Potent biphalin analogs with μ/δ mixed opioid activity: In vivo and in vitro biological evaluation

Article abstract of DOI:10.1002/ardp.201300380

Biphalin [(Tyr-D-Ala-Gly-Phe-NH-)₂] is an octapeptide with mixed μ/δ opioid activity. Its structure is based on two identical enkephalin-like portions linked "tail-to-tail" by a hydrazine bridge. This study presents the synthesis and in vitro and in vivo bioassays of two biphalin analogs that do not present the toxicity connected with the presence of the hydrazine moiety and are able to elicit a higher antinociceptive effect than biphalin.

Full text of DOI:10.1002/ardp.201300380

Arch. Pharm. Chem. Life Sci. 2014, 347, 305–312
305
Full Paper
Potent Biphalin Analogs with m/d Mixed Opioid Activity:
In Vivo and In Vitro Biological Evaluation
Roberto Costante1, Francesco Pinnen1, Azzurra Stefanucci2, and Adriano Mollica1
1
Dipartimento di Farmacia, Università di Chieti-Pescara “G. d’Annunzio”, Chieti, Italy
Dipartimento di Chimica, “Sapienza” Università di Roma, Roma, Italy
2
Biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2] is an octapeptide with mixed m/d opioid activity. Its structure is based
on two identical enkephalin-like portions linked “tail-to-tail” by a hydrazine bridge. This study presents
the synthesis and in vitro and in vivo bioassays of two biphalin analogs that do not present the toxicity
connected with the presence of the hydrazine moiety and are able to elicit a higher antinociceptive
effect than biphalin.
Keywords: Analgesic / Antinociception / Biphalin / Intrathecal administration / Opioids
Received: October 7, 2013; Revised: December 11, 2013; Accepted: December 17, 2013
DOI 10.1002/ardp.201300380
Introduction
barrier after intraperitoneal (i.p.) administration [13–15], it
shows similar potency to that of morphine [16].
Biphalin is a potent opioid peptide agonist with a palindromic
structure, composed of two enkephalin-like active fragments
connected “tail-to-tail” by a hydrazine linker (Tyr-D-Ala-Gly-
Phe-NH-NH<-Phe<-Gly<-D-Ala<-Tyr) (Fig. 1). Biphalin has been
synthesized for the first time by Lipkowski et al. [1] and widely
investigated by Hruby and coworkers [2–8]. Its dimeric
structure greatly enhances the analgesic activity and the
duration of the antinociceptive effect with respect to
enkephalins probably due to a cooperative binding and a
better enzymatic stability [9, 10].
The tendency to induce less physical dependence than
other opioid agonists [17–19] such as morphine represents an
important feature of this dimeric neuropeptide, probably due
to receptor selectivity and cooperative interactions among
different opioid receptors [7]. Several chemical modifications
have been performed in order to investigate the biphalin
structure–activity relationships (SARs) and with the aim to
obtain more potent and stable analogs [2–8, 10, 20–22].
Different molecular fragments, such as piperazine, 1,2-
phenylenediamine, and 1,4-phenylenediamine, were previ-
ously introduced as alternative linkers in place of hydrazine.
The introduction of tertiary amide bonds, obtained by using a
piperazine moiety as linker, and the 1,2-phenylenediamine,
resulted to be the most favorable substitution and led to
analogs with higher affinity and stability [4–6].
After intrathecal (i.t) injection in mice biphalin produces
intense analgesia [11] without showing toxicity [12]. Although
only a small fraction of biphalin crosses the blood–brain
Encouraged by these results, as continuation of our works,
we further investigated the in vivo and in vitro properties of 9
and 10, two of the more promising compounds of the series
(Fig. 2).
Correspondence: Dr. Adriano Mollica, Dipartimento di Farmacia,
Università di Chieti-Pescara “G. d’Annunzio”, Via dei Vestini 31, 66100
Chieti, Italy
E-mail: a.mollica@unich.it
Fax: þ39 0871 3554476
The peptides synthesis was improved with respect to the
original work, obtaining better overall yields and avoiding
the saponification step, which may lead to racemization as
discussed below. The biological evaluation of the compounds
has been enriched with in vitro [35S]GTP-g-S binding studies
in presence of pertussin toxin (PTX) and in vivo through
the tail-flick test, carried out in rats following intrathecal
administration (i.t.).
