chart 2. Synthesis of Dipeptide 9 (General Method of Synthesis for
Dipeptides 9 and 10, and 20, Method B)
1070 and 2170 nM, respectively) for the MC4 receptor, com-
pared to the corresponding tripeptide 2. These findings sug-
gest that Trp can be replaced with 2-Nal or D-2-Nal without
any significant decrease in binding affinity for the MC4 re-
ceptor in these tripeptide analogues. Therefore, both 2-Nal
and D-2-Nal were employed in further research efforts to de-
sign tripeptides for MC4 receptor ligands.
We next considered that the D-Phe in these tripeptides
could be replaced with D-Nal, based on previous SAR obser-
vations of cyclic peptides, MT-II and SHU-9119 (Fig. 1).10,11)
We prepared 3c—f as shown in Chart 1 and evaluated the
affinity for the MC4 receptor. The results indicated that both
3e and 3f containing D-2-Nal showed a much higher affinity
(IC50ꢀ15.4 and 36.5 nM, respectively) for the MC4 receptor
than the D-Phe analogues, 3a and 3b. In contrast, both 3c and
3d containing D-1-Nal exhibited a slightly higher affinity
(IC50ꢀ327 and 690 nM, respectively) for the MC4 receptor
than the corresponding 3a and 3b. These findings suggest
that, in the case of these tripeptide analogues, the substitution
of D-2-Nal at position 7 (a-MSH numbering; Fig. 1) resulted
in a drastic increase in the binding affinity for the MC4 re-
ceptor.
0.95
0.99
3f
36.5
117
3g
3h
7230
9
543.0
3940
10
20
1610
a) The fold increase vs. basal level at a ligand concentration of 10 mM.
To better understand the minimal structure fragment re-
quired for binding affinity for the MC4 receptor, we initially
investigated the effect of removing the N-terminus from 3c relatively high-affinity dipeptide 3g, and evaluated the affin-
and 3e on the binding affinity for the receptor. We prepared ity for the MC4 receptor. Surprisingly, compound 20 exhib-
dipeptides 9 and 10, as shown in Chart 2, and evaluated the ited a moderate affinity for the MC4 receptor (IC50ꢀ
binding affinity for the MC4 receptor. The results indicated 1610 nM), and the affinity was almost the same as that of
that the resultant 9 and 10 showed a much lower affinity tetrapeptide 1 and tripeptide 2. Thus, analogues like 20,
(IC50ꢀ543 and 3940 nM, respectively) for the MC4 receptor which have a significantly reduced peptidic character, might
than the corresponding tripeptides 3c and 3e. Next, we also also be useful as new frameworks for MC4 ligands.
investigated the effect of removing the C-terminus from 3a
We previously reported that 3e was selective for the MC4
and 3e on binding affinity for the MC4 receptor. The results receptor and acted as an antagonist at the MC4 receptor. 3e
indicated that the dipeptide 3g showed a relatively high affin- was also found to exhibit antidepressant and anxiolytic activ-
ity (IC50ꢀ117 nM) for the MC4 receptor. This finding sug- ities in various rodent models.26) To further investigate the in
gests that dipeptide analogues, as exemplified by 3g, might vitro profiles of the compounds described above, the effects
serve as new templates for the design of potent MC4 ligands of several compounds on cAMP formation at a ligand con-
with reduced peptidic character.
centration of 10 mM in MC4 receptor expressing COS-1 cells
As an additional structural conversion to further reduce the were evaluated (Table 1). The results indicated that the
peptidic character and molecular weight, we prepared 20, tripeptides 3c and 3d containing D-1-Nal at position 7 (a-
which was simplified by removing the N-terminus from the MSH numbering) increased cAMP formation, suggesting

August 2007
1235
(2.16 g, 3.98 mmol), H-2-Nal-NH2 hydrochloride 11a (1.00 g, 3.99 mmol),
N-methyl morpholine (0.42 g, 4.2 mmol) and HOBt·H2O (0.92 g, 6.0 mmol)
in DMF (20 ml) was added EDC·HCl (0.96 g, 5.0 mmol) with ice-cooling,
and the reaction mixture was stirred at room temperature for 72 h. The mix-
ture was partitioned between EtOAc and H2O. The separated organic phase
that these compounds act as agonists at the MC4 receptor. In
contrast, the tripeptides 3e and 3f containing D-2-Nal at posi-
tion 7 did not influence the stimulation of cAMP, suggesting
that these compounds act as antagonists at the MC4 receptor.
These findings suggest that, in the case of these tripeptide was washed with 5% aq. KHSO4, saturated aq. NaHCO3 and brine, dried
over Na2SO4, filtered, concentrated in vacuo. The residue was crystallized
analogues, the substituted position (1ꢁ vs. 2ꢁ) of the naphthyl
ring of Nal residue at position 7 appears to be important for
the determining of agonist and antagonist activity at the MC4
from AcOEt, and the precipitate was collected by filtration to give 5a (2.4 g,
81%) as a solid. 1H-NMR (200 MHz, CDCl3) d: 1.05—1.70 (13H, m),
3.01—3.32 (2H, m), 3.38—3.77 (2H, m), 3.99—4.13 (1H, m), 4.50—4.64
receptor; these results were supported by SAR research on
(1H, m), 5.02—5.23 (5H, m), 5.55 (1H, d, Jꢀ7.3 Hz), 6.24 (1H, br s),
6.89 (1H, d, Jꢀ8.1 Hz), 7.17—7.52 (14H, m), 7.57—7.76 (3H, m), 9.00—
9.35 (2H, m). MS (ESI, Pos) m/z: 739 (MꢂH)ꢂ. Anal. Calcd for
C40H46N6O8·0.5H2O: C, 64.24; H, 6.33; N, 11.24. Found: C, 63.87; H, 6.22;
N, 11.14. Rf 0.60 (CHCl3 : MeOHꢀ9 : 1).
the cyclic peptides, MT-II and SHU-9119.10,11)
Conclusion
We have reported on the synthesis and SARs of a new se-
ries of tripeptides that act as MC4 receptor ligands. The lead
compound 3a, obtained by replacing a Trp residue of tripep-
tide 2 with 2-Nal, showed a moderate affinity for the MC4
receptor. Structural conversion led to the identification of a
compound with a high affinity for the MC4 receptor, namely,
3e (IC50ꢀ15.4 nM), which showed a 70-fold higher affinity
for the MC4 receptor than the lead compound 3a. We also
found a dipeptide analogue, 3g, with a reduced peptidic char-
acter that showed a relatively high affinity (IC50ꢀ117 nM) for
the MC4 receptor. Furthermore, in the case of these tripep-
Boc-D-2-Nal-Arg(Z)2-2-Nal-NH2 (6a-1) To
a suspension of Boc-
Arg(Z)2-2-Nal-NH2 5a (500 mg, 0.676 mmol) in CH2Cl2 (5 ml) was added
TFA (5 ml) and the reaction mixture was stirred at room temperature for 2 h.
