New potent biphalin analogues containing p-fluoro-L-phenylalanine at the 4,4′ positions and non-hydrazine linkers

Article abstract of DOI:10.1007/s00726-010-0760-7

We report the synthesis and the biological evaluation of two new analogues of the potent dimeric opioid peptide biphalin. The performed modification is based on the replacement of two key structural elements of the native biphalin, namely: the hydrazine b

Full text of DOI:10.1007/s00726-010-0760-7

Amino Acids (2011) 40:1503–1511
DOI 10.1007/s00726-010-0760-7
ORIGINAL ARTICLE
New potent biphalin analogues containing
p-fluoro-L-phenylalanine at the 4,40 positions
and non-hydrazine linkers
•
Adriano Mollica Francesco Pinnen Federica Feliciani Azzurra Stefanucci
•
•
•
•
•
•
•
•
Gino Lucente Peg Davis Frank Porreca Shou-Wu Ma Josephine Lai
Victor J. Hruby
Received: 23 July 2010 / Accepted: 21 September 2010 / Published online: 6 October 2010
Ó Springer-Verlag 2010
Abstract We report the synthesis and the biological
evaluation of two new analogues of the potent dimeric
opioid peptide biphalin. The performed modification is
based on the replacement of two key structural elements of
the native biphalin, namely: the hydrazine bridge which
joins the two palindromic moieties and the phenylalanine
residues at the 4,40 positions of the backbone. The new
analogues 9 and 10 contain 1,2-phenylenediamine and
piperazine, respectively, in place of the hydrazidic linker
and p-fluoro-L-phenylalanine residues at 4 and 40 positions.
Binding values are: Kil ¼ 0:51 nM and Kid ¼ 12:8 nM for
Abbreviations
Boc
tert-Butyloxycarbonyl
[3H]DAMGO [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]
enkephalin
[3H]DPDPE
DCM
[3H]-c[D-Pen2, D-Pen5]enkephalin
Dichloromethane
DIPEA
DMAP
DMF
N,N-Diisopropylethylamine
4-(Dimethylamino)pyridine
N,N-Dimethyl formamide
Dimethylsulfoxide
DMSO
EDC
1-Ethyl-(3-
compound 9, Kil ¼ 0:09 nM and Kid ¼ 0:11 nM for ana-
logue 10.
dimethylaminopropyl)carbodiimide
Guinea pig ileum/longitudinal muscle
myenteric plexus (l-opioid receptors)
Human l-opioid receptor
1-Hydroxybenzotriazole
Mouse vas deferens (d-opioid receptors)
N-Methyl morpholine
GPI/LMMP
hMOR
HOBt
MVD
NMM
rDOR
TEA
Keywords Activity ꢀ Biphalin ꢀ Dimeric peptide ligands ꢀ
Opioid peptides ꢀ Synthesis
Rat d-opioid receptor
Triethylamine
TFA
Trifluoroacetic acid
A. Mollica (&) ꢀ F. Pinnen ꢀ F. Feliciani ꢀ A. Stefanucci
Dipartimento di Scienze del Farmaco,
`
Universita di Chieti-Pescara ‘‘G. d’Annunzio’’,
Via dei Vestini 31, 66100 Chieti, Italy
e-mail: a.mollica@unich.it
Introduction
G. Lucente
Istituto di Chimica Biomolecolare (CNR) c/o Dipartimento di
Chimica e Tecnologie del Farmaco, ‘‘Sapienza’’,
The opioid pentapeptide enkephalins (Tyr-Gly-Gly-Phe-
Met and Tyr-Gly-Gly-Phe-Leu) were originally isolated
from pig and cow brain (Hughes et al. 1975). Enkephalins
are endogenous d ligands of the opioid multiple receptor
family located in neuronal cell membranes which express
the extensively studied l-, d- and j-opioid receptors. As
expected for simple linear oligopeptides, enkephalins are
highly sensitive to enzymatic degradation and, in addition
`
Universita di Roma, P.le A.Moro, 00185 Rome, Italy
P. Davis ꢀ F. Porreca ꢀ S.-W. Ma ꢀ J. Lai
Department of Pharmacology, University of Arizona,
Tucson, AZ 85719, USA
V. J. Hruby
Department of Chemistry and Biochemistry,
University of Arizona, Tucson, AZ 85721, USA
123

1504
A. Mollica et al.
to this, exhibit low receptor binding selectivity and mini-
mal capacity to cross the phospholipid bilayer (Hruby and
Gehrig 1989). Generally unfit as therapeutic agents, native
enkephalins continue to represent the reference structural
model for studies on recognition and binding mechanisms
of peptide ligands as well as for the development of
secondary effect-free analgesics to be used in place of
morphine.
morphine after i.c.v. and i.t. administration (Lipkowski
et al. 1987; Horan et al. 1993) and less physical depen-
dence than morphine (Horan et al. 1993; Yamazaki et al.
2001). Consequently, biphalin continues to be the object of
chemical modifications and structural studies aimed at
further improving the activity as well as to better under-
stand the molecular bases of its interaction with opioid
receptors.
Modification of the 4,40 residues of biphalin, by sym-
metrical incorporation of para-fluoro or para-nitro phen-
ylalanine residues (see Fig. 1), leads to analogues which
show, although to different degrees, enhancement of
affinity towards d- and l-opioid receptors accompanied by
an increase of d/l selectivity (Misicka et al. 1997; Li et al.
1998). Recently, Hruby and co-workers reported on a series
of biphalin analogues in which the –NH–NH– linker is
replaced by different non-hydrazidic bridges, namely the
residues of 1,4-phenylenediamine, 1,2-phenylenediamine
and piperazine (see Fig. 1, compounds 9a and 10a) (Mol-
lica et al. 2005). These new analogues show better affinity
and in vitro bioactivity than biphalin itself, thus suggesting
that the high activity of biphalin is not critically related to
the structural and conformational properties of the hydra-
zide bridge nor to its capability to form hydrogen bonds
with the opioid receptors.
