Synthesis and biological activity of the first cyclic biphalin analogues

Article abstract of DOI:10.1016/j.bmcl.2005.09.080

Biphalin is a linear octapeptide with strong opioid activity. Its structure is based on two identical sequences derived from enkephalins joined C-terminal to C-terminal by an hydrazide bridge (Tyr-D-Ala-Gly-Phe-NH-NH ← Phe ← Gly ← D-Ala ← Tyr). In this st

Full text of DOI:10.1016/j.bmcl.2005.09.080

Bioorganic & Medicinal Chemistry Letters 16 (2006) 367–372
Synthesis and biological activity of the first cyclic
biphalin analogues
Adriano Mollica,a,c Peg Davis,b Shou-Wu Ma,b Frank Porreca,b
Josephine Laib and Victor J. Hrubya,*
aDepartment of Chemistry, University of Arizona, Tucson, AZ 85721, USA
bDepartment of Pharmacology, University of Arizona, Tucson, AZ 85721, USA
cDipartimento di Studi Farmaceutici, Universitaꢀ di Roma ‘‘La Sapienza,’’ P.le A. Moro 5, Rome 00185, Italy
Received 10 August 2005; revised 23 September 2005; accepted 28 September 2005
Available online 3 November 2005
Abstract—Biphalin is a linear octapeptide with strong opioid activity. Its structure is based on two identical sequences derived from
enkephalins joined C-terminal to C-terminal by an hydrazide bridge (Tyr-D-Ala-Gly-Phe-NH-NH Phe Gly D-Ala Tyr).
In this study we present the design, synthesis, and biological evaluation of the first cyclic biphalin analogues. D-Alanine residues in
positions 2, 20 of the parent peptide were replaced by D- and L-cysteine and an intramolecular disulfide bond between the cysteine
thiol groups was introduced. We obtained two cyclic analogues with quite different biological profiles.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Determining structure–activity relationships (SAR) for
peptides is a stepwise process. The first step may involve
many points: (a) replacement of one or more amino acid
residues in the primary sequence of native peptide; (b)
alanine and D-amino acid scans; (c) truncation, deletion
and then evaluation of the biological effects of those
modifications, etc.1 For small linear peptides with flexi-
ble conformations such as enkephalins and their potent
analogues such as biphalin, the design of conformation-
ally restricted analogues is not obvious. Such a family of
compounds forms more compact conformations inter-
acting with the receptor. In this case, the goal is to deter-
mine which residues, sequentially separated, may
approach each other as a result of the peptide–receptor
interaction. Once a hypothesis regarding this has been
made, the two involved residues can be replaced by
cysteine or penicillamine and then the oxidation of their
side chains will form a cyclic peptide with a disulfide
bond (Fig. 1).
Disulfide bonds are present in natural proteins and
peptides and represent a key structural element in mod-
ulating protein tertiary structure. Although many differ-
ent approaches to cyclization could be adopted, the
disulfide based approach was the preferred choice for
the present study.
In an effort to develop highly potent and selective li-
gands based on the endogenous opioid peptide enkeph-
alins (H-Tyr-Gly-Gly-Phe-Leu-OH and H-Tyr-Gly-Gly-
Phe-Met-OH),2a conformationally and topographically
constrained cyclic analogues of enkephalin such as c[D-
Pen2, D-Pen5] enkephalin (DPDPE) were previously de-
signed.2 In this case, the Gly2 and Met5 (or Leu5) resi-
dues were replaced by D-penicillamine and a 14-
membered ring containing a disulfide bond was formed.
DPDPE was found to be highly potent and selective for
Keywords: Biphalin; Cyclic peptides.