After concentration of the reaction mixture, the residue was partitioned be-
tween CHCl3 and saturated aq. NaHCO3. The separated organic phase was
dried over Na2SO4, filtered, concentrated in vacuo to obtain crude H-
Arg(Z)2-2-Nal-NH2. To a mixture of the crude H-Arg(Z)2-2-Nal-NH2, Boc-
D-2-Nal-OH 12 (235 mg, 0.745 mmol) and HOBt·H2O (156 mg, 1.02 mmol)
in DMF (15 ml) was added EDC·HCl (162 mg, 0.846 mmol) with ice-cool-
ing, and the reaction mixture was stirred at room temperature for 15 h. The
mixture was partitioned between EtOAc and H2O. The separated organic
phase was washed with 5% aq. KHSO4, saturated aq. NaHCO3 and brine,
dried over Na2SO4, filtered, concentrated in vacuo. The residue was chro-
tide analogues, the substituted position (1ꢁ vs. 2ꢁ) of the matographed on Silica gel C-200 (CHCl3/MeOH 50 : 1), and crystallized
from EtOAc to give 6a-1 (227 mg, 38%) as a solid. 1H-NMR (300 MHz,
CDCl3) d: 0.14—0.26 (1H, m), 0.53—0.66 (1H, m), 1.11—1.22 (2H, m),
1.47 (9H, s), 2.36—2.62 (2H, m), 2.82—3.01 (3H, m), 3.51—3.63 (1H, m),
naphthyl ring of Nal residue at position 7 was found to be
important for the differentiation of agonist and antagonist ac-
tivity at the MC4 receptor. These arginine analogues that
4.07—4.30 (2H, m), 4.76—4.83 (1H, m), 5.01 (2H, s), 5.03—5.40 (4H, m),
have been shown to have a high affinity for the MC4 receptor
may be excellent tools for investigating the involvement of
the MC4 receptor in the development of stress-related disor-
ders, such as depression and anxiety, and for elucidating the
actions mediated by the MC4 receptor.
6.76 (1H, br s), 7.04 (1H, d like, Jꢀ8.5 Hz), 7.18—7.64 (25H, m), 9.14 (1H,
br s), 9.42 (1H, br s). MS (ESI, Pos) m/z: 936 (MꢂH)ꢂ. Anal. Calcd for
C53H57N7O9·0.3H2O: C, 67.61; H, 6.17; N, 10.41. Found: C, 67.40; H, 6.15;
N, 10.34. Rf 0.46 (CHCl3 : MeOHꢀ9 : 1).
Ac-D-2-Nal-Arg(Z)2-2-Nal-NH2 (7a-1) To a suspension of Boc-D-2-
Nal-Arg(Z)2-2-Nal-NH2 6a-1 (200 mg, 0.214 mmol) in CH2Cl2 (3 ml) was
added TFA (3 ml) and the reaction mixture was stirred at room temperature
for 1 h. After concentration of the reaction mixture, the residue was parti-
tioned between CHCl3 and saturated aq. NaHCO3. The separated organic
Experimental
Melting points were determined on a Yanaco MP-500D melting point ap-
paratus and are uncorrected. Proton nuclear magnetic resonance (1H-NMR) phase was dried over Na2SO4, filtered, concentrated in vacuo to obtain crude
spectra were obtained using a Varian Gemini 2000 (200 MHz) or Varian
Unity Inova 300 (300 MHz). Chemical shifts are reported in parts per mil-
H-D-2-Nal-Arg(Z)2-2-Nal-NH2. To a solution of the crude H-D-2-Nal-
Arg(Z)2-2-Nal-NH2 in CHCl3 (2 ml) was added a mixture of Ac2O (24 mg,
lion relative to tetramethylsilane (TMS) as an internal standard. Mass spec- 0.235 mmol) and pyridine (19 mg, 0.235 mmol) in CHCl3 (1 ml), and the re-
tra (MS) were obtained on Micromass Platform LC (ESI). High resolution action mixture was stirred at room temperature for 2 h. After concentration
spectra were recorded on a Micromass Q-TOF2 instrument. Elemental of the reaction mixture, the residue was partitioned between EtOAc and 5%
analyses were performed by a Perkin–Elmer 2400 or a Yanaco MT-6. Silica aq. KHSO4. The separated organic phase was washed with saturated aq.
gel C-200 (100—200 mesh, Wako Pure Chemical) and Chromatorex NH NaHCO3 and brine, dried over Na2SO4, filtered, concentrated in vacuo. The
(100—200 mesh, Fuji Silysia Chemical Ltd.) were used for column chro- residue was crystallized from EtOAc to obtain 7a-1 (139 mg, 74%) as a
1
matography, using the solvent systems (volume ratios) indicated below.
solid. H-NMR (300 MHz, CDCl3) d: 0.13—0.28 (1H, m), 0.57—0.69 (1H,
General Method for the Synthesis of 3a—3h (Method A). H-2-Nal- m), 0.86—1.02 (1H, m), 1.22—1.40 (1H, m), 2.02 (3H, s), 2.42—2.78 (2H,
NH2 Hydrochloride (11a) A mixture of Boc-2-Nal-OH (5.00 g, 15.9
m), 3.02—3.21 (3H, m), 3.58—3.68 (2H, m), 4.31—4.42 (1H, m), 4.63—
mmol), methyl iodide (5.00 ml, 80.3 mmol) and NaHCO3 (2.66 g, 31.7 4.77 (1H, m), 5.03 (2H, s), 5.09 (1H, d, Jꢀ12.1 Hz), 5.47 (1H, d,
mmol) in DMF (25 ml) was stirred at room temperature for 72 h. The reac- Jꢀ12.1 Hz), 7.03 (1H, dd, Jꢀ1.7, 6.7 Hz), 7.17—7.76 (27H, m), 7.99—8.04
tion mixture was concentrated in vacuo and partitioned between EtOAc and (1H, m), 9.27 (1H, br s), 9.53 (1H, br s). MS (ESI, Pos) m/z: 900 (MꢂNa)ꢂ.