Based on extensive SAR studies, a variety of synthetic
strategies has been adopted to modify the enkephalin native
structure and most of the relevant opioid peptide ligands so
far described are enkephalin-based. Among consolidated
findings in this field is the role exerted on enkephalin
activity by the phenylalanine residue in position 4. It has
been shown that the aromatic residue at position 4 can
significantly modulate binding to the l- and d-opioid
receptors (Morgan et al. 1976; Chang et al. 1976; Schiller
et al. 1978). Modification of this residue, by introducing
substituents at the para position of the aromatic ring, sig-
nificantly enhances the activity with the highest effect
shown by the introduction of electron-drawing groups
(Schiller et al. 1983). Moreover, it has been found that the
positive consequences of the modification performed at the
Phe4 aromatic ring of enkephalins are maintained in the case
of the highly active synthetic octapeptide biphalin
(Tyr-D-Ala-Gly-Phe-NH-NH\-Phe\-Gly\-D-Ala\-Tyr; Fig. 1;
Abbruscato et al. 1996; Li et al. 1998; Misicka et al. 1999).
Biphalin represents one of the most successful applica-
tions of the dimeric ligand approach to the design of native
bioactive peptide analogues (Coy et al. 1976; Lipkowski
et al. 1982; Shimohigashi et al. 1982; Horan et al. 1993). Its
structure is characterized by two enkephalin-derived bio-
active fragments joined ‘‘tail to tail’’ by a hydrazide bridge.
The presence of two pharmacophores, combined with an
appropriate linker, confers on biphalin highly affinity for
both d- and l-receptors, a potency significantly higher than
With the aim to investigate the effects of the para
substitution at the Phe4 aromatic ring combined with
hydrazine bridge replacements, we report here synthesis
and in vitro assays of two new fluorinated biphalin ana-
logues 9 and 10 containing non-hydrazine linkers.
Chemistry
In the present study, the previously adopted strategy for the
synthesis of non-hydrazine biphalin analogues has been
HO
O
R
O
O
H
O
H
H
N
N
N
H
H2N
N
NH2
N
N
H
N
H
N
H
H
O
O
O
O
R
OH
R = H biphalin
R = F [pF-Phe 4,4'] biphalin
HO
O
O
O
O
O
O
H
O
O
H
H
H
H
H
N
N
N
H
N
H2N
N
N
H2N
N
N
N
NH2
NH2
N
N
H
N
N
N
H
N
H
N
H
H
H
H
O
O
O
O
O
O
O
O
OH
HO
9a
OH
10a
Fig. 1 Structure of biphalin (Bph) and its previously studied 4,40pF-Phe analogue (pF-Bph) (Misicka et al. 1997). For the sake of clarity, the
structure of the non-hydrazine analogues 9a and 10a (Mollica et al. 2005) is also illustrated
123

New potent biphalin analogues
1505
modified. As illustrated in Scheme 1, the amino acid resi-
dues were symmetrically added one by one to the linkers.
This procedure allows an easier isolation and character-
ization of the intermediate products and avoids possible
racemization connected with alkaline hydrolysis of the
intermediate tripeptide ester Boc-Tyr-D-Ala-Gly-OEt.
aromatics]; 7.25 [2H, d, Phe NH]. 13C NMR (CDCl3,
300 MHz) (d, ppm): 28.5, 39.6, 41.6, 45.2, 51.1, 80.3, 115.8,
131.2, 132.1, 155.2, 160.5, 170.3. HRMS (ESI) calcd. for
C32H43F2N4O6 m/z: 617.3151 [M ? H]?; found 617.3155.
(Boc-pF-Phe)2-1,2-phenylenediamine (2)
General procedures
EDCꢀHCl (2.2 eq.), HOBt (2.2 eq.) and DIPEA (6.6 eq.)
were added to a solution of Boc-pF-Phe-OH (2.2 eq.) in
DMF at 0°C. The reaction mixture was stirred for 10 min,
1,2-phenylenediamine (1 eq.) was added, the reaction was
stirred for an additional 10 min at 0°C and then allowed to
warm at r.t. overnight. The solvent was evaporated under
reduced pressure, the residue was precipitated with EtOAc,
and the suspension was filtered through a Buchner funnel
under reduced pressure. The solid residue was washed with
three portions of 5% citric acid, NaHCO3 s.s., brine and
distilled water. Then the solid was dried under reduced
pressure and triturated with diethyl ether to give the desired
product 2 as a crude white solid. RIf ¼ 0:40 (CH2Cl2/
AcOEt 8:2), RIfI ¼ 0:33 (CHCl3/CH3OH 98:2). The product
was purified by silica gel column chromatography
(CH2Cl2/AcOEt 95:5 to CH2Cl2/AcOEt 7:3) to obtain the
pure product 2 (41%). 1H NMR (DMSO-d6, 300 MHz)
(d, ppm): 1.31 [9H, s, (CH3)3–]; 2.97–2.76 [4H, m, Phe
b-CH2]; 4.24 [2H, m, Phe a-CH]; 7.40–6.50 [12H, m,
aromatics]; 6.95 [2H, d, Phe NH]; 9.46 and 9.23 [2H, two
singlets, Ar NH]. 13C NMR (CDCl3, 300 MHz) (d, ppm):
28.5, 37.4, 56.5, 80.8, 115.8, 126.8, 130.3, 131.1, 132.4,
155.8, 160.5, 170.7. HRMS (ESI) calcd. for C34H41F2N4O6
m/z: 639.2994 [M ? H]?; found 639.2991.