Abbreviations: 3H-DAMGO, [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]en-
kephalin; 3H-DPDPE, [3H]-c[2-D-penicillamine,5-D-penicillamine]en-
kephalin; DCM, dichloromethane; DMF, N,N-dimethylformamide;
DMSO, dimethylsulfoxide; EDC, 1-ethyl-(3-dimethyl-aminopro-
pyl)carbodiimide; GPI/LMMP, Guinea pig ileum/longitudinal muscle
myenteric plexus (l opioid receptors); GTP, guanosine triphosphate;
hMOR, human l-opioid receptor; HOBt, 1-hydroxybenzotriazole;
MVD, mouse vas deferens (d-opioid receptors); rDOR, rat d-opioid
receptor; TEA, triethylamine; TFA, trifluoroacetic acid.
H2C
S
S
CH2
N
C
H
CO
peptide
CO
C
H
N
H
H
*
Corresponding author. Tel.: +1 520 621 6332; fax: +1 520 621
8407; e-mail: hruby@u.arizona.edu
Figure 1. Disulfide cyclization.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.080

368
A. Mollica et al. / Bioorg. Med. Chem. Lett. 16 (2006) 367–372
HO
Xaa= Met or Leu
O
O
H
H
N
N
Xaa
H2N
N
H
O
O
Met or Leu Enkefalin
cyclization
OH
H
N
O
H
N
H2N
N
O
H
O
O
HN
S
S
DPDPE
O
HO
Figure 2. Enkephalin and DPDPE.
the d-opioid receptor2d and to be completely stable to
proteolytic breakdown in vitro and in vivo2 (Fig. 2).
However, although a variety of cyclic enkephalin ana-
logues3 have been described, the cyclization approach
has not been applied so far to bivalent analogues such
as biphalin. Due to the entropic factors bound to the
presence of two pharmacophoric moieties in the mole-
cule, bivalent ligands are expected to exhibit biological
potency higher than that of the monomers. Thus, the
synthesis of ligands which combine the favourable prop-
erties of both cyclic and bivalent ligands appears to be a
promising approach.
biphalin heterochiral structure (mixed L and D amino
acids), whereas the Cys containing isomers 7 and 9 pres-
ent the unusual homochiral L-sequence. However, these
new models, at variance with standard cyclopeptides,
present an inversion of the direction of the amide bonds
caused by the hydrazine bridge joining the two Phe res-
idues which behave as gem-diamines in the retro-inverso
peptide isomers. As a consequence of this structural fea-
ture, the two flanking backbone fragments, departing
from the NH–NH moiety of hydrazine, present the same
direction of the peptide bonds (Fig. 4).
Biphalin is a flexible, linear octapeptide (Tyr-D-Ala-
Gly-Phe-NH-NH Phe Gly D-Ala Tyr) which
binds to both l- and d-opioid receptors with an IC50
of about 1–5 nM and exhibits a potent nociceptive effect
and low side-effects, in particular to produce no depen-
dency on chronic use.4c In the light of the above report-
ed data and considerations and taking into account the
interest of obtaining opioid peptides with very high
potency, we report here the design and synthesis of
two 22-membered cyclic analogues of biphalin. They
were obtained by closing a disulfide bridge between
two cysteine residues located in positions 2 and 20 of
the backbone of biphalin as shown in Figure 3. Two dif-
ferent structures were obtained by replacing the D-Ala
residues of biphalin with two D-Cys or two Cys residues.
As a consequence of this design strategy, the D-Cys con-
taining cyclic models 8 and 10 maintain the original
O
O
NH
N
H
HN
O
N
O
H
NH
O
NH
O
HN
S
S
NH
O
O
NH2
HO
NH2
HO
9
O
O
NH
HN
N
H
O
H
O
N
N
H
OH
O
NH
S
Acm
O
NH
O
S
S
O
O
O
O
H
H
HN
H
H
N
N
N
NH2
H2N
N
N
N
N
N
H
NH
H
H
O
O
H
O
O
O
O
O
NH2
HO
H
NH2
N
HO
HO
10
S
Acm
Figure 3. Biphalin. Positions 2, 20 are marked. D-Alanine residues were
substituted with D-cysteine or L-cysteine residues.
Figure 4. Representation of the molecular structure of the final
products 9 and 10.