H2O. The separated organic phase was washed with 5% aq. KHSO4, satu- Anal. Calcd for C50H51N7O8·0.4H2O: C, 67.84; H, 5.90; N, 11.08. Found: C,
rated aq. NaHCO3 and brine, dried over Na2SO4, filtered, and concentrated 67.67; H, 5.83; N, 11.06. Rf: 0.57 (CHCl3 : MeOHꢀ9 : 1).
in vacuo. A solution of the above residue in MeOH (50 ml) was saturated by
NH3 with ice-cooling. The reaction mixture was standed at room tempera-
Ac-D-2-Nal-Arg-2-Nal-NH2 Hydrochloride (3e) A suspension of Ac-
D-2-Nal-Arg(Z)2-2-Nal-NH2 7a-1 (120 mg, 0.137 mmol) and Pd(OH)2/C (20
ture for 24 h, and concentrated in vacuo. To the residue was added a solution wt% Pd on carbon, wet) (100 mg) in a mixture of MeOH (20 ml) and THF
of 4 mol/l HCl in 1,4-dioxane (80 ml), and the reaction mixture was stirred at (10 ml) was stirred under hydrogen atmosphere for 15 h. The mixture was
room temperature for 2 h. After concentration of the reaction mixture, the
residual solid was dissolved in MeOH (50 ml) followed by filtration, and the
filtered through Celite and the filtrate was concentrated in vacuo. To the
residue was added 4 M HCl in 1,4-dioxane (0.10 ml), and the mixture was
filtrate was concentrated in vacuo. To the residual solid was added IPA concentrated in vacuo. To the residual solid was added IPE, and the resulting
(20 ml) and refluxed for 10 min. The resulting suspension was stirred at
suspension was stirred at room temperature for 1 h, and the precipitate was
room temperature for 15 h, and the precipitate was collected by filtration to collected by filtration to obtain 3e (85 mg, 95%) as a solid. 1H-NMR
obtain 11a (3.77 g, 95%) as a solid. 1H-NMR (200 MHz, DMSO-d6) d: (300 MHz, DMSO-d6) d: 0.96—1.08 (2H, m), 1.17—1.59 (2H, m), 1.82
3.13—3.41 (2H, m), 4.08 (1H, t, Jꢀ6.7 Hz), 7.38—7.61 (4H, m), 7.73— (3H, s), 2.70—2.79 (2H, m), 2.95—3.31 (4H, m), 3.88—4.02 (1H, m),
7.96 (4H, m), 8.08 (1H, s), 8.36 (1H, br s). MS (ESI, Pos) m/z: 215 4.38—4.47 (1H, m), 4.53—4.72 (1H, m), 7.00—7.19 (2H, m), 7.37—7.51
(MꢂH)ꢂ. Anal. Calcd for C13H14N2O·HCl: C, 62.28; H, 6.03; N, 11.17. (9H, m), 7.66—7.99 (10H, m), 8.41—8.49 (2H, m). MS (ESI, Pos) m/z: 610
Found: C, 62.24; H, 6.01; N, 11.10.
Boc-Arg(Z)2-2-Nal-NH2 (5a) To
(MꢂH)ꢂ. HR-MS m/z: 610.3148 (Calcd for 610.3142). Rf: 0.73 (n-
BuOH : AcOH : H2Oꢀ4 : 1 : 1).
a mixture of Boc-Arg(Z)2-OH 4

1236
Synthesis of 3a. Boc-D-Phe-Arg(Z)2-2-Nal-NH2 (6a-3) Compound
6a-3 was obtained as a solid from Boc-Arg(Z)2-2-Nal-NH2 5a and Boc-D-
Phe-OH 14 by similar synthetic method of 6a-1. 1H-NMR (200 MHz,
CDCl3) d: 1.02—1.38 (13H, m), 2.67—3.24 (5H, m), 3.51—3.63 (1H, m),
4.02—4.22 (2H, m), 4.70—5.35 (7H, m), 6.70 (1H, br s), 6.92—7.71 (24H,
m), 9.21 (1H, br s), 9.43 (1H, br s). MS (ESI, Pos) m/z: 886 (MꢂH)ꢂ. Anal.
Calcd for C49H55N7O9: C, 66.42; H, 6.26; N, 11.07. Found: C, 66.22; H,
6.26; N, 11.05. Rf: 0.41 (CHCl3 : MeOHꢀ9 : 1).
Ac-D-Phe-Arg(Z)2-2-Nal-NH2 (7a-3) Compound 7a-3 was obtained as
a solid from Boc-D-Phe-Arg(Z)2-2-Nal-NH2 6a-3 by similar synthetic
method of 7a-1. 1H-NMR (300 MHz, CDCl3) d: 0.51—0.70 (1H, m), 1.04—
1.22 (2H, m), 1.42—1.60 (1H, m), 1.98 (3H, s), 2.88—2.99 (2H, m), 3.08—
3.39 (3H, m), 3.58—3.66 (1H, m), 3.69—3.90 (1H, m), 4.21—4.31 (1H, m),
4.63—4.78 (1H, m), 5.04—5.18 (3H, m), 5.38 (1H, d, Jꢀ12.0 Hz), 6.98—
7.50 (17H, m), 7.58—7.94 (10H, m), 9.31 (1H, br s), 9.52 (1H, br s). MS
(ESI, Pos) m/z: 828 (MꢂH)ꢂ. Anal. Calcd for C46H49N7O8·0.5H2O: C,
66.01; H, 6.02; N, 11.71. Found: C, 65.99; H, 5.96; N, 11.67. Rf: 0.46
(CHCl3 : MeOHꢀ9 : 1).
Ac-D-Phe-Arg-2-Nal-NH2 Hydrochloride (3a) Compound 3a was ob-
tained as a solid from Ac-D-Phe-Arg(Z)2-2-Nal-NH2 7a-3 by similar syn-
thetic method of 3e. 1H-NMR (300 MHz, DMSO-d6) d: 1.02—1.19 (2H, m),
1.22—1.58 (2H, m), 1.83 (3H, s), 2.79—2.94 (4H, m), 2.99—3.11 (1H, m),
3.19—3.32 (1H, m), 3.92—4.01 (1H, m), 4.39—4.52 (1H, m), 7.02—7.29
(11H, m), 7.38—7.51 (4H, m), 7.69—7.99 (5H, m), 8.43 (2H, dd, Jꢀ6.6,
8.1 Hz). MS (ESI, Pos). m/z: 560 (MꢂH)ꢂ. HR-MS m/z: 560.2991 (Calcd
for 560.2985). Rf: 0.45 (n-BuOH : AcOH : H2Oꢀ4 : 1 : 1).
Synthesis of 3c. Boc-D-1-Nal-Arg(Z)2-2-Nal-NH2 (6a-2) Compound
6a-2 was obtained as a solid from Boc-Arg(Z)2-2-Nal-NH2 5a and Boc-D-1-
Nal-OH 13 by similar synthetic method of 6a-1. 1H-NMR (200 MHz,
CDCl3) d: 0.90—1.29 (4H, m), 1.47 (9H, s), 2.40—2.90 (2H, m), 3.27—
3.38 (2H, m), 3.49—3.62 (1H, m), 3.99—4.12 (1H, m), 4.40—4.52 (1H, m),
4.75—4.92 (2H, m), 4.97—5.30 (6H, m), 6.68—7.73 (1H, m), 6.98—7.82
(26H, m), 9.01—9.13 (1H, m), 9.29—9.38 (1H, m). MS (ESI, Pos) m/z: 936
(MꢂH)ꢂ. Anal. Calcd for C53H57N7O9: C, 68.01; H, 6.14; N, 10.47. Found:
C, 67.79; H, 6.12; N, 10.40. Rf: 0.73 (CHCl3 : MeOHꢀ9 : 1).