All products were synthesized in solution using the EDC/
HOBt/DIPEA coupling method (Scheme 1). The Na ter-
minal Boc-protected peptides were all deprotected by a
mixture of TFA in DCM 1:1 at r.t. The intermediate TFA
salts were used for subsequent reactions without further
purification. Intermediate products 3 and 5 were purified by
precipitation in EtOAc, the filtrate was washed on a
Buchner funnel with 39 50 mL of NaHCO3 s.s., 39 50 mL
of 5% citric acid, 39 50 mL of deionized water, dried
under vacuum, followed by trituration with diethyl ether
and used in the next step without further purification.
Products 1, 2, 4 and 6 were purified by silica gel column
chromatography. Final products 7, 8, 9 and 10 were puri-
fied by RP-HPLC using a Waters XBridgeTM Prep BEH130
C18, 5.0 lm, 250 mm 9 10 mm column, HPLC solvent A,
0.1% TFA in water; solvent B, acetonitrile; gradient:
5–95% B in A over 45 min, flow rate 5.0 mL/min.
The purity of the Na-Boc-protected products and the
final TFA salts was assessed by analytical RP-HPLC, TLC
confirmed by NMR analysis and mass spectrometry ESI-
HRMS (see Table 1).
(Boc-pF-Phe)2-piperazine (1)
(Boc-Gly-pF-Phe)2-piperazine (3)
EDCꢀHCl (2.2 eq.), HOBt (2.2 eq.) and DIPEA (6.6 eq.)
were added to a solution of Boc-pF-Phe-OH (2.2 eq.) in
DMF at 0°C. The reaction mixture was stirred for 10 min,
piperazine (1 eq.) was added and the reaction was stirred
for an additional 10 min at 0°C and then allowed to warm
at r.t. overnight. The solvent was evaporated under reduced
pressure, the residue was precipitated with EtOAc and the
suspension was filtered through a Buchner funnel under
reduced pressure. The solid residue was washed with three
portions of 5% citric acid, NaHCO3 s.s., brine and distilled
water. The solid was dried under reduced pressure and
triturated with diethyl ether to give the desired product 1 as a
crude white solid. RIf ¼ 0:27 (CH2Cl2/AcOEt 8:2), RIfI ¼
0:71 (CH2Cl2/AcOEt 1:1). The product was purified by silica
gel column chromatography (CH2Cl2/AcOEt 8:2 to CH2Cl2/
Product 1 was deprotected at the Na terminal by TFA in
DCM 1:1 using 1 mL of mixture per 100 mg of Boc-pro-
tected product for 1.5 h at r.t. The mixture was then
evaporated under high vacuum and the TFA salt was used
for the next step without further purification. EDCꢀHCl
(2.2 eq.), HOBt (2.2 eq.) and DIPEA (6.6 eq.) were added
to a solution of Boc-Gly-OH (2.2 eq.) in DMF at 0°C. The
reaction mixture was stirred for 10 min, TFAꢀpF-Phe-Pip-
pF-PheꢀTFA (1 eq.) was added, the reaction was stirred for
an additional 10 min at 0°C then allowed to warm at r.t.
overnight. The solvent was evaporated under reduced
pressure, the residue was precipitated with EtOAc and the
suspension was filtered through a Buchner funnel under
reduced pressure. The solid residue was washed with three
portions of 5% citric acid, NaHCO3 s.s., brine and distilled
water. The solid was dried under reduced pressure and
triturated with diethyl ether to yield the desired product 3
as pure white solid (82%). RIf ¼ 0:52 (AcOEt), RfII ¼ 0:61
1
EtOAc 3:7) to obtain the pure product 1 (92%). H NMR
(DMSO-d6, 300 MHz) (d, ppm): 1.32 [9H, s, (CH3)3–];
2.78–2.70 [4H, m, Phe b-CH2]; 3.41–3.26 [8H, m, –CH2–
CH2–]; 4.55 [2H, m, Phe a-CH]; 7.30–7.07 [8H, m,
123

1506
A. Mollica et al.
F
OH
N
H
Boc
O
a
e
F
F
F
O
H
N
Boc
N
N
H
H
Boc
N
H
N
N
Boc
Boc
N
H
N
O
H
O
O
1
F
2
b
b
F
F
F
O
O
H
H
N
O
O
Boc
N
N
N
N
H
Boc
H
H
H
H
N
H
Boc
N
N
N
N
Boc
O
N
N
O
H
H
O
O
F
3
4
c
c
F
F
F
O
O
O
H
H
H
N
N
Boc
O
N
N
N
N
O
Boc
H
H
H
N
H
N
H
N
H
N
N
N
H
O
Boc
N
H
N
H
O
O
N
H
Boc
N
H
O
O
O
O
F
5
6
d
d
HO
O
F
F
F
O
O
H
O
H
H
H
N
N
Boc
N
N
N
N
N
N
Boc
O
N
O
N
H
O
O
H
H
H
N
H
H
H
H
H
H
O
O
Boc
N
N
N
O
O
N
N
Boc
N
N
N
N
H
H
H
H
O
O
O
F
O
OH
7
OH
8
HO
f
f
HO
O
F
F
F
O
O
O
H
H
H
N
N
H2N
N
N
N
NH2
N
N
H
N
H
O
H
O
O
O
O
O
H
N
H
H
H
O
O
H2N
N
N
N
NH2
N
N
N
H
N
H
H
H
F
O
O
O
2.TFA
O
OH
10
OH
9
HO
2.TFA
Scheme 1 Synthesis of the fluorinated biphalin analogues with non-
hydrazine linker 9 and 10. Reagents and conditions: a EDCꢀHCl
(2.2 eq.), HOBt (2.2 eq.), DIPEA (6.6 eq.), Boc-pF-Phe-OH (2.2 eq.),
piperazine (1 eq.) in DMF at r.t. overnight; b TFA/CH2Cl2 1:1, r.t.
(1.5 h) then EDCꢀHCl (2.2 eq.), HOBt (2.2 eq.), DIPEA (6.6 eq.), Boc-
Gly-OH (2.2 eq.), in DMF at r.t. overnight; c TFA/CH2Cl2 1:1, r.t.