Abbreviations: DOR, d-opioid receptor; EDC, 1-ethyl-(3-dimethylamino-
propyl)-carbodiimide; GPI, guinea pig ileum; GTP, guanosine triphosphate;
[3H]DAMGO, [3H]-[D-Ala(2), N-Me-Phe-(4), Gly-ol(5)] enkephalin; [3H]-
DPDPE, [3H]-c[2-D-penicillamine,5-D-penicillamine] enkephalin; HOBt, 1-
hydroxybenzotriazole; i.p., intraperitoneal; i.t., intrathecal; MOR, m-opioid
receptor; MPE, percentage of maximum effect; MVD, mouse vas deferens;
NMM, N-methylmorpholine; PTX, pertussis toxin; RP-HPLC, reversed
phase high performance liquid chromatography; SEM, standard error of
measurement; DMF, N,N-dimethylformamide; TFA, trifluoroacetic acid.
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OH
O
O
O
O
NH2
H
N
H
N
H
N
H
N
N
H
N
H
N
H
N
H
NH2
O
O
O
O
HO
Biphalin
Figure 1. Representation of the biphalin molecular structure.
Results and discussion
during the saponification step and allows an easier purifica-
tion of the intermediates (i.e., by trituration in EtOAc) with
high overall yields [24, 25].
Synthesis
The previous reported synthesis of compounds 9–10 involved
the preparation of the tripeptide Na-Boc-D-Ala-Gly-OEt by the
asymmetric anhydride method [23] obtained with isobutyl
chloroformate and TEA at ꢀ15°C [2]. In the next step, the
tripeptide ethyl ester was hydrolyzed by 6 N NaOH in
MeOH and coupled with 2TFA·(Phe)2-1,2-phenylenediamine
or 2TFA·(Phe)2-1,2-piperazine, respectively, in order to obtain
the Boc protected final products 8 and 7. In order to avoid the
possible partial loss of chirality due to the saponification step,
and the low-yield reaction between the tripeptide and the
bivalent portions, a different strategy was adopted, by using
the standard coupling method HOBt/EDC/TEA in DMF to
obtain the Na-Boc protected octapeptides 7 and 8. The
synthesis was achieved by reacting 1,2-phenylenediamine
or piperidine with repeated steps of coupling/purification/
deprotection with the single amino acids, until the final
products 9 and 10 were obtained as TFA salts (Scheme 1). This
alternative method avoids the potential loss of chirality
Biological results
In vitro evaluation
The binding affinity of compounds 9 and 10 and biphalin for m
and d opioid receptors has been reported from [2] (Table 1).
Both analogs 9 and 10 show a good opioid receptor affinity,
higher than biphalin, with subnanomolar affinity at the DOR
and MOR. In particular, compound 10 has a high and similar
affinity for DOR and MOR (0.65 and 0.48 nM, respectively),
while 9 shows a discrete selectivity for DOR, with a Kmi 10-fold
higher than Kdi (1.93 vs. 0.19 nM). Isolated tissue-based
functional assay using guinea pig ileum/longitudinal muscle
myenteric plexus (GPI) and mouse vas deferens (MVD) tissues
previously reported by Mollica et al. [2] highlights that
compound 9 is more efficacious toward d opioid receptors
than the m receptor according to the binding assays pattern,
whereas the compound 10 has the opposite behavior, in
analogy with the parent peptide biphalin.