Ac-D-1-Nal-Arg(Z)2-2-Nal-NH2 (7a-2) Compound 7a-2 was obtained
as a solid from Boc-D-1-Nal-Arg(Z)2-2-Nal-NH2 6a-2 by similar synthetic
method of 7a-1. 1H-NMR (300 MHz, CDCl3) d: 0.03—0.21 (1H, m), 0.69—
0.89 (1H, m), 0.91—1.05 (1H, m), 1.21—1.38 (1H, m), 2.05 (3H, s), 2.71—
2.98 (2H, m), 3.06—3.21 (1H, m), 3.46 (2H, d, Jꢀ8.1 Hz), 3.58—3.78 (2H,
m), 4.50—4.62 (1H, m), 4.65—4.79 (1H, m), 5.09—5.30 (4H, m), 7.12—
7.42 (17H, m), 7.45—8.74 (10H, m), 7.78—7.88 (1H, m), 7.94—8.00 (1H,
m), 9.24 (1H, br s), 9.52 (1H, br s). MS (ESI, Pos) m/z: 878 (MꢂH)ꢂ. Anal.
Calcd for C50H51N7O8: C, 68.40; H, 5.85; N, 11.17. Found: C, 68.07; H,
5.87; N, 11.06. Rf: 0.54 (CHCl3 : MeOHꢀ9 : 1).
Ac-D-1-Nal-Arg-2-Nal-NH2 Hydrochloride (3c) Compound 3c was
obtained as a solid from Ac-D-1-Nal-Arg(Z)2-2-Nal-NH2 7a-2 by similar
synthetic method of 3e. 1H-NMR (300 MHz, DMSO-d6) d: 0.91—1.04 (2H,
m), 1.12—1.51 (2H, m), 1.82 (3H, s), 2.76—2.84 (2H, m), 2.99—3.11 (1H,
m), 3.21—3.48 (3H, m), 3.91—4.00 (1H, m), 4.37—4.48 (1H, m), 4.51—
4.61 (1H, m), 7.15—7.21 (2H, m), 7.35—7.59 (10H, m), 7.68—7.94 (9H,
m), 8.12 (1H, d, Jꢀ6.5 Hz), 8.32 (1H, d, Jꢀ6.5 Hz), 8.51 (1H, d, Jꢀ6.5 Hz).
MS (ESI, Pos) m/z: 610 (MꢂH)ꢂ. HR-MS m/z: 610.3152 (Calcd for
610.3142). Rf: 0.55 (n-BuOH : AcOH : H2Oꢀ4 : 1 : 1).
Synthesis of 3f. H-D-2-Nal-NH2 Hydrochloride (11b) Compound
11b was obtained as a solid from Boc-D-2-Nal-OH by similar synthetic
method of 11a. 1H-NMR was accordance to that of 11a. MS (ESI, Pos) m/z:
215 (MꢂH)ꢂ. Anal. Calcd for C13H14N2O·HCl: C, 62.28; H, 6.03; N, 11.17.
Found: C, 62.21; H, 6.01; N, 11.11.
Vol. 55, No. 8
Pos) m/z: 936 (MꢂH)ꢂ. Anal. Calcd for C53H57N7O9: C, 68.00; H, 6.14; N,
10.47. Found: C, 67.61; H, 6.16; N, 10.41. Rf: 0.70 (CHCl3 : MeOHꢀ9 : 1).
Ac-D-2-Nal-Arg(Z)2-D-2-Nal-NH2 (7b-1) Compound 7b-1 was obtained
as a solid from Boc-D-2-Nal-Arg(Z)2-D-2-Nal-NH2 6b-3 by similar synthetic
method of 7a-1. 1H-NMR (300 MHz, CDCl3) d: 0.33—0.48 (1H, m), 0.65—
0.84 (1H, m), 1.13—1.32 (2H, m), 1.90 (3H, s), 2.64—2.88 (2H, m), 3.00—
3.11 (3H, m), 3.46 (1H, dd, Jꢀ4.8, 9.0 Hz), 4.12—4.22 (1H, m), 4.58—4.69
(2H, m), 5.05 (2H, dd, Jꢀ11.2, 12.1 Hz), 5.19 (2H, dd, Jꢀ6.5, 12.1 Hz),
5.39 (1H, br s), 6.24 (1H, d, Jꢀ5.9 Hz), 6.67 (1H, br s), 6.93 (1H, d,
Jꢀ7.5 Hz), 7.05—7.52 (20H, m), 7.57—7.74 (9H, m), 9.21 (1H, br s), 9.42
(1H, br s). MS (FAB, Pos) m/z: 900 (MꢂH)ꢂ. Rf: 0.61 (CHCl3 : MeOHꢀ
9 : 1).
Ac-D-2-Nal-Arg-D-2-Nal-NH2 Hydrochloride (3f) Compound 3f was
obtained as a solid from Ac-D-2-Nal-Arg(Z)2-D-2-Nal-NH2 7b-1 by similar
synthetic method of 3e. 1H-NMR (300 MHz, DMSO-d6) d: 0.83—0.98 (2H,
m), 1.07—1.49 (2H, m), 1.77 (3H, s), 2.59—2.69 (2H, m), 2.92—3.38 (4H,
m), 4.09—4.22 (1H, m), 4.45—4.63 (1H, m), 6.80—7.49 (14H, m), 7.70
(1H, d, Jꢀ8.9 Hz), 7.79—7.89 (10H, m), 8.23 (1H, d, Jꢀ8.1 Hz), 8.32 (1H,
d, Jꢀ7.5 Hz). MS (ESI, Pos) m/z: 610 (MꢂH)ꢂ. HR-MS m/z: 610.3154
(Calcd for 610.3142). Rf: 0.55 (n-BuOH : AcOH : H2Oꢀ4 : 1 : 1).
Synthesis of 3b. Boc-D-Phe-Arg(Z)2-D-2-Nal-NH2 (6b-3) Compound
6b-3 was obtained as a solid from Boc-Arg(Z)2-D-2-Nal-NH2 5b and Boc-D-
Phe-OH 14 by similar synthetic method of 6a-1. 1H-NMR (300 MHz,
CDCl3) d: 0.68—0.78 (1H, m), 1.01—1.48 (12H, m), 2.92—3.01 (3H, m),
3.22—3.33 (1H, m), 3.41—3.60 (2H, m), 4.14—4.30 (2H, m), 4.68—4.81
(1H, m), 4.97 (1H, d, Jꢀ7.0 Hz), 5.03—5.32 (3H, m), 6.71 (1H, br s),
7.02—7.51 (23H, m), 7.62—7.77 (3H, m), 9.22 (1H, br s), 9.41 (1H, br s).
MS (ESI, Pos) m/z: 886 (MꢂH)ꢂ. Anal. Calcd for C49H55N7O9·0.2H2O: C,
66.16; H, 6.28; N, 11.02. Found: C, 65.88; H, 6.29; N, 11.08. Rf: 0.61
(CHCl3 : MeOHꢀ9 : 1).