(1.5 h) then EDCꢀHCl (2.2 eq.), HOBt (2.2 eq.), DIPEA (6.6 eq.), Boc-
D-Ala-OH (2.2 eq.) in DMF at r.t. overnight; d TFA/CH2Cl2 1:1, r.t.
(1.5 h) then EDCꢀHCl (2.2 eq.), HOBt (2.2 eq.), DIPEA (6.6 eq.), Boc-
Tyr-OH (2.2 eq.) in DMF at r.t. overnight; e EDCꢀHCl (2.2 eq.), HOBt
(2.2 eq.), DIPEA (6.6 eq.), Boc-pF-Phe-OH (2.2 eq.), 1,2-phenylene-
diamine (1 eq.) in DMF at r.t. overnight. f TFA/CH2Cl2 1:1, r.t. (1.5 h)
123

New potent biphalin analogues
1507
Table 1 Structures and the physicochemical properties of the biphalin analogues 9–10 and N-Boc derivatives 1–8
No.
Structures
m/z [M ? H]? (ESI)
RP-HPLCa, retention
time (min)
Calcd
Found
617.3155
1
2
3
4
5
6
7
8
9
10
a
(Boc-pF-Phe)2-piperazine
617.3151
639.2994
731.3580
753.3426
873.4322
895.4166
1,199.5589
1,221.5432
1,021.4384
999.4540
39.50
43.79
32.91
39.49
30.74
37.46
29.35
34.15
22.73
20.98
(Boc-pF-Phe)2-1,2-phenylenediamine
639.2991
731.3588
753.3425
873.4318
895.4160
1,199.5586
1,221.5435
1,021.4381
999.4542
(Boc-Gly-pF-Phe)2-piperazine
(Boc-Gly-pF-Phe)2-1,2-phenylenediamine
(Boc-D-Ala-Gly-pF-Phe)2-piperazine
(Boc-D-Ala-Gly-pF-Phe)2-1,2-phenylenediamine
(Boc-Tyr-D-Ala-Gly-pF-Phe)2-piperazine
(Boc-Tyr-D-Ala-Gly-pF-Phe)2-1,2-phenylenediamine
2 TFAꢀ(Tyr-D-Ala-Gly-pF-Phe)2-1,2-phenylenediamine
2 TFAꢀ(Tyr-D-Ala-Gly-pF-Phe)2-piperazine
HPLC column: waters XBridgeTM BEH130 C18, 250 9 4.6 mm, 5.0 lm; HPLC solvent A, 0.1% TFA in water; solvent B, acetonitrile;
gradient: 5–95% B in A over 45 min, flow rate 1.0 mL/min, 50 lL injection of a solution containing 1 mg/mL in acetonitrile/water 2:1
(AcOEt/CH3OH 99:1). 1H NMR (DMSO-d6, 300 MHz)
(d, ppm): 1.35 [9H, s, (CH3)3–]; 2.96–2.75 [4H, m, Phe
b-CH2]; 3.41–3.26 [8H, m, –CH2–CH2–]; 3.49 [4H, m, Gly
CH2]; 4.89 [2H, m, Phe a-CH]; 6.65 [2H br, Gly NH];
7.22–6.98 [8H, m, aromatics]; 7.90 [2H, d, Phe NH]. 13C
NMR (CDCl3, 300 MHz) (d, ppm): 28.5, 38.7, 41.7, 45.2,
49.8, 60.6, 80.6, 115.9, 131.2, 131.7, 156.1, 160.5, 169.7,
171.4. HRMS (ESI) calcd. for C36H46F2N6O8 m/z:
731.3580 [M ? H]?; found 731.3588.
[2H, m, Phe a-CH]; 7.22–6.59 [12H, m, aromatics]; 7.99
[4H, br, Gly NH]; 8.88 [2H, br, Phe NH]; 9.70 [2H, s, Ar
NH]. 13C NMR (CDCl3, 300 MHz) (d, ppm): 28.4, 37.1,
55.3, 60.6, 80.7, 115.9, 125.5, 126.8, 131.1, 132.1, 132.2,
156.7, 160.5, 170.2, 170.6. HRMS (ESI) calcd. for
C38H47F2N6O3 m/z: 753.3426 [M ? H]?; found 753.3425.
(Boc-D-Ala-Gly-pF-Phe)2-piperazine (5)
Product 3 was deprotected at the Na terminal by TFA in
DCM 1:1 using 1 mL of the mixture per 100 mg of Boc-
protected product for 1.5 h at r.t. The mixture was then
evaporated under high vacuum and the TFA salt was used
for the next step without further purification. EDCꢀHCl
(2.2 eq.), HOBt (2.2 eq.) and DIPEA (6.6 eq.) were added
to a solution of Boc-D-Ala-OH (2.2 eq.) in DMF at 0°C.