A. Mollica et al. / Bioorg. Med. Chem. Lett. 16 (2006) 367–372
369
The synthesis of the compounds was performed in solu-
tion following the Na-Boc strategy. Coupling reactions
were performed using the standard method of HOBtÆ-
H2O/EDC/TEA in DMF.5 The symmetric and linear
Na-Boc-protected peptides 5 and 6 were synthesized by
elongation of the amino acid sequence simultaneously
in both directions starting from the hydrazine group
(Scheme 1).
cess iodine, and then the solvent was evaporated in high
vacuum. The orange oily residue was charged into a
semi-preparative HPLC column and the pure Na-Boc-
protected products were collected and lyophilized (yield
40% ca.).6
Deprotection of the tert-butyloxycarbonyl groups (Boc)
was performed by dissolving the products in a mixture
of TFA 50% in DCM for 30 min at rt, under nitrogen
atmosphere. The DCM and the TFA were removed under
vacuum. The resultant intermediate products were used in
the next step without further purification as TFA salts.
The acetamidomethyl (Acm) group was adopted since it
offers an orthogonal protection with the Boc group and
allows, at the same time, a one-pot deprotection-oxida-
tion reaction. Cyclization was obtained by dissolving the
pure Na-Boc, S-Acm-protected linear peptides 5 and 6
(100 mg) in 350 mL of DMSO/water/acetic acid (1:2:3).
I2 (51 mg) was added to the stirred solution at rt. After
3 h, ascorbic acid (60 mg) was added to quench the ex-
All the Na-Boc-protected intermediate products 1–8 were
purified by reverse-phase semi-preparative high-perfor-
mance liquid chromatography (HPLC), isolated and
characterized by mass spectra and NMR. The final prod-
Boc-Phe
a
O
O
O
H
H
b
H
N
H
N
H
N
N
H
Boc
Boc
N
Boc
N
N
N
N
H
H
Boc
N
H
H
O
O
O
2
1
d
c
O
O
NH
NH
S
S
O
O
H
N
H
H
N
H
N
N
Boc
N
H
N
H
Boc
N
H
N
H
O
O
O
O
3
4
2
2
e
e
O
O
NH
NH
S
S
O
O
O
O
H
H
H
N
H
N
H
N
H
Boc
N
N
Boc
N
N
N
N
N
H
H
H
H
O
O
O
O
6
5
OH
OH
2
2
Scheme 1. Reagents and conditions: (a) Boc-Phe-OH (2.2 equiv), EDC (2.2 equiv), HOBtÆH2O (2.2 equiv), TEA (3.2 equiv), NH2 NH2 (1 equiv),
DMF (20 mL), 30 min 0 ꢁC, then 12 h rt; (b) TFA 50 % in DCM 30 min rt under N2 atm, then Boc-Gly-OH (2.2 equiv), EDC (2.2 equiv), HOBtÆH2O
(2.2 equiv), TEA (3.2 equiv), DMF (20 mL), 30 min 0 ꢁC, then 12 h rt; (c) TFA 50% in DCM, 30 min rt under N2 atm, then Boc-D-Cys(Acm)-OH
(2.2 equiv), EDC (2.2 equiv), HOBtÆH2O (2.2 equiv), TEA (3.2 equiv), DMF (20 mL), 30 min 0 ꢁC, then 12 h rt; (d) TFA 50% in DCM, 30 min rt
under N2 atm, then Boc-Cys(Acm)-OH (2.2 equiv), EDC (2.2 equiv), HOBtÆH2O (2.2 equiv), TEA (3.2 equiv), DMF (20 mL), 30 min 0 ꢁC, then 12 h
rt; (e) TFA 50% in DCM, 30 min rt under N2 atm, then Boc-Tyr-OH (2.2 equiv), EDC (2.2 equiv), HOBtÆH2O (2.2 equiv), TEA (3.2 equiv), DMF
(20 mL), 30 min 0 ꢁC, then 12 h rt (overall yield 50–60%).