In order to complete in vitro evaluation, we have reported in
Table 1 the [35S]GTP-g-S binding data from Mollica et al. [2],
which display the capability of the two biphalin derivatives to
induce the intracellular transduction of the signal in cells
expressing hDOR (d-receptor) or rMOR (m-receptor) [4]. The
GTP binding value (EC50) reveals that 10 possesses a lower
capacity to trigger the transduction mechanisms with respect
to biphalin in particular at the DOR (44 vs. 2.5 nM), while
analog 9 showed a transduction efficacy higher than parent
compound in both opioid receptors (1.7 nM for DOR and
2.5 nM for MOR). The Emax% of both the analogs is higher
than that of biphalin, in particular 9, which may be
considered as a full agonist while biphalin and 10 are partial
agonists. To investigate the interaction of biphalin analogs
with Gi-family G-proteins, the transduction of the signal
induced by compounds 9 and 10 in MOR and DOR-expressing
cells was evaluated after PTX overnight (18 h) treatment
(100 ng/mL) [26]. This pretreatment strongly attenuated
the inhibition of maximally effective concentrations of
O
O
O
O
H
N
H
N
H
N
H
N
H2N
NH2
N
H
N
H
N
H
N
H
O
O
O
O
OH
HO
9
HO
O
O
O
O
H
H
N
H
N
H2N
N
N
N
NH2
N
H
N
H
N
H
O
O
O
O
OH
10
Figure 2. Representation of the molecular structures of 9 and 10.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 305–312
Biphalin Analogs with m/d Mixed Opioid Activity
307
OH
N
H
Boc
O
e
a
H
N
H
N
Boc
Boc
N
H
N
H
O
H
O
O
N
Boc
N
N
Boc
N
H
2
O
1
b
b
O
O
H
H
N
H
N
H
N
Boc
N
Boc
N
H
N
H
O
O
O
O
H
N
H
N
Boc
N
N
N
H
Boc
N
4
H
O
O
c
3
c
O
O
H
H
H
H
N
N
N
N
Boc
N
H
N
N
N
H
Boc
H
H
O
O
O
O
O
O
6
O
H
N
H
H
N
Boc
N
N
N
N
H
Boc
N
H
N
H
O
d
O
O
F
5
O
O
O
O
H
N
H
H
N
H
H
H
N
d
Boc
N
N
N
Boc
N
H
N
N
N
H
H
H
O
O
O
O
HO
O
OH
8
HO
O
O
O
H
N
H
H
N
H
N
Boc
N
N
N
N
N
Boc
N
H
N
H
H
f
H
O
O
O
O
OH
7
O
f
O
O
O
H
N
H
H
H
H2N
N
N
NH2
N
N
N
N
H
N
H
HO
H
H
O
O
O
O
O
O
O
O
H
H
N
OH
9
HO
2.TFA
H
N
H2N
N
N
N
N
NH2
N
N
H
H
H
O
O
O
O
2.TFA
OH
10
Scheme 1. Synthesis of products 9 and 10. Reagents and conditions: (a) EDC·HCl (2.2 eq.), HOBt (3.3 eq.), NMM (2.2 eq.), Boc-Phe-OH
(2.2 eq.), piperazine (1 eq.) in DMF at r.t. overnight; (b) TFA/CH2Cl2 1:1, r.t. (1 h) then EDC·HCl (2.2 eq.), HOBt (2.2 eq.), NMM (3.3 eq.), Boc-
Gly-OH (2.2 eq.), in DMF at r.t. overnight; (c) TFA/CH2Cl2 1:1, r.t. (1 h) then EDC·HCl (2.2 eq.), HOBt (2.2 eq.), NMM (3.3 eq.), Boc-D-Ala-OH
(2.2 eq.) in DMF at r.t. overnight; (d) TFA/CH2Cl2 1:1, r.t. (1 h) then EDC·HCl (2.2 eq.), HOBt (2.2 eq.), NMM (3.3 eq.), Boc-Tyr-OH (2.2 eq.) in
DMF at r.t. overnight; (e) EDC·HCl (2.2 eq.), HOBt (2.2 eq.), NMM (3.3 eq.), Boc-Phe-OH (2.2 eq.), 1,2-phenylenediamine (1 eq.) in DMF at r.t.
overnight; (f) TFA/CH2Cl2 1:1, r.t. (1 h).
compounds 9 and 10 toward both receptors (Table 1),
indicating that the in vitro activity of the analogs is
receptor-mediated, dose-dependent, and involves substantial-
ly the Gia/Goa subfamily of G-proteins [27, 28].
In vivo evaluation
The antinociceptive profile of compounds 9 and 10 was
investigated using the rat tail-flick assay, following a local
intrathecal (i.t.) administration. The potency of the analogs
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Arch. Pharm. Chem. Life Sci. 2014, 347, 305–312
Table 1. Binding affinity, GTP binding assay, Emax (%) (net total bound/basal binding ꢃ 100), and in vitro activity.