The reaction mixture was stirred for 10 min, TFAꢀGly-pF-
Phe-Pip-pF-Phe-GlyꢀTFA (1 eq.) was added, the reaction
was stirred for an additional 10 min at 0°C then allowed to
warm at r.t. overnight. The solvent was evaporated under
reduced pressure, the residue was precipitated with EtOAc
and the suspension was filtered through a Buchner funnel
under reduced pressure. The solid residue was washed with
three portions of 5% citric acid, NaHCO3 s.s., brine and
distilled water, then dried under reduced pressure and
triturated with diethyl ether to give the desired product 5 as
a pure white solid (60%). RIf ¼ 0:16 (AcOEt/CH3OH 9:1),
RIfI ¼ 0:80 (AcOEt/CH3OH 8:2). 1H NMR (DMSO-d6,
300 MHz) (d, ppm): 1.16 [6H, m, Ala CH3]; 1.34 [9H, s,
(CH3)3–]; 2.89–2.74 [4H, m, Phe b-CH2]; 3.41–3.10 [8H,
m, –CH2–CH2–]; 3.67–3.55 [4H, m, Gly CH2]; 3.98 [2H,
m, D-Ala a-CH]; 4.83 [2H, m, Phe a-CH]; 6.98 [2H, d,
D-Ala NH]; 7.23–7.04 [8H, m, aromatics]; 7.96 [2H, t, Gly
NH]; 8.21 [2H, d, Phe NH]. 13C NMR (DMSO-d6,
300 MHz) (d, ppm): 18.7, 28.8, 37.3, 42.1, 42.3, 50.2, 50.3,
(Boc-Gly-pF-Phe)2-1,2-phenylenediamine (4)
Product 2 was deprotected at the Na terminal by TFA in
DCM 1:1 using 1 mL of mixture per 100 mg of the Boc-
protected product for 1.5 h at r.t. The mixture was then
evaporated under high vacuum and the TFA salt was used
for the next step without further purification. EDCꢀHCl
(2.2 eq.), HOBt (2.2 eq.), DIPEA (6.6 eq.) were added to a
solution of Boc-Gly-OH (2.2 eq.) in DMF at 0°C and
reaction mixture was stirred for 10 min, TFAꢀpF-Phe-1,2-
phenylenediamine-pF-PheꢀTFA (1 eq.) was added, the
reaction was stirred for an additional 10 min at 0°C then
allowed to warm at r.t. overnight. The solvent was evap-
orated under reduced pressure, the residue was precipitated
with EtOAc, and the suspension was filtered through a
Buchner funnel under reduced pressure. The solid residue
was washed with three portions of 5% citric acid, NaHCO3
s.s., brine and distilled water, then the solid dried under
reduced pressure and triturated with diethyl ether to give
the desired product 4 as a crude white solid. The product
was purified by silica gel column chromatography (AcOEt/
CH2Cl2 1:1 to AcOEt/CH2Cl2 8:2) to obtain the pure
product 4 (77%). RfI ¼ 0:21 (AcOEt/CH2Cl2 1:1), RIfI ¼
1
0:80 (AcOEt). H NMR (DMSO-d6, 300 MHz) (d, ppm):
3.22–2.86 [4H, m, Phe b-CH2]; 3.55 [4H, m, Gly NH]; 4.77
123

1508
A. Mollica et al.
78.7, 115.6, 131.9, 133.9, 155.7, 160.1, 168.8, 169.6,
173.6. HRMS (ESI) calcd. for C42H59F2N8O10 m/z:
873.4322 [M ? H]?; found 873.4318.
reduced pressure. The solid residue was washed with three
portions of 5% citric acid, NaHCO3 s.s., brine and distilled
water, dried under reduced pressure and triturated with
diethyl ether to give the product 7 as crude white solid.
The product was purified on RP-HPLC to give the pure
product 7 (90%). RfI ¼ 0:20 (AcOEt/MeOH 9:1), RIfI ¼
(Boc-D-Ala-Gly-pF-Phe)2-1,2-phenylenediamine (6)
Product 4was deprotectedat the Na terminal by TFA in DCM
1:1 using 1 mL of mixture per 100 mg of Boc-protected
product for 1.5 h at r.t. The mixture was then evaporated
under high vacuum and the TFA salt was used for the next
step without further purification. EDCꢀHCl (2.2 eq.), HOBt
(2.2 eq.) and DIPEA (6.6 eq.) were added to a solution of
Boc-D-Ala-OH (2.2 eq.) in DMF at 0°C and stirred for
10 min. TFAꢀGly-pF-Phe-1,2-phenylenediamine-pF-Phe-
GlyꢀTFA (1 eq.) was added, the reaction was stirred for an
additional 10 min at 0°C, then allowed to warm at r.t.
overnight. The solvent was evaporated under reduced pres-
sure, the residue was precipitated with EtOAc and the sus-
pension was filtered through a Buchner funnel under reduced
pressure. The solid residue was washed with three portions of
5% citric acid, NaHCO3 s.s., brine and distilled water, dried
under reduced pressure and triturated with diethyl ether to
give the desired product 6 as a crude white solid. RIf ¼ 0:34
(AcOEt), RIfI ¼ 0:50 (AcOEt/CH3OH 99:1). The product
was purified by silica gel column chromatography (AcOEt/
CH2Cl2 1:1 to AcOEt/CH2Cl2 94:6) to obtain the pure
product 6 (35%). 1H NMR (DMSO-d6, 300 MHz) (d, ppm):
1.16 [6H, m, Ala CH3]; 1.34 [9H, s, (CH3)3–]; 3.18–2.74 [4H,
m, Phe b-CH2]; 3.81–3.61 [4H, m, Gly CH2]; 3.95 [2H, m,
D-Ala a-CH]; 4.60 [2H, m, Phe a-CH]; 7.35–6.50 [12H, m,
aromatics]; 7.50 [2H, br, D-Ala NH]; 8.05 [2H, t, Gly NH];
8.21 [2H, d, Phe NH]; 9.50 [2H, s, Ar NH]. 13C NMR
(CDCl3, 300 MHz) (d, ppm): 18.7, 28.5, 37.1, 43.1, 50.6,
55.9, 80.4, 115.7, 125.4, 126.6, 130.4, 131.1, 132.5, 156.0,
160.4, 169.9, 170.4, 174.3. HRMS (ESI) calcd. for
C44H57F2N8O10 m/z: 895.4166 [M ? H]?; found 895.4160.
1
0:73 (CHCl3/MeOH 8:2). H NMR (DMSO-d6, 300 MHz)
(d, ppm): 1.15 [6H, m, Ala CH3]; 1.32 [9H, s, (CH3)3–];
2.98–2.54 [8H, m, Phe b-CH2 and Tyr b-CH2]; 3.45–3.10
[8H, m, –CH2–CH2– superimposed to the water signal];
3.65 [4H, m, Gly CH2]; 4.05 [2H, m, D-Ala aCH]; 4.22 [2H,
m, Tyr a-CH]; 4.85 [2H, m, Phe a-CH]; 6.90 [2H, d, Tyr
NH]; 7.25–6.64 [16H, m, aromatics]; 8.05 [4H, br, Phe
NH and D-Ala]; 8.22 [2H, t, Gly NH]; 9.20 [2H, s, Tyr OH].