370
A. Mollica et al. / Bioorg. Med. Chem. Lett. 16 (2006) 367–372
ucts 9 and 10 were purified as TFA salts by reverse-phase
HPLC using a semi-preparative Vydac (C18-bonded,
˚
300 A) column and a gradient elution at a flow rate of
tion cocktail. The data were analyzed using GraphPad
Prism Software (San Diego, CA).
10 mL/min. The gradient used was 10–90% acetonitrile
in 0.1% aq TFA over 50 min. Approximately 20 mg of
crude peptide was injected each time, and the fractions
containing the purified peptide were collected and lyoph-
ilized to dryness. The purity, determined by NMR analy-
sis and by analytic reverse-phase HPLC, monitored at
220, 254, 280 and 350 nm, was found to be >98%.
The in vitro tissue bioassays (MVD and GPI/LMMP)
were performed as described previously.9 IC50 values rep-
resent means of no less than four experiments. IC50 val-
ues, relative potency estimates and their associated
standard errors were determined by fitting the data to
the Hill equation by a computerized non-linear least-
squares method. All biological data are summarized in
Table 1.
In order to confirm the molecular structures, all NMR
spectra were acquired on a Bruker DRX-500 spectrom-
eter at 25 ꢁC using a Nalorac triple-resonance single-axis
gradient 3-mm probe, processed with the Bruker soft-
ware XWINNMR. NMR analyses of the products were
performed in DMSO-d6 at a peptide concentration of
2 mM ca. (for analytical data, see Ref. 10).
In conclusion, we have synthesized the first examples of
cyclic analogues of biphalin. Cyclization of peptides is,
in fact, a useful approach to develop diagnostic and
therapeutic peptidic and peptidomimetic agents. The
new cyclic ligands show high in vitro bioactivity (Table
1). As expected, compound 10, containing D-Cys at the
positions 2 and 20 in place of D-Ala residues of biphalin,
reveals binding affinity and bioactivity higher than those
of the product 9 built with Cys. Although the binding
value of isomer 10 is close to that of biphalin, the
GTP binding and Emax are surprisingly high. In particu-
lar, compound 10 shows a capacity to activate the trans-
duction to the d receptor (Emax = 100%) higher than that
to the l receptor (Emax = 47%).
Receptor binding affinities to the d- and l-opioid recep-
tors were performed using cell membrane preparations
from transfected cells that stably express the respective
receptor type and were evaluated as previously de-
scribed.7 The ligands used were [3H]DPDPE and
[3H]DAMGO for d and l receptors, respectively.
We used [35S]GTP-c-S binding to examine opioid agonist
efficacy, for functional characterization of the ligands at
the d- and l-opioid receptors, which are members of the
seven transmembrane G-protein-coupled receptor super
family. Agonist efficacy can be determined at the level of
receptor G-protein interaction by measuring agonist-
stimulated binding with a non-hydrolysable GTP ana-
logue. The ability of l and d opioid agonists to activate
G-proteins has been demonstrated by studying the bind-
ing of the GTP analogue guanosine-50-O-(3-[35S]thio)tri-
phosphate ([35S]GTP-c-S). The opioid receptor mediated
assay was performed as previously described.8 Cells
expressing hDOR for d receptor (or rMOR for l receptor)
were incubated with increasing concentrations of the test
compounds in the presence of 0.1 nM [35S]GTP-c-S
(1000–1500 Ci/mmol, MEN, Boston, MA) in assay buffer
(total volume of 1 mL, duplicate samples) as a measure of
agonist-mediated G-protein activation. After incubation
(90 min, 30 ꢁC), the reaction was terminated by rapid fil-
tration under vacuum through Whatman GF/B glass fibre
filters, followed by four washes with ice-cold 15 mM Tris/
120 mM NaCl, pH 7.4. Filters were pretreated with assay
buffer prior to filtration to reduce non-specific binding.