Binding Kia),b),c) (nM) GTP bindingc),d) (nM) Bioassaya),c),e) IC50 (nM)
d
PTX
m
PTX
EC50
Emax
Emax
(%)g)
EC50
Emax
Emax
(%)g)
Cpd
Kid
Kim
(nM)a)
(%)a),f)
(nM)a)
(%)a),f)
MVD
GPI
Bphh)
9
10
2.6 ꢁ 0.4
0.19 ꢁ 0.04
0.65 ꢁ 0.35
1.4 ꢁ 0.2
1.93 ꢁ 0.25
0.48 ꢁ 0.06
2.5 ꢁ 0.5
1.7 ꢁ 0.4
44 ꢁ 5.9
27 ꢁ 3
85 ꢁ 9
56 ꢁ 6
–
6.0 ꢁ 0.02
2.5 ꢁ 0.3
13 ꢁ 1.1
25 ꢁ 4
89 ꢁ 8
47 ꢁ 4
–
27 ꢁ 1.5
0.73 ꢁ 0.2
9.3 ꢁ 0.4
8.8 ꢁ 0.3
39.5 ꢁ 13
2.5 ꢁ 0.7
18 ꢁ 6
6 ꢁ 2
14 ꢁ 9
7 ꢁ 3
a)
Previously reported [2, 4].
b)
c)
d)
e)
f)
Displacement of [3H]DAMGO (m-receptor) and [3H]DPDPE (d-receptor).
ꢁSEM.
Reference compound [35S]GTP-g-S.
Concentration at 50% inhibition of muscle contraction in electrically stimulated isolated tissues.
Net total bound/basal binding ꢃ 100 ꢁ SEM.
g)
h)
Emax ꢁ SEM after 18 h cells pretreatment with PTX.
Ref. [10].
was then compared with biphalin and morphine. Co-
administrations of compounds 9 and 10 together with
morphine were also performed in order to investigate a
possible synergic effect with non-peptide opioid drugs. The
analgesic profile of the two derivatives and reference
compounds is shown in Fig. 3.
Following i.t. administration in rats, 9 showed a very
similar antinociceptive profile compared with biphalin and
reached the maximum effect at 30 min after the injection.
In the same way, compound 10 showed in the first 20 min a
similar potency with respect to biphalin, while it induced a
higher and very persistent analgesia later: In particular, it
reached the maximum efficacy from 30 to 45-min after the
administration (99–100% MPE) and it showed the higher
distance from biphalin at 45 min (99.3% vs. 48.6%). It is worth
noting that, in this animal model, all peptides have a higher
antinociceptive effect than morphine.
Although 9 reported the best results for d-receptor in in vitro
evaluation (Ki binding, MVD IC50, and GTP EC50), the 10, which
shows a good in vitro activity/affinity for both the receptors,
resulted more efficacious in eliciting analgesia in thermal
in vivo antinociception test. These results seem to confirm the
role of the m-receptor in eliciting analgesia and confirm that a
well-balanced affinity for both the opioid receptors produces a
greater antinociceptive response. In co-administration experi-
ments, half the previous doses of compounds 9–10 were
injected i.t. together with half the previous dose of morphine.
No substantial additive effect was observed, with a weak
increasing of the antinociceptive effect only 30 and 45 min
after the administration.
Conclusions
This paper represents the continuation of our previous
works [2, 4], with the aim to complete characterization of the
antinociceptive profile of the 9 and 10. Previously reported
interaction with human opioid receptors (MOR and DOR) has
been improved by in vitro PTX-[35S]GTP-g-S binding assay, 9
and 10 have been tested alone and in co-administration
with morphine, through in vivo tail-flick test following i.t.
administration in rats and compared with the parent peptide
biphalin.
This work confirms the importance of mixed m/d agonists
as a future alternative in treatment of severe pain, due to
the synergistic activity of d-receptor activation on m opioid
functional activities [29, 30].
The in vivo hot plate test reveals that products 9 and 10 are
capable of eliciting a similar or improved antinociceptive
Figure 3. Time–response of the antinociceptive activity (tail-flick
test) of morphine (7.8 nmol), biphalin (8.8 nmol), 9 (8.25 nmol), 10
(8.40 nmol), and co-administrations of 9 (4.1 nmol) and 10 (4.2 nmol)
with morphine (3.9 nmol) following i.t. injection in rats (n ¼ 6–8). The
antinociceptive activity is expressed as percentage of the maximum
possible effect (% MPE) ꢁ SEM. ꢂStatistical significance was
assumed at p < 0.05 (ꢂp < 0.05; ꢂꢂꢂp < 0.001).