13C NMR (DMSO-d6, 300 MHz) (d, ppm): 18.7, 28.8, 37.4,
38.4, 45.2, 48.8, 50.1, 56.7, 60.4, 78.8, 115.4, 128.5, 128.9,
130.0, 130.8, 137.8, 155.9, 156.4, 168.6, 169.8, 172.0,
173.0. HRMS (ESI) calcd. for C60H77F2N10O14 m/z:
1,199.5589 [M ? H]?; found 1,199.5586.
(Boc-Tyr-D-Ala-Gly-pF-Phe)2-1,2-phenylenediamine (8)
Product 6 was deprotected at the Na terminal by TFA in
DCM 1:1 using 1 mL of mixture per 100 mg of Boc-pro-
tected product for 1.5 h at r.t. The mixture was then
evaporated under high vacuum and the TFA salt was used
for the next step without further purification. EDCꢀHCl
(2.2 eq.), HOBt (2.2 eq.) and DIPEA (6.6 eq.) were added
to a solution of Boc-Tyr-OH (2.2 eq.) in DMF at 0°C. The
reaction mixture was stirred for 10 min. TFAꢀD-Ala-
Gly-pF-Phe-1,2-phenylenediamine-pF-Phe-Gly-D-AlaꢀTFA
(1 eq.) was added, the reaction was stirred for an additional
10 min at 0°C, then allowed to warm at r.t. overnight. The
solvent was evaporated under reduced pressure, the residue
was precipitated with EtOAc and suspension was filtered
through a Buchner funnel under reduced pressure. The
solid residue was washed with three portions of 5% citric
acid, NaHCO3 s.s., brine and distilled water, dried under
reduced pressure and triturated with diethyl ether to give
the desired product 8 as a crude white solid. RIf ¼ 0:46
(AcOEt/MeOH 9:1), RfII ¼ 0:37 (CHCl3/MeOH 9:1). The
product was purified on RP-HPLC to give the pure product
(Boc-Tyr-D-Ala-Gly-pF-Phe)2-piperazine (7)
Product 5 was deprotected at the Na terminal by TFA in
DCM 1:1 using 1 mL of mixture per 100 mg of Boc-pro-
tected product for 1.5 h at r.t. The mixture was then
evaporated under high vacuum and the TFA salt was used
for the next step without further purification. EDCꢀHCl
(2.2 eq.), HOBt (2.2 eq.) and DIPEA (6.6 eq.) were added
to a solution of Boc-Tyr-OH (2.2 eq.) in DMF at 0°C and
stirred for 10 min. TFAꢀD-Ala-Gly-pF-Phe-Pip-pF-Phe-
Gly-D-AlaꢀTFA (1 eq.) was added, the reaction was stirred
for an additional 10 min at 0°C then allowed to warm at r.t.
overnight. The solvent was evaporated under reduced
pressure, the residue was precipitated with EtOAc and the
suspension was filtered through a Buchner funnel under
1
8 (30%). H NMR (DMSO-d6, 300 MHz) (d, ppm): 1.16
[6H, m, Ala CH3]; 1.32 [9H, s, (CH3)3–]; 3.15–2.42 [8H,
m, Tyr b-CH2 and Phe b-CH2]; 3.70 [4H, m, Gly CH2];
4.03 [2H, m, D-Ala a-CH]; 4.23 [2H, m, Tyr a-CH]; 4.65
[2H, m, Phe a-CH]; 7.42–6.59 [20H, m, aromatics]; 6.85
[2H, d, Tyr NH]; 8.22–8.05 [6H, br, D-Ala NH, Gly
NH and Phe NH]; 9.50 and 9.15 [4H, two singlets, Tyr
OH and Ar NH]. 13C NMR (DMSO-d6, 300 MHz)
(d, ppm): 18.7, 28.7, 37.1, 37.2, 42.5, 48.7, 56.8, 56.9, 78.8,
115.4, 115.6, 120.7, 128.5, 130.7, 130.8, 131.8, 133.3,
123

New potent biphalin analogues
1509
134.3, 156.0, 156.4, 160.1, 169.6, 170.6, 172.2, 173.1.
HRMS (ESI) calcd. for C62H75F2N10O14 m/z: 1,221.5432
[M ? H]?; found 1,221.5435.
transfected cells that stably express the respective receptor
type and were evaluated as previously described (Polt et al.
1994; Misicka et al. 1992). The ligands used were
[3H]DPDPE and [3H]DAMGO for d- and l-receptors,
respectively.
2 TFAꢀ(Tyr-D-Ala-Gly-pF-Phe)2-1,2-phenylenediamine (9)
We used [35S]GTP-c-S binding to examine opioid ago-
nist efficacy and functional characterization of the ligands
at the d- and l-opioid receptors. Agonist efficacy can be
determined at the level of receptor G-protein interaction by
measuring agonist-stimulated binding with a non-hydro-
lyzable GTP analogue. The ability of l- and d-opioid ago-
nists to activate G-proteins has been demonstrated
by studying the binding of the GTP analogue guanosine-50-
O-(3-[35S]thio)triphosphate ([35S]GTP-c-S). The opioid
receptor-mediated assay was performed as previously
described (Szekeres and Traynor 1997). Cells expressing
hDOR for d-receptor (or rMOR for l-receptor) were incu-
bated with increasing concentrations of the test compounds
in the presence of 0.1 nM [35S]GTP-c-S (1,000–1,500 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 filtration 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 pre-treated with assay buffer prior to filtration
to reduce non-specific binding. Bound reactivity was mea-
sured by liquid scintillation spectrophotometry after an
overnight extraction with EcoLite (ICN, Biomedicals,
Costa Mesa, CA) scintillation cocktail. The data were
analysed using GraphPad Prism Software (San Diego, CA).
