Synthesis and biological evaluation of Naloxone and Naltrexone-derived hybrid opioids

Article abstract of DOI:10.2174/157340612801216193

The synthesis and biological evaluation of hybrid opioids consisting of Naloxone or Naltrexone and a partial opioid peptide are described. These compounds were synthesized in a homogeneous solution as well as in solid phase. A hydrazone linkage was employed to connect the alkaloids to the tetrapeptides. In synthesizing the peptides some nontraditional methods, which provided excellent results, were explored. The solid phase synthesis was achieved by anchoring the Fmoc-Phe to the 2-chlorotrityl resin, followed by stepwise addition of two Fmoc-Gly units. Each addition step preceded by standard piperidine removal of the Fmoc from the prior amino acid (AA) residue. The final AA, Tyr, was added as its Boc derivative. The Boc-tetrapeptide was then separated from the resin with a TFE/AcOH/CH₂Cl₂ mixture. In the solution synthesis, each peptide elongation step was accomplished by one-pot removal of the Fmoc from the prior AA residue and addition of the next Fmoc-AA. TBAF-thiol was used to cleanly remove the Fmoc, before adding the next Fmoc-AA in the presence of DIPEA and TBTU. All prepared hybrid ligands exhibited high affinities toward all three opioid receptors; moderate preferences for κ and μ receptors over δ receptor were observed. [ ³⁵S]GTPγS binding assays indicated that these hybrid opioids are δ and μ antagonists but partial κ agonist.

Full text of DOI:10.2174/157340612801216193

Medicinal Chemistry, 2012, 8, 683-689
683
Synthesis and Biological Evaluation of Naloxone and Naltrexone-Derived
Hybrid Opioids
Bahman E. Nassima* and Ming-Lei Wangb
aDepartment of Chemistry, Indiana University Southeast, 4201 Grant Line Road, New Albany, IN 47150, USA
bChemPep, Inc. 3460 Fairlane Farms Road, Suite 10, Wellington, FL 33414, USA
Abstract: The synthesis and biological evaluation of hybrid opioids consisting of Naloxone or Naltrexone and a partial
opioid peptide are described. These compounds were synthesized in a homogeneous solution as well as in solid phase. A
hydrazone linkage was employed to connect the alkaloids to the tetrapeptides. In synthesizing the peptides some non-
traditional methods, which provided excellent results, were explored. The solid phase synthesis was achieved by anchor-
ing the Fmoc-Phe to the 2-chlorotrityl resin, followed by stepwise addition of two Fmoc-Gly units. Each addition step
preceded by standard piperidine removal of the Fmoc from the prior amino acid (AA) residue. The final AA, Tyr, was
added as its Boc derivative. The Boc-tetrapeptide was then separated from the resin with a TFE/AcOH/CH2Cl2 mixture. In
the solution synthesis, each peptide elongation step was accomplished by one-pot removal of the Fmoc from the prior AA
residue and addition of the next Fmoc-AA. TBAF-thiol was used to cleanly remove the Fmoc, before adding the next
Fmoc-AA in the presence of DIPEA and TBTU. All prepared hybrid ligands exhibited high affinities toward all three
opioid receptors; moderate preferences for ꢀ and μ receptors over ꢁ receptor were observed. [35S]GTPꢀS binding assays
indicated that these hybrid opioids are ꢁ and μ antagonists but partial ꢀ agonist.
Keywords: Hybrid opiates, Hybrid opioids, kappa agonist, naloxone derivative, naltrexone derivative, opiate ago-
nist/antagonist, peptide synthesis.
1. INTRODUCTION
biologically active synthetic non-peptide opioids [8-10]. Al-
though opioid peptides are considered to be potential candi-
dates for therapeutic purposes, their tendency to undergo
enzymatic cleavage and their general inability to cross
blood-brain barrier (BBB) limit their use as pharmacological
agents. However, shorter peptides present smaller targets for
the peptidase and their attachment to a non-peptide molecule
is expected to give them added protection. Naloxone and
Naltrexone are non-peptide opiates that are recognized for
their strong affinity for all three receptors and have no
known significant undesirable properties. Thus, in order to
produce high affinity ligands, bearing the opioid tetrapeptide
“message” with anticipated protection from the peptidases
and increased potential for penetration to the central nervous
system, we have constructed hybrid opioids consisting of
either Naloxone or Naltrexone linked to the N-terminal
tetrapeptide (Tyr-Gly-Gly-Phe). These compounds show
excellent promise and provide a mechanism for employing
short therapeutic peptides to be carried and docked at the
receptors by non-peptide ligands. As indicated above, how-
ever, Naloxone and Naltrexone are not purely “carriers” and
participate in transduction as non-selective antagonists. But
their well known properties provide a safe base for examin-
ing the effects of the added peptide and their potential syner-
gistic benefits. It should be noted that similar hybrid struc-
tures, constituting partial “address” peptides as receptor se-
lective modulators, have been studied [11]. But their rates of
metabolism were found to be an obstacle in their utilization
[12]. In the present case the “message” peptide is generally
less functionalize and is anticipated to have improved in vivo
stability.
Opioid receptors and their mediation in the effect of a va-
riety of ligands on the central and peripheral nervous sys-
tems have been studied for over a half a century. Availability
of at least three different opioid receptors (μ, ꢀ, and ꢁ) was
established about thirty five years ago [1]. Since then many
medicinal scientists have sought, and examined the therapeu-
tic usefulness of, agonist and antagonist ligands for these
receptors [2-4].