Ac-D-Phe-Arg(Z)2-D-2-Nal-NH2 (7b-3) Compound 7b-3 was obtained
as a solid from Boc-D-Phe-Arg(Z)2-2-Nal-NH2 6b-3 by similar synthetic
method of 7a-1. 1H-NMR (300 MHz, CDCl3) d: 0.64—0.79 (1H, m), 1.01—
1.49 (3H, m), 1.85 (3H, s), 2.93 (2H, d, Jꢀ7.3 Hz), 2.98—3.08 (1H, m),
3.21—3.38 (1H, m), 3.40—3.56 (2H, m), 4.16—4.24 (1H, m), 4.38—4.48
(1H, m), 4.69—4.81 (1H, m), 5.10—5.31 (5H, m), 6.76 (1H, br s), 6.76—
6.82 (1H, m), 7.04—7.41 (20H, m), 7.67—7.75 (4H, m), 9.26 (1H, br s),
9.43 (1H, br s). MS (ESI, Pos) m/z: 828 (MꢂH)ꢂ. Anal. Calcd for
C46H49N7O8·0.3H2O: C, 66.30; H, 6.00; N, 11.70. Found: C, 66.14; H, 5.98;
N, 11.73. Rf: 0.47 (CHCl3 : MeOHꢀ9 : 1).
Ac-D-Phe-Arg-D-2-Nal-NH2 AcOH (3b) Compound 3b was obtained
as a solid from Ac-D-Phe-Arg(Z)2-D-2-Nal-NH2 7b-3 by similar synthetic
method of 3e. 1H-NMR (300 MHz, DMSO-d6) d: 0.91—1.22 (3H, m),
1.32—1.50 (1H, m), 1.74 (3H, s), 1.76 (3H, s), 2.70—2.99 (4H, m), 3.15—
3.48 (2H, m), 4.02—4.17 (1H, m), 4.39—4.57 (2H, m), 7.11—7.27 (7H, m),
7.30—7.43 (5H, m), 7.65—7.83 (6H, m), 8.22—8.41 (3H, m), 8.89 (1H,
br s). MS (ESI, Pos) m/z: 560 (MꢂH)ꢂ. HR-MS m/z: 560.2977 (Calcd for
560.2985). Rf: 0.42 (n-BuOH : AcOH : H2Oꢀ4 : 1 : 1).
Synthesis of 3d. Boc-D-1-Nal-Arg(Z)2-D-2-Nal-NH2 (6b-2) Com-
pound 6b-2 was obtained as a solid from Boc-Arg(Z)2-D-2-Nal-NH2 5b and
Boc-D-1-Nal-OH 13 by similar synthetic method of 6a-1. 1H-NMR
(300 MHz, CDCl3) d: 0.25—0.42 (1H, m), 0.70—0.87 (1H, m), 0.93—1.24
(2H, m), 1.41 (9H, s), 2.58—2.72 (1H, m), 2.90—3.05 (2H, m), 3.26—3.51
(2H, m), 4.11—4.22 (1H, m), 4.42—4.58 (3H, m), 5.02—5.32 (5H, m),
6.51—6.68 (2H, m), 7.10—7.72 (25H, m), 7.83—7.98 (1H, m), 9.10 (1H,
br s), 9.29 (1H, br s). MS (ESI, Pos) m/z: 936 (MꢂH)ꢂ. Anal. Calcd for
C53H57N7O9·0.3H2O: C, 67.61; H, 6.17; N, 10.41. Found: C, 67.42; H, 6.17;
N, 10.33. Rf: 0.68 (CHCl3 : MeOHꢀ9 : 1).
Ac-D-1-Nal-Arg(Z)2-D-2-Nal-NH2 (7b-2) Compound 7b-2 was ob-
tained as a solid from Boc-D-1-Nal-Arg(Z)2-D-2-Nal-NH2 6b-2 by similar
synthetic method of 7a-1. H-NMR (300 MHz, CDCl3) d: 0.31—0.46 (1H,
m), 0.72—0.89 (1H, m), 1.02—1.19 (2H, m), 1.91 (3H, s), 2.63—2.78
(2H, m), 3.23—3.51 (3H, m), 4.08—4.18 (1H, m), 4.51—4.70 (2H, m),
5.03—5.31 (5H, m), 6.31 (1H, d, Jꢀ6.2 Hz), 6.58 (1H, br s), 6.63—6.74
(1H, m), 7.10—7.77 (24H, m), 8.00 (1H, d, Jꢀ7.9 Hz), 9.17 (1H, br s),
9.32 (1H, br s). MS (ESI, Pos) m/z: 890 (MꢂNa)ꢂ. Anal. Calcd for
C50H51N7O8·0.3H2O: C, 67.98; H, 5.98; N, 11.10. Found: C, 67.79; H, 5.88;
N, 11.04. Rf: 0.60 (CHCl3 : MeOHꢀ9 : 1).
Boc-Arg(Z)2-D-2-Nal-NH2 (5b) Compound 5b was obtained as a solid
from Boc-Arg(Z)2-OH 4, H-D-2-Nal-NH2 hydrochloride 11b by similar syn-
thetic method of 5a. 1H-NMR (200 MHz, CDCl3) d: 1.01—1.32 (4H, m),
1.38 (9H, s), 2.76—2.92 (1H, m), 3.24—3.67 (2H, m), 3.92—4.08 (1H, m),
4.89—5.38 (6H, m), 6.87 (1H, br s), 7.03—7.51 (16H, m), 7.59—7.72 (4H,
m), 9.10—9.40 (2H, m). MS (ESI, Pos) m/z: 739 (MH)ꢂ. Anal. Calcd for
C40H46N6O8: C, 65.03; H, 6.28; N, 11.37. Found: C, 64.93; H, 6.28; N,
11.34. Rf: 0.65 (CHCl3 : MeOHꢀ9 : 1).
Boc-D-2-Nal-Arg(Z)2-D-2-Nal-NH2 (6b-1) Compound 6b-1 was ob-
tained as a solid from Boc-Arg(Z)2-D-2-Nal-NH2 5b and Boc-D-2-Nal-OH
12 by similar synthetic method of 6a-1. 1H-NMR (300 MHz, CDCl3) d:
0.29—0.43 (1H, m), 0.33—0.78 (1H, m), 1.14—1.25 (2H, m), 1.39 (9H, s),
2.71—3.11 (4H, m), 3.43—3.55 (1H, m), 4.09—4.35 (2H, m), 4.54—4.63
(1H, m), 5.00—5.32 (4H, m), 6.61 (1H, br s), 6.77 (1H, br s), 7.16—7.50
(22H, m), 7.59—7.72 (5H, m), 9.19 (1H, br s), 9.39 (1H, br s). MS (ESI,
1
Ac-D-1-Nal-Arg-D-2-Nal-NH2 Hydrochloride (3d) Compound 3d was
obtained as a solid from Ac-D-1-Nal-Arg(Z)2-D-2-Nal-NH2 7b-2 by similar
synthetic method of 3e. 1H-NMR (300 MHz, DMSO-d6) d: 0.75—0.99 (2H,
m), 1.02—1.29 (2H, m), 1.80 (3H, s), 2.60—3.10 (3H, m), 3.17—3.38 (2H,
m), 4.03—4.21 (1H, m), 4.38—4.62 (2H, m), 6.90—7.63 (12H, m), 7.65—
7.96 (7H, m), 8.04—8.29 (4H, m), 8.42 (1H, d, Jꢀ7.3 Hz). MS (ESI, Pos)

August 2007
1237
m/z: 610 (MꢂH)ꢂ. HR-MS m/z: 610.3139 (Calcd for 610.3142). Rf: 0.46 (n-
BuOH : AcOH : H2Oꢀ4 : 1 : 1).