The in vitro tissue bioassays (MVD and GPI/LMMP)
were performed as described previously (Kramer et al.
1993). IC50 values represent means of no less than four
experiments. IC50 values, 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 2.
Product 8 was deprotected at the Na terminal by TFA
in DCM 1:1 using 1 mL of mixture per 100 mg of Boc-
protected product for 1.5 h at r.t. The mixture was then
evaporated under high vacuum and the TFA salt was
purified on RP-HPLC to give the pure product 9 (78%).
RIf ¼ 0:69 (n-Bu-OH/CH3COOH/H2O 4:1:1), RIfI ¼ 0:65
(n-Bu-OH/CH3COOH/AcOEt/H2O 5:1:3:1). 1H NMR
(DMSO-d6, 500 MHz) (d, ppm): 1.05 [6H, m, Ala CH3];
3.20–2.90 [8H, m, Tyr b-CH2 and Phe b-CH2]; 3.60 [4H,
m, Gly CH2]; 4.00 [2H, m, Tyr a-CH]; 4.32 [2H, m, D-Ala
a-CH]; 4.65 [2H, m, Phe a-CH]; 7.50–6.70 [20H, m, aro-
matics]; 8.06 [2H, br, Tyr NH]; 8.15 [4H, br, Gly NH and
Phe NH]; 8.55 [2H, br, D-Ala NH]; 9.56 and 9.35 [4H, two
singlets, Tyr OH and Ar NH]. 13C NMR (DMSO-d6,
300 MHz) (d, ppm): 18.9, 36.9, 37.2, 48.8, 54.2, 55.4, 55.5,
115.9, 125.4, 126.0, 131.1, 131.8, 134.3, 157.2, 158.6,
159.0, 160.1, 163.3, 168.3, 169.5, 170.7, 172.5. HRMS
(ESI) calcd. for C52H59F2N10O10 m/z: 1,021.4384
[M ? H]?; found 1,021.4381.
2 TFAꢀ(Tyr-D-Ala-Gly-pF-Phe)2-piperazine (10)
Product 7 was deprotected at the Na terminal by TFA in
DCM 1:1 using 1 mL of mixture per 100 mg of Boc-pro-
tected product for 1.5 h at r.t. The mixture was then
evaporated under high vacuum and the TFA salt was
purified on RP-HPLC to give the pure product 10 (90%).
RIf ¼ 0:46 (n-Bu-OH/CH3COOH/H2O 4:1:1), RIfI ¼ 0:12
(n-Bu-OH/CH3COOH/AcOEt/H2O 5:1:3:1). 1H NMR
(DMSO-d6, 500 MHz) (d, ppm): 1.08 [6H, m, Ala CH3];
2.98–2.77 [8H, m, Tyr b-CH2 and Phe b-CH2]; 3.35 [8H,
m, piperazine protons under the water signal]; 3.68 [4H, m,
Gly CH2]; 3.99 [2H, m, Tyr a-CH]; 4.35 [2H, m, D-Ala
a-CH]; 4.90 [2H, m, Phe a-CH]; 7.25–6.67 [16H, m, aro-
matics]; 8.15 [2H, br, Tyr NH]; 8.25 [4H, br, Gly NH and
Phe NH]; 8.58 [2H, br, D-Ala NH]; 9.34 [2H, s, Tyr OH].
13C NMR (DMSO-d6, 300 MHz) (d, ppm): 18.9, 36.9,
42.1, 48.9, 50.2, 50.9, 54.2, 58.4, 115.6, 115.9, 125.4,
131.1, 132.0, 133.8, 157.2, 158.5, 158.9, 168.7, 169.9,
172.5, 172.6. HRMS (ESI) calcd. for C50H61F2N10O10 m/z:
999.4540 [M ? H]?; found 999.4542.
Results and discussion
In order to better discuss the consequences of the structural
modifications, Table 2 reports, in addition to the activity data
of the new analogues 9 and 10 and native biphalin, the bio-
logical evaluations previously found for pF-Bph, the pF-Phe
analogue of biphalin (Misicka et al. 1997) and the analogues
9a and 10a (Mollica et al. 2005). The latter, as compared with
9 and 10, maintain the non-hydrazine linker but lack the
para-fluoro (pF) substitution at the 4,40-Phe residues.
Biological assays
Receptor binding affinities to the d- and l-opioid receptors
were performed using cell membrane preparations from
The positive influence of the introduction of a pF-Phe
residue in position 4,40 of biphalin has already been
123

1510
A. Mollica et al.
Table 2 Binding affinity, GTP binding assay, Emax (%) (net total bound/basal binding 9 100), and in vitro activity
Compound Binding Kai ,b (nM)
Kil
GTP bindingb,c (nM)
Functional bioactivityb (nM)
EC50 (nM), hDOR Emax (%)d EC50 (nM), hMOR Emax (%)d MVD
GPI/LMMP
Kid
Bphe
pF-Bphf
9ah
9
2.6 0.4
1.4 0.2
2.5 0.5
g
27
g
3
6.0 0.2
g
25
g
4
27 1.5
1.3 0.1
8.8 0.3
2.14 0.6
40 13
0.31 0.1
0.64 0.3
1.9 0.2
–
–
–
–
0.19 0.04
1.7 0.2
83
7
6
2.5 0.7
2.9 0.5
89 10
0.72 0.21
0.66 0.17
9.3 0.4
13
0.65 0.3
0.09 0.01 0.11 0.01 0.80 0.05
1
0.51 0.07 0.56 0.04
72
44
47
77
3
5
8
3.7 1.0
2.5 0.7
0.48 0.07
10ah
0.48 0.06 44
2
56.9
94
3
13
1
10
8
1.0 0.02
2.7 1.7
a
Displacement of [3H]DAMGO (l-selective) and [3H]DPDPE (d-selective) using membrane preparations from transfected cells expressing rat
l-opioid receptor and human d-opioid receptor, respectively
b
SEM
Reference compound: [35S]GTP-c-S
c
d
Net total bound/basal binding 9 100 SEM
e
Data from Lipkowski et al. (1999)
f
Data from Misicka et al. (1997)
g
Data not present in the original paper
h
Radiolabeled binding and MVD GPI functional bioassays from Mollica et al. (2005)
reported and is analogous to that previously observed in the
field of enkephalins (Toth et al. 1990). In particular, the pF-
Phe containing biphalin analogue (see pF-Bph in Table 2),
when compared to the parent, exhibits ten to two times
higher affinity to d- and l-receptor types, respectively
(Misicka et al. 1997).