In 1975 the discovery and the structures of enkephalins,
as endogenous opioid peptides, were reported [5]. Following
that, a number of other endogenous opioid peptides, with
differing preferences for the three opioid receptors, were
identified and characterized [6]. Most of these endogenous
opioids contain the same tetrapeptide fragment (Tyr-Gly-
Gly-Phe) at their N-terminal but have different C-terminals
with varying lengths. Biological studies indicate that the N-
terminal tetrapeptide fragment carries the message, which
imparts the opiate properties, and the variable C-terminals
confer selectivity for the receptors [7]. Thus the N-terminal
“message” segment is thought to be involved in signal trans-
duction at the receptor while the C-terminal “address” pro-
vides for recognition of the receptor and binding but is not
considered essential to the transduction process. This mes-
sage-address concept has also been shown to apply to some
*Address correspondence to this author at the Department of Chemistry,
Indiana University Southeast, 4201 Grant Line Road, New Albany, IN
47150, USA; Tel: (812) 941-2305; Fax: (812) 941-2637;
E-mail: bnassim@ius.edu
1875-6638/12 $58.00+.00
© 2012 Bentham Science Publishers

684 Medicinal Chemistry, 2012, Vol. 8, No. 4
Nassim and Wang
c
a
b
FmocGlyGlyPhe
3
O
FmocGlyPhe
2
O
Cl
FmocPhe
1
O
O
f
BocTyrGlyGlyPhe-OCH3
6
e
d
BocTyrGlyGlyPhe-OH
5
BocTyrGlyGlyPhe
4
h
g
BocTyrGlyGlyPhe-NHNH2
7
HO
HO
HO
O
OH
i
O
OH
O
OH
N
R
N
R
H
N
N
R
N
BocTyrGlyGlyPheNHN
TyrGlyGlyPhe
N
TyrGlyGlyPhe NH
10a = E isomer
10b = E isomer
H2CHC CH2
8a =
8b =
9a = Z isomer
9b = Z isomer
H2C
Scheme 1. Reagents and conditions: (a) Fmoc-Phe/DIPEA/CH2Cl2/DMF; (b) 20% piperidine/DMF, Fmoc-Gly/DIC/HOBt/DIPEA/CH2Cl2;
(c) 20% piperidine/DMF, Fmoc-Gly/DIC/HOBt/DIPEA/DMF; (d) 20% piperidine/DMF, Boc-Tyr/DIC/HOBt/DIPEA/DMF; (e)
TFE/AcOH/CH2Cl2 (1/1/8); (f) Trimethylsilyl diazomethane; (g) H2NNH2/EtOH; (h) Naloxone or Naltrexone/AcOH/MeOH; (i) 15%
TFA/CH2Cl2, Prepararive TLC, CH2Cl2/MeOH (10:1).
The total solid phase syntheses of our ligands have al-
ready been reported [13]. Here we present their receptor
binding and functional evaluation along with alternative syn-
theses methods, employing partial and total solution synthe-
ses.
corresponding hydrazide derivative by treatment with
Trimethylsilyldiazomethane (TMSCHN2) [15] and anhy-
drous hydrazine [17,18]. Condensation between the hydraz-
ide and the ketone functional group provided the hybrid
structures 8a and 8b [19,20] Deprotection of the Boc-
protecting group by 15% TFA in CH2Cl2 followed by prepa-
rative TLC led to the separation of the corresponding Z and
E isomers of the hybrid opiates 9a, 10a, and 9b, 10b. How-
ever, NMR studies showed that the E isomers spontaneously
change to their corresponding Z isomers, on standing in solu-
tion.
2. RESULTS AND DISCUSSION
2.1. Chemistry
In a previous publication on this work, we reported the
solid phase synthesis of our hybrid opioids, which was ac-
complished by employing Fmoc strategy, using Wang resin
as the polymeric support [13]. This was done by loading the
alkaloids onto Wang resin, and the Fmoc-protected amino
acids were coupled to the resin-bound moiety via a hydra-
zone functional group at the alkaloids’ 6-position.
The Fmoc strategy, which does not use strong acids for
deprotection, was the preferred method in solid phase pep-
tide synthesis. However, the use of excess secondary amine
for deprotection was not amenable to the solution phase con-
dition. The amine in solution interferes with the next cou-
pling step, and its removal is difficult [21]. Possible subse-
quent reactions of liberated amino components with dibenzo-
fulvene, the cleavage product of the Fmoc group, is another
problem [22]. To overcome these problems in our solution
synthesis, we applied the TBAF-thiol system to the cleavage
of the Fmoc group [23]. As shown in Scheme 2, we em-
ployed a general one-pot procedure in all the elongations
steps, to remove the Fmoc from the preceding amino acid
residue and to add the next Fmoc-amino acid. Thus, the syn-
thesis began with addition of excess 1-octanethiol and TBAF
to a solution of Fmoc-Phe-OCH3 ester in DMF. Then, bis(1-
methyl-1H-tetrazol-5-yl)disulfide was added to oxidize the
remaining thiol, in a thiol-disulfide exchange reaction. In
addition, the resulting 1-methyl-1H-tetrazol-5-thiol (MTZ-
SH) deactivated the TBAF. Addition of Fmoc-Gly with
DIPES and TBTU to the mixture resulted in the Fmoc-
dipeptide, which was purified by chromatography. Each ad-
ditional Fmoc-protected amino acid was coupled in a similar
way to produce the Fmoc protected tetrapeptide fragment 14.
The C-terminal of the N-Fmoc-protected tetrapeptide was
converted to the hydrazide derivative (15) and condensed
Alternatively, here we discuss approaches which began
with preparation of the peptide segment followed by its at-
tachment to the alkaloids to create the hybrid structures. The
attachment was made by converting the C-terminus of a pep-
tide to a hydrazide, which was then reacted with the 6-ketone
functional group of the respective alkaloids, in a homogene-
ous solution.