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Arch. Pharm. Chem. Life Sci. 2014, 347, 305–312
Biphalin Analogs with m/d Mixed Opioid Activity
309
effect than the parent compound biphalin. While analogue 9
has a very similar in vivo profile in comparison with biphalin,
compound 10 showed a higher efficacy. This is probably due
to the presence of the piperazine linker, which strongly
influences the conformational rigidity of the molecule, in
particular with an increased affinity for m receptor. The
absence of an in vivo synergic effect after combination of half-
doses of 9 and 10 with morphine and the loss of in vitro activity
after cells pretreatment with PTX confirm that the analgesic
activity of these compound is due to m/d receptors activation
through G protein pathway [31].
In conclusion, compounds 9 and 10 are good candidates for
further studies, since they have shown some improvements
versus native biphalin: (i) a better capability to trigger the
transduction mechanisms; (ii) an increased antinociceptive
effect, and (iii) reduced toxicity connected with the absence of
the hydrazine linker [32–34].
The solid was dried under reduced pressure and triturated with
diethyl ether to give the desired product 1 as a crude white solid,
in 95% yield. Rf 0.56 (EtOAc). The product was used for the next
step without further purification. 1H NMR (DMSO-d6, 300 MHz) (d,
ppm): 1.32 [18H, s, (CH3)3–]; 2.75–2.89 [4H, m, Phe b-CH2]; 2.86–
2.93 [8H, m, –CH2–CH2– Pip]; 4.57 [2H, m, Phe a-CH]; 7.15 [2H, br,
Phe NH]; 7.18–7.26 [10H, m, aromatics]. HR-MS (ESI) calcd. for
C
32H44N4O6 m/z: 581.334 [MþH]þ; found 581.342.
(Boc-Phe)2-1,2-phenylenediamine (2)
EDC·HCl (2.2 eq.), HOBt (2.2 eq.), and NMM (3.3 eq.) were added to
a solution of Boc-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 precipitat-
ed 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. Rf 0.54 (CH2Cl2/EtOAc 2:1). The
product was purified by silica gel column chromatography
(CH2Cl2/EtOAc 98:2 to CHCl3/EtOAc 90:10) to obtain the pure
product 2 (88%). 1H NMR (DMSO-d6, 300 MHz) (d, ppm): 1.30
[18H, s, (CH3)3–]; 2.88–2.95 [4H, m, Phe b-CH2]; 4.40 [2H, m, Phe
a-CH]; 7.05 [2H, d, Phe NH]; 7.15–7.55 [14H, m, aromatics]; 9.46
[2H, s, Ar NH]. HR-MS (ESI) calcd. for C34H42N4O6 m/z: 603.318
[MþH]þ; found 603.330.
Experimental
Chemistry
Synthesis of all new analogs was performed in solution phase,
using the Na-Boc strategy. All synthesis began with the
appropriate diamine, performing repeated steps of coupling/
purification/deprotection of the intermediate products, until the
final products were obtained as TFA salts (Scheme 1). All coupling
reactions were performed with the standard method of HOBt/
EDC/NMM in DMF [8]. Deprotection of Na-tert-butyloxycarbonyl
group was performed using TFA/CH2Cl2 1:1 for 1 h, under
nitrogen atmosphere. The intermediate TFA salts were used for
subsequent reactions without further purification. Boc protected
intermediate products were purified by silica gel column
chromatography, or in case of scarcely soluble products, the
purification was performed by trituration in EtOAc. Final
products 9 and 10 were purified by RP-HPLC using a Waters
XBridge Prep BEH130 C18, 5.0 mm, 250 mm ꢃ 10 mm column at a
flow rate of 4 mL/min on a Waters Binary pump 1525, using as
eluent a linear gradient of H2O/acetonitrile 0.1% TFA ranging
from 5% acetonitrile to 90% acetonitrile in 45 min. The purity of
the Na-Boc-protected products was confirmed by NMR analysis on
a Varian VXR 300 MHz and mass spectrometry ESI-HRMS. The
purity of final TFA salts was confirmed by NMR analysis, ESI-HRMS
and by analytical RP-HPLC (C18-bonded 4.6 ꢃ 150 mm) at a flow
rate of 1 mL/min using as eluent a gradient of H2O/acetonitrile
0.1% TFA ranging from 5% acetonitrile to 95% acetonitrile in
50 min and was found to be not <95%.