fluorinated analogues, which share with 9 and 10 a non-
hydrazine linker, show in fact greater binding affinities to
both d- and l-receptors with respect to biphalin (Mollica
et al. 2005). In analogue 10a, which possesses a piperazine
linker, the pF substitution improves all of the measured
parameters and efficacy (see in vitro bioassays, binding
assays, and GTP binding in Table 2). On the contrary, in the
1,2-phenylenediamine containing analogue 9a, the pF sub-
stitution was detrimental for the d binding assays (0.19 vs.
13) and three to four times better for l binding assays (1.9 vs.
0.51). Concerning the GTP-c-35S binding assays, the pF
substitution leads to a 1.2–2-fold drop in efficacy for
both receptors (Emax (%) is 83 and 89 for 9a and 71.6 and
43.8 for 9).
Examination of the biological data reported in Table 2
confirms that, as already demonstrated (Mollica et al.
2005), the hydrazine bridge is not essential for binding and
activity and that the pF substitution on the phenylalanine
side chain maintains or improves the interaction of the
ligands with both d- and l-opioid receptors.
When compared to biphalin, the fluorinated piperazinic
derivative 10 shows improved binding values for both
l- and d-receptors (0.11 vs. 1.4 nM and 0.09 vs. 2.6 nM,
respectively). Furthermore, the GTP binding value (EC50;
1.0 nM) reveals an extremely high capacity to trigger the
transduction mechanisms with an efficacy three to six times
higher than biphalin for both receptors (0.8 vs. 2.5 and 1.0
vs. 6.0, for d- and l-receptors, respectively). In particular,
the Emax exhibited by 10 (94.1%) is, to the best of our
knowledge, the highest value so far reported for a non-
cyclic biphalin analogue. Table 2 shows that the new
analogues 9 and 10 are both more active than biphalin: the
IC50 (nM) of 10 is in fact 1:10 for l/GPI receptors (2.7 vs.
27) and ca. 1:20 for d/MVD (0.48 vs. 8.8). The corre-
sponding values of 9 are ca. 1:30 for l/GPI (0.7 vs. 27) and
ca. 1:2 for d/MVD (3.7 vs. 8.8).
In summary, the reported results indicate that the
improvement of the activity due to the replacement of the
native hydrazine linker and those derived from the pF
substitution on Phe aromatic ring at positions 4,40 are in
part additive and synergistic, in particular for the stimula-
tion of the second messenger system. As data in Table 2
show, the piperazine linker, as compared to the 1,2-phe-
nylenediamine linker, confirms more favourable properties
in bridging the two palindromic arms of biphalin. The
absence in the piperazine moiety of the two CO–NH
groups, available for H-bond donor interactions as well as
its more flexible and less planar structure, is probably at the
basis of the different behaviours observed. The influence
on the activity of the analogue 10 appears remarkable as
the introduction of the piperazine link and the pF substi-
tution leads to the most potent non-cyclic biphalin ana-
logue so far described. This result can be only compared to
that previously achieved with the synthesis of the cyclic
It should be noted that, when compared with native
biphalin, the activity data of the non-fluorinated analogues
9a and 10a (Fig. 1; Table 2) are in general comparable to
those of corresponding analogues 9 and 10. The non-
123

New potent biphalin analogues
1511
Lipkowski AW, Konecka AM, Sroczynska I, Przewlocki R, Stala L,
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Lipkowski AW, Misicka A, Davis P, Stropova D, Janders J, Lachwa
M, Porreca F, Yamamura HI, Hruby VJ (1999) Biological
activity of fragments and analogues of the potent dimeric opioid
peptide, biphalin. Bioorg Med Chem Lett 9:2763–2766
Misicka A, Lipkowski AW, Horvath R, Davis P, Kramer TH,
Yamamura HI, Hruby VJ (1992) Topographical requirements for
delta opioid ligands: common structural features of dermen-
kephalin and deltorphin. Life Sci 51:1025–1103
biphalin analogue containing a disulphide Cys-Cys bridge
(Mollica et al. 2006) which shows a Emax (%) value of 100.
In the case of the opioid peptide 10 described here, the
synthesis of a linear potent full agonist which escapes the
problems and low yields often associated with the cycli-
zation reactions may be a valuable advantage.
Acknowledgments This study was supported in part by a grant
from the USDHS, NIDA (DA06284).
Misicka A, Lipkowski AW, Horvath R, Davis P, Porreca F, Yamamura
HI, Hruby VJ (1997) Structure–activity relationship of biphalin.
The synthesis and biological activities of new analogs with
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Mollica A, Davis P, Ma S-W, Lai J, Porreca F, Hruby VJ (2005)
Synthesis and biological evaluation of new biphalin analogs with
non-hydrazine linkers. Bioorg Med Chem Lett 15:2471–2475
Mollica A, Davis P, Ma S-W, Lai J, Porreca F, Hruby VJ (2006)
Synthesis and biological activity of the first cyclic biphalin
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Conflict of interest The authors declare that they have no conflict
of interest.
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