The syntheses of the peptide was carried out in two dif-
ferent ways. As shown in Scheme 1, the first method started
with anchoring of the Fmoc-protected C-terminal amino acid
onto the 2-chlorotrityl resin with excess DIPEA [14]. The
Fmoc-protecting group was removed by the standard
piperidine procedure. Subsequent coupling of various Fmoc
and Boc-protected amino acids onto the solid support pro-
ceeded in the presence of DIC, HOBt, and DIPEA in DMF.
Kaiser ninhydrin test was used on the beads of the resin to
determine the progress of the coupling. The N-Boc-protected
tetrapeptide was cleaved from the resin smoothly by using
the mixture of TFE/AcOH/CH2Cl2 [15,16]. The C-terminal
end of the N-Boc-protected tetrapeptide was converted to the

Synthesis and Biological Evaluation of Naloxone and Naltrexone-Derived
Medicinal Chemistry, 2012, Vol. 8, No. 4 685
j
k
FmocGlyGlyPhe OCH3
13
FmocPhe OCH3
11
FmocGlyPhe OCH3
12
m
n
l
FmocTyrGlyGlyPhe-OCH3
14
FmocTyrGlyGlyPhe-NHNH2
15
HO
O
HO
HO
O
OH
o
O
OH
OH
N
R
N
R
H
N
R
N
N
FmocTyrGlyGlyPheNHN
TyrGlyGlyPhe
N
TyrGlyGlyPhe NH
18a = E isomer
18b = E isomer
H2CHC CH2
16a =
16b
17a = Z isomer
17b = Z isomer
H2C
Scheme 2. Reagents and conditions: (j) TBAF/1-octanethiol/DMF, bis(1-methyl-1H-tetrazol-5-yl)disulfide, Fmoc-Gly/DIPEA/TBTU/ DMF;
(k) TBAF/1-octanethiol/DMF, bis(1-methyl-1H-tetrazol-5-yl)disulfide, Fmoc-Gly/DIPEA/TBTU/DMF; (l) TBAF/1-octanethiol /DMF,
bis(1-methyl-1H-tetrazol-5-yl)disulfide, Fmoc-Tyr/DIPEA/TBTU/DMF; (m) H2NNH2/EtOH; (n) Naloxone or Naltrexone /AcOH/MeOH;
(o) TBAF/1-octanethiol/DMF, bis(1-methyl-1H-tetrazol-5-yl)disulfide, Prepararive TLC, CH2Cl2/MeOH (10:1).
with the 6-ketone functional group of the respective alka-
loids by the same method described for Scheme 1. Deprotec-
tion of Fmoc group followed by preparative TLC gave the
same Z and E isomers of the hybrid opioids. The methods
used to synthesize our peptides present a variation to the
traditional methods. These reactions proceeded smoothly and
could provide an alternative to the established standard
methods.
some drug abuse patients[26-29]; yet, traditional treatments
for drug abuse have included the use of Naloxone and
Naltrexone as nonspecific antagonists. In addition, some
pain studies indicate a potential superiority in analgesic
agents with ꢁ-agonist and μ-antagonist properties [30-32].
Therefore, it is possible that our products, which display
both properties, could potentially be useful for treatment of
drug abuse victims as well as in treatment of pain.
2.2. Biology
3. EXPERIMENTAL SECTION
3.1. Materials and Abbreviations
The new ligands were evaluated in receptor binding as-
says on human opioid receptors transfected into Chinese
hamster ovary (CHO) cells by displacement of [3H]Cl-
DPDPE (ꢀ), [3H]U69593 (ꢁ), and [3H]DAMGO (μ) [24]. As
expected, all ligands have high affinities for all three opioid
receptors (Table 1). Ki values of the ligands indicated mod-
erate preference for ꢁ and μ over ꢀ opioid receptors. How-
ever, there does not seem to be a significant difference be-
tween the binding of Z and E (9a-10a / 17a-18a and 9b-10b /
17b-18b) stereoisomer pairs. This is likely due to the spon-
taneous conversion of E to the Z isomers, as observed in the
NMR studies.
[35S]GTPꢀS binding assays were applied for functional
characterization of the analogues at ꢀ, ꢁ, and μ receptors
[25]. All compounds tested in these assays are ꢀ and μ an-
tagonists but partial ꢁ agonists (Tables 2 and 3). Once again,
differences between the stereo isomers (9a-10a / 17a-18a
and 9b-10b / 17b-18b) seem to be negligible. The nonspe-
cific binding of these ligands are in line with our expecta-
tion, based on the lack of specificity of naloxone and
naltrexon. The fact that their functional examination show
agonistic properties towards the ꢁ receptor is interesting and
may indicate possession of properties similar to that of
dynorphine [26]. Furthermore, the antagonist and agonist
properties of these ligands may present a useful combination.
This is because during the past twenty years or so, ꢁ-agonists
have generated some interest for their use in treatment of
The reagents and supplies used in hybrid opioids synthe-
sis were as follows: 2-chlorotrityl resin (1%DVB, 100-200
mesh, 1.1-1.5 mmol/g, AlbatrossChem.Inc. Montreal, Can-
ada); Wang resin (1%DVB, 100-200 mesh, 1.0 mmol/g,
Sigma); Fmoc-protected amino acids (SynPep, Dublin, CA);
Boc-protected amino acid (Sigma); diisopropylcarbodiimide
(DIC); 1-hydroxybenzotriazole (HOBt); N,N-diisopropyl-
ethylamine (DIPEA); trifluoroethanol (TFE); trifluoroacetic
acid (TFA); acetic acid (AcOH); anhydrous hydrazine (H2N
NH2); piperidine; tetrabutylammonium fluoride (TBAF); O-
(benzotriazol-1-yl)-N,N,N',N'-tetramethyluro-nium tetrafluo-
roborate (TBTU); trimethylsilyldiazomethane (TMSCHN2);
dimethyl formamide (DMF); dichloromethane (CH2Cl2);
1
methanol (MeOH); ethanol (EtOH), Aldrich. H NMR spec-
tra were obtained on a Varian Gemini 2000 300 MHz NMR
spectrometer.