Bound reactivity was measured by liquid scintillation
spectrophotometry after an overnight extraction with
EcoLite (ICN, Biomedicals, Costa Mesa, CA) scintilla-
This is in contrast with its binding values which are very
similar for both the receptors. The analysis of the data
indicates that biphalin and compound 9 are partial ago-
nists at both l and d receptors. Compound 10 is a full
agonist at the d receptor and a partial agonist at the l
receptor. Its ability to partially stimulate the l-opioid
receptor is also appreciable by taking into account the
fact that the high biphalin analgesic activity seems to
be related to its ability to bind both d- and l-opioid
receptors.4c Furthermore, although a certain degree of
selectivity is observed for both the isomers 9 and 10, this
is certainly low as already found in the case of biphalin.
In summary, the in vitro studies reported here are cer-
tainly promising. However, the evaluation of the thera-
peutic interest bound to compound 10 high ability to
activate the transduction mechanism requires further
in vivo studies.
Acknowledgment
This work was supported by a grant from the U.S., Pub-
lic Health Service, National Institute of Drug Abuse,
DA 06284.
Table 1. Binding affinity, GTP binding assay, Emax % (net total bound/basal binding · 100), and in vitro activity
a
Binding IC50 (nM)
a,b
GTP binding IC50 (nM)
a
Bioassay IC50 (nM)
Drugs
d
l
d
Emax (%)
l
Emax (%)
MVD GPI/LMMP
Biphalin
2.6 0.4c
53 10.5
0.87 0.1
1.4 0.2c
2.5 0.5
260 100
0.87 0.3
27 3.5
58 18
100 2.3
6
0.2
25 4.7
57 2.2
47 5.7
27 1.5c
570 130
9.9 1.3
8.8 0.3c
420 48
25 7.9
9
10
130 230
0.60 0.2
120 34
0.2 0.1
a
SEM.
b Reference compound: cold [35S]GTP-c-S.
c Data according to Ref. 9.

A. Mollica et al. / Bioorg. Med. Chem. Lett. 16 (2006) 367–372
371
NMR d: 1.40 (18H, s, Boc), 2.80–3.05 (4H, m, Phe
bCH2), 3.44–3.59 (4H, m, Gly aCH2), 4.61 (2H, m, Phe
aCH), 6.86 (2H, m, Gly NH), 7.18–7.27 (10H, m, Ar), 8.03
(2H, d, Phe NH), 10.18 (2H, s, NH–NH). FAB-MS m/e
641.0 (M+). Boc-Cys(Acm)-Gly-Phe-NH-NH Phe -
Gly Cys(Acm)-Boc (4): 1H NMR d: 1.40 (18H, s, Boc),
1.85 (6H, s, CH3-CO), 2.65 and 2.88 (4H, m, Cys bCH2),
2.75 and 3.08 (4H, m, Phe bCH2), 3.65–3.78 (4H, m, Gly
aCH2), 4.18–4.28 (6H, m, Acm CH2 and Cys aCH), 4.60