(Boc-Gly-Phe)2-piperazine (3)
Product 1 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 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. EDC·HCl (2.2 eq.), HOBt (2.2 eq.), and NMM (3.3 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, then TFA·Phe-Pip-Phe·TFA
(1 eq.) was added, and 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 (93%). Rf 0.63 (EtOAc/MeOH 9:1). 1H NMR (DMSO-
d6, 300 MHz) (d, ppm): 1H NMR (DMSO-d6, 300 MHz) (d, ppm): 1.32
[18H, s, (CH3)3–]; 2.75–2.90 [4H, m, Phe b-CH2]; 2.86–2.93 [8H, m,
–CH2–CH2– Pip]; 3.55 [4H, m, Gly b-CH2]; 4.50 [2H, m, Phe a-CH];
6.90 [2H, t, Gly NH]; 7.20–7.32 [10H, m, aromatics]; 8.25 [2H, br,
Phe NH]. HR-MS (ESI) calcd. for C36H50N6O8 m/z: 695.377 [MþH]þ;
found 695.381.
(Boc-Phe)2-piperazine (1)
EDC·HCl (2.2 eq.), HOBt (2.2 eq.), and NMM (3.3 eq.) were added to a
solution of Boc-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.
(Boc-Gly-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 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. EDC·HCl (2.2 eq.), HOBt (2.2 eq.), NNM (3.3 eq.) were
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R. Costante et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 305–312
added to a solution of Boc-Gly-OH (2.2 eq.) in DMF at 0°C and
reaction mixture was stirred for 10 min, TFA·Phe-1,2-phenylenedi-
amine-Phe·TFA (1 eq.) was added, and 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, and then the
solid dried under reduced pressure and triturated with diethyl
ether to give the desired product 4 as a pure white solid. (97%).
Rf 0.66 (EtOAc). 1H NMR (DMSO-d6, 300 MHz) (d, ppm): 1.32
[18H, s, (CH3)3–]; 2.88–2.95 [4H, m, Phe b-CH2]; 3.50 [4H, m, Gly
b-CH2]; 4.65 [2H, m, Phe a-CH]; 6.85 [2H, t, Gly NH]; 7.15–
7.55 [14H, m, aromatics]; 8.40 [2H, d, Phe NH]; 9.46 [2H, s, Ar NH].
HR-MS (ESI) calcd. for C38H48N6O8 m/z: 717.361 [MþH]þ; found
717.369.
b-CH2]; 4.00 [2H, m, D-Ala a-CH]; 4.60 [2H, m, Phe a-CH]; 6.80 [2H,
d, D-Ala NH]; 7.15–7.55 [14H, m, aromatics]; 8.15 [2H, t, Gly NH];
8.35 [2H, d, Phe NH]; 9.50 [2H, s, Ar NH]. HR-MS (ESI) calcd. for
C44H58N8O10 m/z: 859.435 [MþH]þ; found 859.443.
(Boc-Tyr-D-Ala-Gly-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-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. EDC·HCl (2.2 eq.), HOBt (2.2 eq.), and NMM (3.3 eq.)
were added to a solution of Boc-Tyr-OH (2.2 eq.) in DMF at 0°C
and stirred for 10 min. TFA·D-Ala-Gly-Phe-Pip-Phe-Gly-D-Ala·TFA
(1 eq.) was added, and 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, dried under reduced
pressure, and triturated with diethyl ether to give the product 7
as crude white solid.