3.2. Procedures for Route 1
The tetrapeptide fragment was synthesized on a 2-
chlorotrityl resin using Fmoc and Boc protected amino acids.
Fmoc-L-phenylalanine (1.92g, 5.0 mmol) and DIPEA (1.74
mL, 10.0 mmol) were dissolved in dry CH2Cl2 (20 mL). A
small amount of DMF was added if the amino acid was not
completely dissolved in CH2Cl2 (just enough to facilitate
dissolution of the amino acid). The resulting solution was

686 Medicinal Chemistry, 2012, Vol. 8, No. 4
Nassim and Wang
Table 1. Opioid Receptor Binding Affinities of Target Ligands
IC50 (nM)
Ki (nM ± SEM)
Hill Slope ± SEM
Compd.
ꢀ
ꢁ
μ
ꢀ
ꢁ
μ
ꢀ
ꢁ
μ
9a
10a
9b
11.88
12.26
5.14
7.15
1.86
2.37
1.22
1.18
5.28
4.99
1.49
1.74
8.18 ± 2.49
8.44 ± 2.69
3.54 ± 1.15
4.93 ± 0.80
1.36 ± 0.48
1.76 ± 0.34
0.90 ± 0.24
0.87 ± 0.16
3.22 ± 0.30
1.15 ± 0.08
1.09 ± 0.06
1.16 ± 0.06
1.42 ± 0.11
1.36 ± 0.35
1.40 ± 0.15
1.67 ± 0.05
1.38 ± 0.20
1.11 ± 0.07
1.07 ± 0.04
1.09 ± 0.09
1.06 ± 0.00
3.05 ± 0.14
0.91 ± 0.09
1.06 ± 0.02
10b
Table 2. Opioid Agonist Properties of Target Ligands in the [35S]GTPꢁS Assay
Agonist
EC50 (nM) ± SEM
%Stim ± SEM
Compd.
___________________________________________ _____________________________________
ꢀ
ꢁ
μ
ꢀ
ꢁ
μ
9a
10a
9b
> 10,000
> 10,000
> 10,000
> 10,000
10.34 ± 1.39
7.96 ± 2.70
1.54 ± 0.32
3.32 ± 0.77
> 10,000
> 10,000
> 10,000
> 10,000
52.07 ± 1.42
55.84 ± 2.70
68.65 ± 11.36
50.26 ± 13.36
10b
Table 3. Opioid Antagonist Properties of Target Ligands in the [35S]GTPꢁS Assay
Antagonist
Ke (nM) ± SEM
pA2 ± SEM
Slope ± SEM
n
Compd.
____________________________ ____________________________ _______________________________ ___________
ꢀ
ꢁ
μ
ꢀ
ꢁ
μ
ꢀ
ꢁ
μ
ꢀ
ꢁ
μ
9a
10a
9b
11.33 ± 1.30
6.23 ± 0.87
3.56 ± 0.31
4.49 ± 0.36
2.76 ± 0.05
2.73 ± 0.24
0.40 ± 0.03
0.61 ± 0.08
7.99 ± 0.05
8.19 ± 0.06
8.44 ± 0.04
8.44 ± 0.03
8.59 ± 0.04
8.65 ± 0.04
9.43 ± 0.03
9.23 ± 0.05
-0.92 ± 0.03
-1.07 ± 0.03
-1.02 ± 0.03
-0.87 ± 0.03
-0.95 ± 0.04
-0.87 ± 0.03
-0.97 ± 0.03
-1.00 ± 0.02
5
5
5
4
5
5
5
6
10b
added to 2-chlorotrityl resin (1g, 1.0-1.5 mmol/g). The sus-
pension was shaken for 1h at rt. Subsequently MeOH (10
mL) was added, and shaking was continued for another 20
min. The resin was filtered, washed with CH2Cl2 (15 mL),
DMF (15 mL), and MeOH (15 mL). The first Fmoc depro-
tection could be carried out using 5% piperidine in
CH2Cl2/DMF (1/1) for 10 min. (this gives better resin swel-
ling) followed by 20% piperidine in DMF for 15 min. All
subsequent Fmoc deprotections could be done in the usual
way with 20% piperidine. Elongation of the peptide chain
could be achieved by using Fmoc-Glycine (890 mg, 3.0
mmol) or Boc-L-Tyrosine (843 mg, 3.0 mmol) with DIC
(0.47 mL, 3.0 mmol), HOBt (405 mg, 3.0 mmol) and DIPEA
(0.87 mL, 5.0 mmol) in DMF. Boc-protected tetrapeptide
fragment 5 (355 mg) could be obtained by treating the resin
ArH of Tyr), 6.61 (2H, d, J = 8.3 Hz, ArH of Tyr), 1.25 (9H,
s, 3 CH3 of BOC).