(2H, m, Phe aCH), 6.96 (2H, d, Cys NH) 7.18–7.31 (10H,
m, Ar), 7.95 (2H, m, Gly NH), 8.16 (2H, d, Phe NH), 8.45
(2H, m, Acm NH), 10.18 (2H, s, NH–NH). FAB-MS m/e
989.0 (M+). Boc-Tyr-Cys(Acm)-Gly-Phe-NH-NH
Phe Gly Cys(Acm) Tyr- Boc (6): 1H NMR d:
1.30 (18H, s, Boc), 1.82 (6H, s, CH3CO), 2.63 and 2.89
(4H, m, Tyr bCH2), 2.74 and 2.90 (4H, m, Cys bCH2),
2.81–3.05 (4H, m, Phe bCH2), 3.65–3.80 (4H, m, Gly
aCH2), 4.12 (4H, m, Acm CH2), 4.15 (2H, m, Tyr aCH),
4.51 (2H, m, Cys aCH), 4.62 (2H, m, Phe aCH), 6.62 and
7.05 (8H, dd, Tyr aromatic), 6.82 (2H, m, Tyr NH), 7.15–
7.32 (10H, m, Ar), 8.07–8.12 (4H, m, Gly NH and Cys
NH), 8.20 (2H, d, Phe NH), 8.46 (2H, m, Acm NH), 9.12
(2H, br, Tyr OH), 10.22 (2H, s, NH–NH). FAB-MS m/e
1315.0 (M+). Boc-Tyr-Cys(Acm)-Gly-Phe-NH-NH
Phe Gly Cys(Acm) Tyr- Boc (6): 1H NMR d:
1.30 (18H, s, Boc), 1.82 (6H, s, CH3CO), 2.63 and 2.89
(4H, m, Tyr bCH2), 2.74 and 2.90 (4H, m, Cys bCH2),
2.81–3.05 (4H, m, Phe bCH2), 3.65–3.80 (4H, m, Gly
aCH2), 4.12 (4H, m, Acm CH2), 4.15 (2H, m, Tyr aCH),
4.51 (2H, m, Cys aCH), 4.62 (2H, m, Phe aCH), 6.62 and
7.05 (8H, dd, Tyr aromatic), 6.82 (2H, m, Tyr NH), 7.15–
7.32 (10H, m, Ar), 8.07–8.12 (4H, m, Gly NH and Cys
NH), 8.20 (2H, d, Phe NH), 8.46 (2H, m, Acm NH), 9.12
(2H, br, Tyr OH), 10.22 (2H, s, NH–NH). FAB-MS m/e
1315.0 (M+). Boc-Tyr-c[Cys-Gly-Phe-NH-NH Phe -
Gly Cys] Tyr-Boc (7): 1H NMRd: 1.32 (18H, s, Boc),
2.60 and 2.85 (4H, m, Tyr bCH2), 2.78 and 3.10 (4H, m,
Cys bCH2), 2.86–3.08 (4H, m, Phe bCH2), 3.55–3.70 (4H,
m, Gly aCH2), 4.15 (2H, m, Tyr aCH), 4.55 (4H, m, Phe
aCH and Cys aCH), 6.62 and 7.05 (8H, dd, Tyr aromatic),
6.76 (2H, m, Tyr NH), 7.18–7.32 (10H, m, Ar), 8.07 (2H,
d, Phe NH), 8.15 (2H, d, Cys NH), 8.25 (2H, br, Gly NH),
9.15 (2H, br, Tyr OH), 10.18 (2H, s, NH–NH). FAB-MS
m/e 1193.3 (M+ Na). TFAÆTyr-c[Cys-Gly-Phe-NH-
NH Phe-Gly] Cys-TyrÆTFA (9): 1H NMR d: 1.78
and 3.15 (4H, m, Cys bCH2), 2.80 and 3.05 (4H, m, Tyr
bCH2), 2.83 and 3.05 (4H, m, Phe bCH2), 3.50 and 3.81
(4H, m, Gly aCH2), 3.98 (2H, br, Tyr aCH), 4.55 (4H, m,
Phe aCH and Cys aCH), 6.62 and 7.05 (8H, dd, Tyr
aromatic), 7.18–7.32 (10H, m, Ar), 8.03 (2H, br, Tyr NH),
8.05 (2H, d, Phe NH), 8.40 (2H, m, Gly NH), 8.83 (2H, d,
Cys NH), 9.35 (2H, s, Tyr OH), 10.10 (2H, s, NH–NH).