Boc-Arg(Z)2-2-Nal-NH2 5a and 2-naphthyl-(CH2)2-CO2H 15 by similar syn-
thetic method of 6a-1. H-NMR (300 MHz, DMSO-d6) d: 1.31—1.57 (4H,
1
m), 2.37—2.46 (2H, m), 2.82—3.02 (3H, m), 3.16 (1H, dd, Jꢀ5.0, 9.0 Hz),
3.67 (1H, br s), 4.14—4.23 (1H, m), 4.48—4.58 (1H, m), 5.03 (2H, s), 5.19
(2H, s), 7.12 (1H, s), 7.21—7.46 (17H, m), 7.61 (1H, s), 7.66 (1H, s),
7.69—7.83 (6H, m), 7.90 (1H, d, Jꢀ8.2 Hz), 8.01 (1H, d, Jꢀ8.2 Hz),
9.13 (2H, br s). MS (ESI, Pos) m/z: 821 (MꢂH)ꢂ. Anal. Calcd for
C48H48N6O7·1.6H2O: C, 67.85; H, 6.07; N, 9.89. Found: C, 67.54; H, 5.74;
N, 9.75. Rf: 0.36 (CHCl3 : MeOHꢀ20 : 1).
2-Naphthyl-(CH2)2-CO-Arg-2-Nal-NH2 (9) Compound 9 was obtained
as a solid from 2-naphthyl-(CH2)2-CO-Arg(Z)2-2-Nal-NH2 8-1 by similar
synthetic method of 3e. 1H-NMR (300 MHz, DMSO-d6) d: 1.18—1.59 (4H,
m), 2.58—2.66 (2H, m), 2.79—3.04 (5H, m), 3.08—3.31 (1H, m), 4.10—
4.22 (1H, m), 4.49—4.56 (1H, m), 6.80—6.90 (1H, m), 6.95—7.84 (21H,
m). MS (ESI, Pos) m/z: 553 (MꢂH)ꢂ. HR-MS m/z: 553.2919 (Calcd for
553.2927). Rf: 0.74 (n-BuOH : AcOH : H2Oꢀ4 : 1 : 1).
Synthesis of 3g. Boc-Arg(Z)2-NH-(CH2)2-2-naphthyl (5c) Com-
pound 5c was obtained as a solid from Boc-Arg(Z)2-OH 4 and 2-naphthyl-
(CH2)2-NH2 17 by similar synthetic method of 5a. 1H-NMR (200 MHz,
CDCl3) d: 1.20—1.49 (11H, m), 1.55—1.69 (2H, m), 2.71 (2H, d,
Jꢀ6.9 Hz), 3.03—3.62 (3H, m), 3.78—3.99 (1H, m), 4.10—4.24 (1H, m),
5.09 (2H, dd, Jꢀ7.5, 12.0 Hz), 5.18 (2H, s), 6.61—7.73 (1H, m), 7.14 (2H,
dd, Jꢀ1.8, 6.6 Hz), 7.25—7.48 (13H, m), 7.60—7.78 (3H, m), 9.02—9.25
(2H, m). MS (ESI, Pos) m/z: 696 (MꢂH)ꢂ. Anal. Calcd for C39H45N5O7: C,
67.32; H, 6.52; N, 10.07. Found: C, 67.22; H, 6.53; N, 10.06.
Boc-D-2-Nal-Arg(Z)2-NH-(CH2)2-2-naphthyl (6c-1) Compound 6c-1
was obtained as a solid from Boc-Arg(Z)2-NH-(CH2)2-2-naphthyl 5c and
Boc-D-2-Nal-OH 12 by similar synthetic method of 6a-1. 1H-NMR
(200 MHz, CDCl3) d: 0.30—0.45 (1H, m), 0.62—0.78 (1H, m), 1.02—1.21
(1H, m), 1.39 (9H, s), 1.62—1.73 (1H, m), 2.35—3.20 (6H, m), 3.25—3.46
(1H, m), 4.36—4.63 (2H, m), 4.96—5.38 (6H, m), 6.83—6.96 (1H, m), 7.05
(2H, dd, Jꢀ1.6, 6.8 Hz), 7.17—7.48 (18H, m), 7.52—7.73 (5H, m), 9.03
(1H, br s), 9.32 (1H, br s). MS (ESI, Pos) m/z: 915 (MꢂNa)ꢂ. Anal. Calcd
for C52H56N6O8: C, 69.94; H, 6.32; N, 9.41. Found: C, 69.75; H, 6.38; N,
9.33. Rf: 0.44 (hexane : EtOAcꢀ1 : 1).
Ac-D-2-Nal-Arg(Z)2-NH-(CH2)2-2-naphthyl (7c-1) Compound 7c-1
was obtained as a solid from Boc-D-2-Nal-Arg(Z)2-NH-(CH2)2-2-naphthyl
6c-1 by similar synthetic method of 7a-1. 1H-NMR (200 MHz, CDCl3) d:
0.23—0.37 (1H, m), 0.61—0.77 (1H, m), 1.06—1.20 (1H, m), 1.57—1.70
(1H, m), 1.91 (3H, s), 2.37—2.76 (4H, m), 2.91—3.03 (2H, m), 3.08 (1H,
dd, Jꢀ5.0, 8.2 Hz), 3.39—3.43 (1H, m), 4.52—4.61 (1H, m), 4.63—4.72
(1H, m), 5.02 (1H, dd, Jꢀ9.0, 12.0 Hz), 5.14 (1H, d, Jꢀ12.1 Hz), 5.31 (1H,
d, Jꢀ12.1 Hz), 6.21 (1H, d, Jꢀ7.2 Hz), 6.84 (1H, t, Jꢀ5.8 Hz), 7.05 (1H, dd,
Jꢀ1.7, 6.7 Hz), 7.19—7.43 (18H, m), 7.52—7.69 (7H, m), 9.02 (1H, br s),
9.29 (1H, br s). MS (ESI, Pos) m/z: 835 (MꢂH)ꢂ. Anal. Calcd for
C49H50N6O7: C, 70.49; H, 6.04; N, 10.07. Found: C, 70.16; H, 6.06; N, 9.92.
Rf: 0.59 (CHCl3 : MeOHꢀ9 : 1).