N-(t-Butyloxycarbonyl)tyrosylglycylglycylphenylalanine
Methyl Ester (6) To a solution of 5 (355 mg, 0.65 mmol) in
MeOH (5 mL) was added an excess of trimethylsilyldia-
zomethane (TMSCHN2). After stirring for 1 h, 1 drop of
acetic acid was added and the solvent was evaporated under
1
reduce pressure to yield 6 (353 mg, 97%). H NMR (300
MHz, DMSO) ꢀ 8.25-8.15 (4H, m, 4 NH of Tyr, Gly and
Phe), 7.33 (5H, m, ArH of Phe), 7.14 (2H, d, J = 8.1 Hz,
ArH of Tyr), 6.85 (2H, d, J = 8.1 Hz, ArH of Tyr), 3.41 (3H,
s, OCH3), 1.40 (9H, s, 3 CH3 of BOC).
N-(t-Butyloxycarbonyl)tyrosylglycylglycylphenylalanine
Hydrazide (7) A mixture of 6 (353 mg, 0.635 mmol) and
hydrazine hydrate 99% (200 ꢀL, 4 mmol)) in absolute EtOH
(5 mL) was stirred at rt overnight. A white precipitate was
filtered off, washed with cold EtOH and dried to give the
1
with TFE/AcOH/DCM (1/1/8) at rt for 30 min. H NMR
(300 MHz, DMSO) ꢀ 8.18-7.95 (4H, m, 4 NH of Tyr, Gly
and Phe), 7.21 (5H, m, ArH of Phe), 7.00 (2H, d, J = 8.3 Hz,

Synthesis and Biological Evaluation of Naloxone and Naltrexone-Derived
Medicinal Chemistry, 2012, Vol. 8, No. 4 687
1
corresponding hydrazide 7 (293 mg, 83%). H NMR (300
N-(9-Fluorenylmethoxycarbonyl)tyrosylglycylgly-
cylphenylalanine Methyl Ester (14) 640 mg of 14 (75%)
was prepared by the same procedure with 13 (650 mg, 1.26
mmol) as the starting material. H NMR (300 MHz, DMSO)
ꢀ 8.39 (4H, m, 4NH of Tyr, Gly and Phe), 3.67 (3H, s,
OCH3).
MHz, DMSO) ꢀ 9.18 (1H, s, CONHNH2), 8.18-8.01 (4H, m,
4 NH of Tyr, Gly and Phe), 7.21 (5H, m, ArH of Phe), 7.01
(2H, d, J = 7.9 Hz, ArH of Tyr), 6.61 (2H, d, J = 7.9 Hz,
ArH of Tyr), 4.20 (2H, m, CONHNH2), 1.30 (9H, s, 3 CH3
of BOC).
1
Tyrosylglycylglycylphenylalanine(hydrazino) Naloxone
Derivative (9a) and (10a) 145 mg of hydrazide 7 (0.27
mmol) was added to Naloxne (132 mg, 0.4 mmol) in abso-
lute EtOH (5 mL) containing 2 drops of glacial acetic acid.
The mixture was refluxed for 2 h and then stirred at rt over-
night. A solid product was separated by filtration, washed
with EtOH and dried by vacuum to yield 197 mg 8a (85%).
This intermediate was added to 15% TFA solution in CH2Cl2
(4 mL) and stirred for 10 min. Aqueous workup followed by
preparative TLC purification with CH2Cl2/MeOH (10/1) of
the crude product led to the separation of the corresponding
N-(9-Fluorenylmethoxycarbonyl)tyrosylglycylglycy-
lphenylalanine Hydrazide (15) 536 mg of 15 (86%) was
prepared by the same method of 7 as described above with
1
14 (625 mg, 0.92 mmol) as the starting material. H NMR
(300 MHz, DMSO) ꢀ 9.25 (1H, s, CONHNH2), 8.30-8.19
(4H, m, 4NH of Tyr, Gly and Phe), 4.42 (1H, m,
CONHNH2).
Tyrosylglycylglycylphenylalanine(hydrazino) Naloxone
Derivative (17a) and (18a) 250 mg of hydrazide 15 (0.36
mmol) was added to Naloxne (176 mg, 0.54 mmol) in abso-
lute EtOH (5 mL) containing 2 drops of glacial acetic acid.
The mixture was refluxed for 2 h and then stirred at rt over-
night. A solid product was separated by filtration, washed
with EtOH, and dried by vacuum to yield 309 mg 16a
(87%). 1-octanethiol (520 ꢀL, 3.0 mmol) followed by TBAF
(189 mg, 0.6 mmol) was then added to a solution of interme-
diate 16a (300 mg, 0.30 mmol) in DMF (5 mL) under the
nitrogen atmosphere. The mixture was stirred for 10 min. To
this was added bis(1-methyl-1H-tetrazol-5-yl) disulfide (414
mg, 1.8 mmol) and stirring was continued for 3 min. Then
5% NaHCO3 was added and the mixture was extracted with
EtOAc. Purification of the crude product by preparative TLC
with CH2Cl2/MeOH (10/1) yielded the corresponding Z and
1
Z and E hybrid opiates isomers. 9a (87 mg, 57%) H NMR
(300 MHz, DMSO) ꢀ 10.61 (1H, s, hydrazone hydrogen),
8.30-8.00 (4H, m, 4 NH of Tyr, Gly and Phe), 5.90 (1H, m,
H-18), 5.59 (2H, m, H-19), 4.98 (1H, s, H-5). 10a (58 mg,
1
28%) H NMR (300 MHz, DMSO) ꢀ 10.39 (1H, s, hydra-
zone hydrogen), 8.35-8.01 (4H, m, 4 NH of Tyr, Gly and
Phe), 5.82 (1H, m, H-18), 5.20 (2H, m, H-19), 4.79 (1H, s,
H-5).