(Boc-D-Ala-Gly-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 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. EDC·HCl (2.2 eq.), HOBt (2.2 eq.), and NMM (3.3 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, TFA·Gly-Phe-Pip-Phe-
Gly·TFA (1 eq.) was added, and 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, and then dried under
reduced pressure and triturated with diethyl ether to give the
desired product 5 as a pure white solid (80%). Rf 0.22 (EtOAc/
CH3OH 9:1). 1H NMR (DMSO-d6, 300 MHz) (d, ppm): 1.32 [18H, s,
(CH3)3–]; 2.75–2.90 [4H, m, Phe b-CH2]; 2.86–2.93 [8H, m,
–CH2–CH2– Pip]; 3.65 [4H, m, Gly b-CH2]; 4.52 [2H, m, Phe
a-CH]; 6.90 [2H, br, D-Ala a-CH]; 7.20–7.32 [10H, m, aromatics]; 8.15
[2H, t, Gly NH]; 8.24 [2H, br, Phe NH]. HR-MS (ESI) calcd. for
The product was purified by silica gel column chromatography
(CH2Cl2/EtOAc 20:80 to EtOAc/MeOH 86:14) to obtain the pure
product 7 (82%). Rf 0.3 (EtOAc/MeOH 85:15). 1H NMR (DMSO-d6,
300 MHz) (d, ppm): 1.08 [6H, d, Ala CH3]; 1.38 [9H, s, (CH3)3–]; 2.84–
3.29 [8H, m, Phe b-CH2 and Tyr b-CH2]; 3.65–3.89 [8H, m,
–CH2–CH2–]; 3.71 [4H, m, Gly CH2]; 4.15 [2H, m, D-Ala a-CH]; 4.22
[2H, m, Tyr a-CH]; 4.85 [2H, m, Phe a-CH]; 6.90 [2H, d, Tyr NH];
7.28–6.68 [18H, m, aromatics]; 8.05 [4H, br, Phe NH and D-Ala];
8.22 [2H, t, Gly NH]; 9.20 [2H, s, Tyr OH]. HR-MS (ESI) calcd. for
C
60H78N10O14 m/z: 1163.578 [MþH]þ; found 1163.585.
(Boc-Tyr-D-Ala-Gly-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-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. EDC·HCl (2.2 eq.), HOBt (2.2 eq.), and NMM (3.3 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. TFA·D-Ala-Gly-Phe-1,2-
phenylenediamine-Phe-Gly-D-Ala·TFA (1 eq.) was added, and 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. The product was purified
by silica gel column chromatography (EtOAc/CHCl3 80:20 to
EtOAc/MeOH 95:5) to obtain the pure product 8 (79%). Rf 0.4
(EtOAc/MeOH 9:1). 1H NMR (DMSO-d6, 300 MHz) (d, ppm): 1.22 [6H,
d, Ala CH3]; 1.30 [18H, s, (CH3)3–]; 2.65 and 2.90 [2H, m, Tyr b-CH2];
2.70 and 3.18 [4H, m, Phe b-CH2]; 3.75 [4H, m, Gly CH2]; 4.10
[2H, m, Tyr a-CH]; 4.28 [2H, m, D-Ala a-CH]; 4.68 [2H, m, Phe a-CH];
6.62 and 7.01 [8H, dd, Tyr aromatics]; 6.78 [2H, d, Tyr NH]; 7.15–
7.35 [14H, m, aromatics]; 8.05 [2H, d, D-Ala NH]; 8.19 [2H, t, Gly
NH]; 8.21 [2H, d, Phe NH]; 9.15 and 9.45 [4H, two singlets, Tyr OH
and Ar NH]. HR-MS (ESI) calcd. for C62H76N10O14 m/z: 1185.562
[MþH]þ; found 1185.575.
C
42H60N8O10 m/z: 837.451 [MþH]þ; found 837.460.
(Boc-D-Ala-Gly-Phe)2-1,2-phenylenediamine (6)
Product 4 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 used for the next step without further
purification. EDC·HCl (2.2 eq.), HOBt (2.2 eq.), and NMM (3.3 eq.)
were added to a solution of Boc-D-Ala-OH (2.2 eq.) in DMF at 0°C
and stirred for 10 min. TFA·Gly-Phe-1,2-phenylenediamine-Phe-
Gly·TFA (1 eq.) was added, and 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, dried under reduced
pressure, and triturated with diethyl ether to give the desired
product 6 as a pure white solid (92%). Rf 0.70 (EtOAc/MeOH 9:1).