Synthesis of 10. 1-Naphthyl-(CH2)2-CO-Arg(Z)2-2-Nal-NH2 (8-2)
Compound 8-2 was obtained as a solid from Boc-Arg(Z)2-2-Nal-NH2 5a and
1
1-naphthyl-(CH2)2-CO2H 16 by similar synthetic method of 6a-1. H-NMR
(300 MHz, DMSO-d6) d: 1.30—1.56 (4H, m), 2.39—2.47 (2H, m), 2.97
(1H, dd, Jꢀ5.3, 9.0 Hz), 3.09—3.22 (2H, m), 3.59—3.82 (2H, m), 4.16—
4.24 (1H, m), 4.47—4.57 (1H, m), 5.00 (2H, s), 5.20 (2H, s), 7.12 (1H, s),
7.20—7.52 (17H, m), 7.65 (1H, s), 7.68—7.95 (6H, m), 8.00 (1H, d,
Jꢀ8.4 Hz), 9.15 (2H, br s). MS (ESI, Pos) m/z: 821 (MꢂH)ꢂ. Anal. Calcd
for C48H48N6O7·0.4H2O: C, 69.62; H, 5.94; N, 10.15. Found: C, 69.46; H,
5.94; N, 10.25. Rf: 0.25 (CHCl3 : MeOHꢀ20 : 1).
1-Naphthyl-(CH2)2-CO-Arg-2-Nal-NH2 (10) Compound 10 was ob-
tained as a solid from 1-naphthyl-(CH2)2-CO-Arg(Z)2-2-Nal-NH2 8-2 by
similar synthetic method of 3e. 1H-NMR (300 MHz, DMSO-d6) d: 1.22—
1.61 (4H, m), 2.78—3.30 (8H, m), 4.16—4.28 (1H, m), 4.49—4.58 (1H, m),
6.80—7.60 (14H, m), 7.69 (1H, s), 7.72—7.94 (6H, m), 8.07 (1H, d,
Jꢀ7.8 Hz). MS (ESI, Pos) m/z: 553 (MꢂH)ꢂ. HR-MS m/z: 553.2914 (Calcd
for 553.2927). Rf: 0.74 (n-BuOH : AcOH : H2Oꢀ4 : 1 : 1).
The Synthesis of 20. 2-Naphthyl-(CH2)2-CO-Arg(Z)2-NH-(CH2)2-2-
naphthyl (19) Compound 19 was obtained as a solid from Boc-Arg(Z)2-
NH-(CH2)2-2-naphthyl 5c and 2-naphthyl-(CH2)2-CO2H 15 by similar syn-
Ac-D-2-Nal-Arg-NH-(CH2)2-2-Naphthyl Hydrochloride (3g) Com-
pound 3g was obtained as a solid from Ac-D-2-Nal-Arg(Z)2-NH-(CH2)2-2-
naphthyl 7c-1 by similar synthetic method of 3e. 1H-NMR (300 MHz,
DMSO-d6) d: 1.04—1.20 (2H, m), 1.24—1.43 (1H, m), 1.50—1.68 (1H, m),
1.78 (3H, s), 2.79—3.01 (6H, m), 3.10 (1H, dd, Jꢀ5.9, 8.2 Hz), 3.30—3.42
(2H, m), 4.08—4.18 (1H, m), 4.56—4.66 (1H, m), 6.80—7.30 (5H, m), 7.37
(1H, dd, Jꢀ1.6, 6.8 Hz), 7.40—7.52 (6H, m), 7.72 (1H, d, Jꢀ12.7 Hz),
7.79—7.90 (5H, m), 8.07 (1H, t, Jꢀ6.8 Hz), 8.30—8.38 (2H, m). MS (ESI,
Pos) m/z: 567 (MꢂH)ꢂ. HR-MS m/z: 567.3067 (Calcd for 567.3084). Rf:
0.63 (n-BuOH : AcOH : H2Oꢀ4 : 1 : 1).
Synthesis of 3h. Boc-D-Phe-Arg(Z)2-NH-(CH2)2-2-naphthyl (6c-3)
Compound 6c-3 was obtained as a solid from Boc-Arg(Z)2-NH-(CH2)2-2-
naphthyl 5c and Boc-D-Phe-OH 14 by similar synthetic method of 6a-1. 1H-
NMR (200 MHz, CDCl3) d: 1.01—1.26 (2H, m), 1.38 (9H, s), 1.51—1.75
(2H, m), 2.72 (2H, q, Jꢀ7.4 Hz), 2.92 (2H, d, Jꢀ7.3 Hz), 3.09—3.60 (4H,
m), 4.22—4.41 (1H, m), 4.43—5.58 (1H, m), 4.99—5.21 (5H, m), 6.85—
7.49 (21H, m), 7.58—7.76 (3H, m), 9.16 (1H, br s), 9.35 (1H, br s). MS
(ESI, Pos) m/z: 843 (MꢂH)ꢂ. Anal. Calcd for C48H54N6O8: C, 68.39; H,
6.46; N, 9.97. Found: C, 68.11; H, 6.49; N, 10.02. Rf: 0.39 (hexane :
EtOAcꢀ1 : 1).
Ac-D-Phe-Arg(Z)2-NH-(CH2)2-2-naphthyl (7c-3) Compound 7c-3 was
obtained as a solid from Boc-D-Phe-Arg(Z)2-NH-(CH2)2-2-naphthyl 6c-3 by
similar synthetic method of 7a-1. 1H-NMR (200 MHz, CDCl3) d: 1.08—
1.49 (2H, m), 1.52—1.63 (2H, m), 1.88 (3H, s), 2.72 (2H, q, Jꢀ6.6 Hz),
2.94 (2H, t like, Jꢀ7.4 Hz), 3.11—3.55 (4H, m), 4.41—4.64 (2H, m),
5.08—5.24 (4H, m), 6.00 (1H, d, Jꢀ7.5 Hz), 6.80—7.44 (21H, m), 7.59—
7.78 (3H, m), 9.16 (1H, br s), 9.32 (1H, br s). MS (ESI, Pos) m/z: 785
(MꢂH)ꢂ. Anal. Calcd for C45H48N6O7: C, 68.86; H, 6.16; N, 10.71. Found:
C, 68.67; H, 6.19; N, 10.65. Rf: 0.55 (CHCl3 : MeOHꢀ9 : 1).
Ac-D-Phe-Arg-NH-(CH2)2-2-naphthyl Hydrochloride (3h) Com-
pound 3h was obtained as a solid from Ac-D-Phe-Arg(Z)2-NH-(CH2)2-2-
naphthyl 7c-3 by similar synthetic method of 3e. 1H-NMR (300 MHz,
DMSO-d6) d: 1.06—1.22 (2H, m), 1.24—1.42 (1H, m), 1.50—1.74 (1H, m),
1.79 (3H, s), 2.69—3.03 (6H, m), 3.30—3.45 (2H, m), 4.04—4.17 (1H, m),
4.43—4.52 (1H, m), 6.90—7.25 (9H, m), 7.38 (1H, dd, Jꢀ1.7, 6.7 Hz),
7.40—7.45 (2H, m), 7.60 (1H, t, Jꢀ5.9 Hz), 7.70 (1H, s), 7.79—7.89 (3H,
m), 8.04 (1H, t, Jꢀ5.9 Hz), 8.20—8.29 (2H, m). MS (ESI, Pos) m/z: 517
(MꢂH)ꢂ. HR-MS m/z: 517.2919 (Calcd for 517.2927). Rf: 0.55 (n-
BuOH : AcOH : H2Oꢀ4 : 1 : 1).