Tyrosylglycylglycylphenylalanine(hydrazino) Naltrex-
one Derivativ (9b) and (10b) Naltrexone derived intermedi-
ate 8b (186 mg, 79%) was prepared from the condensation
of 145 mg hydrazide 7 (0.27 mmol) and Naltrexone (136 mg,
0.4 mmol). Z and E isomers of 9b and 10b were prepared by
1
E hybrid opiates isomers. 17a (118 mg, 52%) H NMR (300
1
the same procedure as describe above. 9b (83 mg, 56%) H
MHz, DMSO) ꢀ 10.85 (1H, s, hydrazone hydrogen), 8.20-
NMR (300 MHz, DMSO) ꢀ 10.87 (1H, s, hydrazone hydro-
gen), 8.14-7.95 (4H, m, 4 NH of Tyr, Gly and Phe), 5.21
(1H, s, H-5). 10b (55 mg, 28%) 1H NMR (300 MHz,
DMSO) ꢀ 10.28 (1H, s, hydrazone hydrogen), 8.22-7.90 (4H,
m, 4 NH of Tyr, Gly and Phe), 4.69 (1H, s, H-5).
7.95 (4H, m, 4 NH of Tyr, Gly and Phe), 5.81 (1H, m, H-18),
1
5.19 (2H, m, H-19), 4.87 (1H, s, H-5). 18a (65 mg, 28%) H
NMR (300 MHz, DMSO) ꢀ 10.42 (1H, s, hydrazone hydro-
gen), 8.35-8.09 (4H, m, 4 NH of Tyr, Gly and Phe), 5.88
(1H, m, H-18), 5.30 (2H, m, H-19), 4.85 (1H, s, H-5).
Tyrosylglycylglycylphenylalanine(hydrazino) Naltrex-
one Derivative (17b) and (18b) Naltrexone derived interme-
diate 16b (291 mg, 81%) was prepared from the condensa-
tion of 250 mg of hydrazide 15 (0.36 mmol) and Naltrexone
(184 mg, 0.54 mmol). Z and E isomers of 17b and 18b were
3.3. Procedures for Route 2: N-(9-Fluorenylmethoxy-
carbonyl)Glycylphenylalanine Methyl Ester (12)
1-octanethiol (3.8 mL, 22 mmol) followed by TBAF
(1.38 g, 4.4 mmol) was added to a solution of 11 (900 mg,
2.2 mmol) in DMF (10 mL) under the nitrogen atmosphere.
The mixture was stirred for 10 min. To this was added bis(1-
methyl-1H-tetrazol-5-yl) disulfide (3.0 g, 13.2 mmol) and
stirring was continued for 3 min. Then, DIPEA (1.4 mL, 8.0
mmol), Fmoc-Gly-OH (980 mg, 3.3 mmol), and TBTU (1.06
g, 3.3 mmol) were added in this order. The mixture was
stirred for 10 min. To this 5% NaHCO3 was added and the
mixture was extracted with EtOAc and isolated by column
chromatography on silica gel using gradient elution with
CHCl3 to CHCl3-CH3OH (20:1) to afford 12 (836 mg, 83%).
1H NMR (300 MHz, CDCl3) ꢀ 6.39 (1H, m, NH of Gly),
5.40 (1H, m, NH of Phe), 3.76 (3H, s, OCH3).
1
prepared by the same procedure. 17b (115 mg, 51%) H
NMR (300 MHz, DMSO) ꢀ 10.75 (1H, s, hydrazone hydro-
gen), 8.01-7.85 (4H, m, 4 NH of Tyr, Gly and Phe), 5.08
(1H, s, H-5). 18b (63 mg, 27%) 1H NMR (300 MHz,
DMSO) ꢀ 10.27 (1H, s, hydrazone hydrogen), 8.21-7.87 (4H,
m, 4 NH of Tyr, Gly and Phe), 4.68 (1H, s, H-5).
3.4. Binding Assays
Receptor binding studies were conducted on human
opioid transfected into Chinese hamster ovary (CHO) Cells.
The μ cell line was maintained in Ham’s F-12 supplemented
with 10% fetal bovine serum (FBS) and 400 μg/mL Ge-
neticin (G418). The ꢀ and the ꢁ cell lines were maintained in
Dulbecco’s minimal essential medium (DMEM) supple-
mented with 10% FBS, 400 μg/mL G418, and 0.1% penicil-
lin/streptomycin. All cell lines were grown to confluence and
then harvested for membrane preparation. The membrane for
functional assay was prepared in buffer A (20 mM HEPES,
10 mM MgCl2, and 100 mM NaCl at pH 7.4); the membrane
N-(9-Fluorenylmethoxycarbonyl)glycylglycylphenyl-
alanine Methyl Ester (13) 711 mg of 13 (79%) was prepared
by the same procedure with 12 (800 mg, 1.75 mmol) as the
starting material. 1H NMR (300 MHz, CDCl3) ꢀ 6.90 (1H, m,
NH of Gly), 6.75 (1H, m, NH of Gly), 5.64 (1H, m, NH of
Phe), 3.68 (3H, s, OCH3).

688 Medicinal Chemistry, 2012, Vol. 8, No. 4
Nassim and Wang
[2]
[3]
[4]
[5]
Zimmerman, D. M.; Leander, J. D. Selective opioid receptor ago-
nist and antagonist: research tools and potential therapeutic agents.
J. Med. Chem., 1990, 33, 895-902.
Aldrich, J. V. Analgesics. In Burger’s Medicinal Chemistry and
Drug Discovery, 5th ed.; Wolf, M. e., Ed.; John Wiley and Sons:
New York. 1996; Vol. 3. Pp. 321-441.
Schmidhammer, H. S. Opioid receptor antagonists. In Progress in
Medicinal Chemistry, Ellis, G. P., Luscombe, D. K., Oxford, A. W.