FAB-HRMS m/e 971.3472 (M+). Boc-D-Cys(Acm)-Gly-
Phe-NH-NH Phe Gly D-Cys(Acm)-Boc (3): 1H
NMR d: 1.35 (18H, s, Boc), 1.85 (6H, s, CH3CO), 2.64
and 2.88 (4H, m, Cys bCH2), 2.80 and 3.08 (4H, m, Phe
bCH2), 3.60–3.88 (4H, m, Gly aCH2), 4.12–4.28 (6H, m,
Acm CH2 and Cys aCH), 4.62 (2H, m, Phe aCH), 6.98
(2H, d, Cys NH) 7.19–7.30 (10H, m, Ar), 7.93 (2H, m, Gly
NH), 8.20 (2H, d, Phe NH), 8.48 (2H, m, Acm NH), 10.22
(2H, s, NH–NH). FAB-MS m/e 988.9 (M+). Boc-Tyr-D-
Cys(Acm)-Gly-Phe-NH–NH Phe Gly D-Cys-
(Acm) Tyr-Boc (5): 1H NMR d: 0.30 (18H, s, Boc), 1.82
(6H, s, CH3CO), 2.63 and 2.89 (4H, m, Tyr bCH2), 2.69
and 2.89 (4H, m, Cys b CH2), 2.81–3.06 (4H, m, Phe
bCH2), 3.62–3.82 (4H, m, Gly aCH2), 4.16–4.28 (6H, m,
Acm CH2 and Tyr aCH), 4.55 (2H, m, Cys aCH), 4.62
(2H, m, Phe aCH), 6.63 and 7.05 (8H, dd, Tyr aromatic),
6.80 (2H, m, Tyr NH), 7.15–7.32 (10H, m, Ar), 8.07 (2H,
References and notes
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10. NMR and MS data: Boc-Phe-NH-NH Phe-Boc (1): 1H
NMR d: 1.27 (18H, s, Boc), 2.76–3.05 (4H, m, Phe bCH2),
4.26 (2H, m, Phe aCH), 6.95 (2H, d, Phe NH), 7.19–7.38
(10H, m, Ar), 10.15 (2H, s, NH–NH). FAB-MS m/e 527.1
(M+). Boc-Gly-Phe-NH-NH Phe Gly-Boc (2): 1H

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A. Mollica et al. / Bioorg. Med. Chem. Lett. 16 (2006) 367–372
m, Gly NH), 8.22 (4H, m, Phe NH and Cys NH), 8.48
(2H, m, Acm NH), 9.12 (2H, br, Tyr Tyr OH), 10.22 (2H,
Tyr OH), 10.05 (2H, s, NH–NH). FAB-MS m/e 1170.8
(M+).
TFAÆTyr-c[D-Cys-Gly-Phe-NH–NH Phe
s, NH–NH). FAB-MS m/e 1314.9 (M+). Boc-Tyr-c[D-Cys-
Gly D-Cys] TyrcdotTFA (10): 1H NMR d: 1.79
and 3.15 (4H, m, Cys bCH2), 2.80 and 3.05 (4H, m, Tyr
bCH2), 2.85 and 3.05 (4H, m, Phe bCH2), 3.55 and 4.05
(4H, m, Gly a CH2), 3.95 (2H, m, Tyr aCH), 4.60 (2H, m,
Phe aCH), 4.68 (2H, m, Cys aCH), 6.65 and 7.08 (8H, dd,
Tyr aromatic), 7.18–7.30 (10H, m, Ar), 7.90 (2H, d, Phe
NH), 7.97 (2H, br, Tyr NH), 8.68 (2H, br, Gly NH), 8.94
(2H, d, Cys NH), 9.34 (2H, s, Tyr OH), 10.15 (2H, s, NH–
NH). FAB-HRMS m/e 971.3471 (M+).
Gly-Phe-NH-NH Phe-Gly D-Cys] Tyr-Boc
(8)
1H NMR d: 0.28 (18H, s, Boc), 2.65 and 2.89 (4H, m,
Tyr bCH2), 2.81–3.04 (4H, m, Cys bCH2), 2.87 and 3.14
(4H, m, Phe bCH2), 3.35 and 3.88 (4H, m, Gly aCH2),
4.12 (2H, m, Tyr aCH), 4.58 (4H, m, Cys aCH and Phe
aCH), 6.62 and7.05 (8H, dd, Tyr aromatic), 6.80 (2H, m,
Tyr NH), 7.15–7.30 (10H, m, Ar), 7.81 (2H, d, Phe NH),
8.32 (2H, d, Cys NH), 8.46 (2H, br, Gly NH), 9.15 (2H, br,