1
thetic method of 6a-1. H-NMR (200 MHz, CDCl3) d: 1.01—1.43 (2H, m),
1.51—1.72 (2H, m), 2.38—2.46 (2H, m), 2.63—2.78 (2H, m), 2.96—3.33
(6H, m), 4.43—4.58 (1H, m), 4.99—5.12 (4H, m), 6.52—6.72 (1H, m),
7.02—7.42 (19H, m), 7.58—7.77 (6H, m), 9.15 (1H, br s), 9.34 (1H, br s).
MS (ESI, Pos) m/z: 788 (MꢂH)ꢂ. Anal. Calcd for C47H47N5O6: C, 72.57; H,
6.09; N, 9.00. Found: C, 72.35; H, 6.13; N, 9.03.
2-Naphthyl-(CH2)2-CO-Arg-NH-(CH2)2-2-naphthyl Hydrochloride
(20) Compound 20 was obtained as a solid from 2-naphthyl-(CH2)2-CO-
Arg(Z)2-NH-(CH2)2-2-naphthyl 19 by similar synthetic method of 3e. 1H-
NMR (300 MHz, DMSO-d6) d: 1.24—1.51 (3H, m), 1.54—1.63 (1H, m),
2.56 (1H, dd, Jꢀ2.8, 5.8 Hz), 2.85 (2H, d, Jꢀ7.2 Hz), 2.93—3.03 (3H, m),
3.29—3.41 (2H, m), 3.50—3.71 (2H, m), 4.19—4.27 (1H, m), 6.80—7.25
(5H, m), 7.33—7.50 (6H, m), 7.59—7.72 (3H, m), 7.77—7.90 (5H, m), 8.03
(1H, t, Jꢀ5.5 Hz), 8.10 (1H, d, Jꢀ8.2 Hz). MS (ESI, Pos) m/z: 510
(MꢂH)ꢂ. HR-MS m/z: 510.2859 (Calcd for 510.2869). Rf: 0.77 (n-
BuOH : AcOH : H2Oꢀ4 : 1 : 1).
Binding Test. Material
[
125I][Nle4,D-Phe7]a-Melanocyte stimulating
hormone ([Nle4,D-Phe7]a-MSH) (specific radioactivity: 81.4 TBq/mmol)
was purchased from Amersham International (Buckinghamshire, England).
COS-1 cells were purchased from American Type Culture Collection
(Rocksville, MD, U.S.A.). [Nle4,D-Phe7]a-MSH was purchased from Penin-
sula Laboratories (Belmont, CA, U.S.A.). All other chemicals used in this
study were obtained commercially, and all were of the highest purity avail-
able.
[
125I][Nle4,D-Phe7]a-MSH Binding to Recombinant the MC4 Receptor
COS-1 cells expressing the MC4 receptor, prepared according to the method
reported previously,26) were washed with phosphate buffered saline, scraped
and pelleted by centrifugation. Cell pellets were homogenized with 50 mM
Tris–HCl buffer (pH 7.4) containing 2 mM EDTA, 10 mM CaCl2, and 100 mM
phenylmethylsulfonylfluoride, and centrifuged at 48000ꢃg for 20 min at
4 °C. The pellet was washed twice with the buffer, and the final pellet was
suspended in an assay buffer (50 mM Tris–HCl buffer (pH 7.4) containing
2 mM EDTA, 10 mM CaCl2, 100 mM phenylmethylsulfonylfluoride and 0.1%
bovine serum albumin (BSA)), and served as crude membrane preparation
for binding studies. Binding assays of [125I][Nle4,D-Phe7]a-MSH were per-
formed according to Chaki et al. (2003).27) Membranes were incubated with
[
125I][Nle4,D-Phe7]a-MSH (0.2 nM) for 120 min at 25 °C, and the reaction
was terminated by rapid filtration over a GF/C filter presoaked with 0.5%
The Synthesis of 9, 10 and 20 (Method B). 2-Naphthyl-(CH2)2-CO-
Arg(Z)2-2-Nal-NH2 (8-1) Compound 8-1 was obtained as a solid from

1238
Vol. 55, No. 8
BSA, after which the filters were washed three times with the buffer. Ra-
dioactivity was quantified in a g-counter. Nonspecific binding was deter-
mined in the presence of 1 mM [Nle4,D-Phe7]a-MSH. Specific binding was
determined by subtracting nonspecific from total binding. In the competition
Zeng H., Joseph I. B. J. K., Rodriguez L. E., Maguire M. P., Patane M.
A., Claiborne C. F., J. Med. Chem., 47, 1602—1604 (2004).
22) Marks D. L., Ling N., Cone R. D., Cancer Res., 61, 1432—1438
(2001).
assay, concentration of the test compound that caused 50% inhibition of the 23) Adan R. A., Szklarczyk A. W., Oosterom J., Brakkee J. H., Nijenhuis
specific binding (IC50 value) was determined from each concentration-re-
sponse curve.
W. A., Schaaper W. M., Meloen R. H., Gispen W. H., Eur. J. Pharma-
col., 378, 249—258 (1999).
Determination of cAMP COS-1 cells transiently expressing the MC4 24) Vergoni A. V., Bertolini A., Wikberg J. E., Schioth H. B., Eur. J. Phar-
receptor and grown in a six-well plate were used. The culture medium was macol., 369, 11—15 (1999).
removed, the cells were washed with phosphate buffered saline, and 1 ml of 25) Von Frijtag J. C., Croiset G., Gispen W. H., Adan R. A., Wiegant V.
DMEM containing 1 mM isobutylmethylxanthine, a phosphodiesterase in-
hibitor, was added. The cells were incubated with 10 mM concentration of the
test compound for 15 min at 37 °C. The culture medium was then aspirated
M., Br. J. Pharmacol., 123, 1503—1508 (1998).
26) Chaki S., Ogawa S., Toda Y., Funakoshi T., Okuyama S., Eur. J. Phar-
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and the cells were washed with phosphate buffered saline. Two milliliters of 27) Chaki S., Hirota S., Funakoshi T., Suzuki Y., Suetake S., Okubo T.,
ice-cold 65% ethanol were added, and the cells were scraped from the wells.
The supernatant was collected by centrifugation at 15000 rpm for 15 min at
Ishii T., Nakazato A., Okuyama S., J. Pharmcol. Exp. Ther., 304,
818—826 (2003).
4 °C. cAMP formed in the cells was determined using a commercially avail- 28) Chaki S., Oshida Y., Ogawa S., Funakoshi T., Shimazaki T., Okubo T.,
able cAMP EIA system.
Nakazato A., Okuyama S., Pharmacol. Biochem. Behav., 82, 621—
626 (2005).
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