Eds.; Elsevier: New York, 1998; Vol. 35, pp. 83-132.
Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill, L. A.;
Morgan, B. A.; Morris, H. R. Identification of two related pen-
tapeptides from the brain with potent opiate agonist activity.
Nature, 1975, 258, 577-579.
for binding assays was prepared in 50 mM Tris buffer (pH
7.7). Cells were scraped from the plates and centrifuged at
500 ꢀ g for 10 minutes. The cell pellet was homogenized in
buffer with a polytron, centrifuged at 20, 000 ꢀ g for 20
min., washed and recentrifuged, and finally resuspended at 3
mg protein/mL in buffer to determine protein content. The
homogenate was then stored at -70°C in 1 mL aliquots.
Binding assays were conducted using [3H] DAMGO, [3H]
DPDPE, and [3H] U69, 593 at theμ, ꢀ, and ꢁ receptors, re-
spectively. The assay was performed in triplicate in a 96-
well plate. Nonspectific binding was determined with 1.0
μM of the unlabeled counterpart of each radioligand. Cell
membranes were incubated with the appreciate radioligand
and test compound at 25°C for 60 min. The incubation was
terminated by rapid filtration through glass fiber filter paper
on a Tomtec cell harvester. The filters were dried overnight
and bagged with 10 mL scitillation cocktail before counting
for 2 min on a Wallac Betaplate 1205 liquid scintillation
counter. Full characterization of compounds includes analy-
sis of the data for IC50 values and Hill coefficients by using
the program PRISM. Ki values were calculated using the
Cheng Prusoff transformation: Ki = IC50/ (1 + L/Kd) where
“L” is the radioligand concentration and “Kd” is the binding
affinity of the radioligand.
[6]
[7]
Morley, J. C. Chemistry of opioid peptides. Br. Med. Bull., 1983,
39, 5-10.
Chavkin, C.; Goldstein, A. Specific receptor for the opioid peptide
dynorphin: structure-activity relationships. Proc. Natl. Acad. Sci.
USA, 1981, 78, 6543-6547.
[8]
Metzger, T. G.; Paterlini, M. G.; Portoghese, P. S.; Ferguson, D. M.
Application of the message-address concept to the docking of
Naltrexone and selective Naltrexone-derived opioid antagonists
into opioid receptor models. Neurochem. Res., 1996, 21, 1287-
1294.
Gao, P.; Larson, D.L.; Portoghese, P.S. Synthesis of 7-aryl-
morphinans. Probing the “address” requirements for selectivity at
opioid ꢀ-recptors. J. Med. Chem., 1998, 41, 3091-3098.
Ananthan, S.; Kezar III, H. S.; Carter, R. L.; Saini, S. K.; Rice, K.
C.; Wells, J. L.; Davis, P.; Xu, H.; Dersch, C. M.; Bilsky, E. J.;
Porreca, F.; Rothman, R. B. Synthesis, opioid receptor binding, and
biological activities of Naltrexon-derived pyrido- and pyrimido-
morphinans. J. Med. Chem., 1999, 42, 3527-3538.
Lipkowski, A.W.; Tam, S.W.; Portoghese, P.S. Peptides as receptor
selectivity modulators of opiate pharmacophores. J. Med. Chem.,
1986, 29, 1222-1225.
Lipkowski, A.W.; Pasternak, G.W.; Misterek, K.; Gumulka, S.W.;
Pachocka, M. Synthesis and biological properties of new naltrex-
one-peptide hybrids. Regulatory Peptides, 1994, 53 (Suppl 1),
S127-S128.
[9]
[10]
[35S]GTPꢂS Binding Assays
[11]
[12]
Membrane prepared as described above was incubated
with [35S]GTPꢁS (50 pM), GDP (usually 10 μM), and the
test compound, in a total volume of 1mL, for 60 min at 25°C
(Traynor and Nahorski, 1995). Samples were filtered over
glass fiber filters and counted as described for the binding
assays. A dose response curve with a prototypical full ago-
nist (DAMGO, DPDPE, and U69, 593 for μ, ꢀ, and ꢁ recep-
tors, respectively) was conducted in each experiment to iden-
tify full and partial agonist compounds. High affinity com-
pounds (Ki value is 200 nM or less) that demonstrate no ago-
nist activity were tested as antagonists. A Schild analysis
(Schild, 1949) was conducted, using a full agonist dose re-
sponse curve in the presence of at least three concentrations
of the experiments. If the Schild slope was significantly dif-
ferent from -1.00, the antagonist activity was non-
[13]
[14]
Wang, M. L.; Nassim, B. E. Solid phase synthesis of novel hybrid
opiates. Tetrahedron Lett., 2003, 44, 1487-1489.
Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. 2-Chlorotrityl
chloride resin. Studies on anchoring of Fmoc-amino acids peptide
cleavage. Int. J. Pept. Protein Res., 1991, 37, 513-520.
Barlos, K.; Gatos, D.; Kapolos, S.; Poulos, C.; Schafer, W.; Yao W.
Q. Application of 2-chlorotrityl resin in solid phase synthesis of
[15]
[16]
(Leu15)-gastrin
I and unsulfated cholecystokinin octapeptide.
Selective O-deprotection of tyrosine. Int. J. Peptide protein Res.,
1991, 38, 555-561.
Barlos, K.; Gatos, D.; Kutsogianni, S.; Papaphotiou, G.; Poulos, C.;
Tsegenidis, T. Solid phase synthesis of partially protected and free
peptides containing disulphide bonds by simultaneous cysteine
oxidation-release from 2-chlorotrityl resin. Int. J. Peptide protein
Res., 1991, 38, 562-568.