1H NMR (DMSO-d6, 300 MHz) (d, ppm): 1.16 [6H, m, Ala CH3]; 1.34
[18H, s, (CH3)3–]; 2.78–3.05 [4H, m, Phe b-CH2]; 3.70 [4H, m, Gly
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2014, 347, 305–312
Biphalin Analogs with m/d Mixed Opioid Activity
311
TFA·(Tyr-D-Ala-Gly-Phe)2-1,2-phenylenediamine (9)
Community ethical regulations for the use and care of animals
for scientific research (European Economic Community Council
89/609; Italian D.L. 22-1-92 No. 116), and were approved by the
Ethical Committee on Animal Health and Care of G. d’Annunzio
University (Project n. 35, prot. N. 2913, 17/09/2007).
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 (quantitative). Rf 0.7 (n-Bu-OH/CH3COOH/H2O
8:1:1). 1H NMR (DMSO-d6, 500 MHz) (d, ppm): 1.05 [6H, m, Ala
CH3]; 2.80–3.10 [8H, m, Tyr b-CH2 and Phe b-CH2]; 3.75 [4H, m,
Gly CH2]; 4.00 [2H, m, Tyr a-CH]; 4.32 [2H, m, D-Ala, a-CH]; 4.68
[2H, m, Phe a-CH]; 6.70–7.50 [22H, m, aromatics]; 8.06 [6H, br,
Tyr NH3þ]; 8.20 [4H, br, Gly NH and Phe NH]; 8.50 [2H, br, D-Ala
NH]; 9.30 and 9.55 [4H, two singlets, Tyr OH and Ar NH. HR-MS
Surgery for i.t. injections
Rats were implanted with chronic indwelling intrathecal
catheters using a modification [36] of the method of Yaksh and
Rudy [37]. Rats were anesthetized with 10% isoflurane. Catheters
were made of silastic tubing (ID ¼ 0.30 mm; OD ¼ 0.64 mm) and
had a dead volume of 10 mL. Catheters measured a total of 12 cm
with 7.5 cm inserted into the intrathecal space to the level of
T13-L1. The catheter was inserted through the atlanto-occipital
membrane and into the intrathecal space using a guide wire.
Sutures were used to secure the placement of the catheter. All
rats were allowed to recover from the surgery for 3 days. Rats
exhibiting any sign of neurological or motor impairment
(paralysis, abnormal gait, weight loss, or negligent grooming)
were excluded from the experiment. Seventy hours after surgery,
peptides solutions were freshly prepared using saline containing
0.9% NaCl and rats (6–8 per group) were injected i.t. with
biphalin (8.8 nmol), 9 (8.25 nmol), 10 (8.40 nmol), morphine
sulfate (7.8 nmol), saline (control rats), and 9 (4.10 nmol) and 10
(4.20 nmol) together with morphine sulfate (3.9 nmol) in a
volume of 5 mL. After completion of drug testing, the catheter
position was verified in each animal by injection of 20 mg of
biphalin. Animals that did not show transient, opioid-induced
rigidity of the hind paws were excluded from analyses.
(ESI) calcd. for
985.462.
C
52H60N10O10 m/z: 985.457 [MþH]þ; found
TFA·(Tyr-D-Ala-Gly-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-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
10 (quantitative). Rf 0.5 (n-Bu-OH/CH3COOH/H2O 8:1:1). 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 [18H, m, aromatics]; 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].
HR-MS (ESI) calcd. for C50H62N10O10 m/z: 963.473 [MþH]þ; found
963.477.
Data analysis and statistics
In vitro biological assays
Experimental data were expressed as mean ꢁ SEM. The signifi-
cance among groups was evaluated with the analysis of variance
(one-way ANOVA) followed by Student’s t-test or Bonferroni’s post
hoc comparisons using the statistical software SPSS. Statistical
significance was assumed at p < 0.05 (ꢂp < 0.05; ꢂꢂꢂp < 0.001).
Statistical analysis was performed using GraphPad Prism
Software (GraphPad Software, San Diego, CA).
GTP binding assay
Cells expressing hDOR for d receptor (or rMOR for m receptor)
were incubated with increasing concentrations of the test
compounds in the presence of 0.1 nM
[
35S]GTP-g-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 filtration under vacuum through
Whatman GF/B glass fiber 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) scintillation cocktail. The data
were analyzed using GraphPad Prism Software (San Diego, CA).
For PTX control test, cells were treated overnight (18 h) with PTX
at concentration of 100 ng/mL and were washed twice with ice-
cold phosphate-buffered saline following the procedure described
by Clark et al. [35] before incubation with test compounds as
described above.
The authors have declared no conflict of interest.
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