Hashimoto, N.; Aoyma, T.; Shiori, T. A simple efficient prepara-
tion of methyl esters with trimethylsilyldiazomethane (TMSCHN2)
and its application to gas chromatographic analysis of fatty acids.
Chem. Pharm. Bull., 1981, 29, 1475-1478.
Al-afaleq, E. I.; Abubshait, A. L-amino acid esters studies: Part II:
Synthesis of N-(dimethoxy/3, 5-diacetoxybenzoyl)-L-amino acid
hydrazides and their reactions with aldehydes and ketones. Syn.
Commun., 1999, 29, 1317-1331.
competitive; in such cases, the A2 value was not reported.
P
Equilibrium dissociation constants (Ke values) were calcu-
lated as: Ke = a/(DR-1) where “a” is the nanomolar concen-
tration of the antagonist and “DR” is the shift of the agonist
concentration-response curve to the right in the presence of a
given concentration of antagonist.
[17]
[18]
ACKNOWLEDGEMENT
This project was supported by Indiana University Office
of Research & Graduate School, IU Southeast Office of Re-
search, and the James Y. McCullough Memorial Endow-
ment. Ligand binding and [35S]GTPꢁS assay data were pro-
vided by NIDA-OTDP.
[19]
[20]
[21]
Palla, G.; Predieri, G.; Domiano, P. Conformational behavior and
E/Z isomerization of N-acyl and N-aroylhydrazones. Tetrahedron,
1986, 42, 3649-3654.
Luke, M.G.; Hahun, E.F.; Price, M.; Pasternal, G.W. Irreversible
opiate agonists and antagonists: V. Hydrazone and acylhydrazone
derivatives of naltrexone. Life Sci., 1988, 43, 1249-1256.
Carpino, L.A.; Cohen, B.J.; Stephens, K.E.; Sadat-Aalaee, S.Y.;
Tien, J.H.; Langridge, D.C. (Fluoren-9-ylmethyoxy)carbonyl
(Fmoc)amino acid chlorides. Synthesis, characterization, and appli-
cation to the rapid synthesis of short peptide segments. J. Org.
Chem., 1986, 51, 3732-3734.
REFERENCES
[1]
Dhawan, B. N.; Cesselin, F.; Raghubir, R.; Reisine, T.; Bradley, P.
B.; Portoghese, P. S.; Hamon, M. International Union of Pharma-
cology. XII. Classification of opioid receptors. Pharmacol. Rev.,
1996, 48, 567-592.
[22]
Carpino, L.A. The 9-fluorenylmethyloxycarbonyl family of base-
sensitive amino-protecting groups. Acc. Chem. Res., 1987, 20, 401-
407.

Synthesis and Biological Evaluation of Naloxone and Naltrexone-Derived
Medicinal Chemistry, 2012, Vol. 8, No. 4 689
[23]
[24]
Ueki, M.; Nishigaki, N.; Aoki, H.; Tsurusaki, T.; Katoh, T. One-pot
deprotection and coupling of peptides by the Fmoc strategy. Chem.
Lett., 1993, 721-724.
Toll, L.; Berzetei-Gurske, I. P.; Polar, W. E.; Brandt, S. R.; Adapa,
I. D.; Rodriguez, L.; Schwartz, R. W.; Haggart, D.; O’Brien, A.;
White, A.; Kennedy, J. M.; Craymer, K.; Farrington, L.; Auh, J. S.
Standard binding and functional assays related to (NIDA) Medica-
tions Development Division testing for potential cocaine and nar-
cotic treatment programs. NIDA Res. Monogr. Series, 1998, 178,
440-466.
[27]
Heidbreder, C. A.; Babovic-Vuksanovic, D.; Shoaib, M.; Shippen-
berg, T. S. Development of behavioral sensitisation to cocaine: In-
fluence of kappa opioid receptor agonists. J. Pharmacol. Exp.
Ther., 1995, 275, 150-163.
Archer, S.; Glick, S. D.; Bidlack, J. Cyclazocine revisited. Neuro-
chem. Res., 1996, 21, 1369-1373.
Spealman, R. D.; Bergman, J. Opioid modulation of the discrimina-
tive stimulus effects of cocaine: Comparison of mu, kappa and
delta agonists in squirrel monkeys discriminating low doses of co-
caine. Behav. Pharmacol., 1994, 5, 21-31.
[28]
[29]
[25]
[26]
Traynor, J. R.; Nahorski, S. R. Modulation by mu-opioid agonists
of guanosine-5ꢀ-O- (3-[S-35] thio) triphosphate binding to mem-
branes from human neuroblastoma SH-SY5Y cells. Mol. Pharma-
col., 1995, 47, 848-854.
Mysels, D.; Sullivan, M.A. The Kappa-opiate receptor impacts the
pathology and behavior of substance use. Am. J. Addict., 2009,
18(4), 271-276.
[30]
[31]
Riviere, P.J.-M. Peripheral kappa-opioid agonists for visceral pain.
British J. Pharm., 2004, 141, 1331-1334.
Portenoy, R. K.; Caraceni, A.; Cherny, N. I.; Goldblum, R.; Ing-
ham, J.; Inturrisi, C. E.; Johnson, J. H.; Lapin, J.; Tiseo, P. J.;
Kreek, M. J. Dynorphin A(1-13) Analgesia in opioid-treated pa-
tients with chronic pain. Clin. Drug Invet., 1999, 17, 33-42.
Pan, Z. Z. μ–Opposing action of the ꢀ–opioid receptors. Trends
Pharmacol. Sci., 1998, 19, 94-98.
[32]
Received: January 19, 2011
Revised: July 01, 2011
Accepted: July 05, 2011